Information

How exactly is glyceraldehyde 3-Phosphate reverted to ribulose 1,5-bisphosphate for the continuation of the calvin cycle?


Around 6 molecules of G3P is produced at the end of the Calvin Cycle (light independent reactions of photosynthesis), and 5 of which are reverted back to RuBp.

The general equation that I read is 5 G3P -----> 3 RuBp using 3 ATP.

It however doesn't seem to add up (regarding balancing of the atoms) as 5 phosphates would be converted to 6.

I would appreciate a link to any resource explaining this process in detail.

Thank you and have a nice day ^^


You need to account for free phosphates (Pi) that derive from ATP and are released in phosphatase reactions. The regeneration of 3 ribulose-1,5-2P has the overall reaction

5 glyceraldehyde-3P + 3 ATP $ ightarrow$ 3 ribulose-1,5-2P + 3 ADP + 2 Pi

So in total eight phosphates (here counting ATP as 1) are redistributed, 6 of which end up in ribulose-1,5-2P, and two are released as free phosphates. (These Pi are then recaptured during ATP synthesis in the light-dependent reactions.)

The above is a summary reaction of course; the complete scheme involves 10 enzymatic reactions that rearrange carbons in various ways to form 5-carbon sugars from 3-carbon ones (which takes a bit of juggling of atoms). Release of free Pi occurs in the fructose biphosphatase and seduheptulose biphosphatase steps.

A detailed account can be found in any major biochemistry textbook, like Stryer's Biochemistry. Online resources like MetaCyc can also be helpful.


The nitrate and ammonium nitrate supply have a major influence on the response of photosynthesis, carbon metabolism, nitrogen metabolism and growth to elevated carbon dioxide in tobacco

The effect of elevated [CO2] on biomass, nitrate, ammonium, amino acids, protein, nitrate reductase activity, carbohydrates, photosynthesis, the activities of Rubisco and six other Calvin cycle enzymes, and transcripts for Rubisco small subunit, Rubisco activase, chlorophyll a binding protein, NADP-glyceraldehyde-3-phosphate dehydrogenase, aldolase, transketolase, plastid fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase was investigated in tobacco growing on 2, 6 and 20 m M nitrate and 1, 3 and 10 m M ammonium nitate. (i) The growth stimulation in elevated [CO2] was attenuated in intermediate and abolished in low nitrogen. (ii) Elevated [CO2] led to a decline of nitrate, ammonium, amino acids especially glutamine, and protein in low nitrogen and a dramatic decrease in intermediate nitrogen, but not in high nitrogen. (iii) Elevated [CO2] led to a decrease of nitrate reductase activity in low, intermediate and high ammonium nitrate and in intermediate nitrate, but not in high nitrate. (iii) At low nitrogen, starch increased relative to sugars. Elevated [CO2] exaggerated this shift. ADP-glucose pyrophosphorylase transcript increased in low nitrogen, and in elevated [CO2]. (iv) In high nitrogen, sugars rose in elevated [CO2], but there was no acclimation of photosynthetic rate, only a small decrease of Rubisco and no decrease of other Calvin cycle enzymes, and no decrease of the corresponding transcripts. In lower nitrogen, there was a marked acclimation of photosynthetic rate and a general decrease of Calvin cycle enzymes, even though sugar levels did not increase. The decreased activities were due to a general decrease of leaf protein. The corresponding transcripts did not decrease except at very low nitrogen. (v) It is concluded that many of the effects of elevated [CO2] on nitrate metabolism, photosynthate allocation, photosynthetic acclimation and growth are due to a shift in nitrogen status.


I. Introduction

The 14 inorganic elements required by plants to complete a full life cycle are coined the essential plant nutrients, and grouped into macronutrients and micronutrients on the basis of their concentration in plant dry matter. The macronutrients are comprised of nitrogen (N), phosphorus (P), sulphur (S), potassium (K), calcium (Ca) and magnesium (Mg). In situations in which a nutrient is not present in sufficient amounts to support its functional roles, it will lead to a state of deficiency, with specific responses characteristic for each nutrient (van Maarschalkerweerd & Husted, 2015 ). The severity of a nutrient deficiency can range from mild and transient to severe and chronic, and plants may experience multiple deficiencies during their lifespan, some even occurring simultaneously. In addition, widespread interactions with a range of different abiotic and biotic growth factors such as water, light, pests and pathogens, may lead to atypical symptoms of nutrient-related disorders.

On a global scale, various nutrient deficiencies have major negative consequences for crop production, resulting in reduced yields and a poor quality of food and feed. At the same time, inappropriate use of fertiliser may lead to pollution of terrestrial and aquatic environments, while fertiliser production itself is highly energy demanding, thus contributing to climate change (Sharpley et al., 2018 Thompson et al., 2019 ). In natural terrestrial ecosystems, nutrient availability is, next to that of water, the environmental factor that most strongly affects the responses of plants to climate change and their ability to cope with environmental stress, thus having a major effect on species biodiversity (Isbell et al., 2013 Lambers & Oliveira, 2019 Terrer et al., 2019 ).

A large body of knowledge concerning the functional properties of macronutrients in plants already exists (Hawkesford et al., 2012 ). However, in the recent years significant new knowledge on the molecular processes underlying the direct responses of plants to limitation of a given nutrient has appeared. In addition, starvation networks, describing how individual nutrients interact and cross-talk along the pathway from uptake to integration in metabolism, have been unravelled (Kopriva et al., 2015 Dong et al., 2017 Maeda et al., 2018 Courbet et al., 2019 Hu et al., 2019 Medici et al., 2019 ).

In this review, we highlight recent insights into the physiological and molecular functions of plant macronutrients. Special emphasis is given to linking their physiological functions with the typical deficiency symptoms that may appear when nutrient availability is suboptimal for proper growth. Understanding the coupling between visual deficiency symptoms and their interactions with the functional properties of the triggering nutrient(s) is relevant for breeding of new nutrient-efficient genotypes that are able to withstand unfavourable climatic events and adverse soil conditions. Moreover, a proper understanding and interpretation of visual deficiency symptoms will support the potential for sustainable intensification of crop production systems by enabling development of new technologies that support automatised and data-driven management practices based on bioimaging, remote sensing, in-field spectrometers and/or sensors, thereby providing a basis for timely application of nutrients to crops.


RESULTS

To improve our understanding of how plants respond to changes in the gaseous environment in which they grow, Arabidopsis thaliana ecotype Col-0 grown under standard growth conditions were transferred to different conditions anticipated to influence photosynthesis and photorespiration and analyzed at physiological, transcriptional, and primary metabolite levels. These measurements were carried out over a 24 h time course (for the experimental set-up see Figure 1) to obtain temporal resolution of the responses. The plants were grown in a 10/14 h day/night photoperiod under a light intensity of 150 µmol/m 2 per s and at a temperature of 20/16 °C (day/night) before being transferred to identical conditions with the exception of different CO2 concentrations of 100 and 155 Pa or different O2 concentrations of 14 or 40 kPa.

Following growth under standard growth conditions, plants were transferred at 07.00 a.m. to different growth conditions. Samples were harvested from the plants at seven consecutive time points at 2 h intervals.

High CO2 partial pressure results in higher rate of CO2 assimilation and decreased photorespiration rates

As a first experiment, we determined net CO2 assimilation rate (A), stomatal conductance to water vapor (gs), and internal CO2 concentration (Ci)/external CO2 concentration (Ca) as well as measuring the rates of dark respiration (Rd). Additionally, we calculated the rates of photorespiration (PR) under all appropriate experimental conditions (Tables 1, S1). The control condition showed relatively stable levels of photosynthesis as well as in the deduced rates of photorespiration. The rate of CO2 assimilation was elevated at all time points in both CO2 treatments and a reduction of photorespiration rates was also observed across the entire experiment (Table 1). Correspondingly, the deduced A/PR ratio was significantly higher at all time points for both treatments in comparison to those deduced for the control conditions. Furthermore, the Rd was initially increased in high CO2 conditions but only significantly so in the higher CO2 condition and the values were not significantly different from the control at the end of the night. In parallel to the increased assimilation rates, gs was reduced during the day and the night in plants exposed to high CO2 however, this was only significant at the 4 h time point (Table S1). The ratio of internal to ambient CO2 concentration (Ci/Ca) was invariant across the CO2 treatments (Table S1).

2 h 4 h 6 h 8 h 10 h 12 h 24 h
A (µmol CO2/m 2 per s)
CC 40 Pa CO2 and 21 kPa O2 4.69 ± 0.28 4.08 ± 0.06 4.60 ± 0.22 4.49 ± 0.08 4.44 ± 0.07
100 Pa CO2 7.56 ± 0.24 6.63 ± 0.42 7.31 ± 0.54 7.48 ± 0.16 7.61 ± 0.24
155 Pa CO2 9.63 ± 0.26 9.98 ± 0.47 9.57 ± 0.07 9.30 ± 0.64 9.84 ± 0.49
40 kPa O2 3.57 ± 0.22 3.61 ± 0.17 3.54 ± 0.10 3.58 ± 0.14 3.67 ± 0.12
14 kPa O2 5.23 ± 0.41 5.69 ± 0.18 5.88 ± 0.21 6.13 ± 0.19 6.13 ± 0.11
PR (µmol CO2/m 2 per s)
CC 40 Pa CO2 and 21 kPa O2 0.75 ± 0.03 0.70 ± 0.05 0.82 ± 0.09 0.75 ± 0.02 0.74 ± 0.04
100 Pa CO2 0.50 ± 0.05 0.42 ± 0.05 0.46 ± 0.08 0.45 ± 0.03 0.48 ± 0.03
155 Pa CO2 0.36 ± 0.06 0.38 ± 0.03 0.36 ± 0.03 0.40 ± 0.04 0.35 ± 0.01
40 kPa O2 0.63 ± 0.04 0.59 ± 0.05 0.56 ± 0.04 0.57 ± 0.05 0.59 ± 0.05
14 kPa O2 0.90 ± 0.09 0.98 ± 0.06 1.02 ± 0.03 1.09 ± 0.08 1.07 ± 0.06
Rd (µmol CO2/m 2 per s)
CC 40 Pa CO2 and 21 kPa O2 −0.49 ± 0.12 −0.60 ± 0.18
100 Pa CO2 −0.77 ± 0.14 −1.20 ± 0.45
155 Pa CO2 −1.71 ± 0.11 −0.41 ± 0.13
40 kPa O2 −0.30 ± 0.03 −0.13 ± 0.05
14 kPa O2 −0.28 ± 0.01 −0.57 ± 0.15
  • Rates of CO2 assimilation (A), photorespiration rates (PR), and dark respiration (Rd) analyzed during a day cycle. CC represents the control condition of the individual experiments. Values presented are means ± SE (n = 3). Bold values are significantly different to the corresponding control harvest point according to a two-way ANOVA (P < 0.05) for A and PR or to two-tailed Student's t-test (P < 0.05) for Rd.

Changes in external CO2 concentrations result in considerable metabolic changes

In general, elevated atmospheric CO2 stimulates C3 photosynthesis and inhibits photorespiration because high CO2 around Rubisco accelerates the carboxylation reaction while suppressing the competing oxygenation reaction and subsequently reducing the CO2 loss and energy costs associated with photorespiration (Long et al. 2004 Leakey et al. 2009 ). Given this fact, the changes in gas-exchange parameters documented above may well result in metabolic alterations. For this reason, we next analyzed the major pools of primary metabolites (Figure 2 Tables S2–S4). As would be expected, we could observe decreased levels of photorespiratory intermediates in both elevated CO2 treatments (100 and 155 Pa). Especially, significant decreased levels of glycolate, glycine, and glycerate were detected during the day in both treatments (Tables S3, S4). Additionally, a decrease of serine could be observed in plants treated with 155 Pa CO2 (Table S4). These changes are in agreement with the gas-exchange data. This could indicate that the photorespiration pathway is largely minimized under the employed conditions. Exceptions to this statement were the level of hydroxypyruvate (HP), which showed strong oscillations during the day but more or less no significant differences in comparison to the control condition. It is interesting to note that in both CO2 treatments decreased levels of organic acids (e.g. succinate, fumarate, and malate) could be observed at some harvest points (Tables S3, S4). Additionally, significantly decreased levels of 2-oxoglutarate were observed in the 100 Pa CO2 treatment. When examining sugars, a significant increase of maltose at both elevated CO2 partial pressures was observed (Tables S3, S4).

Analyzed metabolites of the photorespiratory pathway (A), tricarboxylic acid cycle and the closely related amino acid GABA (B), sugar metabolism (C), and other amino acids (D). The values represent relative metabolite levels compared to the control condition of 40 Pa CO2 of determinations of six independent samplings. Relative metabolite levels were obtained as element-wise ratios of the normalized mean profiles. Yellow presents the treatment of 100 Pa CO2 and green the 155 Pa CO2 treatment. The control condition corresponds to a value of 1 for every time series due to the normalization, and is omitted from the plots.

Conversely, amino acid contents showed little variation following exposure to 100 Pa CO2, with significant changes only observed in tryptophan (Table S3). Plants exposed to 155 Pa CO2, however, displayed significantly higher levels of glutamate, aspartate, tryptophan, and β-alanine in at least some harvest points in comparison to the control condition (Table S4). Additionally, decreased levels of isoleucine, threonine, and proline were observed in these plants, while slightly decreased levels of inositol and dehydroascorbate were additionally observed at the end of the day under this condition (Table S4).

Starch levels followed typical diel cycle (Smith and Stitt 2007 ), both under control and elevated CO2 conditions with the high CO2 treatment not resulting in any alteration in starch levels at any time point (Figure 3).

Starch contents in µmol/g fresh weight. Values presented are means ± SE of determinations of three independent samplings. Black represents the control condition of 40 Pa CO2, yellow the treatment of 100 Pa CO2, and green the 155 Pa CO2 treatment.

Changes of gene expression in different CO2 concentrations

To provide more information concerning possible adjustments of primary metabolism in response to changes in the CO2 partial pressure we next used a quantitative real-time polymerase chain reaction (Qrt-PCR) approach to analyze transcript levels of 25 enzymes involved in the major pathways of plant metabolism including photorespiration, tricarboxylic acid (TCA) cycle, nitrogen metabolism, and sucrose and starch metabolism (Figure 4 Tables S5–S7). In general, few significant changes could be observed and only small variations in transcript levels were detected under the different CO2 partial pressures. However, decreased values of transcripts after 2 h were observed following transfer to both new CO2 regimes in comparison to the control condition. The levels of transcripts such as adenosine 5′-diphosphate glucose pyrophosphorylase (AGPase, At5g48300), catalase 2 (CAT2, At4g35090), ferredoxin-dependent glutamate synthase (Fd-GOGAT, At5g04140), glutamate dehydrogenase (GDH, At5g18170), glycolate oxidase 1 (GOX1, At3g14415), peroxisomal, cytosolic, and plastidial HP reductase (HPR1, At1g68010 HPR2, At1g79870 HPR3, At1g12550), Rubisco activase (AT2G39730), serine:glyoxylate aminotransferase (SGT, At2g13360), serine hydroxymethyltransferase 1 and 2 (SHM1, At4g37930 SHM2, At5g26780), and sucrose phosphate synthase (SPS, At5g20280) all decreased (Figure 4 Tables S6, S7). Additionally, the photorespiratory genes analyzed were largely unaltered in comparison to the control with the exception of the transcript levels of glycine decarboxylase H-protein (GLDH1, At2g35370) which were decreased in nearly all harvest points in 155 Pa CO2-treated plants (Figure 4 Tables S6, S7). Moreover, it was observed that the GDH transcripts were increased in both CO2 treatments in comparison to the control levels but only in the night.

Values represent relative changes of transcripts to the control condition of 40 Pa CO2 of three biological replicates. Relative transcriptomic data were obtained as element-wise differences of the normalized mean profiles. Yellow represents the CO2 treatment of 100 Pa and green the CO2 treatment of 155 Pa. The control condition corresponds to a value of 0 for every time series due to the normalization, and is omitted from the plots.

Changes in O2 partial pressure results in expected changes in photosynthesis and photorespiration

As mentioned before, Rubisco cannot completely discriminate between CO2 and O2 (Woodrow and Berry 1988 ) and the gases thus compete with one another at the active site of Rubisco. The ratio between intrachloroplastic concentrations of CO2 and O2 defines the in vivo reactions catalyzed by Rubisco. Therefore, to alter the rate of photorespiration, different O2 concentrations were tested. Wild-type Col-0 plants growing under normal growth conditions were transferred to a high O2 partial pressure of 40 kPa in order to upregulate the photorespiratory pathway, and to a low O2 partial pressure of 14 kPa, in attempt to inhibit the pathway.

First, we determined A, gs, Ci/Ca, Rd and calculated PR under different O2 partial pressure conditions (Tables 1, S1). As anticipated, plants exposed to 40 kPa O2 showed reduced rates of A whereas those exposed to reduced O2 partial pressure exhibited increased A values (Table 1). The computed photorespiration rates showed largely unaltered values following exposure to 14 kPa O2 and were significantly increased in comparison to the control condition in plants exposed to 40 kPa. However, the deduced A/PR ratios were significantly increased when plants were treated with 14 kPa O2 and decreased in 40 kPa O2 conditions compared with that deduced for the control. Additionally, an increase in the gs was observed at the low O2 partial pressure, however, this was only significant at the 10 h time point (Table S1). As for the CO2 treatments, the ratio of internal to ambient CO2 concentration (Ci/Ca) was invariant across the O2 treatments (Table S1).

Sudden variations in O2 concentrations lead to metabolic reprogramming

It was shown before, that the computed photorespiratory rates showed significant increased rates in plants which were treated with high O2 partial pressure of 40 kPa in comparison to the control condition. Surprisingly, the rates of photorespiration were largely unaltered following exposure to 14 kPa O2.

As a first step, we again analyzed the major pools of primary metabolites (Figure 5 Tables S8–S10). As expected, the high O2 partial pressure results in increased levels of photorespiratory intermediates such as glycolate, glycine, and glycerate, while serine showed increased levels during the night, but decreased levels during the light period (Figure 5A Table S9). In addition, HP showed significantly decreased values in the 40 kPa O2 partial pressure in comparison to the control condition. In the 14 kPa O2 partial pressure treatment, not many changes could be observed in comparison to the control condition, in which only decreased levels of glycerate were detected (Table S10).

Analyzed metabolites of photorespiratory pathway (A), tricarboxylic acid cycle and the closely related amino acid GABA (B), sugar metabolism (C), and some amino acids (D). The values represent relative metabolite levels compared to the control condition of 21 kPa O2 of determinations of six independent samplings. Relative metabolite levels were obtained as element-wise ratios of the normalized mean profiles. Red represents the O2 treatment of 40 kPa, and blue the O2 treatment of 14 kPa. The control condition corresponds to a value of 1 for every time series due to the normalization, and is omitted from the plots.

On analyzing the TCA cycle intermediates, no consistent behavior could be detected. On one hand, pyruvate levels were slightly reduced in the 40 kPa O2 concentration in comparison to the control condition. On the other hand, increased levels of succinate and malate were detected as well as increased levels of 2-oxoglutarate in all harvest points in the 40 kPa O2 treatment (Figure 5B Table S9). The low O2 partial pressure treatment resulted in very few changes in TCA cycle intermediates in comparison to the control condition. Only slightly increased values of succinate in some harvest points were observed (Table S10). A general decrease in sugar levels was observed at the high O2 partial pressure (Figure 5C Table S9). This is clearly observed for glucose and sucrose but also a trend of reduction in the values of fructose was detected (Figure 5C Table S9). Additionally, maltose also showed reduced levels during the night at the 40 kPa O2 partial pressure. In contrast, the low O2 concentration resulted in slightly increased sugar values, especially glucose, maltose, fructose, and trehalose, at least in some harvest points (Figure 5C Table S10). By contrast, amino acids showed very strong variations following this treatment in comparison to the control condition (Figure 5D Tables S9, S10). Namely, high O2 levels resulted in significantly decreased levels of glutamine, aspartate, asparagine, alanine, proline, and threonine. The strongest effects were observed for aspartate (reduced to approximately one-fifth of the control content) and alanine (also reduced to approximately one-fifth of the control content). Having said that, increased levels of isoleucine, phenylalanine, tyrosine, and valine were also observed in the high O2 concentration. The low O2 treatment seemed to have little effect on primary metabolic pathways in comparison to the high O2 treatment and showed very few changes in the levels of amino acids.

As for the CO2 treatment, starch levels in the O2 experiment followed typical diel transitions (Figure 6). However, the effect of the change in gas environment invoked greater effects in this instance with the high O2 treatment displaying decreased starch levels at four time points and the low O2 treatment displaying elevated starch content at two time points (but also a decreased starch content at one time point). It is well known that a higher concentration of O2 leads to higher oxygenation and reduced carboxylation and thus reduces photosynthetic carbon fixation and reduced starch contents (Ogren 1984 Moroney et al. 2013 ). The opposite can be observed by reducing the O2 concentration, which results in slightly increased starch levels in comparison to the control condition because the oxygenation reaction of Rubisco is inhibited and the carboxylation reaction is increased.

Starch contents in µmol/g fresh weight. Values presented are means ± SE of determinations of three independent samplings. Black represents the control condition of 21 kPa O2, red the O2 treatment of 40 kPa, and blue the O2 treatment of 14 kPa. Asterisks demarcate significant changes to the corresponding control harvest point according to two-tailed Student's t-test (P < 0.05).

Changes in gene expression following exposure to different O2 concentrations

When analyzing the gene expression of 25 enzymes involved in the major pathways of plant metabolism, we observed a general decrease in transcript levels in most of the measured genes at least in samples which were harvested during the light period when plants were transferred to elevated O2 (Figure 7 Tables S11, S12). In contrast, not many changes were observed in transcript levels relative to the control condition in plants transferred to 14 kPa O2 (Figure 7 Table S13). In several measured genes, an increase of transcripts after 2 h was observed under low O2 conditions, for example, AGPase, aspartate aminotransferase (AspAT, At2g30970), CAT2, Fd-GOGAT, fumarase (FUM, At2g47510), glycine decarboxylase P-protein (GLDP, At4g33010), HPR1, HPR3, nicotinamide adenine dinucleotide phosphate-dependent isocitrate dehydrogenase (NADP-ICDH, At1g54340), 2PG phosphatase (PGLP, AT5g36790), Rubisco activase, serine decarboxylase (SDC, At1g43710), serine glyoxylate aminotransferase (SGT, At2g13360), SHM2, and SPS (Figure 7 Table S13). However, after this time point, the levels of these transcripts reverted to resemble their levels in the control condition.

Values represent relative changes of transcripts to the control condition of 21 kPa O2 of three biological replicates. Relative transcriptomic data were obtained as element-wise differences of the normalized mean profiles. Red represents the high O2 treatment of 40 kPa and blue the low O2 treatment of 14 kPa. The control condition corresponds to a value of 0 for every time series due to the normalization, and is omitted from the plots.

Statistical analyses of control experiments and the global datasets

Before carrying out the clustering analyses of the combined datasets from the different treatments, we investigated the data from the two independent controls. Because the corresponding profiles from the two controls could not be overlaid, we first determined a simple normalization strategy, which renders the independent controls comparable. To this end, we first determined the similarity between the two datasets by using the Rv coefficient (see “Materials and Methods” section) in the following four scenarios: (i) raw data (ii) data normalized to the mean of the first time point (2 h) (iii) data normalized to the mean of the last time point (24 h) and (iv) data normalized to the mean over the entire time domain. For the metabolic profiles, the Rv coefficients for these four scenarios were 0.01, 0.12, 0.37*, and 0.51*, respectively, while for the transcript profiles, the Rv coefficients were 0.34, 0.26, 0.11, and 0.46*, respectively (*denotes the statistically significant). As the data normalized to the mean over the entire time domain resulted in the highest Rv coefficient (i.e. similarity) for both types of data, we used this normalization in the remainder of the integrative data analysis.

This normalization also suggests that the majority of metabolites share the same temporal shape of profiles, thus supporting the qualitative correspondence between the two independent controls. To make this more precise, for each metabolite we determined the correlations with all other metabolic profiles in each control. We then found the correlation between the vectors of Pearson correlation coefficients resulting from the two controls. Over all of the metabolites, the largest correlation was 0.43, while the smallest was −0.48. The presence of a negative correlation indicates that the shape of the profiles for some metabolites was different between the two controls. We then selected all 35 metabolites for which the correlations, determined by the outlined procedure, were higher than 0. Interestingly, when the same procedure was repeated only on the so selected metabolites, the correlations ranged 0–0.73 with a mean of 0.43, supporting the claim. The metabolites that showed highest divergence between the two controls included aspartate, fumarate, GABA, glutamate, homoserine, inositol, spermidine, succinate, trehalose, and tryptophan.

In order to evaluate the relationship between the levels of photosynthetic parameters and the relative levels of both transcripts and metabolites in the experiments of different CO2 and O2 concentrations, K-medoid clustering analysis was used (Figures 8, S1). Two separate clusterings were carried out, one in which the assimilation rate and deduced rate of photorespiration were included and, therefore, only time points receiving illumination were clustered (Figure 8), and the second using all time points but not including the assimilation rate and deduced rate of photorespiration (Figure S1). Interestingly, when the illuminated time points are compared, the photorespiratory metabolites glycine and glycolate co-cluster with the assimilation rate and the estimated photorespiratory rate. There was, however, little co-regulation between photorespiratory metabolites and transcripts with the exception of HP clustering with the majority of photorespiratory transcripts measured. Exceptions are the glycerate kinase (GLYK) and the glycine decarboxylase H-protein (GLDH). In this analysis, GLDH co-clustered with the photorespiratory intermediate glycerate in cluster (F) (Figure 8). On addition of the data from samples harvested in the dark (and omission of the rates of assimilation and photorespiration), the clustering pattern is largely similar however, in this instance glycine co-clusters with glycerate but not any more with glycolate. Additionally, in this analysis, the photorespiratory intermediates glycolate, glycine, serine, and glycerate are not co-clustered with any of the transcripts measured while HP clusters to all of the measured transcripts with the exception of GLYK (Figure S1). These data indicate that the majority of the regulation of photorespiratory pathway is not mediated at the level of transcription. Intriguingly, however, the level of HP was co-clustered with the expression level of photorespiratory genes.

K-medoid clustering of the relative metabolite and transcript levels for different CO2 and O2 concentrations was conducted by the cluster package in the R statistical environment with the distance matrix derived from the matrix of Pearson correlations. Text color indicates that blue presents relative metabolite levels, green presents relative transcript levels, and red indicates photosynthetic parameters. Line colors indicate corresponding to different clusters (A–F).


DISCUSSION

During the last years, the core enzymes of the photorespiratory cycle have been placed under intensive analyses at both the molecular and biochemical levels (for review, see Bauwe et al., 2010). With the exception of the multigene-encoded glycolate oxidase, Arabidopsis mutants could be isolated for all of these enzymes. Taken together, such mutants share one common feature, their strong sensitivity to air, which can easily be reverted by cultivation in elevated concentrations of CO2. This situation is long known from studies with chemically generated mutants ( Somerville, 2001), but, more recently, it was shown that several exceptions exist. First, high-CO2 conditions cannot rescue mutants without Gly decarboxylase activity ( Engel et al., 2007). This is because this particular enzyme not only functions in the photorespiratory cycle but also in one-carbon metabolism and therefore is absolutely necessary for plant development under any conditions. Second, we previously showed that, contrary to what was commonly thought, photorespiratory HP reduction is not restricted to peroxisomes but, via HPR2, can also occur in the cytosol, resulting in an atypically low air sensitivity of hpr1 mutants ( Timm et al., 2008). Third, photorespiration has specific links to folate ( Collakova et al., 2008) and energy metabolism ( Sweetlove et al., 2006). The photorespiratory pathway is thus integrated into whole-cell metabolism in a more complex manner than previously considered. While most of the other photorespiratory mutants are not very useful to investigate connections with other pathways, on account of their conditional lethal phenotypes in ambient air, the HPR mutants are not constrained in this manner. Utilizing the moderate phenotype of HPR mutants, connections to other pathways could be studied and compared with the wild-type situation. Using this approach, we observed that knocking out HPR1 and HPR2 invokes effects that are not restricted to photorespiration. In this previous study, we observed links to tricarboxylic acid cycle intermediates, amino acid metabolism, and ethanolamine, which acts as a precursor of choline biosynthesis ( Timm et al., 2008). Such cross talk and connections are still under investigation, but the HPR mutants appear to be a very useful tool to address such interactions.

This study presents evidence that HP reduction is not even restricted to peroxisomes and cytosol but involves chloroplasts too, albeit to a lesser extent. By the identification of a novel 2-hydroxyacid dehydrogenase as an enzyme with HP and Glx reductase activity ( Fig. 1), we could enlarge the general importance of the reduction of these two metabolites. Since the newly identified HPR3 is predicted to be located in chloroplasts (http://aramemnon.botanik.uni-koeln.de/tm_sub.ep?GeneID=13660&modelID=0 Schwacke et al., 2003 Yu et al., 2008), a third compartment is likely involved in the reduction of these photorespiratory intermediates. As we show by metabolite analyses of HPR3 single knockouts ( Fig. 3), some photorespiratory intermediates (glycolate, Gly, Ser, 3HP, and glycerate) are slightly altered, which indicates a disruption of the photorespiratory cycle, thus further cementing the association of HPR3 function to that of photorespiration. Furthermore, we discovered changes in the levels of α-ketoglutarate, Glu, and Gln, which indicate a shift in the carbon-nitrogen balance of the mutants. These effects are consistent with those observed following the knockout of other photorespiratory genes ( Igarashi et al., 2006 Eisenhut et al., 2008 Timm et al., 2008). The higher levels of Ala, Asn, and Asp additionally indicate an impairment in nitrogen assimilation and may be regulated by the increased levels of Gln. The altered Thr level could further support this notion or alternatively could be due to the conversion of the Ser, which accumulates, to alternative metabolites. Moreover, the combined deletion of HPR1, HPR2, and HPR3 leads to strong air sensitivity, which, as mentioned above, is a cardinal property of all other photorespiratory mutants. In this triple mutant, not only the CO2 compensation point and net CO2 uptake were strongly inversely influenced ( Fig. 9) but also the photochemical efficiency of PSII after transfer of the mutant from elevated to air levels of CO2 ( Fig. 8).

This latter observation corresponds well to a previous report that impairment of the photorespiratory cycle and, as a consequence, the Calvin cycle leads to pronounced photoinhibition by suppression of the synthesis of the D1 subunit of PSII ( Takahashi et al., 2007a). Apart from this impact on linear electron transport, elevated levels of photorespiratory metabolites can inhibit carbon flux through the Calvin cycle. This is not restricted to the 2PG effects mentioned in the introduction. It is also known, for example, that Glx inhibits Rubisco activase and hence Rubisco ( Chastain and Ogren, 1989 Campbell and Ogren, 1990 Häusler et al., 1996), and Gly accumulation is toxic due to binding of Mg 2+ by complex formation ( Eisenhut et al., 2007). In addition, the elevated oxygen dependency of γ in all HPR mutants indicates higher rates of decarboxylation of photorespiratory metabolites, for example Ser ( Rontein et al., 2003) or HP ( Hedrick and Sallach, 1961), outside the core photorespiratory cycle. This is because the slope γ of the γ-versus-oxygen response curve is essentially codetermined by two factors, the Rubisco specificity factor, which is invariable in a given species, and the stoichiometry (i.e. tightness) of the photorespiratory cycle ( Farquhar et al., 1980). This stoichiometry of ribulose 1,5-bisphosphate oxygenation versus photorespiratory CO2 release, which would be exactly 2 if only Gly becomes decarboxylated, apparently shows some variation dependent on the fraction of additional decarboxylation reactions. This fraction could well be enhanced by environmental conditions such as higher light intensities and/or temperatures ( Hanson and Peterson, 1986) and the intactness of photorespiratory metabolism (this work). Interestingly, mutants of the peroxisomal malate dehydrogenases support these results, because oxygen-dependent γ values are also changed in these plants ( Cousins et al., 2008). Concerning the fact that both isoforms are responsible for NADH supply of the peroxisomes, the double knockout is quite similar to HPR1 mutants and seems to be a good confirmation of the results presented here. Unfortunately, no other defined photorespiratory mutants of Arabidopsis have been analyzed in this context, and it remains open if this is an exclusive feature of mutants with a defective HP-to-glycerate conversion step.

The above-mentioned conclusion is further substantiated by the analysis of the leaf amino acid content of the mutants, which revealed a distinct accumulation of the photorespiratory intermediate Ser ( Fig. 10). This is perhaps not overly surprising, since this amino acid is the direct precursor of HP ( Liepman and Olsen, 2001), but the enhanced levels could indeed trigger additional decarboxylating reactions, most prominently to ethanolamine, as we have shown previously ( Timm et al., 2008), and therefore affect γ in HPR mutants. Nevertheless, this enhancement is not restricted only to Ser but also includes the metabolically related amino acids Thr and Trp and additionally influences the steady-state level of Gly. The latter is consistently increased in all mutants but showed no response to the level of HPR mutation.

In addition to increased decarboxylating reactions of photorespiratory metabolites, the ratio of respiration in the light to the maximum rate of Rubisco carboxylation could also influence γ. This also seems to be a likely explanation for the altered values in HPR mutants. This notion is supported by a previously radiogasometric study of HPR1 mutants, which show higher respiratory rates of primary and stored photosynthates in the dark and the light ( Supplemental Table S1 in Timm et al., 2008 Pärnik and Keerberg, 2007). It seems reasonable that these reactions become pronounced with the level of HPR mutation and therefore lead to increased values of γ.

As we previously demonstrated, knocking out HPR1 does not lead to the typical phenotype associated with the knockout of most other photorespiratory enzymes ( Timm et al., 2008). Interestingly, given the daylength-dependent phenotype we demonstrated here ( Figs. 5 and 7), it seems likely that gene redundancy is only part of the reason behind this phenomenon. It is a general observation that plants that are grown in short days become carbon starved during a prolonged night ( Matt et al., 1998 Graf et al., 2010). This effect is exacerbated in HPR1 mutants, also indicating that there is a possible connection of photorespiration and carbon fixation during the Calvin cycle. Most likely, this is mediated by reduced 3PGA recycling resulting from the disrupted photorespiratory flux in combination with an outflow of metabolites from the cycle. The photorespiratory flux is clearly not optimal in hpr1 mutants under these conditions, which enhances the general short-day effect described above and as suggested by decreased Glc levels at the end of the night under short-day conditions ( Fig. 7).

Here, we report to our knowledge a previously unknown enzyme that is able to reduce both HP and Glx, two of the key metabolites within the photorespiratory cycle, and hence could directly or indirectly contribute to photorespiration in higher plants. Considering its dual substrate specificity and its preference for NADPH as the cosubstrate, the identified HPR3 could support peroxisomal and cytosolic HPRs ( Timm et al., 2008) as well as chloroplastidial and cytosolic glyoxylate reductase ( Allan et al., 2009) for an optimal reduction of these intermediates. Given the reported chloroplastic localization of HPR3 ( Yu et al., 2008), its exact physiological role is not absolutely clear however, the observed effects on photosynthesis ( Fig. 9) and the normalization of the elevated levels of several diagnostic amino acids by elevated CO2 ( Fig. 10) clearly suggest that its function is indeed associated with photorespiration. Unfortunately, no HP transporter was identified yet ( Reumann and Weber, 2006) therefore, it is not known how this photorespiratory intermediate gets into chloroplast. Since the chemical structure of HP is similar to glycerate, it can be hypothesized that import could possibly occur via the reported glycerate/glycolate transporter ( Howitz and McCarty, 1986). In addition to the reduction of HP, the newly identified HPR3 could also contribute to the reduction of chloroplastidially generated Glx, which, as discussed above, is an inhibitor of several enzymes involved in photosynthetic CO2 fixation. This, however, is not very likely, since a plastidial Glx reductase exists ( Simpson et al., 2008) and the single knockout of HPR3 does not cause drastic effects ( Fig. 5).

Leaf amino acid analysis reveals strong accumulation of Ser and related metabolites. Plants were grown in elevated CO2 (0.15%) and in normal air with a 10/14-h day/night cycle. Leaves from six individual plants at developmental stage 5.1 ( Boyes et al., 2001) were harvested in the middle of the photoperiod, and soluble amino acids were analyzed by HPLC. Mean amino acid contents ± sd are shown. Asterisks indicate significant changes according to Student’s t test (P < 0.05 for the corresponding wild-type sample [*], hpr1-1 [**], and hpr1xhpr2 [***]). FW, Fresh weight.

Leaf amino acid analysis reveals strong accumulation of Ser and related metabolites. Plants were grown in elevated CO2 (0.15%) and in normal air with a 10/14-h day/night cycle. Leaves from six individual plants at developmental stage 5.1 ( Boyes et al., 2001) were harvested in the middle of the photoperiod, and soluble amino acids were analyzed by HPLC. Mean amino acid contents ± sd are shown. Asterisks indicate significant changes according to Student’s t test (P < 0.05 for the corresponding wild-type sample [*], hpr1-1 [**], and hpr1xhpr2 [***]). FW, Fresh weight.

The construction of a chloroplast protein interaction network in Arabidopsis emphasizes an additional role of HPR3 in plant metabolism ( Yu et al., 2008). From these analyses, it appears that HPR3 could interact with three other chloroplast proteins, two of which, 3PGA dehydrogenase (At1g17745) and phosphoserine aminotransferase (At4g35630), are related to Ser synthesis. The third interacting protein, Ser hydroxymethyltransferase (At4g32520), is related to Gly synthesis and one-carbon metabolism ( Zhang et al., 2010). Thus, HP could be generated in glycolytic routes from 3PGA, most likely the phosphoserine pathway. This pathway is best described in bacteria but also is found in plants, mainly in heterotrophic issue or during the night when the photorespiratory flux is not active ( Ho et al., 1998 Muñoz-Bertomeu et al., 2009). Supportive evidence for this hypothesis is the accumulation of Gly, which we observed in the HPR3 single mutants. If HPR3 works in the phosphoserine pathway, the deletion of this enzyme would be expected to lead to a disrupted flow, which would explain the altered Gly-to-Ser interconversion in the mutants. However, the exact mechanism behind these changes is not yet known, and it would be very informative to analyze mutants within the phosphoserine pathway under nonphotorespiratory and photorespiratory conditions as well as in combination with different HPR mutations. It seems likely that alternative Ser-synthesizing pathways, like the phosphoserine-metabolizing route, interact with the photorespiratory pathway and, therefore, the HPR triple mutant shows further growth retardation.

In conclusion, although the role for HPR3 will need further investigation, this study presents strong indications that the enzyme is associated with the photorespiratory process and can at least partially complement the function of the peroxisomal HPR1 and the cytosolic HPR2. Moreover, HPR3 could display a possible link of photorespiration to other Ser-synthesizing pathways.


4. Cofactors, and the emergence and centralization of metabolic control

Cofactors form a unique and essential class of components within biochemistry, both as individual molecules and as a distinctive level in the control over metabolism. In synthesis and structure they tend to be among the most complex of the metabolites, and unlike amino acids, nucleotides, sugars and lipids, they are not primary structural elements of the macromolecular components of cells. Instead, cofactors provide a limited but essential inventory of functions, which are used widely and in a variety of macromolecular contexts. As a result they often have the highest connectivity (forming topological 'hubs') within metabolic networks, and are required in conjunction with key inputs or enzymes [200–202] to complete the most elaborate metabolisms.

Cofactor chemistry is in its own right an essential component of the logic underlying metabolic architecture and evolution. We argued in section 2.2 that part of the structure of the small-molecule substrate network is explained by reaction mechanisms and autocatalysis in short-loop pathways, which may once have been supported by external mineral catalysts. At least since the first cells, however, all such pathways have been realized only with the essential participation of intermediates from the hierarchically and functionally distinct cofactor class, which add a second layer of network-catalytic feedback. The more structurally complex cofactors tend to be associated with more catalytically complex functions within carbon fixation. Their long synthetic pathways result in long feedback loops, creating new needs for pathway stabilization and control. Because cofactors often mediate kinetic bottlenecks in metabolism, their inventory of functions may constrain the evolutionary possibilities for new pathways, so innovations in cofactor synthesis can have dramatic consequences for the large-scale structure of evolution.

As we note below, cofactors are among the less well-understood components of metabolism. Our ability to decompose cofactor functions and reconstruct the likely history of their elaboration is therefore less comprehensive than the analysis we have given of the small-molecule substrate. However, many functions that divide the cofactors into groups, which seem also to have been responsible for cases of convergent evolution and have perhaps stabilized major functional categories, relate directly to properties of particular chemical elements. Others are molecular properties shared as a consequence of derivation from a common precursor. In this section we select aspects of cofactor chemistry that seem to us most essential to overall metabolic architecture and evolution, with the goal of framing as much as of answering questions. As our understanding of cofactor chemistry improves through laboratory studies, so will our ability to integrate the observations in this section into a more complete theory of metabolic architecture and evolution.

4.1. Introduction to cofactors as a group, and why they define an essential layer in the control of metabolism

4.1.1. Cofactors as a class in extant biochemistry

The biosynthesis of cofactors involves some of the most elaborate and least understood organic chemistry used by organisms. The pathways leading to several major cofactors have only very recently been elucidated or remain to be fully described, and their study continues to lead to the discovery of novel reaction mechanisms and enzymes that are unique to cofactor synthesis [203–205]. While cofactor biosynthetic pathways often branch from core metabolic pathways, their novel reactions may produce special bonds and molecular structures not found elsewhere in metabolism. These novel bonds and structures are generally central in their catalytic functions.

Structurally, many cofactors form a class in transition between the core metabolites and the oligomers. They contain some of the largest directly-assembled organic monomers (pterins, flavins, thiamin, tetrapyrroles), but many also show the beginnings of polymerization of standard amino acids, lipids or ribonucleotides. These may be joined by the same phosphate ester bonds that link RNA oligomers or aminoacyl-tRNA, or they may use distinctive bonds (e.g. 5'–5' esters) found only in the cofactor class [206].

The polymerization exhibited within cofactors is distinguished from that of oligomers by its heterogeneity. Srinivasan and Morowitz [51] have termed cofactors 'chimeromers', because they often include monomeric components from several molecule classes. Examples are CoA, which includes several peptide units and an ATP folates, which join a pterin moiety to para-aminobenzoic acid (PABA) quinones, which join a PABA derivative to an isoprene lipid tail and a variety of cofactors assembled on phosphoribosyl-pyrophosphate (PRPP) to which RNA 'handles' are esterified.

We may understand the border between small and large molecules, where most cofactors are found, as more fundamentally a border between the use of heterogeneous organic chemistry to encode biological information in covalent structures, and the transition to homogeneous phosphate chemistry, with information carried in sequences or higher-order non-covalent structures. The chemistry of the metabolic substrate is mostly the chemistry of organic reactions. Phosphates and thioesters may appear in intermediates, but their role generally is to provide energy for leaving groups, enabling formation of the main structural bonds among C, N, O and H. One of the striking characteristic scales in metabolism is that its organic reactions, the near-universal mode of construction for molecules of 20 to 30 carbons or less, cease to be used in the synthesis of larger molecules. Even siderophores, among the most complex of widely-used organic compounds, are often elaborations of functional centers that are small core metabolites, such as CIT [207, 208]. Large oligomeric macromolecules are almost entirely synthesized using the dehydration potential of phosphates [209] to link monomers drawn from the inventory [50] of small core metabolites. Many cofactors have structure of both kinds, and they are the smallest molecules that as a class commonly use phosphate esters as permanent structural elements [210].

Finally, cofactors are distinguished by structure–function relations determined mostly at the single-molecule scale. The monomers that are incorporated into macromolecules are often distinguished by general properties, and only take on more specific functional roles that depend strongly on location and context [211, 212]. In contrast, the functions of cofactors are specific, often finely tuned by evolution [93], and deployable in a wide range of macromolecular contexts. Usually they are carriers or transfer agents of functional groups or reductants in intermediary metabolism [213]. Nearly half of enzymes require cofactors as coenzymes [210, 213]. If we extend this grouping to include chelated metals [214, 215] and clusters, ranging from common iron–sulfur centers to the elaborate metal centers of gas-handling enzymes [104, 150], more than half of enzymes require coenzymes or metals in the active site.

The universal reactions of intermediary metabolism depend on only about 30 cofactors [213] (though this number depends on the specific definition used). Major functional roles include (1) transition-metal-mediated redox reactions (heme, cobalamin, the nickel tetrapyrrole F430, chlorophylls 6 ), (2) transport of one-carbon groups that range in redox state from oxidized (biotin for carboxyl groups, methanofurans for formyl groups) to reduced (lipoic acid for methylene groups, SAM, coenzyme-M and cobalamin for methyl groups), with some cofactors spanning this range and mediating interconversion of oxidation states (the folate family interconverting formyl to methyl groups), (3) transport of amino groups (pyridoxal phosphate, glutamate, glutamine), (4) reductants (nicotinamide cofactors, flavins, deazaflavins, lipoic acid and coenzyme-B), (5) membrane electron transport and temporary storage (quinones), (6) transport of more complex units such as acyl and amino-acyl groups (pantetheine in CoA and in the acyl-carrier protein (ACP), lipoic acid, thiamine pyrophosphate), (7) transport of dehydration potential from phosphate esters (nucleoside di- and tri-phosphates), and (8) sources of thioester bonds for substrate-level phosphorylation and other reactions (pantetheine in CoA).

4.1.2. Roles as controllers, and consequences for the emergence and early evolution of life

Cofactors fill roles in network or molecular catalysis below the level of enzymes, but they share with all catalysts the property that they are not consumed by participating in reactions, and therefore are key loci of control over metabolism. Cofactors as transfer agents are essential to completing many network-catalytic loops. In association with enzymes, they can create channels and active sites, and thus they facilitate molecular catalysis. An example of the creation of channels by cofactors is given by the function of cobalamin as a C1 transfer agent to the nickel reaction center in the acetyl-CoA synthase from a corrinoid iron–sulfur protein [216–218]. An example of cofactor incorporation in active sites is the role of TPP as the reaction center in the pyruvate-ferredoxin oxidoreductase (PFOR), which lies at the end of a long electron-transport channel formed by Fe–S clusters [108]. Through the limits in their own functions or in the functional groups they transport through networks, they may impose constraints on chemical diversity or create bottlenecks to evolutionary innovation. The previous sections have shown that many module boundaries in carbon fixation and core metabolism are defined by idiosyncratic reactions, and we have noted that many of these idiosyncrasies are associated with specific cofactor functions.

Cofactors, as topological hubs, and participants in reactions at high-flux boundaries in core and intermediary metabolism, are focal points of natural selection. The adaptations available to key atoms and bonds include altering charge or pKa, changing energy level spacing through non-local electron transport, or altering orbital geometry through ring strains. Divergences in low-level cofactor chemistry may alter the distribution of functional groups and thereby change the global topology of metabolic networks, and some of these changes map onto deep lineage divergences in the tree of life. A well-understood example is the repartitioning of C1 flux from methanopterins versus folates [22, 93]. The same adaptation that enables formylation of methanopterins within an exclusively thioester system, where the homologous folate reaction requires ATP, reduces the potential for methylene-group transfer, and necessitates the oxidative formation of serine from 3PG in methanogens, which is not required of acetogens.

Most research on the origin of life has focused either on the metabolic substrate [6, 219] or catalysis by RNA [193], but we believe the priority of cofactors deserves (and is beginning to receive) greater consideration [89, 220]. In the expansion of metabolic substrates from inorganic inputs, the pathways to produce even such complex cofactors as folates et alia are comparable in position and complexity to those for purine RNA, while some for functional groups such as nicotinamide [89] or chorismate are considerably simpler. Therefore, even though it is not known what catalytic support or memory mechanisms enabled the initial elaboration of metabolism, any solutions to this problem should also support the early emergence of at least the major redox and C- and N-transfer cofactors. Conversely, the pervasive dependence of biosynthetic reactions on cofactor intermediates makes the expansion of protometabolic networks most plausible if it was supported by contemporaneous emergence and elaboration of cofactor groups. In this interpretation cofactors occupy an intermediate position in chemistry and complexity, between the small-metabolite and oligomer levels [89]. They were the transitional phase when the reaction mechanisms of core metabolism came under selection and control of organic as opposed to mineral-based chemistry, and they provided the structured foundation from which the oligomer world grew.

We argue next that a few properties of the elements have governed both functional diversification and evolutionary optimization of many cofactors, especially those associated with core carbon fixation. We focus on heterocycles with conjugated double bonds incorporating nitrogen, and on the groups of functions that exploit special properties of bonds to sulfur atoms. The recruitment of elements or special small-molecule contexts constitutes an additional distinct form of modularity within metabolism. Like the substrate network, cofactor groups often share or re-use synthetic reaction sequences. However, unlike the small-molecule network, cofactors can also be grouped by criteria of catalytic similarity that are independent of pathway recapitulation. For example, alkyl-thiol cofactors, which comprise diverse groups of molecules, all make essential use of distinctive properties of the sulfur bond to carbon, which appear nowhere else in biochemistry. As an example involving elements in specific contexts, a large group of cofactors employing C-N heterocycles all arise from a single sub-network whose reactions are catalyzed by related enzymes, and the transport and catalytic functions performed by the heterocycles are distinctive of this cofactor group.

4.2. The cofactors derived from purine RNA

Most of the cofactors that use heterocycles for their primary functions have biosynthetic reactions closely related to those for purine RNA. These reactions are performed by a diverse class of cyclohydrolase enzymes, which are responsible for the key ring-formation and ring-rearrangement steps. The cyclohydrolases can split and reform the ribosyl ring in PRPP, jointly with the 5- and 6-membered rings of guanine and adenine. Five biosynthetically related cofactor groups are formed in this way. Three of these—the folates, flavins and deazaflavins—are formed from GTP, while one—thiamin—is formed from a direct precursor to GTP, as shown in figure 14.

Figure 14. Key molecular re-arrangements in the network leading from AIR to purines and the purine-derived cofactors. The 3.5.4 class of cyclohydrolases (red) convert FAICAR to IMP (precursor to purines), and subsequently convert GTP to folates and flavins by opening the imidazole ring. Acting on the six-member ring of ATP and on a second attached PRPP, the enzyme 3.5.4.19 initiates the pathway to histidinol. The thiamin pathway, which uses the unclassified enzyme ThiC to hydrolyze imidazole and ribosyl moieties, is the most complex, involving multiple group rearrangements (indicated by colored atoms). This complexity, together with the subsequent attachment of a thiazole group, lead us to place thiamin latest in evolutionary origin among these cofactors.

Folates. The folates are structurally most similar to GTP, but have undergone the widest range of secondary specializations, particularly in the Archaea. They are primarily responsible for binding C1 groups during reduction from formyl to methylene or methyl oxidation states, and their secondary diversifications are apparently results of selection to tune the free-energy landscape of these oxidation states.

Flavins and deazaflavins. The flavins are tricyclic compounds formed by condensation of two pterin groups, while deazaflavins are synthesized through a modified version of this pathway, in which one pterin group is replaced by a benzene ring derived from chorismate. Flavins are general-purpose reductants, while deazaflavins are specifically associated with methanogenesis.

Thiamin. Thiamin combines a C–N heterocycle common to the GTP-derived cofactors with a thiazole group (so incorporating sulfur), and shares functions with both the purine cofactor group and the alkyl-thiol group reviewed in the next subsection.

Histidine. The last 'cofactor' in this group is the amino acid histidine, synthesized from ATP rather than GTP but using similar reactions. Histidine is a general acid–base catalyst with unique pKa, which in many ways functions as a 'cofactor in amino acid form' [51].

We will first describe in detail the remarkable role of the folate group in the evolutionary diversification of the WL pathway, and then return to general patterns found among the purine-derived cofactors, and their placement within the elaboration of metabolism and RNA chemistry.

4.2.1. Folates and the central superhighway of C1 metabolism

Members of the folate family carry C1 groups bound to either the N 5 nitrogen of a heterocycle derived from GTP, an exocyclic N 10 nitrogen derived from a PABA, or both. The two most common folates are THF, ubiquitous in bacteria and common in many archaeal groups, and tetrahydromethanopterin (H4MPT), essential for methanogens and found in a small number of late-branching bacterial clades. Other members of this family are exclusive to the archaeal domain and are structural intermediates between THF and H4MPT. Two kinds of structural variation are found among folates, as shown in figure 15. First, only THF retains the carbonyl group of PABA, which shifts electron density away from N 10 via the benzene ring and lowers its pKa relative to N 5 of the heterocycle. All other members of the family lack this carbonyl. Second, all folates besides THF incorporate one or two methyl groups that impede rotation between the pteridine and aryl-amine planes, changing the relative entropies of formation among different binding states for the attached C1 [22, 93, 221].

Figure 15. Structural variants among cofactors in the folate family, shown with the biosynthetic pathways that produce these variations, from [22]. Pteridine and benzene groups are shown in blue, active nitrogens are shown in green, electron-withdrawing carbonyl groups are shown in red and methyl groups that regulate steric hindrance are shown in purple.

Folates mediate a diverse array of C1 chemistry, various parts of which are essential in the biosynthesis of all organisms [93]. The collection of reactions, summarized in figure 5, has been termed the 'central superhighway' of one-carbon metabolism. Functional groups supplied by folate chemistry, connected by interconversion of C1-oxidation states along the superhighway, include (1) formyl groups for synthesis of purines, formyl-tRNA, and formylation of methionine (fMet) during translation, (2) methylene groups to form thymidilate, which are also used in many deep-branching organisms to synthesize glycine and serine, forming the ancestral pathway to these amino acids [22], and (3) methyl groups which may be transferred to SAM as a general methyl donor in anabolism, to the acetyl-CoA synthase to form acetyl-CoA in the WL pathway, or to coenzyme-M where the conversion to methane is the last step in the energy system of methanogenesis.

The variations among folates, shown in figure 15, leave the charge, pKa and resulting C–N bond energy at N 5 roughly unaffected, while the N 10 charge, pKa and C–N bond energy change significantly across the family. This charge effect, together with entropic effects due to steric hindrance from methyl groups, can sharply vary the functional roles that different folates play in anabolism.

The biggest difference lies between THF and H4MPT. In THF, the N 10 pKa is as much as 6.0 natural-log units lower than that of N 5 [222]. The resulting higher-energy C-N bond cannot be formed without hydrolysis of one ATP, either to bind formate to N 10 of THF, or to cyclize N 5 -formyl-THF to form N 5 ,N 10 -methenyl-THF (see figure 5). This latter reaction is the mirror image of the cyclization of N 10 -formyl-THF, and as we will argue below, a plausibly conserved evolutionary intermediate in the attachment of formate onto folates. After further reduction, the resulting methylene is readily transferred to lipoic acid to form glycine and serine, in what we have termed the 'glycine cycle' [22] (the lipoyl-protein based cycle on the right in figure 5).

In contrast, in H4MPT the difference in pKa between N 10 and N 5 is only 2.4 natural-log units. The lower C–N 10 bond energy permits spontaneous cyclization of N 5 -formyl-H4MPT, following (also ATP-independent) transfer of formate from a formyl-methanofuran cofactor. Through this sequence, methanogens fix formate in an ATP-independent system using only redox chemistry. The initial free energy to attach formate to methanofuran is provided by the terminal methane released in methanogenesis (the Co-M/Co-B cycle in figure 5). The resulting downstream methylene group, however, has too little energy as a leaving group to transfer to an alkyl-thiol cofactor, so methanogens sacrifice the ability to form glycine and serine by direct reduction of formate.

The reconstructed ancestral use of the 7–9 reactions in figure 5 is to reduce formate to acetyl-CoA or methane. However, the reversibility of many reactions in the sequence, possibly requiring substitution of reductant/oxidant cofactors, allows folates to accept and donate C1 groups in a variety of oxidation states, from and into many pathways including salvage pathways. Methylotrophic proteobacteria which have obtained H4MPT through horizontal gene transfer [195, 196] may run the full reaction sequence in reverse. They may use either H4MPT to oxidize formaldehyde or THF to oxidize various methylated C1 compounds, in both cases leading to formate, or other intermediary oxidation states (from THF) as inputs to anabolic pathways. In many late-branching bacteria, some archaea and eukaryotes, the THF based pathway may run in part oxidatively and in part reductively, through connections to either gluconeogenesis/glycolysis or glyoxylate metabolism. In these organisms serine (derived through oxidation, amination and dephosporylation from 3-phosphoglycerate) or glycine (derived through amination of glyoxylate) become the sources of transferable methyl groups in anabolism. This versatility has preserved the folate pathway as an essential module of biosynthesis in all domains of life, and at the same time has made it a pivot of evolutionary variation.

4.2.2. Refinement of folate-C1 chemistry maps onto lineage divergence of methanogens

The structural and functional variation within the folate family illustrates the way that selection, acting on cofactors, can create large-scale re-arrangements in metabolism, enabling adaptations that are reflected in lineage divergences. The free-energy cascade described in the last section, linking ATP hydrolysis, the charge and pKa of the N 10 nitrogen, and the leaving-group activity of the resulting bound carbon for transfer to alkyl-thiol cofactors or other anabolic pathways, is a fundamental long-range constraint of folate-C1 chemistry. A comparative analysis of gene profiles in pathways for glycine and serine synthesis, explained in [22], shows that while the constraint cannot be overcome, its impact on the form of metabolism can vary widely depending on the structure of the mediating folate cofactor.

The annotated role for ATP hydrolysis in WL autotrophs is to attach formate to N 10 of THF, initiating the reduction sequence. However, many deep-branching bacteria and archaea show no gene for this reaction, while multiple lines of evidence indicate that THF nonetheless functions as a carbon-fixation cofactor in these organisms [22]. In almost all cases where an ATP-dependent N 10 -formyl-THF synthase is absent, an ATP-dependent N 5 -formyl-THF cycloligase [223, 224] is found. This is another case where a broad evolutionary context allows an alternate interpretation. N 5 -formyl-THF cycloligase was originally discovered in mammalian systems, where its function has been highly uncertain and hypothesized to be the salvage mechanism as part of a futile cycle [223, 224], before being found to be widespread across the tree of life [22]. If we deduce by reconstruction, however, that ancestral folate chemistry operated in the fully reductive direction, and that in H4MPT systems formate is attached at the N 5 position, while in THF systems formate is attached at the N 10 position, the widespread distribution of the cycloligase takes on a different possible meaning. It is plausible that the N 5 -formyl-THF cycloligase allows a formate incorporation pathway that is an evolutionary intermediate between the commonly recognized pathway using THF and its evolutionary derivative using H4MPT (see figure 5). The ATP-dependent cycloligase produces N 5 ,N 10 -methenyl-THF from N 5 -formyl-THF, which may potentially form spontaneously due to the higher N 5 -pKa [224]. ATP hydrolysis is thus specifically linked to the N 10 -carbon bond, which is the primary donor for carbon groups from folates. Methanogens, in contrast, escape the dependence on ATP hydrolysis by decarboxylating PABA before it is linked to pteridine to form methanopterin (see figure 15), but they sacrifice methyl-group donation from H4MPT to most anabolic pathways, making methanogenesis viable only in clades that evolved the oxidative pathway to serine from 3-phosphoglycerate.

We noted in section 3.4 that the elimination of one ATP-dependent acyl-CoA synthase in acetogens reduces the free energy cost of carbon fixation relative to rTCA cycling. The decoupling of the formate-fixation step on methanopterins from ATP hydrolysis is a further significant innovation, lowering the ATP cost for uptake of CO2. This divergence of H4MPT from THF, and a related divergence of deazaflavins from flavins (see figure 16), follow phylogenetically (and we believe, were responsible for) the divergence of the methanogens from other euryarcheota [22].

Figure 16. The substrate modifications leading from GTP to the four major cofactors H4MPT, THF, riboflavin (in FAD) and the archaeal homologue deazaflavin F420. The branches indicating substrate diversification may also reflect evolutionary lineage.

We regard this example as representative of the way that innovations in cofactor chemistry more generally mediated large-scale rearrangements in metabolism, and corresponding evolutionary (and ecological) divergences of clades. Another similar example comes from the quinones, a diverse family of cofactors mediating membrane electron transport [225]. [114] found that the synthetic divergence of mena- and ubiquinone follows the pattern of phylogenetic diversification within proteobacteria. δ- and -proteobacteria use menaquinone, γ-proteobacteria use both mena- and ubiquinone, and α- and β-proteobacteria use only ubiquinone. Because mena- and ubiquinone have different midpoint potentials, it was suggested that their distribution reflects changes in environmental redox state as the proteobacteria diversified during the rise of oxygen [114, 226].

4.2.3. Relation of the organic superhighway to minerals

An interpretive frame for many of these observations is the proposal that metabolism is an outgrowth of geochemistry [41, 74, 149], which came under the control of living organisms [58] (see section 7 for dedicated discussion). If we wish to judge this proposal, then it is informative to look for parallels and differences between biochemical and plausible geochemical reaction sequences. The distinctive features of biochemical C1 reduction are the attachment of formate to tuned heterocyclic or aryl-amine nitrogen atoms for reduction, and the transfer of reduced C1 groups to sulfhydryl groups (of SAM, lipoic acid or CoM). In the mineral-origin hypothesis for direct reduction, the C1 were adsorbed at metals and either reduced through crystal oxidation [227] or by reductant in solution. The transfer of reduced C1 groups to alkyl-thiol cofactors may show continuity with reduction on metal-sulfide minerals. However, the mediation of reduction by nitrogens appears to be a distinctively biochemical innovation.

4.2.4. Cyclohydrolases as the central enzymes in the family, and the resulting structural homologies among cofactors

The common reaction mechanism unifying the purine-derived cofactors is an initial hydrolysis of both purine and ribose rings performed by cyclohydrolases assigned EC numbers 3.5.4 (see figure 14). All cyclohydrolases within this EC family are used for biosynthesis or conversions within this class of molecules. They are responsible for the synthesis of inosine-monophosphate (IMP, precursor to AMP and GMP) from 5-formamidoimidazole-4-carboxamide ribonucleotide (FAICAR), for the first committed steps in the syntheses of both folates and flavins from GTP, and for the initial ring-opening step in the synthesis of histidine from ATP and PRPP. Figure 14 shows the key steps in the network synthesizing both purines and the pterins, folates, flavins, thiamin and histidine.

The common function of the 3.5.4 cyclohydrolases is hydrolysis of rings on adjacent nucleobase and ribose groups, or the formation of cycles by ligation of ring fragments. In all cases, the ribosyl moieties come from PRPP. In the synthesis of pterins from GTP and of histidinol from ATP, both a nucleobase cycle and a ribose are cleaved. In pterin synthesis, the imidazole of guanine and the purine ribose are cleaved. In histidine synthesis, the six-membered ring of adenine is cleaved (at a different bond than the one synthesized from FAICAR), and the ribose comes from a secondary PRPP.

By far the most complex synthesis in this family is that of thiamin from aminoimidazole ribonucleotide (AIR). This sequence begins with an elaborate molecular rearrangement, performed in a single step by the enzyme ThiC [205]. (Eukaryotes use an entirely different pathway, in which the pyrimidine is synthesized from histidine and pyridoxal-5-phosphate [228].) While the ThiC enzyme is unclassified, and its reaction mechanism incompletely understood, it shares apparent characteristics with members of the 3.5.4 cyclohydrolases. As in the first committed steps in the synthesis of folates and flavins from GTP, both a ribose ring and a 5-member heterocycle are cleaved and subsequently (as in folate synthesis) recombined into a 6-member heterocycle. The complexity of this enzymatic mechanism makes a pre-enzymatic homologue to ThiC difficult to imagine, and suggests that thiamin is both of later origin, and more highly derived, than other cofactors in this family. This derived status is supported by the fact that the resulting functional role of thiamin is not performed on the pyrimidine ring itself, but rather on the thiazole ring to which it is attached, and which is likewise created in an elaborate synthetic sequence [205].

Figure 16 shows the detailed substrate re-arrangement in the sub-network leading from GTP to methanopterins, folates, riboflavin and the archaeal deazaflavin F420. In the pterin branch, both rings of neopterin are synthesized directly from GTP, and an aryl-amine originating in PABA provides the second essential nitrogen atom. PABA is either used directly (in folates) or decarboxylated with attachment of a PRPP (in methanopterins) to vary the pKa of the amine. In contrast, the flavin branch is characterized by the integration of either ribulose (in riboflavin) or chorismate (in F420) to form the internal rings. Two 6,7-dimethyl-8-(D-ribityl)lumazine are condensed to form riboflavin, whereas a single GTP with chorismate forms F420.

The cyclohydrolase reactions can be considered the key innovation enabling the biosynthesis of this whole family of cofactors and, importantly, of purine RNA itself. The heterocycles that are formed or cleaved by these reactions provide the central structural components of the active parts of the final cofactor molecules. In this sense, except for TPP, the distinctions among purine-derived cofactors can be considered secondary modifications on a background structured by PRPP and C–N heterocycles. If we consider sub-networks of metabolism as producing key structural or functional components, in this case for the synthesis of cofactors, then this family draws on only two such developed sub-networks. The first of these is purine synthesis and the other is synthesis of chorismate, the precursor to PABA and the unique source of single benzene rings in biochemistry [229]. Flexibility in the ways that chorismate is modified to control electron density, and the way the benzene ring is combined with other heterocycles, contributes to the combinatorial elaboration within the family.

4.2.5. Placing the members of the class within the network expansion of metabolism

The following observations suggest to us the possibility that most of the purine-derived cofactors (perhaps excepting thiamin) were available contemporaneously with monomer purine RNA.

For some reactions, the abstraction of enzyme mechanisms is advanced enough to identify small-molecule organocatalysts that could have provided similar functions [230, 231]. The current understanding of cyclohydrolase proteins, however, does not suggest other simpler mechanisms by which similar reactions might first have been catalyzed, leaving us almost wholly uncertain about how RNA was first formed. Unless non-enzymatic mechanisms are discovered which are both plausible and selective, our previous arguments about the permissiveness of crude catalysts lead us to expect that, at whatever stage catalysts capable of interconverting AIR, AICAR, FAICAR and IMP first became available, pteridines would have been formed contemporaneously and possibly played a role in the elaboration of the metabolic network. (See section 5 for further discussion on promiscuous versus selective enzymes.) If the chorismate pathway (which begins in the sugar-phosphate network) had also arisen by that stage, the same arguments suggest that folates and flavins may also have been available. In this supposition we are treating the first three EC numbers as an appropriate guide to reaction mechanism without restriction of the molecular substrate. Whether the first RNA were produced in this way, or through structurally very dissimilar stages, is a currently active question [232].

As in our discussion of the root node in section 3.4.4, we consider it important to apply ubiquitously the premise that enabling network throughput and pruning network diversity were concurrent ongoing requirements in the co-evolution of substrate reactions and their catalysts. Most often [160, 190, 233], the inability to prune networks is recognized as a problem for the early formation of order. In the case of the purine-derived cofactors, it may offer both clues to help explain the structure of the biosynthetic network, and a way to break down the problem of early metabolic evolution into simpler steps with intermediate criteria for selection.

The patterns that characterize current metabolism as a recursive network expansion [200, 201] about inorganic inputs are most easily understood as a reflection of the organic-chemical possibilities opened by cofactors. Pterins, as donors of activated formyl groups, support (among other reactions) the synthesis of purines, forming a short autocatalytic loop. Similarly, flavins would have augmented redox reactions. Finally, it has long been recognized that acid/base catalysis is uniquely served by histidine, which has a pKa ≈ 6.5 on the ε-nitrogen, a property not found among any biological ribonucleotides (though possible for some substituted adenine derivatives) [234].

Within the class of GTP-derived cofactors, a sub-structure may perhaps be suggested: the dimer condensation that forms riboflavin is a hierarchical use of building blocks formed from GTP. Although simple and consisting of a single key reaction, this could reflect a later stage of refinement. It is recognized [235] that flavins are somewhat specialized reductants, both biosynthetically and functionally more specific than the much simpler nicotinamide cofactors, which plausibly preceded them [89].

4.2.6. Purine-derived cofactors selected before RNA itself, as opposed to having descended from an RNA world defined through base pairing? The overlap between RNA and cofactor biosynthesis, and the incorporation of AMP in several cofactors (where is serves primarily as a 'handle' for docking), has been noticed and given the interpretation that cofactors are a degenerated relic of an oligomer RNA world [210]. While monomer RNA is of comparable complexity to small-molecule cofactors, oligomer RNA is significantly more complex. The only significant logical motivation to place oligomer RNA prior to small-molecule cofactors, is therefore the premise that RNA base pairing and replication is the least-complex plausible mechanism supporting (specifically, Darwinian) selection and persistence of catalysts that are hypothesized to have been required for the elaboration of biosynthesis.

This is still a complex premise, however, as it requires not only organosynthesis of oligomer RNA, but also chiral selection and mechanisms to enable base pairing and (presumably template-directed) ligation [236]. A particular problem for RNA replication is the steric restriction to 3'-5' phosphate esters, over the kinetically favored 2'-5' linkage. In comparison, small-molecule catalysis by either RNA [237] or related cofactors may be considered in any context that supports their synthesis 7 . If chemical mechanisms are found which support structured organosynthesis and selection—a requirement for any metabolism-first theory of the origin of life—the default premise may favor simplicity: that heterocycles were first selected as cofactors, and that purine RNA, only one among many species maintained by the same generalized reactions, was subsequently selected for chirality, base-pairing and ligation.

4.3. The alkyl-thiol cofactors

The major chemicals in this class include the sulfonated alkane-thiols coenzyme-B (CoB) and coenzyme-M (CoM), cysteine and homocysteine including the activated forms S-adenosyl-homocysteine (which under methylation becomes SAM), lipoic acid and pantetheine or pantothenic acid, including pantetheine-phosphate. The common structure of the alkyl-thiol cofactors is an alkane chain terminated by one or more sulfhydryl (SH) groups. In all cases except lipoic acid, a single SH is bound to the terminal carbon in lipoic acid two SH groups are bound at sub-adjacent carbons. Differences among the alkyl-thiol cofactors arise from their biosynthetic context, the length of their alkane chains, and perhaps foremost the functional groups that terminate the other ends of the chains. These may be as simple as sulfones (in CoB) or as complex as peptide bonds (in CoA).

Cofactors in this class serve three primary functions, as reductants (cysteine, CoB, pantetheine and one sulfur on lipoic acid), carriers of methyl groups (CoM, SAM), and carriers of larger functional groups such as acyl groups (lipoic acid in lipoyl protein, phosphopantetheine in ACP). A highly specialized role in which H is a leaving group is the formation of thioesters at carboxyl groups (pantethenic acid in CoA, lipoic acid in lipoyl protein) This function is essential to substrate-level phosphorylation [241], and appears repeatedly in the deepest and putatively oldest reactions in core metabolism. A final function closely related to reduction is the formation and cleavage of S–S linkages by cysteine in response to redox state, which is a major controller of both committed and plastic tertiary structure in proteins. The sulfur atoms on cysteine often form coordinate bonds to metals in metallo-enzymes, a function that we may associate with protein ligands, in contrast to the more common nitrogen atoms that coordinate metals in pyrrole cofactors.

The properties of the alkyl-thiol cofactors derive largely from the properties of sulfur, which is a 'soft' period-3 element [242] that forms relatively unstable (usually termed 'high-energy') bonds with the hard period-2 element carbon. For the alkyl-thiol cofactors in which sulfur plays direct chemical roles, three main bonds dictate their chemistry: S–C, S–S, and S–H. Sulfur can also exist in a wide range of oxidation states, and for this reason often plays an important role in energy metabolism [243], particularly for chemotrophs, and due to its versatility has been suggested to precede oxygen in photosynthesis [244]. The electronic versatility of sulfur and the high-energy C–S bonds combine with the large atomic radius of sulfur to give access to additional geometrical, electronic and ring-straining possibilities not available to CHON chemistry.

Although not alkyl-thiol compounds as categorized above, two additional cofactors that make important indirect use of sulfur are thiamin and biotin. In neither case is sulfur the element to which transferred C1 groups are bound. For reactions involving TPP the C1-unit is bound to the carbon between sulfur and the positively charged nitrogen, while in biotin C1-units are bound to the carboxamide nitrogen in the (non-aromatic) heterocycle opposite the sulfur-containing ring. It seems likely, however, that the sulfur indirectly contributes to the properties of the binding carbon or nitrogen, through some combination of electrostatic, resonance or possibly ring-straining interactions. The importance of the sulfur to the focal carbon or nitrogen atom is suggested by the complexity of the chemistry and enzymes involved in its incorporation into these two cofactors [205, 245].

4.3.1. Biochemical roles and phylogenetic distribution

Transfer of methyl or methylene groups. The S atoms of CoM, lipoic acid, and S-adenosyl-homocysteine accept methyl or methylene groups from the nitrogen atoms of pterins. Considering that transition-metal sulfide minerals are the favored substrates for prebiotic direct-C1 reduction [147, 149, 246], a question of particular interest is how, in mineral scenarios for the emergence of carbon fixation, the distinctive relation between tuned nitrogen atoms in pterins as carbon carriers, and alkyl-thiol compounds as carbon acceptors, would have formed.

Reductants and co-reductants. CoB and CoM act together as methyl carrier and reductant to form methane in methanogenesis. In this complex transfer [150], the fully-reduced (Ni + ) state of the nickel tetrapyrrole F430 forms a dative bond to –CH3 displacing the CoM carrier, effectively re-oxidizing F430 to Ni 3 + . Reduced F430 is regenerated through two sequential single-electron transfers. The first, from CoM–SH, generates a Ni 2 + state that releases methane, while forming a radical CoB –S–S–CoM intermediate with CoB. The radical then donates the second electron, restoring Ni + . The strongly oxidizing heterodisulfide CoB–S–S–CoM is subsequently reduced with two NADH, regenerating CoM–SH and CoB–SH.

A similar role as methylene carrier and reductant is performed by the two SH groups in lipoic acid. CoM is specific to methanogenic archaea [247], while lipoic acid and S-adenosyl-homocysteine are found in all three domains [22, 248]. Lipoic acid is formed from octanoyl-CoA, emerging from the biotin-dependent malonate pathway to fatty acid synthesis, and along with fatty acid synthesis [109], may have been present in the universal common ancestor. The previously noted universal distribution of the glycine cycle supports this hypothesis.

Role in the reversal of citric-acid cycling. Lipoic acid becomes the electron acceptor in the oxidative decarboxylation of α-ketoglutarate and pyruvate in the oxidative Krebs cycle, replacing the role taken by reduced ferredoxin in the rTCA cycle. Thus the prior availability of lipoic acid was an enabling precondition for reversal of the cycle in response to the rise of oxygen.

Carriers of acyl groups. Transport of acyl groups in the ACP proceeds through thioesterification with pantetheine phosphate, similar to the thioesterification in fixation pathways. In fatty acid biosynthesis acyl groups are further processed while attached to the pantetheine phosphate prosthetic group.

Electron bifurcation. The heterodisulfide bond of CoB–S–S–CoM has a high midpoint potential (E'0 = −140 mV), relative to the H + /H2 couple (E'0 = −414 mV), and its reduction is the source of free energy for the endergonic production of reduced ferredoxin (Fd 2 − , E'0 in situ unknown but between −520 mV and −414 mV) [249], which in turn powers the initial uptake of CO2 on H4MPT in methanogens. The remarkable direct coupling of exergonic and endergonic redox reactions through splitting of binding pairs into pairs of radicals, which are then directed to paired high-potential/low-potential acceptors, is known as electron bifurcation [140]. Variant forms of bifurcation are coming to be recognized as a widely-used strategy of metal-center enzymes, either consuming oxidants as energy sources to generate uniquely biotic low-potential reductants such as Fd 2 − [249, 250–252], or to 'titrate' redox potential to minimize dissipation and achieve reversibility of redox reactions involving reductants at diverse potentials, e.g. by combining low-potential (Fd 2 − , E'0 = −420 mV) and high-potential (NADH, E'0 = −300 mV) reductants to produce intermediate-potential reductants (NADPH, E'0 = −360 mV) [253]. Together with substrate-level phosphorylation (SLP), electron bifurcation may be the principal chemical mechanism (contrasted with membrane-mediated oxidative phosphorylation) for interconverting biological energy currencies, and along with SLP [241], a mechanism of central importance in the origin of metabolism [254]. Small metabolites including such heterodisulfides of cofactors, which can form radical intermediates exchanging single electrons with Fe–S clusters (typically via flavins) are essential sources and repositories of free energy in pathways using bifurcation. Both electron bifurcation and the stepwise reduction of F430 (above) illustrate the central role of metals as mediators of single-electron transfer processes in metabolism.

4.3.2. Participation in carbon fixation pathway modules

The similarity between the glycine cycle and methanogenesis in figure 5 emphasizes the convergent roles of alkyl-thiol cofactors. In the glycine cycle, methylene groups are accepted by the terminal sulfur on lipoic acid, and the subadjacent SH serves as reductant when glycine is produced, leaving a disulfide bond in lipoic acid. The disulfide bond is subsequently reduced with NADH. In methanogenesis, a methyl group from H4MPT is transferred to CoM, with the subsequent transfer to F430, and the release from F430 as methane in the methyl-CoM reductase, coupled to formation of CoB–S–S–CoM. The heterodisulfide is again reduced with NADH, but employs a pair of electron bifurcations to retain the excess free energy in the production of Fd 2 − rather than dissipating it as heat [249]. Methanogenesis is thus associated with seven distinctive cofactors beyond even the set known to have diversified functions within the archaea [5], again suggesting the derived and highly optimized nature of this Euryarchaeal phenotype. The striking similarity of these two methyl-transfer systems, mediated by independently evolved and structurally quite different cofactors, suggests evolutionary convergence driven specifically by properties of alkyl thiols.

A curious pattern, which we note but do not attempt to interpret, is the association of non-sulfur, nitrogen-heterocycle cofactors with WL carbon fixation, contrasted with the use of sulfur-containing heterocycles in carboxylation reactions of the rTCA cycle. The non-sulfur cofactors THF and H4MPT are used in the reactions of the WL pathway, while the biosynthetically-related but sulfur-containing cofactor thiamin mediates the carbonyl insertion (at a thioester) in rTCA [108, 255]. Biotin—which has been generally associated with malonate synthesis in the fatty-acid pathway (and derivatives such as propionate carboxylation to methyl-malonate in 3HP [109])—mediates the subsequent β-carboxylation of pyruvate and of α-ketoglutarate [110, 256, 257]. Thus the two cofactors we have identified as using sulfur indirectly to tune properties of carbon or nitrogen C1-bonding atoms mediate the two chemically quite different sequential carboxylations in rTCA.

4.4. Carboxylation reactions in cofactor synthesis

Carboxylation reactions can be classified as falling into two general categories: those used in core carbon 'uptake', and those used exclusively in the synthesis of specific cofactors. In addition to carboxylation reactions in carbon-fixation pathways, the former category includes the carboxylation of crotonyl-CoA in the glyoxylate regeneration cycle. This cycle is a mixotrophic rather than an autotrophic pathway, but this reaction does form a distinct entry point for CO2 into the biosphere. The carboxylation of acetyl-CoA to malonyl-CoA further serves a dual purpose, in being both the starting point for fatty acid synthesis, as well as a key step in the 3HP pathway used in several carbon-fixation pathways. All these carboxylation reactions thus have in common that they are used at least in some organism as the central source for cellular carbon. All other carboxylation reactions that are not used as part of core carbon uptake, are used in the synthesis of the biotin cofactor, and the purine and pyrimidine nucleotides (see figure 17).

Figure 17. Carboxylation reactions in the synthesis of cofactors. The sequences show the immediate amination of the carboxyl group to a carboxamide group, which is then preserved into the final heterocyclic structure. As the only carboxylations not used in core carbon uptake, these reaction sequences form a distinct class of chemistry. Amination reactions are shown as net additions of ammonia, which may be derived from other sources (such as glutamine, aspartate or SAM). Abbreviations: alanine (ALA) aspartate (ASP) phosphoribosyl pyrophosphate (PRPP).

If we consider the sequences in which these carboxylation reactions are used to synthesize biotin, purine and pyrimidine, they also form a distinct class of chemistry. In all three cases the resulting carboxyl group is immediately aminated, either as part of the carboxylation reaction, or in the following reaction, and the carboxamide group is subsequently maintained into the final heterocyclic structure. In addition we previously saw that IMP becomes the source for the folate and flavin family (through GTP). Carboxylation reactions are thus either a general source for cellular carbon in core metabolism, or a specific source of carboxamide groups in the synthesis of cofactors that are part of the catalytic control of core metabolism.

4.5. The chorismate pathway in both amino acid and cofactor synthesis

Chorismate is the sole source of single benzene rings in biochemistry [229]. The non-local π-bond resonance is used in a variety of charge-transfer and electron transfer and storage functions, in functional groups and cofactors derived from chorismate. We have noted the charge-transfer function of PABA in tuning N 10 of folates, and its impact on C1 chemistry. The para-oriented carbonyl groups of quinones may be converted to partially- or fully-resonant orbitals in the benzene ring, enabling fully oxidized (quinone), half-reduced (semiquinone), or fully reduced (hydroquinone) states [235]. Finally, the aromatic ring in tryptophan (a second amino acid which behaves in many ways like a cofactor) has at least one function in the active sites of enzymes as a mediator of non-local electron transfers [258].


DISCUSSION

The initial reason for testing the interaction between GA regime and CO2 was the observation that at ambient [CO2], PAC uncoupled growth from carbon availability (Ribeiro et al., 2012). So far, there is a general consensus that elevated [CO2] increases carbon uptake and foliar carbohydrate content and, thereby, stimulates plant growth (Ainsworth et al., 2002 Kirschbaum, 2011). Here, we found that growth inhibition induced by PAC is overcome by elevated [CO2]. Although GA homeostasis as determined at the transcript level is hardly affected by high [CO2], reprogramming at the metabolic level occurs concomitantly with the induction of GA-responsive growth-related genes. Taken together, our observations suggest that increased [CO2] stimulates growth at least in part in a GA-independent manner.

Expression of Growth-Related Genes Is Uncoupled from GA Status at Elevated [CO2]

In an attempt to clarify the effect of elevated [CO2] on GA metabolism and growth, the expression of GA biosynthesis and signaling genes was analyzed in plants treated with PAC and/or GA grown under ambient or elevated [CO2]. PAC treatment at both ambient [CO2] and elevated [CO2] resulted in a similar activation of GA biosynthesis gene expression, suggesting that GA biosynthesis is still affected by PAC at elevated [CO2]. Similarly, the feedback repression of GID1B, encoding a GA receptor protein (Ueguchi-Tanaka et al., 2007), as well as RGL2 and RGL3, encoding DELLA proteins, by PAC is observed under both ambient and elevated [CO2]. Moreover, the addition of external GA to plants grown under elevated [CO2] provokes a similar expressional response to that under ambient levels. Thus, increased [CO2] does not affect the transcriptional responsiveness of the plants toward this phytohormone. Moreover, elevated [CO2] alone did not significantly affect the expression level of GA-related genes as compared with ambient [CO2], indicating that the effect of [CO2] on plant growth might be independent of changes in the transcription of GA biosynthesis genes.

Cell growth involves the selective loosening and rearrangement of the cell wall to provoke a turgor-driven expansion (Marga et al., 2005). Expansins (Goh et al., 2012) and XTHs (Van Sandt et al., 2007) are the best characterized protein classes known to drive this process. Previously, we found that GA and PAC have opposing effects on the expression of these genes under ambient [CO2], fitting to the expansion rates of the plants (Ribeiro et al., 2012). In contrast, a remarkable increase in the expression of these genes was observed after PAC treatment under elevated [CO2], while GA treatment no longer induced the expression of these genes when plants were grown at high [CO2]. Thus, under high [CO2], the GA regime no longer controls the expression of these growth-related genes. Interestingly, a similar observation was made for genes involved in lipid metabolism. Recently, it was found that brassinosteroids are the main effectors that induce growth-related genes, whereas GA quantitatively enhances brassinosteroid-potentiated growth (Bai et al., 2012), suggesting that the induction of these genes during elevated [CO2] might depend on brassinosteroids. Taken together, the expressional analysis suggests that under high carbon availability, plant growth might be uncoupled from GA status.

Growth and Source versus Sink Limitation

The decreases in relative growth rate, relative expansion rate, fresh weight, and dry weight in shoots of PAC-treated plants under ambient [CO2] were overcome when PAC-treated plants were grown in elevated [CO2]. These results indicate an involvement of elevated [CO2] in alleviating GA deficiency-induced responses. Leaf growth is known to be highly dependent on carbon availability (Wiese et al., 2007 Pantin et al., 2011). Three of the five Calvin-Benson cycle enzymes that we investigated (Rubisco, PGK, and TK) showed an increase in activity in low-GA plants under elevated [CO2]. Although this suggests that photosynthetic carbon assimilation is increased in PAC-treated plants, it is the developmental program itself that determines how efficiently carbon is converted into biomass (Sulpice et al., 2010). Still, low-GA plants grown under elevated [CO2] maintained carbon gain. Furthermore, the activity of NR also remained high in plants treated with PAC under elevated [CO2]. In ambient [CO2], the level of nitrate accumulated in plants treated with PAC but not in other treatments. On the other hand, at elevated [CO2], nitrate remained at the same level in low- or high-GA plants as compared with control plants. This highlights the ability of plants growing under the low-GA regime to invest inorganic nitrogen to support the light-dependent use of CO2 to produce sugars, organic acids, and amino acids, the basic building blocks of biomass accumulation.

It is known that malate and fumarate serve as alternative and flexible sinks for photosynthate in Arabidopsis (Chia et al., 2000 Pracharoenwattana et al., 2010 Zell et al., 2010). Two sets of observations showed that diurnal malate and fumarate turnover was highly regulated in low-GA plants grown at ambient [CO2]. First, in low-GA plants, only small amounts of malate and fumarate were left at the end of the night. Second, a larger proportion of the organic acids accumulated during the day in low-GA plants grown under ambient [CO2], although these plants also exhibited the greatest growth inhibition. These observations indicate that organic acid synthesis corresponds to changes of the sink-source balance. In agreement with this model, GA was able to recover the growth of plants treated with PAC, and this was accompanied by decreases in malate and fumarate levels in plants grown under ambient [CO2]. Furthermore, the high levels of organic acid during the day in plants treated with PAC grown under ambient [CO2] were not maintained under elevated [CO2], correlating with the differences in growth rate.

Rubisco adds CO2 to ribulose-1,5-bisphosphate to form glycerate-3-P in the first step of carbon fixation. The majority of the NADPH and ATP produced in the light reactions are used to reduce glycerate-3-P to triose phosphates. Triose phosphates are used to regenerate ribulose-1,5-bisphosphate and to synthesize end products such as Suc, starch, and amino acids (Stitt et al., 2010). Thus, the increased level of NADP observed in plants treated with PAC grown at elevated [CO2] indicates that NADPH is being utilized more rapidly when low-GA plant growth is stimulated by elevated [CO2]. In agreement with this model, there were no significant differences in Rubisco activities (initial and total) and NADP(H) levels in plants treated with PAC and/or GA under ambient [CO2]. The turnover of soluble sugars and starch that are usually considered to be diagnostic for the regulation of photosynthesis (Stitt et al., 2010) was not affected by GA level (PAC and/or GA treatment) both in plants grown in ambient and elevated [CO2]. Thus, the carbon status in shoots of low-GA plants grown under ambient [CO2] can be interpreted as the result of the rosette expansion being more reduced than carbon inflow. These data indicate that carbon only leads to extra growth if plants have a use for it (e.g. for the growth of new sinks such as leaves or roots). GA addition creates such an additional developmental force and completely rescues the rosette growth of PAC-treated plants in ambient [CO2]. At elevated [CO2], there was a substantial stimulation of sugar and starch synthesis in plants treated with PAC and/or GA, similar to the control. The stimulation of starch synthesis is accompanied by an increase of AGPase activity, irrespective of the GA regime. Most importantly, elevated [CO2] increased the biomass accumulation of plants with the restriction of GA biosynthesis, and the turnover of soluble sugars and starch was increased as compared with plants at ambient [CO2]. These results provide compelling evidence that under elevated [CO2], the carbohydrates are readily used for growth or export in plants with low GA levels, as indicated by the expression of cell wall-related genes. Thus, growth is faster at elevated [CO2] in GA-limited plants, because the storage and allocation of assimilates are altered. We cannot, however, formally exclude the possibility that elevated [CO2] regulates growth by linking primary metabolism with enhanced GA biosynthesis, since there is evidence that elevated [CO2] increases the level of GA in plants such as Arabidopsis (Teng et al., 2006) and orchids (Li et al., 2002).

Metabolic Reprogramming during Growth at Elevated [CO2] Might Circumvent GA Biosynthesis

As indicated above, the growth of plants with differing GA regimes under elevated [CO2] only partially restores the metabolic profile, indicating a potential metabolic flexibility to restore plant growth. Previously, it has been reported that the metabolite levels of putrescine and trehalose positively correlate with plant biomass in Arabidopsis (Meyer et al., 2007). Our analysis here indicates that both trehalose and putrescine act independently of GA level and are both induced specifically by elevated [CO2] (Supplemental Table S1), correlating with the increased growth phenotype. Trehalose-6-P has been put forward as a signaling molecule of both carbon metabolism and plant development (Meyer et al., 2007 Paul et al., 2008).

The accumulation of Orn upon elevated [CO2] might be representative for the different water use efficiencies (Gonzàlez-Meler et al., 2009) of plants grown at high [CO2] and the relationship between Orn and water status (Kalamaki et al., 2009). For other metabolites, a negative correlation has been described between biomass accumulation and their levels (Meyer et al., 2007). Here, we found that three of them are specifically controlled by [CO2]: glyceraldehyde-3-P, Phe, and Tyr (Meyer et al., 2007). Of these growth markers, PAC treatment at ambient [CO2] only negatively affects the accumulation of trehalose, while the other [CO2]-regulated markers remain unchanged.

Of the GA-specific metabolites found here, including erythritol, Rha, threitol, and tartaric acid, none has so far been correlated with plant biomass accumulation. Rha is a major component of pectin (Ridley et al., 2001). As cell growth is to a large extent determined by the extension rate of the cell wall (Brown et al., 2001), it is not surprising to see a correlation between GA regime and Rha level. Erythritol represents an intermediate in the methylerythritol phosphate pathway (Suzuki et al., 2009), which is used to synthesize plastidic isoprenoids (e.g. carotenoids and the side chains of chlorophylls and plastoquinones) and some isoprenoid-type phytohormones (e.g. GAs, abscisic acid, and cytokinins). Therefore, the effect of GA on the erythritol level might represent a metabolic feedback regulation. That said, the limited number of GA-specific metabolites indicates that the effect of GA on carbon metabolism is probably additive or redundant with other major regulating factors.

A restoration of several metabolites affected by PAC treatment under ambient [CO2] is observed upon elevated [CO2]. However, other metabolites are affected by PAC and GA only at high [CO2]. Although no obvious phenotypic differences exist between control and PAC-treated plants at elevated [CO2], their metabolic profiles are clearly distinctive. This might indicate a certain metabolic flexibility at high [CO2] that allows for plant growth even under low-GA regimes. Most GA oxidases involved in controlling GA homeostasis are 2-oxoglutarate dependent, including the Arabidopsis GA3ox, GA20ox, and GA2ox enzymes, which use it as a cosubstrate (Hedden and Phillips, 2000 Mitchum et al., 2006). The dependence on 2-oxoglutarate thereby links GA biosynthesis directly with primary metabolism (Lancien et al., 2000). Interestingly, 2-oxoglutarate is a central regulator of metabolism and the integration point of carbon and nitrogen metabolism. 2-Oxoglutarate is not only a central metabolite in the tricarboxylic acid cycle but is also important for primary amino acid metabolism (Nunes-Nesi et al., 2010), nitrate assimilation, and linked to NR activity (Kinoshita et al., 2011) and the photorespiratory glyoxylate cycle (Igarashi et al., 2003). Altered uptake of 2-oxoglutarate into the chloroplast results in growth retardation (Taniguchi et al., 2002), emphasizing the role of 2-oxoglutarate in coupling plant growth with metabolism. Elevated [CO2] did not affect the level of the potential nitrogen storage molecule Arg (Llr et al., 2008) however, PAC treatment resulted in a lower level of this compound under ambient [CO2], which is restored by high [CO2]. We believe that the restoration of growth by elevated [CO2] of PAC-treated plants occurs not merely by complementing the metabolic changes induced by PAC but more likely by a mechanism of metabolic flexibility (Eberhard et al., 2008 Hebbelmann et al., 2012). By contrast, 2-oxoglutarate-dependent GA metabolism would seem likely to be strictly controlled at ambient CO2 and, as such, may mediate the growth-promoting effect of GA under these conditions. Recently, it was found that reducing the activity of the enzyme 2-oxoglutarate dehydrogenase in tomato (Solanum lycopersicum) results in different levels of bioactive GA and corresponding developmental phenotypes (Araújo et al., 2012). That said, considerable further research on the interaction between 2-oxoglutarate, primary metabolism, and GAs is required to determine the precise mechanisms underlying this linkage.


Abstract

Although the positive effect of elevated CO2 concentration [CO2] on plant growth is well known, it remains unclear whether global climate change will positively or negatively affect crop yields. In particular, relatively little is known about the role of hormone pathways in controlling the growth responses to elevated [CO2]. Here, we studied the impact of elevated [CO2] on plant biomass and metabolism in Arabidopsis (Arabidopsis thaliana) in relation to the availability of gibberellins (GAs). Inhibition of growth by the GA biosynthesis inhibitor paclobutrazol ( PAC) at ambient [CO2] (350 µmol CO2 mol −1 ) was reverted by elevated [CO2] (750 µmol CO2 mol −1 ). Thus, we investigated the metabolic adjustment and modulation of gene expression in response to changes in growth of plants imposed by varying the GA regime in ambient and elevated [CO2]. In the presence of PAC (low-GA regime), the activities of enzymes involved in photosynthesis and inorganic nitrogen assimilation were markedly increased at elevated [CO2], whereas the activities of enzymes of organic acid metabolism were decreased. Under ambient [CO2], nitrate, amino acids, and protein accumulated upon PAC treatment however, this was not the case when plants were grown at elevated [CO2]. These results suggest that only under ambient [CO2] is GA required for the integration of carbohydrate and nitrogen metabolism underlying optimal biomass determination. Our results have implications concerning the action of the Green Revolution genes in future environmental conditions.

The worldwide emission of gases derived from fossil fuel burning is seen as a major cause of global climate change mainly visible as an increase in atmospheric CO2 concentration [CO2] ( Baker and Allen, 1994), which could rise to more than 750 µL L −1 by the end of this century ( Meehl et al., 2007). Since a higher [CO2] increases photosynthesis, development, and in many cases yield, it has often been considered as a positive factor for future crop agriculture ( Baker, 2004 Ainsworth, 2008). On the other hand, cereals grown under elevated [CO2] show a decreased harvest index (i.e. the ratio of grain yield to total biomass Meehl et al., 2007) and decreased seed quality ( Taub et al., 2008). Elevated [CO2] leads to a stimulation of leaf growth by triggering both cell expansion and cell division ( Masle, 2000 Taylor et al., 2003 Luomala et al., 2005). Elongation growth is strongly influenced by GAs ( Richards et al., 2001). During the Green Revolution, breeding programs focused on increasing the harvest index by selecting semidwarf cereal varieties that invest relatively few resources in vegetative growth but support increased partition to grains ( Khush, 2001 Phillips, 2004). It was subsequently discovered that most Green Revolution varieties, selected for their semidwarf phenotype, harbored mutations affecting GA biosynthesis ( Hedden, 2003). Such mutants include Semidwarf1 (SD1) in rice (Oryza sativa), which encodes a GA 20-oxidase ( Spielmeyer et al., 2002), and the wheat (Triticum aestivum) Reduced-height1B and Reduced-height1D and maize (Zea mays) dwarf d8 genes, which encode orthologs of the Arabidopsis (Arabidopsis thaliana) Gibberellin Insensitive gene ( Peng et al., 1999). However, relatively little is known concerning the action of GA in determining plant growth at elevated [CO2]. For example, the yield of the miracle rice line IR8, the original sd1 mutant, has dropped by 15% compared with the levels achieved in the 1960s ( Peng et al., 2010), in part due to global climate change, including increased temperature and [CO2], but also due to changed soil properties and increased biotic stress.

GA biosynthesis and signaling pathways have been studied intensively, particularly in Arabidopsis ( Hedden and Phillips, 2000 Olszewski et al., 2002 Yamaguchi, 2008 Middleton et al., 2012). Early steps in GA biosynthesis involve the cyclization of geranylgeranyl diphosphate to ent-copalyl diphosphate, which in turn is converted to ent-kaurene. The enzymes that catalyze these reactions are ent-copalyl diphosphate synthase and ent-kaurene synthase, respectively ( Sun and Kamiya, 1997). Subsequent reactions catalyzed by the cytochrome P450 enzymes ent-kaurene oxidase and ent-kaurenoic acid oxidase produce ent-kaurenoic acid and GA12 from ent-kaurene ( Helliwell et al., 2001 Yamaguchi, 2008). In the final steps of the pathway, GA12 is converted to GA4 through oxidations on C-20 and C-3 by GA 20-oxidase (GA20ox) and GA 3-oxidase (GA3ox), respectively ( Hedden and Phillips, 2000 Olszewski et al., 2002). The content of bioactive GA is negatively regulated by GA 2-oxidases (GA2ox Martin et al., 1999), which at the transcriptional level are induced by elevated levels of GA ( Thomas et al., 1999 Elliott et al., 2001). Furthermore, GA homeostasis is also achieved by transcriptional negative feedback regulation of GA20ox and GA3ox biosynthesis genes ( Yamaguchi, 2008). In principle, the expression of GA biosynthetic genes correlates with the level of GA ( Cowling et al., 1998 Ogawa et al., 2003 Zhao et al., 2007 Yamaguchi, 2008). In addition to homeostatic mechanisms that control GA levels, the metabolism of GAs is particularly sensitive to developmental and environmental factors ( Sun and Gubler, 2004 Fleet and Sun, 2005). The increase of atmospheric [CO2] during recent decades has promoted a growing interest in the function of this environmental factor on plant growth.

Previously, we investigated the modulation of gene regulation and metabolic acclimation during altered plant growth imposed by a change of the GA regime ( Ribeiro et al., 2012). Comparison of gene expression and metabolite profiles demonstrated that low GA level uncouples growth from carbon and nitrogen availability, while under normal or high GA levels a tight relationship exists between carbon availability and growth. Since the atmospheric [CO2] is steadily increasing ( Solomon et al., 2007) and the GA biosynthesis pathway has been the major driver for the spectacular increases in yield during the Green Revolution ( Peng et al., 1999), we investigated how elevated [CO2] affects plant growth and metabolism of Arabidopsis grown under different GA regimes.


Abstract

Volatile compounds ( VCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth, and the accumulation of high levels of starch in leaves through cytokinin ( CK)-regulated processes. In Arabidopsis (Arabidopsis thaliana) plants not exposed to VCs, plastidic phosphoglucose isomerase (pPGI) acts as an important determinant of photosynthesis and growth, likely as a consequence of its involvement in the synthesis of plastidic CKs in roots. Moreover, this enzyme plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in leaves. To elucidate the mechanisms involved in the responses of plants to microbial VCs and to investigate the extent of pPGI involvement, we characterized pPGI-null pgi1-2 Arabidopsis plants cultured in the presence or absence of VCs emitted by Alternaria alternata. We found that volatile emissions from this fungal phytopathogen promote growth, photosynthesis, and the accumulation of plastidic CKs in pgi1-2 leaves. Notably, the mesophyll cells of pgi1-2 leaves accumulated exceptionally high levels of starch following VC exposure. Proteomic analyses revealed that VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism, and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants can respond to VCs emitted by phytopathogenic microorganisms by triggering pPGI-independent mechanisms.

It is well known that volatile compounds ( VCs) emitted by beneficial rhizosphere bacteria and fungi can promote plant growth ( Ryu et al., 2003 Hung et al., 2013 Kanchiswamy et al., 2015). We recently showed that this action is not only restricted to beneficial microorganisms but extends to pathogens and microbes that do not normally interact mutualistically with plants ( Sánchez-López et al., 2016). When Arabidopsis (Arabidopsis thaliana) plants were exposed to VCs emitted by the fungal phytopathogen Alternaria alternata, growth promotion was accompanied by enhanced intracellular levels of plastid-type, 2-C-methyl- d -erythritol 4-phosphate ( MEP) pathway-derived cytokinins ( CKs), augmented photosynthesis, and the accumulation of exceptionally high levels of starch in leaves ( Ezquer et al., 2010 Li et al., 2011 Sánchez-López et al., 2016). Furthermore, mutants with reduced CK content or sensitivity responded poorly to VCs ( Sánchez-López et al., 2016). Because CKs are major determinants of growth, photosynthesis, and starch accumulation in mature leaves ( Riefler et al., 2006 Werner et al., 2008 Kieber and Schaller, 2014 Bahaji et al., 2015b), we postulated that a plant’s response to VCs involves CK action ( Sánchez-López et al., 2016). The transcriptome changes of plants exposed to VCs emitted by phylogenetically distant microbial species, such as A. alternata and the beneficial plant growth-promoting rhizobacterium Bacillus subtilis GB03, were strikingly similar ( Sánchez-López et al., 2016), indicating that plants react to microbial VCs through highly conserved regulatory mechanisms. We have proposed that VC-promoted plant growth and metabolic changes prepare the plant to host the microorganism, which, in the case of phytopathogenic microorganisms, ensures proper continuation into the pathogenic phase ( Sánchez-López et al., 2016).

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of Glc-6-P and Fru-6-P. This enzyme is involved in glycolysis and in the regeneration of Glc-6-P molecules in the oxidative pentose phosphate pathway. In mesophyll chloroplasts of illuminated leaves, the plastidic isoform of PGI (pPGI) also plays a fundamental role in starch biosynthesis, connecting the Calvin-Benson cycle ( CBC) with the starch biosynthetic pathway that encompasses plastidic phosphoglucomutase (pPGM), ADP-Glc pyrophosphorylase (AGP), and starch synthase (SS Bahaji et al., 2014b). Recent studies have shown that the leaves of pPGI-null pgi1-2 mutants accumulate low levels of plastidic CKs ( Bahaji et al., 2015b). These plants display reduced photosynthetic capacity and slow growth phenotypes and accumulate low levels of starch in the mesophyll cells of leaves ( Bahaji et al., 2015b). However, this phenotype can be reverted to the wild type by exogenous CK supplementation ( Bahaji et al., 2015b). Thus, pPGI is an important determinant of photosynthesis, starch accumulation, and growth, most likely as a consequence of its involvement in the production of oxidative pentose phosphate pathway/glycolysis intermediates that are required to synthesize plastidic CKs in roots, assimilate nitrogen, and/or maintain plastid redox homeostasis ( Bahaji et al., 2015b).

A. alternata emits highly reactive VCs such as sesquiterpenes ( Weikl et al., 2016). These compounds are known to function as infochemicals, playing crucial roles in plant-microbe interactions ( Peñuelas et al., 2014 Ditengou et al., 2015). Although there has been a considerable increase in our knowledge regarding the importance of metabolic adjustments to changing environmental conditions in recent years, little is known about the adjustments that occur in plants following exposure to microbial VCs. Thus, to obtain insights into the mechanisms involved in the A. alternata VC-promoted growth, photosynthesis, and accumulation of CKs and starch, and to investigate the extent to which pPGI is involved in these responses, in this work we characterized pgi1-2 plants exposed to A. alternata VCs. Our findings show that Arabidopsis is capable of responding to microbial volatile emissions by triggering pPGI-independent mechanisms and raise important questions regarding the basic mechanisms of starch biosynthesis in leaves exposed to VCs.


How exactly is glyceraldehyde 3-Phosphate reverted to ribulose 1,5-bisphosphate for the continuation of the calvin cycle? - Biology

Vesley, Donald Langholz, Ann C. Rohlfing, Stephen R. Foltz, William E.

A biological indicator based on fluorimetric detection within 60 min of a Bacillus stearothermophilus spore-bound enzyme , α-d-glucosidase, has been developed. Results indicate that the enzyme survived slightly longer than spores observed after 24 h of incubation. The new system shows promise for evaluating flash sterilization cycles within 60 min compared with conventional 24-h systems. PMID:16348654

Solar cycle predictions are needed to plan long-term space missions just like weather predictions are needed to plan the launch. Fleets of satellites circle the Earth collecting many types of science data, protecting astronauts, and relaying information. All of these satellites are sensitive at some level to solar cycle effects. Predictions of drag on LEO spacecraft are one of the most important. Launching a satellite with less propellant can mean a higher orbit, but unanticipated solar activity and increased drag can make that a Pyrrhic victory as you consume the reduced propellant load more rapidly. Energetic events at the Sun can produce crippling radiation storms that endanger all assets in space. Solar cycle predictions also anticipate the shortwave emissions that cause degradation of solar panels. Testing solar dynamo theories by quantitative predictions of what will happen in 5-20 years is the next arena for solar cycle predictions. A summary and analysis of 75 predictions of the amplitude of the upcoming Solar Cycle 24 is presented. The current state of solar cycle predictions and some anticipations how those predictions could be made more accurate in the future will be discussed.

Angeler, David G. Allen, Craig R. Garmestani, Ahjond S. Gunderson, Lance H. Hjerne, Olle Winder, Monika

The adaptive cycle was proposed as a conceptual model to portray patterns of change in complex systems. Despite the model having potential for elucidating change across systems, it has been used mainly as a metaphor, describing system dynamics qualitatively. We use a quantitative approach for testing premises (reorganisation, conservatism, adaptation) in the adaptive cycle , using Baltic Sea phytoplankton communities as an example of such complex system dynamics. Phytoplankton organizes in recurring spring and summer blooms, a well-established paradigm in planktology and succession theory, with characteristic temporal trajectories during blooms that may be consistent with adaptive cycle phases. We used long-term (1994–2011) data and multivariate analysis of community structure to assess key components of the adaptive cycle . Specifically, we tested predictions about: reorganisation: spring and summer blooms comprise distinct community states conservatism: community trajectories during individual adaptive cycles are conservative and adaptation: phytoplankton species during blooms change in the long term. All predictions were supported by our analyses. Results suggest that traditional ecological paradigms such as phytoplankton successional models have potential for moving the adaptive cycle from a metaphor to a framework that can improve our understanding how complex systems organize and reorganize following collapse. Quantifying reorganization, conservatism and adaptation provides opportunities to cope with the intricacies and uncertainties associated with fast ecological change, driven by shifting system controls. Ultimately, combining traditional ecological paradigms with heuristics of complex system dynamics using quantitative approaches may help refine ecological theory and improve our understanding of the resilience of ecosystems.

Angeler, David G Allen, Craig R Garmestani, Ahjond S Gunderson, Lance H Hjerne, Olle Winder, Monika

The adaptive cycle was proposed as a conceptual model to portray patterns of change in complex systems. Despite the model having potential for elucidating change across systems, it has been used mainly as a metaphor, describing system dynamics qualitatively. We use a quantitative approach for testing premises (reorganisation, conservatism, adaptation) in the adaptive cycle , using Baltic Sea phytoplankton communities as an example of such complex system dynamics. Phytoplankton organizes in recurring spring and summer blooms, a well-established paradigm in planktology and succession theory, with characteristic temporal trajectories during blooms that may be consistent with adaptive cycle phases. We used long-term (1994-2011) data and multivariate analysis of community structure to assess key components of the adaptive cycle . Specifically, we tested predictions about: reorganisation: spring and summer blooms comprise distinct community states conservatism: community trajectories during individual adaptive cycles are conservative and adaptation: phytoplankton species during blooms change in the long term. All predictions were supported by our analyses. Results suggest that traditional ecological paradigms such as phytoplankton successional models have potential for moving the adaptive cycle from a metaphor to a framework that can improve our understanding how complex systems organize and reorganize following collapse. Quantifying reorganization, conservatism and adaptation provides opportunities to cope with the intricacies and uncertainties associated with fast ecological change, driven by shifting system controls. Ultimately, combining traditional ecological paradigms with heuristics of complex system dynamics using quantitative approaches may help refine ecological theory and improve our understanding of the resilience of ecosystems.

Enzymes have been used for the production and processing of fish and seafood for several centuries in an empirical manner. In recent decades, a growing trend toward a rational and controlled application of enzymes for such goals has emerged. Underlying such pattern are, among others, the increasingly wider array of enzyme activities and enzyme sources, improved enzyme formulations, and enhanced requirements for cost-effective and environmentally friendly processes. The better use of enzyme action in fish- and seafood-related application has had a significant impact on fish-related industry. Thus, new products have surfaced, product quality has improved, more sustainable processes have been developed, and innovative and reliable analytical techniques have been implemented. Recent development in these fields are presented and discussed, and prospective developments are suggested. PMID:27458583

The relationship between allopurinol oxidizing enzyme and aldehyde oxidase was investaged in mice. The oxidation of both N-methylnicotinamide and allopurinol appears to be catalized by a single enzyme , aldehyde oxidase (aldehyde-oxygen oxidoreductase EC, 1.2.3.1.). This conclusion is based on the following evidence The postnatal changes of allopurinol and N-methylnicotinamide oxidizing activities were similar during growth and the levels of both activities increased in a parallel fashion upon the attainment of sexual maturity. The rates of loss of the activities of both enzymes by heat denaturation as well as dexamethasone administration were similar. The inhibitors of allopurinol oxidizing enzyme also suppressed N-methylnicotinamide oxidation. Competition of N-methylnicotineamide and allopurinol for oxidation was demonstrated. The rate of increase of the activities in both enzymes was almost parallel during each step of the purification from mouse liver supernatant. It was ascertained that xanthine oxidase in the enzyme preparation does not influence allopurinol oxidation.

Green, Brett J. Beezhold, Donald H.

Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes . PMID:21747869

Cardinale, Daniela Carette, Noëlle Michon, Thierry

The cooperative organization of enzymes by cells is a key feature for the efficiency of living systems. In the field of nanotechnologies, effort currently aims at mimicking this natural organization. Nanoscale resolution and high-registration alignment are necessary to control enzyme distribution in nano-containers or on the surface of solid supports. Virus capsid self-assembly is driven by precise supramolecular combinations of protein monomers, which have made them attractive building blocks to engineer enzyme nano-carriers (ENCs). We discuss some examples of what in our opinion constitute the latest advances in the use of plant viruses, bacteriophages and virus-like particles (VLPs) as nano-scaffolds for enzyme selection, enzyme confinement and patterning, phage therapy, raw material processing, and single molecule enzyme kinetics studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Zheng, Yi-Hua Hua, Tse-Chao Xu, Fei

Application of the thermal biosensor as analytical tool is promising due to advantages as universal, simplicity and quick response. A novel thermal biosensor based on enzyme reaction has been developed. This biosensor is a flow injection analysis system and consists of two channels with enzyme reaction column and reference column. The reference column, which is set for eliminating the unspecific heat, is inactived on special enzyme reaction of the ingredient to be detected. The special enzyme reaction takes places in the enzyme reaction column at a constant temperature realizing by a thermoelectric thermostat. Thermal sensor based on the thermoelectric module containing 127 serial BiTe-thermocouples is used to monitor the temperature difference between two streams from the enzyme reaction column and the reference column. The analytical example for dichlorvos shows that this biosensor can be used as analytical tool in medicine and biology.

Chen, Ming Zeng, Guangming Xu, Piao Lai, Cui Tang, Lin

Enzymes are fundamental biological catalysts responsible for biological regulation and metabolism. The opportunity for enzymes to 'meet' nanoparticles and nanomaterials is rapidly increasing due to growing demands for applications in nanomaterial design, environmental monitoring, biochemical engineering, and biomedicine. Therefore, understanding the nature of nanomaterial- enzyme interactions is becoming important. Since 2014, enzymes have been used to modify, degrade, or make nanoparticles/nanomaterials, while numerous nanoparticles/nanomaterials have been used as materials for enzymatic immobilization and biosensors and as enzyme mimicry. Among the various nanoparticles and nanomaterials, metal nanoparticles and carbon nanomaterials have received extensive attention due to their fascinating properties. This review provides an overview about how enzymes meet nanoparticles and nanomaterials. Copyright © 2017 Elsevier Ltd. All rights reserved.

Marine actinobacteria are well recognized for their capabilities to produce valuable natural products, which have great potential for applications in medical, agricultural, and fine chemical industries. In addition to producing unique enzymes responsible for biosynthesis of natural products, many marine actinobacteria also produce hydrolytic enzymes which are able to degrade various biopolymers, such as cellulose, xylan, and chitin. These enzymes are important to produce biofuels and biochemicals of interest from renewable biomass. In this chapter, the recent reports of novel enzymes produced by marine actinobacteria are reviewed, and advanced technologies that can be applied to search for novel marine enzymes as well as for improved enzyme production by marine actinobacteria are summarized, which include ribosome engineering, genome mining, as well as synthetic biology studies. © 2016 Elsevier Inc. All rights reserved.

Ayyagari, M. Kamtekar, S. Pande, R.

A methodology is described for immobilizing the enzyme alkaline phosphatase onto a glass surface using a novel biotinylated copolymer, poly(3-undecylthiophene-co-3- methanoithiophene). A streptavidin conjugate of alkaline phosphatase is used in this study. The biotinylated polymer is attached to the silanized glass surface via hydrophobic interactions and the enzyme is interfaced with the polymer through the classical biotin- streptavidin interaction. Alkaline phosphatase catalyzes the dephosphorylation of a macrocyclic compound, chloro-3-(4-methoxy spiro) (1,2 dioxetane-3-2`-tricyclo-) (3.3.1.1 )-(decani-4-yl) phenyl phosphate, to a species which emits energy by chemiluminescence. This chemiluminescence signal can be detected with a photomultiplier tube for enzymatic catalysis with the biocatalystmore » both in solution and immobilized on a glass surface. The signal generation is inhibited by the organophosphorus based insecticides such as paraoxon as well as nerve agents. We demonstrate in this study that a number of organophosphorus based insecticides inhibit the enzyme -mediated generation of chemiluminescence signal. This is true for the enzyme conjugate both free in solution and immobilized on a glass surface. In solution, the inhibition resembles the case of a partially uncompetitive system. By this type of inhibition we are able to detect pesticides down to about 50 ppb for the enzyme in solution. The pesticide detection limit of immobilized enzyme is currently being investigated. The enzyme is capable of a number of measurement cycles without significant loss of signal level.« less

Iglesias, Jose Lamontagne, Julien Erb, Heidi Gezzar, Sari Zhao, Shangang Joly, Erik Truong, Vouy Linh Skorey, Kathryn Crane, Sheldon Madiraju, S R Murthy Prentki, Marc

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle , encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes , including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes . We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme . Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Iglesias, Jose Lamontagne, Julien Erb, Heidi Gezzar, Sari Zhao, Shangang Joly, Erik Truong, Vouy Linh Skorey, Kathryn Crane, Sheldon Madiraju, S. R. Murthy Prentki, Marc

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle , encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes , including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes . We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme . Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. PMID:26423520

Schüürmann, Jan Quehl, Paul Festel, Gunter Jose, Joachim

Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes .

Deconstructs the "kiddie porn" media frame used by the industry and mainstream media to characterize Klein's ad campaign. Extends scholarship on the construction of youth in the media, showing how the kiddie-porn frame produces and reproduces common-sense beliefs about the nature of youth. Suggests a metadiscourse encompassing the…

Taoka, Yousuke Nagano, Naoki Okita, Yuji Izumida, Hitoshi Sugimoto, Shinichi Hayashi, Masahiro

Extracellular enzymes produced by six strains of thraustochytrids, Thraustochytrium, Schizochytrium, and Aurantiochytrium, were investigated. These strains produced 5 to 8 kinds of the extracellular enzymes , depending on the species. Only the genus Thraustochytrium produced amylase. When insoluble cellulose was used as substrate, cellulase was not detected in the six strains of thraustochytrids. This study indicates that marine eukaryotes, thraustochytrids, produced a wide variety of extracellular enzymes .

Jolly, Clifford D. Schussel, Leonard J. Carter, Layne

Fixed-bed reactors have been used at NASA-Marshall to purify wastewater generated by an end-use equipment facility, on the basis of a combination of multifiltration unibeds and enzyme unibeds. The enzyme beds were found to effectively remove such targeted organics as urea, alcohols, and aldehydes, down to levels lying below detection limits. The enzyme beds were also found to remove organic contaminants not specifically targeted.

Hou, Feng-Hua Ye, Jian-Qing Chen, Zuan-Guang Cheng, Zhi-Yi

With the continuous development in microfluidic fabrication technology, microfluidic analysis has evolved from a concept to one of research frontiers in last twenty years. The research of enzymes and enzyme inhibitors based on microfluidic devices has also made great progress. Microfluidic technology improved greatly the analytical performance of the research of enzymes and enzyme inhibitors by reducing the consumption of reagents, decreasing the analysis time, and developing automation. This review focuses on the development and classification of enzymes and enzyme inhibitors research based on microfluidic devices.

Mattiasson, B Borrebaeck, C Sanfridson, B Mosbach, K

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

Nanda, Vikas Koder, Ronald L.

The rational design of artificial enzymes either by applying physio-chemical intuition of protein structure and function or with the aid of computation methods is a promising area of research with the potential to tremendously impact medicine, industrial chemistry and energy production. Designed proteins also provide a powerful platform for dissecting enzyme mechanisms of natural systems. Artificial enzymes have come a long way, from simple α-helical peptide catalysts to proteins that facilitate multi-step chemical reactions designed by state-of-the-art computational methods. Looking forward, we examine strategies employed by natural enzymes which could be used to improve the speed and selectivity of artificial catalysts. PMID:21124375

Shearer, Alexander G. Altman, Tomer Rhee, Christine D.

Despite advances in sequencing technology, there are still significant numbers of well-characterized enzymatic activities for which there are no known associated sequences. These ‘orphan enzymes’ represent glaring holes in our biological understanding, and it is a top priority to reunite them with their coding sequences. Here we report a methodology for resolving orphan enzymes through a combination of database search and literature review. Using this method we were able to reconnect over 270 orphan enzymes with their corresponding sequence. This success points toward how we can systematically eliminate the remaining orphan enzymes and prevent the introduction of future orphan enzymes . PMID:24826896

Illanes, Andrés Wilson, Lorena

Temperature is a very relevant variable for any bioprocess. Temperature optimization of bioreactor operation is a key aspect for process economics. This is especially true for enzyme -catalyzed processes, because enzymes are complex, unstable catalysts whose technological potential relies on their operational stability. Enzyme reactor design is presented with a special emphasis on the effect of thermal inactivation. Enzyme thermal inactivation is a very complex process from a mechanistic point of view. However, for the purpose of enzyme reactor design, it has been oversimplified frequently, considering one-stage first-order kinetics of inactivation and data gathered under nonreactive conditions that poorly represent the actual conditions within the reactor. More complex mechanisms are frequent, especially in the case of immobilized enzymes , and most important is the effect of catalytic modulators (substrates and products) on enzyme stability under operation conditions. This review focuses primarily on reactor design and operation under modulated thermal inactivation. It also presents a scheme for bioreactor temperature optimization, based on validated temperature-explicit functions for all the kinetic and inactivation parameters involved. More conventional enzyme reactor design is presented merely as a background for the purpose of highlighting the need for a deeper insight into enzyme inactivation for proper bioreactor design.

Díaz, Sebastián. A. Breger, Joyce C. Malanoski, Anthony Claussen, Jonathan C. Walper, Scott A. Ancona, Mario G. Brown, Carl W. Stewart, Michael H. Oh, Eunkeu Susumu, Kimihiro Medintz, Igor L.

Enzymes are important players in multiple applications, be it bioremediation, biosynthesis, or as reporters. The business of catalysis and inhibition of enzymes is a multibillion dollar industry and understanding the kinetics of commercial enzymes can have a large impact on how these systems are optimized. Recent advances in nanotechnology have opened up the field of nanoparticle (NP) and enzyme conjugates and two principal architectures for NP conjugate systems have been developed. In the first example the enzyme is bound to the NP in a persistent manner, here we find that key factors such as directed enzyme conjugation allow for enhanced kinetics. Through controlled comparative experiments we begin to tease out specific mechanisms that may account for the enhancement. The second system is based on dynamic interactions of the enzymes with the NP. The enzyme substrate is bound to the NP and the enzyme is free in solution. Here again we find that there are many variables , such as substrate positioning and NP selection, that modify the kinetics.

Arkesteijn, Marco Jobson, Simon Hopker, James Passfield, Louis

Previous research has shown that cycling in a standing position reduces cycling economy compared with seated cycling . It is unknown whether the cycling intensity moderates the reduction in cycling economy while standing. The aim was to determine whether the negative effect of standing on cycling economy would be decreased at a higher intensity. Ten cyclists cycled in 8 different conditions. Each condition was either at an intensity of 50% or 70% of maximal aerobic power at a gradient of 4% or 8% and in the seated or standing cycling position. Cycling economy and muscle activation level of 8 leg muscles were recorded. There was an interaction between cycling intensity and position for cycling economy (P = .03), the overall activation of the leg muscles (P = .02), and the activation of the lower leg muscles (P = .05). The interaction showed decreased cycling economy when standing compared with seated cycling , but the difference was reduced at higher intensity. The overall activation of the leg muscles and the lower leg muscles, respectively, increased and decreased, but the differences between standing and seated cycling were reduced at higher intensity. Cycling economy was lower during standing cycling than seated cycling , but the difference in economy diminishes when cycling intensity increases. Activation of the lower leg muscles did not explain the lower cycling economy while standing. The increased overall activation, therefore, suggests that increased activation of the upper leg muscles explains part of the lower cycling economy while standing.

Dimroth, Peter von Ballmoos, Christoph Meier, Thomas

Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in cell metabolism include the tricarboxylic acid and the urea cycle , in which a specific carrier substrate undergoes a sequence of chemical transformations and is regenerated at the end. Other examples include the interconversions of cofactors, such as NADH or ATP, which are present in the cell in limiting amounts and have to be recycled effectively for metabolism to continue. Every living cell performs a rapid turnover of ATP to ADP to fulfil various energetic demands and effectively regenerates the ATP from ADP in an energy-consuming process. The turnover of the ATP cycle is impressive a human uses about its body weight in ATP per day. Enzymes perform catalytic reaction cycles in which they undergo several chemical and physical transformations before they are converted back to their original states. The ubiquitous F1F(o) ATP synthase is of particular interest not only because of its biological importance, but also owing to its unique rotational mechanism. Here, we give an overview of the membrane-embedded F(o) sector, particularly with respect to the recent crystal structure of the c ring from Ilyobacter tartaricus, and summarize current hypotheses for the mechanism by which rotation of the c ring is generated.

Razavi, Bahar S. Zarebanadkouki, Mohsen Blagodatskaya, Evgenia Kuzyakov, Yakov

Extracellular enzymes are important for decomposition of many biological macromolecules abundant in soil such as cellulose, hemicelluloses and proteins. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling . So far acquisition of in situ data about local activity of different enzymes in soil has been challenged. That is why there is an urgent need in spatially explicit methods such as 2-D zymography to determine the variation of enzymes along the roots in different plants. Here, we developed further the zymography technique in order to quantitatively visualize the enzyme activities (Spohn and Kuzyakov, 2013), with a better spatial resolution We grew Maize (Zea mays L.) and Lentil (Lens culinaris) in rhizoboxes under optimum conditions for 21 days to study spatial distribution of enzyme activity in soil and along roots. We visualized the 2D distribution of the activity of three enzymes :β-glucosidase, leucine amino peptidase and phosphatase, using fluorogenically labelled substrates. Spatial resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography shows different pattern of spatial distribution of enzyme activity along roots and soil of different plants. We observed a uniform distribution of enzyme activities along the root system of Lentil. However, root system of Maize demonstrated inhomogeneity of enzyme activities. The apical part of an individual root (root tip) in maize showed the highest activity. The activity of all enzymes was the highest at vicinity of the roots and it decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify

Jacobberger, James W Sramkoski, R Michael Stefan, Tammy Woost, Philip G

Cell cycle cytometry and analysis are essential tools for studying cells of model organisms and natural populations (e.g., bone marrow). Methods have not changed much for many years. The simplest and most common protocol is DNA content analysis, which is extensively published and reviewed. The next most common protocol, 5-bromo-2-deoxyuridine S phase labeling detected by specific antibodies, is also well published and reviewed. More recently, S phase labeling using 5'-ethynyl-2'-deoxyuridine incorporation and a chemical reaction to label substituted DNA has been established as a basic, reliable protocol. Multiple antibody labeling to detect epitopes on cell cycle regulated proteins, which is what this chapter is about, is the most complex of these cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. However, because this knowledge is relatively well presented methodologically in many papers and reviews, this chapter will present a minimal Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature. Most of the presented work involves how to segment the data to produce a complete, progressive, and compartmentalized cell cycle analysis from early G1 to late mitosis (telophase). A more recent development, using fluorescent proteins fused with proteins or peptides that are degraded by ubiquitination during specific periods of the cell cycle , termed "Fucci" (fluorescent, ubiquitination-based cell cycle indicators) provide an analysis similar in concept to multiple antibody labeling, except in this case cells can be analyzed while living and transgenic organisms can be created to perform cell cycle analysis ex or in vivo (Sakaue-Sawano et al., Cell 132:487-498, 2007). This technology will not be discussed.

Burrage, Lindsay C. Sun, Qin Elsea, Sarah H. Jiang, Ming-Ming Nagamani, Sandesh C.S. Frankel, Arthur E. Stone, Everett Alters, Susan E. Johnson, Dale E. Rowlinson, Scott W. Georgiou, George Lee, Brendan H.

Arginase deficiency is caused by deficiency of arginase 1 (ARG1), a urea cycle enzyme that converts arginine to ornithine. Clinical features of arginase deficiency include elevated plasma arginine levels, spastic diplegia, intellectual disability, seizures and growth deficiency. Unlike other urea cycle disorders, recurrent hyperammonemia is typically less severe in this disorder. Normalization of plasma arginine levels is the consensus treatment goal, because elevations of arginine and its metabolites are suspected to contribute to the neurologic features. Using data from patients enrolled in a natural history study conducted by the Urea Cycle Disorders Consortium, we found that 97% of plasma arginine levels in subjects with arginase deficiency were above the normal range despite conventional treatment. Recently, arginine-degrading enzymes have been used to deplete arginine as a therapeutic strategy in cancer. We tested whether one of these enzymes , a pegylated human recombinant arginase 1 (AEB1102), reduces plasma arginine in murine models of arginase deficiency. In neonatal and adult mice with arginase deficiency, AEB1102 reduced the plasma arginine after single and repeated doses. However, survival did not improve likely, because this pegylated enzyme does not enter hepatocytes and does not improve hyperammonemia that accounts for lethality. Although murine models required dosing every 48 h, studies in cynomolgus monkeys indicate that less frequent dosing may be possible in patients. Given that elevated plasma arginine rather than hyperammonemia is the major treatment challenge, we propose that AEB1102 may have therapeutic potential as an arginine-reducing agent in patients with arginase deficiency. PMID:26358771

Fries, Pascal Nikolić, Danko Singer, Wolf

Activated neuronal groups typically engage in rhythmic synchronization in the gamma-frequency range (30-100 Hz). Experimental and modeling studies demonstrate that each gamma cycle is framed by synchronized spiking of inhibitory interneurons. Here, we review evidence suggesting that the resulting rhythmic network inhibition interacts with excitatory input to pyramidal cells such that the more excited cells fire earlier in the gamma cycle . Thus, the amplitude of excitatory drive is recoded into phase values of discharges relative to the gamma cycle . This recoding enables transmission and read out of amplitude information within a single gamma cycle without requiring rate integration. Furthermore, variation of phase relations can be exploited to facilitate or inhibit exchange of information between oscillating cell assemblies. The gamma cycle could thus serve as a fundamental computational mechanism for the implementation of a temporal coding scheme that enables fast processing and flexible routing of activity, supporting fast selection and binding of distributed responses. This review is part of the INMED/TINS special issue Physiogenic and pathogenic oscillations: the beauty and the beast, based on presentations at the annual INMED/TINS symposium (http://inmednet.com).

A unique process cycle and apparatus design separates the consumer (cryogenic) load return flow from most of the recycle return flow of a refrigerator and/or liquefier process cycle . The refrigerator and/or liquefier process recycle return flow is recompressed by a multi-stage compressor set and the consumer load return flow is recompressed by an independent consumer load compressor set that maintains a desirable constant suction pressure using a consumer load bypass control valve and the consumer load return pressure control valve that controls the consumer load compressor's suction pressure. The discharge pressure of this consumer load compressor is thereby allowed to float at the intermediate pressure in between the first and second stage recycle compressor sets. Utilizing the unique gas management valve regulation, the unique process cycle and apparatus design in which the consumer load return flow is separate from the recycle return flow, the pressure ratios of each recycle compressor stage and all main pressures associated with the recycle return flow are allowed to vary naturally, thus providing a naturally regulated and balanced floating pressure process cycle that maintains optimal efficiency at design and off-design process cycle capacity and conditions automatically.

A unique process cycle and apparatus design separates the consumer (cryogenic) load return flow from most of the recycle return flow of a refrigerator and/or liquefier process cycle . The refrigerator and/or liquefier process recycle return flow is recompressed by a multi-stage compressor set and the consumer load return flow is recompressed by an independent consumer load compressor set that maintains a desirable constant suction pressure using a consumer load bypass control valve and the consumer load return pressure control valve that controls the consumer load compressor's suction pressure. The discharge pressure of this consumer load compressor is thereby allowed to float at the intermediate pressure in between the first and second stage recycle compressor sets. Utilizing the unique gas management valve regulation, the unique process cycle and apparatus design in which the consumer load return flow is separate from the recycle return flow, the pressure ratios of each recycle compressor stage and all main pressures associated with the recycle return flow are allowed to vary naturally, thus providing a naturally regulated and balanced floating pressure process cycle that maintains optimal efficiency at design and off-design process cycle capacity and conditions automatically.

Pectinolytic enzymes of four rumen fungi have been described. Three fungal species were monocentric Neocallimastix spp. H15, JL3 and OC2, and one isolate was a polycentric strain of Orpinomyces joyonii, A4. They differed in degree of pectin degradation and utilization. Only the strain Neocallimastix sp. H15 and partially Orpinomyces joyonii A4 were able to utilize pectin to a higher extent. The most important pectinolytic activity in all these isolates represented pectin lyase (EC 4.2.2.10) and polygalacturonase (EC 3.2.1.15). Their specific activities were in the range of 100-900 and 10-450 micrograms galacturonic acid h-1 mg protein-1 for pectin lyase and polygalacturonase, respectively. Polygalacturonase, located mainly in the endocellular fraction, was inhibited by calcium ions and had the main pH optimum at pH 6.0. All strains produced pectate lyase (EC 4.2.2.2). None of the strains tested produced pectinesterase (EC 3.1.1.11).

Gussin, Arnold E. S. McCormack, Jeffrey H. Waung, Lucille Yih-Lo Gluckin, Doreen S.

Pollen from 5 plant species (Lycopersicon pimpinellifolium Mill., Hermerocallis minor Mill., Galtonia condicans Decne., Camellia japonica L., and Lathyrus odoratus L.) representing 4 families germinated well in media containing trehalose as the sole carbon source. Data are presented indicating that pollen metabolized this disaccharide for germination and subsequent pollen-tube growth the sugar was not merely an osmoregulator. An inhibitor of trehalase activity depressed germination in trehalose but not in sucrose. Phloridzin dihydrate, an inhibitor of glucose transport, depressed germination in both disaccharides. Biochemical tests demonstrated that a pollen extract was capable of hydrolyzing trehalose to its constituent glucose monomers. Heat inactivation experiments confirmed the presence of a distinct trehalase having a rigid specificity for its substrate. By this method, trehalase activity was completely distinguishable from the activities of other α- and β-glucosidases and β-galactosidases. Localization data indicated that the enzyme diffused from intact grains and was probably soluble. The presence of its substrate could not be demonstrated in pollen or in stigmatic or stylar tissues. PMID:5379538

Gussin, A E McCormack, J H Waung, L Y Gluckin, D S

Pollen from 5 plant species (Lycopersicon pimpinellifolium Mill., Hermerocallis minor Mill., Galtonia condicans Decne., Camellia japonica L., and Lathyrus odoratus L.) representing 4 families germinated well in media containing trehalose as the sole carbon source. Data are presented indicating that pollen metabolized this disaccharide for germination and subsequent pollen-tube growth the sugar was not merely an osmoregulator. An inhibitor of trehalase activity depressed germination in trehalose but not in sucrose. Phloridzin dihydrate, an inhibitor of glucose transport, depressed germination in both disaccharides. Biochemical tests demonstrated that a pollen extract was capable of hydrolyzing trehalose to its constituent glucose monomers. Heat inactivation experiments confirmed the presence of a distinct trehalase having a rigid specificity for its substrate. By this method, trehalase activity was completely distinguishable from the activities of other alpha- and beta-glucosidases and beta-galactosidases. Localization data indicated that the enzyme diffused from intact grains and was probably soluble. The presence of its substrate could not be demonstrated in pollen or in stigmatic or stylar tissues.

Elms, J Robinson, E Mason, H Iqbal, S Garrod, A Evans, G S

Enzymes are commonly used in the baking industry, as they can improve dough quality and texture and lengthen the shelf life of the final product. There is little published information highlighting exposure to enzymes (other than fungal alpha-amylase) in the baking industry, therefore the purpose of this study was to identify antibodies and develop assays for the measurement of a variety of such enzymes in samples of airborne flour dust. Polyclonal antibodies to bacterial amylase, glucose oxidase and amyloglucosidase were identified and developed into ELISA assays. The assays showed limited cross-reactivity with other enzymes commonly used in the baking industry. We measured levels of airborne enzymes in 195 personal air samples taken from a sample of 55 craft baking establishments. We were able to detect amyloglucosidase in 9% (16/184) of the samples, fungal alpha-amylase in 6% (11/171), bacterial alpha-amylase in 7% (13/195). However, we were unable to detect glucose oxidase in any of the samples. Measurements for protease enzymes were not carried out. Median levels in detectable samples of amyloglucosidase, fungal alpha-amylase and bacterial amylase were similar at 10.3, 5.3 and 5.9 ng/m(3), respectively. These figures represent the total enzyme protein (active and inactive) measured. There are few data in the literature regarding sensitization and exposure-response relationships to these enzymes , and indeed there is often a lack of information within the industry as to the precise enzyme content of particular baking ingredients. As a precautionary measure, all enzymes are regarded as having the potential to cause respiratory sensitization. Consequently, exposures need to be controlled to as low a level as reasonably practicable, and future investigation may highlight the importance of measuring a variety of enzyme exposures and standardizing these methodologies to inform approaches to adequate control.

Microbes are the engines driving biogeochemical cycles . Microbial extracellular enzymatic activities (EEAs) are the "gatekeepers" of the carbon cycle . The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes . Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell's fate. In contrast, cell-free enzymes belong to a kind of "living dead" realm because they are not attached to a living cell but still are able to perform their function away from the cell and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes . This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go "beyond the living things," studying the response of cells/organisms to different stressors, but also to study cell-free enzymes , in order to fully constrain the future and evolution of marine biogeochemical cycles .

Microbes are the engines driving biogeochemical cycles . Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle . The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes . Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes . This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes , in order to fully constrain the future and evolution of marine biogeochemical cycles . PMID:29354095

Mata-Sotres, José Antonio Moyano, Francisco Javier Martínez-Rodríguez, Gonzalo Yúfera, Manuel

In order to identify daily changes in digestive physiology in developing gilthead seabream larvae, the enzyme activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try, chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2 and α-amylase-amy2a) were measured during a 24h cycle in larvae reared under a 12h light/12h dark photoperiod. Larvae were sampled at 10, 18, 30 and 60days post-hatch. In each sampling day, larvae were sampled every 3h during a complete 24h cycle . The enzyme activity and gene expression exhibited a marked dependent behavior to the light/darkness cycle in all tested ages. The patterns of activity and expression of all tested enzymes were compared to the feeding pattern found in the same larvae, which showed a rhythmic feeding pattern with a strong light synchronization. In the four tested ages, the activities of trypsin, and to a lesser extent lipases and amylase, were related to feeding activity. Molecular expression of the pancreatic enzymes tended to increase during the night, probably as an anticipation of the forthcoming ingestion of food that will take place during the next light period. It follows that the enzymatic activities are being regulated at translational and/or post-translational level. The potential variability of enzyme secretion along the whole day is an important factor to take into account in future studies. A particularly striking consequence of the present results is the reliability of studies based in only one daily sample taken at the same hour of the day, as those focused to assess ontogeny of digestive enzymes . Copyright © 2016 Elsevier Inc. All rights reserved.

Using NSO/KP magnetograms, the pattern and rate of the emergence of magnetic flux and the development of the large-scale patterns of unipolar fields are considered in terms of the solar magnetic cycle . Magnetic flux emerges in active regions at an average rate of 2 x 10(exp 21) Mx/day, approximately 10 times the estimated rate in ephemeral regions. Observations are presented that demonstrate that the large-scale unipolar fields originate in active regions and activity nests. For cycle 21, the net contribution of ephemeral regions to the axial dipole moment of the Sun is positive, and is of opposite sign to that of active regions. Its amplitude is smaller by a factor of 6, assuming an average lifetime of ephemeral regions of 8 hours. Active regions larger than 4500 Mm(sup 2) are the primary contributor to the cycle variation of Sun's axial dipole moment.