The activities of PEP carboxylase and the C4 acid decarboxylases are little changed by drought stress in three C4 grasses of different subtypes

The C(4) photosynthetic pathway involves the assimilation of CO(2) by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C(4) acids. The enzymes of the CO(2) concentrating mechanism could be affected under water deficit and limit C(4) photosynthesis. Three different C(4) grasses were submitted to gradually induced drought stress conditions: Paspalum dilatatum (NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and Zoysia japonica (PEP carboxykinase, PEPCK). Moderate leaf dehydration affected the activity and regulation of PEPC in a similar manner in the three grasses but had species-specific effects on the C(4) acid decarboxylases, NADP-ME, NAD-ME and PEPCK, although changes in the C(4) enzyme activities were small. In all three species, the PEPC phosphorylation state, judged by the inhibitory effect of L: -malate on PEPC activity, increased with water deficit and could promote increased assimilation of CO(2) by the enzyme under stress conditions. Appreciable activity of PEPCK was observed in all three species suggesting that this enzyme may act as a supplementary decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.     

Activity of C<Subscript>4</Subscript> enzymes in C<Subscript>3</Subscript>-type herbaceous plants

The activity of enzymes characteristic for C₄-type photosynthesis was determined in different organs of two herbaceous plants: Reynoutria japonica Houtt. and Helianthus tuberosus L. The activity of phosphoenolpyruvate carboxylase (PEPC) was usually higher in the roots, some of the stem tissues and petioles in comparison to the leaf blades. The highest activity of malic enzymes (NAD-ME, NADP-ME) and phosphoenolpyruvate carboxykinase (PEPCK) was in the petioles and stem tissues of both plants and the lowest in the leaf blades and the pith of Helianthus tuberosus L.     

The effect of pCO2 on carbon acquisition and intracellular assimilation in four marine diatoms

The effect of pCO₂ on carbon acquisition and intracellular assimilation was investigated in the three bloom-forming diatom species, Eucampia zodiacus (Ehrenberg), Skeletonema costatum (Greville) Cleve, Thalassionema nitzschioides (Grunow) Mereschkowsky and the non-bloom-forming Thalassiosira pseudonana (Hust.) Hasle and Heimdal. In vivo activities of carbonic anhydrase (CA), photosynthetic O₂ evolution, CO₂ and HCO₃⁻ uptake rates were measured by membrane-inlet mass spectrometry (MIMS) in cells acclimated to pCO₂ levels of 370 and 800 μatm. To investigate whether the cells operate a C₄-like pathway, activities of ribulose-1,5-bisphosphate carboxylase (RubisCO) and phosphoenolpyruvate carboxylase (PEPC) were measured at the mentioned pCO₂ levels and a lower pCO₂ level of 50 μatm. In the bloom-forming species, extracellular CA activities strongly increased with decreasing CO₂ supply while constantly low activities were obtained for T. pseudonana. Half-saturation concentrations (K_1/2) for photosynthetic O₂ evolution decreased with decreasing CO₂ supply in the two bloom-forming species S. costatum and T. nitzschioides, but not in T. pseudonana and E. zodiacus. With the exception of S. costatum, maximum rates (V_max) of photosynthesis remained constant in all investigated diatom species. Independent of the pCO₂ level, PEPC activities were significantly lower than those for RubisCO, averaging generally less than 3%. All examined diatom species operate highly efficient CCMs under ambient and high pCO₂, but differ strongly in the degree of regulation of individual components of the CCM such as C_i uptake kinetics and extracellular CA activities. The present data do not suggest C₄ metabolism in the investigated species.     

Posttranslational regulation of phosphoenolpyruvate carboxylase in c(4) and crassulacean Acid metabolism plants

Control of C₄ photosynthesis and Crassulacean acid metabolism (CAM) is, in part, mediated by the diel regulation of phosphoenolpyruvate carboxylase (PEPC) activity. The nature of this regulation of PEPC in the leaf cell cytoplasm of C₄ and CAM plants is both metabolite-related and posttranslational. Specificially, the regulatory properties of the enzyme vary in accord with the physiological activity of C₄ photosynthesis and CAM: PEPC is less sensitive to feedback inhibition by l-malate under light (C₄ plants) or at night (CAM plants) than in darkness (C₄) or during the day (CAM). While the view that a light-induced change in the aggregation state of the holoenzyme is a general mechanism for the diel regulation of PEPC activity in CAM plants is currently in dispute, there is no supportive in vivo evidence for such a tetramer/dimer interconversion in C₄ plants. In contrast, a wealth of in vitro and in vivo data has accumulated in support of the view that the reversible phosphorylation of a specific, N-terminal regulatory serine residue in PEPC (e.g. Ser-15 or Ser-8 in the maize or sorghum enzymes, respectively) plays a key, if not cardinal, role in the posttranslational regulation of the carboxylase by light/dark or day/night transitions in both C₄ and CAM plants, respectively.     

Coordinate regulation of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase by light and CO2 during C4 photosynthesis

The aim of this study was to investigate the relationship between the phosphorylation and activation states of phosphoenolpyruvate carboxykinase (PEPCK) and to investigate how the phosphorylation states of PEPCK and phosphoenolpyruvate carboxylase (PEPC) are coordinated in response to light intensity and CO(2) concentration during photosynthesis in leaves of the C(4) plant Guinea grass (Panicum maximum). There was a linear, reciprocal relationship between the phosphorylation state of PEPCK and its activation state, determined in a selective assay that distinguishes phosphorylated from nonphosphorylated forms of the enzyme. At high photon flux density and high CO(2) (750 microL L(-1)), PEPC was maximally phosphorylated and PEPCK maximally dephosphorylated within 1 h of illumination. The phosphorylation state of both enzymes did not saturate until high light intensities (about 1,400 micromol quanta m(-2) s(-1)) were reached. After illumination at lower light intensities and CO(2) concentrations, the overall change in phosphorylation state was smaller and it took longer for the change in phosphorylation state to occur. Phosphorylation states of PEPC and PEPCK showed a strikingly similar, but inverse, pattern in relation to changes in light and CO(2). The protein phosphatase inhibitor, okadaic acid, promoted the phosphorylation of both enzymes. The protein synthesis inhibitor, cycloheximide, blocked dark phosphorylation of PEPCK. The data show that PEPC and PEPCK phosphorylation states are closely coordinated in vivo, despite being located in the mesophyll and bundle sheath cells, respectively.     

Oligomeric enzymes in the C4 pathway of photosynthesis

This review deals with the factors controlling the aggregation-state of several enzymes involved in C₄ photosynthesis, namely phosphoenolpyruvate carboxylase, NAD-and NADP-malic enzyme, NADP-malic dehydrogenase and pyruvate, phosphate dikinase and its regulatory protein. All of these enzymes are oligomeric and have been shown to undergo changes in their quaternary structure in vitro under different conditions. The activity changes linked to variations in aggregation-state are discussed in terms of their putative physiological role in the regulation of C₄ metabolism.Abbreviations P-enolpyruvate phosphoenolpyruvate - NAD-ME NAD-dependent malic enzyme - NADP-ME NADP-dependent malic enzyme - NADP-MDH NADP-dependent malic dehydrogenase - PPDK pyruvate, phosphate dikinase - PPDK-RP pyruvate, phosphate dikinase regulatory protein - V_max maximal velocity - K_m Michaelis constant - CAM Crassulacean acid metabolism     

干旱胁迫对2种光合类型C4荒漠植物叶片光合特征酶和抗氧化酶活性的影响

以荒漠C₄草本植物蔷薇猪毛菜(NADP苹果酸酶型,NADP-ME）和粗枝猪毛菜（NAD苹果酸酶型,NAD-ME）为研究对象,采用盆栽控水试验设置正常供水和轻度、中度、重度干旱处理（土壤含水量分别为田间持水量80%、60%、45%和35%）,通过测定不同程度干旱胁迫下叶片含水量、C₄光合特征酶和抗氧化酶活性等指标,探讨不同类型C₄荒漠植物光合特征酶和抗氧化系统对干旱逆境的适应机制。结果显示：（1）2种植物叶片含水量均随干旱胁迫的加剧不同程度降低。（2）叶片磷酸烯醇式丙酮酸羧化酶（PEPC）活性在中度干旱胁迫下显著增加而在重度干旱胁迫下急剧下降;蔷薇猪毛菜NAD-ME活性和粗枝猪毛菜NADP-ME活性都很低,且它们基本不受干旱胁迫的影响;随干旱胁迫的加剧,蔷薇猪毛菜NADP-ME活性呈下降趋势,而粗枝猪毛菜NAD-ME活性先显著增加而在重度干旱胁迫下显著降低。（3）随着干旱胁迫的加剧,叶片超氧化物歧化酶（SOD）活性呈下降趋势,过氧化物酶（POD）活性在不同程度干旱胁迫下均有不同程度增加;过氧化氢酶（CAT）活性在中度干旱胁迫下均有不同程度的增加,但在重度干旱胁迫下蔷薇猪毛菜CAT活性降低,而粗枝猪毛菜CAT活性显著增加;丙二醛（MDA）含量随干旱胁迫的加剧均有不同程度的增加。研究认为,一定程度干旱胁迫下,2种荒漠植物的PEPC活性均有增加;不同光合类型C₄植物叶片脱羧酶（NADP-ME和NAD-ME）对干旱胁迫的响应有明显的差异。POD和CAT是这两种C₄植物适应干旱胁迫的主要抗氧化酶,但蔷薇猪毛菜CAT在重度干旱胁迫下没有起到积极保护作用。     

Nitrogen metabolism-related enzymes in <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> after <Emphasis Type="Italic">Botrytis cinerea</Emphasis> infection

We compared C₃ and CAM (crassulacean acid metabolism) states in Mesembryanthemum crystallinum, a facultative CAM species, with respect to the involvement of phosphoenolpyruvate carboxylase (PEPC) and nitrogen metabolismrelated enzymes in plant response to Botrytis cinerea infection. The enzyme activities were monitored both in pathogeninoculated 2^nd leaf pair and non-inoculated 3^rd leaf pair. The control activities of most studied enzymes were dependent on the mode of photosynthesis. Compared to C₃ plants, those performing CAM exhibited higher PEPC, nitrate reductase (NR), and deaminating glutamate dehydrogenase (NAD-GDH) activities but lower glutamine synthetase (GS) and alanine aminotransferase (ALT) activities. Regardless of the mode of photosynthetic carbon assimilation, the plants responded to infection with enhancement of PEPC and inhibition of NR activities in the inoculated leaves. Whereas the activity of GS remained unaffected, those of all glutamate-yielding enzymes, namely ferredoxin-dependent glutamate synthase (Fd-GOGAT), aspartate aminotransferase (AST), ALT, and aminating glutamate dehydrogenase (NADHGDH) were altered after infection. However, the time-course and extent of the observed changes differed in C₃ and CAM plants. In general, CAM plants responded to infection with an earlier increase in PEPC and Fd-GOGAT activities as well as later inhibition of NR activity. Contrary to C₃ plants, in those performing CAM the activities of PEPC, Fd-GOGAT, NADH-GDH, and AST in the non-inoculated 3^rd leaf pair were similarly influenced by infection as in leaves directly inoculated with the pathogen. This implies that the local infection induced an alteration of carbon/nitrogen status in healthy upper leaves. This reprogramming resulting from changes in PEPC and nitrogen metabolism-related enzymes was C₃- and CAM-specific.     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

Structural and biochemical bases of photorespiration in C<Subscript>4</Subscript> plants: quantification of organelles and glycine decarboxylase

In C₄ plants, photorespiration is decreased relative to C₃ plants. However, it remains unclear how much photorespiratory capacity C₄ leaf tissues actually have. We thoroughly investigated the quantitative distribution of photorespiratory organelles and the immunogold localization of the P protein of glycine decarboxylase (GDC) in mesophyll (M) and bundle sheath (BS) cells of various C₄ grass species. Specific differences occurred in the proportions of mitochondria and peroxisomes in the BS cells (relative to the M cells) in photosynthetic tissues surrounding a vein: lower in the NADP-malic enzyme (NADP-ME) species having poorly formed grana in the BS chloroplasts, and higher in the NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) species having well developed grana. In all C₄ species, GDC was localized mainly in the BS mitochondria. When the total amounts of GDC in the BS mitochondria per unit leaf width were estimated from the immunogold labeling density and the quantity of mitochondria, the BSs of NADP-ME species contained less GDC than those of NAD-ME or PCK species. This trend was also verified by immunoblot analysis of leaf soluble protein. There was a high positive correlation between the degree of granal development (granal index) in the BS chloroplasts and the total amount of GDC in the BS mitochondria. The variations in the structural and biochemical features involved in photorespiration found among C₄ species might reflect differences in the O₂/CO₂ partial pressure and in the potential photorespiratory capacity of the BS cells.Abbreviations BS Bundle sheath - GDC Glycine decarboxylase - M Mesophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK Phosphoenolpyruvate carboxykinase     

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis

Background

Diatoms are unicellular algae responsible for approximately 20% of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids.

Methodology/Principal Findings

The whole genome sequence of the diatom Phaeodactylum tricornutum has recently been completed. We identified and annotated genes for enzymes involved in carbohydrate pathways based on extensive EST support and comparison to the whole genome sequence of a second diatom, Thalassiosira pseudonana. Protein localization to mitochondria was predicted based on identified similarities to mitochondrial localization motifs in other eukaryotes, whereas protein localization to plastids was based on the presence of signal peptide motifs in combination with plastid localization motifs previously shown to be required in diatoms. We identified genes potentially involved in a C4-like photosynthesis in P. tricornutum and, on the basis of sequence-based putative localization of relevant proteins, discuss possible differences in carbon concentrating mechanisms and CO₂ fixation between the two diatoms. We also identified genes encoding enzymes involved in photorespiration with one interesting exception: glycerate kinase was not found in either P. tricornutum or T. pseudonana. Various Calvin cycle enzymes were found in up to five different isoforms, distributed between plastids, mitochondria and the cytosol. Diatoms store energy either as lipids or as chrysolaminaran (a β-1,3-glucan) outside of the plastids. We identified various β-glucanases and large membrane-bound glucan synthases. Interestingly most of the glucanases appear to contain C-terminal anchor domains that may attach the enzymes to membranes.

Conclusions/Significance

Here we present a detailed synthesis of carbohydrate metabolism in diatoms based on the genome sequences of Thalassiosira pseudonana and Phaeodactylum tricornutum. This model provides novel insights into acquisition of dissolved inorganic carbon and primary metabolic pathways of carbon in two different diatoms, which is of significance for an improved understanding of global carbon cycles.     

Phosphoenolpyruvate metabolism in Teladorsagia circumcincta: A critical junction between aerobic and anaerobic metabolism

Nematodes which have adapted to an anaerobic lifestyle in their adult stages oxidise phosphoenolpyruvate (PEP) to oxaloacetate rather than pyruvate as the final product of glycolysis. This adaptation involves selective expression of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), instead of pyruvate kinase (PK). However, such adaptation is not absolute in aerobic nematode species. We have examined the activity and kinetics of PEPCK and PK in larvae (L₃) and adults of Teladorsagia circumcincta, a parasite known to exhibit oxygen uptake. Results revealed that PK and PEPCK activity existed in both L₃s and adults. The enzymes had differing affinity for nucleotide diphosphates: while both can utilise GDP, only PK utilised ADP and only PEPCK utilised IDP. In both life cycle stages, enzymes showed similar affinity for PEP. PK activity was predominant in both stages, although activity of this enzyme was lower in adults. When combined, both the activity levels and the enzyme kinetics showed that pyruvate production is probably favoured in both L₃ and adult stages of T. circumcincta and suggest that metabolism of PEP to oxaloacetate is a minor metabolic pathway in this species.