Phosphorylation of phosphoenolpyruvate carboxylase is not essential for high photosynthetic rates in the C4 species Flaveria bidentis

Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) plays a key role during C(4) photosynthesis. The enzyme is activated by metabolites such as glucose-6-phosphate and inhibited by malate. This metabolite sensitivity is modulated by the reversible phosphorylation of a conserved serine residue near the N terminus in response to light. The phosphorylation of PEPC is modulated by a protein kinase specific to PEPC (PEPC-PK). To explore the role PEPC-PK plays in the regulation of C(4) photosynthetic CO(2) fixation, we have transformed Flaveria bidentis (a C(4) dicot) with antisense or RNA interference constructs targeted at the mRNA of this PEPC-PK. We generated several independent transgenic lines where PEPC is not phosphorylated in the light, demonstrating that this PEPC-PK is essential for the phosphorylation of PEPC in vivo. Malate sensitivity of PEPC extracted from these transgenic lines in the light was similar to the malate sensitivity of PEPC extracted from darkened wild-type leaves but greater than the malate sensitivity observed in PEPC extracted from wild-type leaves in the light, confirming the link between PEPC phosphorylation and the degree of malate inhibition. There were, however, no differences in the CO(2) and light response of CO(2) assimilation rates between wild-type plants and transgenic plants with low PEPC phosphorylation, showing that phosphorylation of PEPC in the light is not essential for efficient C(4) photosynthesis for plants grown under standard glasshouse conditions. This raises the intriguing question of what role this complexly regulated reversible phosphorylation of PEPC plays in C(4) photosynthesis.     

Interplay of light and temperature during the in planta modulation of C4 phosphoenolpyruvate carboxylase from the leaves of Amaranthus hypochondriacus L.: diurnal and seasonal effects manifested at molecular levels

The interactive effects of light and temperature on C(4) phosphoenolpyruvate carboxylase (PEPC) were examined both in vivo and in situ using the leaves of Amaranthus hypochondriacus collected at different times during a day and in each month during the year. The maximum activity of PEPC, least inhibition by malate, and highest activation by glucose-6-phosphate were at 15.00 h during a typical day, in all the months. This peak was preceded by maximum ambient light but coincided with high temperature in the field. The highest magnitude in such responses was in the summer (e.g. May) and least in the winter (e.g. December). Light appeared to dominate in modulating the PEPC catalytic activity, whereas temperature had a strong influence on the regulatory properties, suggesting interesting molecular interactions. The molecular mechanisms involved in such interactive effects were determined by examining the PEPC protein/phosphorylation/mRNA levels. A marked diurnal rhythm could be seen in the PEPC protein levels and phosphorylation status during May (summer month). In contrast, only the phosphorylation status increased during the day in December (winter month). The mRNA peaks were not as strong as those of phosphorylation. Thus, the phosphorylation status and the protein levels of PEPC were crucial in modulating the daily and seasonal patterns in C(4) leaves in situ. This is the first detailed study on the diurnal as well as seasonal patterns in PEPC activity, its regulatory properties, protein levels, phosphorylation status, and mRNA levels, in relation to light and temperature intensities in the field.     

Immunological analysis of the phosphorylation state of maize C4-form phosphoenolpyruvate carboxylase with specific antibodies raised against a synthetic phosphorylated peptide

The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme's sensitivity to its feedback inhibitor, L-malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state-specific antibodies to the C4-form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15-mer peptide with a sequence corresponding to that flanking the specific site of regulatory phosphorylation (Ser15) and subsequently purified by affinity-chromatography. Specificity of the resulting antibodies to the C4-form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root-form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi-quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13-week-old) maize plants grown in the field, phosphorylation started before dawn, reached a maximum by mid-morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C4-form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen-limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.     

Dramatic difference in the responses of phosphoenolpyruvate carboxylase to temperature in leaves of C3 and C4 plants

Temperature caused phenomenal modulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in leaf discs of Amaranthus hypochondriacus (NAD-ME type C(4) species), compared to the pattern in Pisum sativum (a C(3) plant). The optimal incubation temperature for PEPC in A. hypochondriacus (C(4)) was 45 degrees C compared to 30 degrees C in P. sativum (C(3)). A. hypochondriacus (C(4)) lost nearly 70% of PEPC activity on exposure to a low temperature of 15 degrees C, compared to only about a 35% loss in the case of P. sativum (C(3)). Thus, the C(4) enzyme was less sensitive to supra-optimal temperature and more sensitive to sub-optimal temperature than that of the C(3) species. As the temperature was raised from 15 degrees C to 50 degrees C, there was a sharp decrease in malate sensitivity of PEPC. The extent of such a decrease in C(4) plants (45%) was more than that in C(3) species (30%). The maintenance of high enzyme activity at warm temperatures, together with a sharp decrease in the malate sensitivity of PEPC was also noticed in other C(4) plants. The temperature-induced changes in PEPC of both A. hypochondriacus (C(4)) and P. sativum (C(3)) were reversible to a large extent. There was no difference in the extent of phosphorylation of PEPC in leaves of A. hypochondriacus on exposure to varying temperatures, unlike the marked increase in the phosphorylation of enzyme on illumination of the leaves. These results demonstrate that (i) there are marked differences in the temperature sensitivity of PEPC in C(3) and C(4) plants, (ii) the temperature induced changes are reversible, and (iii) these changes are not related to the phosphorylation state of the enzyme. The inclusion of PEG-6000, during the assay, dampened the modulation by temperature of malate sensitivity of PEPC in A. hypochondriacus. It is suggested that the variation in temperature may cause significant conformational changes in C(4)-PEPC.     

In Vivo Regulatory Phosphorylation of Soybean Nodule Phosphoenolpyruvate Carboxylase

In this report we provide evidence that cytosolic phosphoenolpyruvate carboxylase (PEPC) in soybean (Glycine max L.) root nodules is regulated in vivo by a seryl-phosphorylation cycle, as with the C4, Crassulacean acid metabolism, and C3 leaf isoforms. Pretreatment of parent plants by stem girdling for 5 or 14 h caused a significant decrease in the apparent phosphorylation state of nodule PEPC, as indicated by the 50% inhibition constant (L-malate) and specific activity values assayed at suboptimal conditions, whereas short-term darkness alone was without effect. However, extended (26 h) darkness led to the formation of a relatively dephosphorylated nodule PEPC, an effect that was reversed by illuminating the darkened plants for 3 h. This reversal of the apparent phosphorylation state in the light was prevented by concomitant stem girdling. In contrast, the optimal activity of nodule PEPC and its protein level showed little or no change in all pretreated plants. These results suggest that the phosphorylation state of PEPC in soybean root nodules is possibly modulated by photosynthate transported recently from the shoots. In situ [32P]orthophosphate labeling, immunoprecipitation, and phosphoamino acid analyses confirmed directly that PEPC in detached intact soybean nodules is phosphorylated on a serine residue(s).     

转C4光合酶基因水稻的CO2交换和荧光特性

用转PEPC、PPDK、NADP-ME、PEPC+PPDK酶基因水稻(Oryza sativa L.)及原种为材料 ,研究了光合作用对光照、温度、CO2的响应和光抑制条件下的叶绿素荧光特性,结果如下: 1.转C4光合酶基因水稻的饱和光合速率比原种高,其中转PEPC、PEPC+PPDK双基因水稻的光饱和点比原种高200 μmol*m-2*s-1,饱和光合速率比原种分别高51.6%和 58.5%;转PEPC基因水稻的羧化效率比原种高49.3%,CO2补偿点降低26.2%;在高温(35 ℃)下,转PEPC基因水稻的光合速率比原种高17.5%.2.经光抑制处理8 d后,转PEPC、PEPC +PPDK酶基因水稻的PSⅡ光化学效率(Fv/Fm)和光化学猝灭(qP)下降20%- 30%,非光化学猝灭(qN)增加了约30%;但原种的Fv/Fm和qP下降了5 0%多,qN变化不明显,表明转C4光合基因水稻耐光抑制能力增强.这些结果为用生物技术提高水稻光合效率研究提供了新的依据和途径.     

Mutual stimulation of temperature and light effects on C(4) phosphoenolpyruvate carboxylase in leaf discs and leaves of Amaranthus hypochondriacus

This article reports marked modulation of the activity and regulatory properties of phosphoenolpyruvate carboxylase (PEPC) by temperature and light in leaf discs as well as leaves of Amaranthus hypochondriacus. The activity of PEPC increased by 1.7-fold at 45 degrees C over 25 degrees C. Warm temperature also stimulated the photoactivation of PEPC. The activation by light of PEPC was 1.9-fold at 25 degrees C and increased to 2.2-fold at 45 degrees C. The sensitivity of PEPC to its inhibitor malate was less and the activation by glucose-6-phosphate (G-6-P) or inorganic phosphate (Pi) was more at 45 degrees C than that at 25 degrees C. These effects of temperature were quite pronounced in light. Similar responses were observed when detached leaves were exposed to varying ambient temperature (dry heat). The activity of PEPC increased by 1.6-fold at 45 degrees C over 25 degrees C in the dark. The activation of PEPC by light was 2.1-fold at 25 degrees C and increased to 2.6-fold at 45 degrees C. Inhibition by malate was less and activation by G-6-P or Pi was more at 45 degrees C than that at 25 degrees C. Thus, there was a marked modulation of not only the activity but also the regulatory properties of the enzyme by temperature and light, independently as well as cooperatively with each other. Further experiments suggested that PEPC was able to memorize to a significant extent the changes induced by warm temperature and that these changes were complemented by subsequent illumination. These effects were not due to changes in PEPC protein levels. We conclude that temperature and light can modulate PEPC activity and regulatory properties not only individually but also in a significantly cooperative manner with each other. As significant increases in temperature are common during daytime in tropical or subtropical conditions, we suggest that the synergistic effects of temperature and light are quite relevant in optimizing the activity of PEPC in leaves of C(4) plants.     

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.     

Activity regulation and physiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plants

Phosphoenolpyruvate carboxylase (PEPC) was overproduced in the leaves of rice plants by introducing the intact maize C₄-specific PEPC gene. Maize PEPC in transgenic rice leaves underwent activity regulation through protein phosphorylation in a manner similar to endogenous rice PEPC but contrary to that occurring in maize leaves, being downregulated in the light and upregulated in the dark. Compared with untransformed rice, the level of the substrate for PEPC (phosphoenolpyruvate) was slightly lower and the product (oxaloacetate) was slightly higher in transgenic rice, suggesting that maize PEPC was functioning even though it remained dephosphorylated and less active in the light. ¹⁴CO₂ labeling experiments indicated that maize PEPC did not contribute significantly to the photosynthetic CO₂ fixation of transgenic rice plants. Rather, it slightly lowered the CO₂ assimilation rate. This effect was ascribable to the stimulation of respiration in the light, which was more marked at lower O₂ concentrations. It was concluded that overproduction of PEPC does not directly affect photosynthesis significantly but it suppresses photosynthesis indirectly by stimulating respiration in the light. We also found that while the steady-state stomatal aperture remained unaffected over a wide range of humidity, the stomatal opening under non-steady-state conditions was destabilized in transgenic rice. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ecotypic variations in phosphoenolpyruvate carboxylase activity of the cordgrass Spartina densiflora throughout its latitudinal distribution range

This study compared the specific activity of phosphoenolpyruvate carboxylase (PEPC) of Spartina densiflora Brongn., collected from four populations along its latitudinal distribution range. Spartina densiflora is a halophyte with C₄ photosynthesis that has a very wide latitudinal distribution, from Patagonia to the southwest Iberian Peninsula. The basis of intraspecific differences in PEPC activity were analysed by recording the phosphorylation state and amount of the enzyme, comparing leaf anatomy and evaluating leaf gas exchange. S. densiflora individuals from Patagonia had 60% higher PEPC specific activity than plants from the other three populations due to higher levels of PEPC protein that coincided with lower activation mediated by phosphorylation, yielding similar net photosynthesis rate ( c . 29 μmol CO₂·m⁻²·s⁻¹). Patagonian plants had a higher area of photosynthetic mesophyll relative to total chlorophyll than plants from north Argentina and the southwest Iberian Peninsula. Ecotypic differentiation in PEPC activity and leaf anatomy were found, distinguishing a higher-latitude ecotype from lower-latitude populations. The higher PEPC protein levels of the Patagonian ecotype seemed to be a response to lower light activation level of the enzyme, as judged by the low PEPC phosphorylation state.     

CO2-concentrating mechanisms in Egeria densa,a submersed aquatic plant

Egeria densa is an aquatic higher plant which has developed different mechanisms to deal with photosynthesis under conditions of low CO₂ availability. On the one hand it shows leaf pH-polarity, which has been proposed to be used for bicarbonate utilization. In this way, at high light intensities and low dissolved carbon concentration, this species generates a low pH at the adaxial leaf surface. This acidification shifts the equilibrium HCO₃^–/CO₂ towards CO₂, which enters the cell by passive diffusion. By this means, E. densa increases the concentration of CO₂ available for photosynthesis inside the cells, when this gas is limiting. On the other hand, under stress conditions resulting from high temperature and high light intensities, it shows a biochemical adaptation with the induction of a C₄-like mechanism but without Kranz anatomy. Transfer from low to high temperature and light conditions induces increased levels of phospho enol pyruvate carboxylase (PEPC, EC 4.1.1.31) and NADP-malic enzyme (NADP-ME, EC 1.1.1.40), both key enzymes participating in the Hatch-Slack cycle in plants with C₄ metabolism. Moreover, one PEPC isoform, whose synthesis is induced by high temperature and light, is phosphorylated in the light, and changes in kinetic and regulatory properties are correlated with changes in the phosphorylation state of this enzyme. In the present review, we describe these two processes in this submersed angiosperm that appear to help it perform photosynthesis under conditions of extreme temperatures and high light intensities.     

Requirements for activation of the signal-transduction network that leads to regulatory phosphorylation of leaf guard-cell phosphoenolpyruvate carboxylase during fusicoccin-stimulated stomatal opening

Leaves regulate gas exchange through control of stomata in the epidermis. Stomatal aperture increases when the flanking guard cells accumulate K+ or other osmolytes. K+ accumulation is stoichiometric with H+ extrusion, which is compensated for by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31)-mediated malate synthesis. Plant PEPCs are regulated allosterically and by phosphorylation. Aspects of the signal-transduction network that control the PEPC phosphorylation state in guard cells are reported here. Guard cells were preloaded with [32P]orthophosphate (32Pi); then stomata were incubated with fusicoccin (FC), which activates the guard-cell plasma membrane H+-ATPase. [32P]PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. In -FC controls, stomatal size, guard-cell malate, and [32P]PEPC were low; maximum values for these parameters were observed in the presence of FC after a 90-min incubation and persisted for an additional 90 min. This high steady-state phosphorylation status resulted from continuous phosphorylation and dephosphorylation, even after the malate-accumulation phase. PEPC phosphorylation was diminished by approximately 80% when K+ uptake was associated with Cl- uptake and was essentially abolished when stomatal opening was sucrose--rather than K+--dependent. Finally, alkalinization by NH4+ in the presence of K+ did not cause PEPC phosphorylation (as it does in C4 plants). As discussed, a role for cytoplasmic protons cannot be completely excluded by this result. In summary, activation of the plasma membrane H+-ATPase was essential, but not sufficient, to cause phosphorylation of guard-cell PEPC. Network components downstream of the H+-ATPase influence the phosphorylation state of this PEPC isoform.     

Identification and expression analysis of a gene encoding a bacterial-type phosphoenolpyruvate carboxylase from Arabidopsis and rice

Phosphoenolpyruvate carboxylase (PEPC) is distributed in plants and bacteria but is not found in fungi and animal cells. Important motifs for enzyme activity and structure are conserved in plant and bacterial PEPCs, with the exception of a phosphorylation domain present at the N terminus of all plant PEPCs reported so far, which is absent in the bacterial enzymes. Here, we describe a gene from Arabidopsis, stated as Atppc4, encoding a PEPC, which shows more similarity to Escherichia coli than to plant PEPCs. Interestingly, this enzyme lacks the phosphorylation domain, hence indicating that it is a bacterial-type PEPC. Three additional PEPC genes are present in Arabidopsis, stated as Atppc1, Atppc2, and Atppc3, encoding typical plant-type enzymes. As most plant PEPC genes, Atppc1, Atppc2, and Atppc3 are formed by 10 exons interrupted by nine introns. In contrast, Atppc4 gene has an unusual structure formed by 20 exons. A bacterial-type PEPC gene was also identified in rice (Oryza sativa), stated as Osppc-b, therefore showing the presence of this type of PEPC in monocots. The phylogenetic analysis suggests that both plant-type and bacterial-type PEPCs diverged early during the evolution of plants from a common ancestor, probably the PEPC from gamma-proteobacteria. The diversity of plant-type PEPCs in C3, C4, and Crassulacean acid metabolism plants is indicative of the evolutionary success of the regulation by phosphorylation of this enzyme. Although at a low level, the bacterial-type PEPC genes are expressed in Arabidopsis and rice.     

On the Mechanism of Reinitiation of Endogenous Crassulacean Acid Metabolism Rhythm by Temperature Changes

Under continuous light the endogenous Crassulacean acid metabolism (CAM) rhythm of Kalanchoe daigremontiana Hamet et Perrier de la Bathie disappears at high (>29.0[deg]C) or low (<8.0[deg]C) temperatures. We investigated the reinitiation of rhythmicity when temperature was reduced from above the upper and increased from below the lower threshold level via measurements of (a) short-term changes in carbon-isotope discrimination to illustrate shifts between C3 and C4 carboxylation in vivo, and (b) the malate sensitivity of phosphoenolpyruvate carboxylase (PEPC) in vitro. When the net CO2-exchange rhythm disappears at both temperatures, the instantaneous discrimination indicates low PEPC activity. Leaf malate concentration and osmolarity attain high and low values at low and high temperatures, respectively. After small temperature increases or reductions from the low and high temperatures, respectively, the rhythm is reinitiated, with phases shifted by 180[deg] relative to each other. This can be related to the contrasting low and high leaf malate concentrations due to direct inhibition of PEPC and possibly also of the phosphorylation of PEPC by malate. The experimental results were satisfactorily simulated by a mathematical CAM-cycle model, with temperature acting only on the passive efflux of malate from the vacuole. We stress the important role of the tonoplast in malate compartmentation and of malate itself for the reinitiation and generation of endogenous CAM rhythmicity.     

In vivo regulatory phosphorylation of novel phosphoenolpyruvate carboxylase isoforms in endosperm of developing castor oil seeds

Our previous research characterized two phosphoenolpyruvate (PEP) carboxylase (PEPC) isoforms (PEPC1 and PEPC2) from developing castor oil seeds (COS). The association of a shared 107-kD subunit (p107) with an immunologically unrelated bacterial PEPC-type 64-kD polypeptide (p64) leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. Here, we describe the production of antiphosphorylation site-specific antibodies to the conserved p107 N-terminal serine-6 phosphorylation site. Immunoblotting established that the serine-6 of p107 is phosphorylated in COS PEPC1 and PEPC2. This phosphorylation was reversed in vitro following incubation of clarified COS extracts or purified PEPC1 or PEPC2 with mammalian protein phosphatase type 2A and is not involved in a potential PEPC1 and PEPC2 interconversion. Similar to other plant PEPCs examined to date, p107 phosphorylation increased PEPC1 activity at pH 7.3 by decreasing its K(m)(PEP) and sensitivity to L-malate inhibition, while enhancing glucose-6-P activation. By contrast, p107 phosphorylation increased PEPC2's K(m)(PEP) and sensitivity to malate, glutamic acid, and aspartic acid inhibition. Phosphorylation of p107 was promoted during COS development (coincident with a >5-fold increase in the I(50) [malate] value for total PEPC activity in desalted extracts) but disappeared during COS desiccation. The p107 of stage VII COS became fully dephosphorylated in planta 48 h following excision of COS pods or following 72 h of dark treatment of intact plants. The in vivo phosphorylation status of p107 appears to be modulated by photosynthate recently translocated from source leaves into developing COS.     

Induction of a C(4)-like mechanism of CO(2) fixation in Egeria densa, a submersed aquatic species

The expression of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME) in Egeria densa leaves was studied under low temperature and light (LTL) following incubation under high temperature and light (HTL), conditions previously shown to induce high and low CO(2) compensation points, respectively. Transfer from LTL to HTL conditions induced increases in the activities and amounts of both enzymes. One NADP-ME isoform was observed in induced and uninduced samples. Two isoforms of PEPC were expressed, with the lower M(r) isoform being induced by HTL. NADP-ME showed properties similar to those of the isoform in C(3) species. The inducible PEPC isoform has a low K(m) for both substrates. PEPC kinetic and regulatory properties (V(max) and K(m) for phosphoenolpyruvate, and I(50) for L-malate) are different in samples taken in the dark from those in the light, indicating that some modification of PEPC may be occurring during the day. Finally, abscisic acid induced the expression of PEPC and NADP-ME in a manner similar to temperature induction, except that the activities of both PEPC isoforms were increased. A different signaling system may exist in this species in response to high temperature or abscisic acid, both of which induce changes in photosynthetic metabolism.     

Comparative studies of phosphoenolpyruvate carboxylase from c(3) and c(4) plants

Phosphoenolpyruvate carboxylase (PEPC) from several C₃ plants was compared to maize PEPC by immunoblotting using an antibody against maize PEPC and by peptide mapping. In C₃ gramineous plants, PEPCs of slightly different monomeric sizes were detected as two bands for wheat and barley leaves, as three bands for etiolated maize leaves and as four bands for rice leaves by SDS-polyacrylamide gel electrophoresis and immunoblotting, whereas only one PEPC band was detected for maize leaves, a C₄ plant, or tobacco leaves, a dicotyledonous C₃ plant. The peptide fragment patterns of the lower molecular weight PEPC (major band in immunoblotting) in wheat leaves was similar to that of maize PEPC in peptide mapping by protein staining or by immunological detection, but the upper one (minor band) had a different pattern from the lower one in peptide mapping by immunological detection and few peptide fragments from this were recognized by the anti-(maize) PEPC antibody. These results suggest that there are multiple forms of PEPC subunits in the gramineous plants tested, and the major PEPC has a primary structure similar to that of maize PEPC. To obtain information about the expression of PEPCs in C₃ plants, changes in the amount of PEPC protein were investigated during the greening of rice and wheat seedlings. Judging from the regulation by light, there were two types of PEPCs in greening rice seedlings, one induced by light and the other reduced by it. Greening wheat seedlings also show a PEPC band induced by light. These findings indicate that some PEPCs in C₃ gramineous plants not only have structures similar to that of maize PEPC, but also are regulated by light in a similar manner.     

Regulation of phosphoenolpyruvate carboxylase phosphorylation by metabolites and abscisic acid during the development and germination of barley seeds

During barley (Hordeum vulgare) seed development, phosphoenolpyruvate carboxylase (PEPC) activity increased and PEPC-specific antibodies revealed housekeeping (103-kD) and inducible (108-kD) subunits. Bacterial-type PEPC fragments were immunologically detected in denatured protein extracts from dry and imbibed conditions; however, on nondenaturing gels, the activity of the recently reported octameric PEPC (in castor [Ricinus communis] oil seeds) was not detected. The phosphorylation state of the PEPC, as judged by l-malate 50% inhibition of initial activity values, phosphoprotein chromatography, and immunodetection of the phosphorylated N terminus, was found to be high between 8 and 18 d postanthesis (DPA) and during imbibition. In contrast, the enzyme appeared to be in a low phosphorylation state from 20 DPA up to dry seed. The time course of 32/36-kD, Ca(2+)-independent PEPC kinase activity exhibited a substantial increase after 30 DPA that did not coincide with the PEPC phosphorylation profile. This kinase was found to be inhibited by l-malate and not by putative protein inhibitors, and the PEPC phosphorylation status correlated with high glucose-6-phosphate to malate ratios, thereby suggesting an in vivo metabolic control of the kinase. PEPC phosphorylation was also regulated by photosynthate supply at 11 DPA. In addition, when fed exogenously to imbibing seeds, abscisic acid significantly increased PEPC kinase activity. This was further enhanced by the cytosolic protein synthesis inhibitor cycloheximide but blocked by protease inhibitors, thereby suggesting that the phytohormone acts on the stability of the kinase. We propose that a similar abscisic acid-dependent effect may contribute to produce the increase in PEPC kinase activity during desiccation stages.