Maize Phosphoenolpyruvate Carboxylase Research Articles

ZmOrphan94 Transcription Factor Downregulates ZmPEPC1 Gene Expression in Maize Bundle Sheath Cells.

Spatial separation of the photosynthetic reactions is a key feature of C4 metabolism. In most C4 plants, this separation requires compartmentation of photosynthetic enzymes between mesophyll (M) and bundle sheath (BS) cells. The upstream region of the gene encoding the maize PHOSPHOENOLPYRUVATE CARBOXYLASE 1 (ZmPEPC1) has been shown sufficient to drive M-specific ZmPEPC1 gene expression. Although this region has been well characterized, to date, only few trans-factors involved in the ZmPEPC1 gene regulation were identified. Here, using a yeast one-hybrid approach, we have identified three novel maize transcription factors ZmHB87, ZmCPP8, and ZmOrphan94 as binding to the ZmPEPC1 upstream region. Bimolecular fluorescence complementation assays in maize M protoplasts unveiled that ZmOrphan94 forms homodimers and interacts with ZmCPP8 and with two other ZmPEPC1 regulators previously reported, ZmbHLH80 and ZmbHLH90. Trans-activation assays in maize M protoplasts unveiled that ZmHB87 does not have a clear transcriptional activity, whereas ZmCPP8 and ZmOrphan94 act as activator and repressor, respectively. Moreover, we observed that ZmOrphan94 reduces the trans-activation activity of both activators ZmCPP8 and ZmbHLH90. Using the electromobility shift assay, we showed that ZmOrphan94 binds to several cis-elements present in the ZmPEPC1 upstream region and one of these cis-elements overlaps with the ZmbHLH90 binding site. Gene expression analysis revealed that ZmOrphan94 is preferentially expressed in the BS cells, suggesting that ZmOrphan94 is part of a transcriptional regulatory network downregulating ZmPEPC1 transcript level in the BS cells. Based on both this and our previous work, we propose a model underpinning the importance of a regulatory mechanism within BS cells that contributes to the M-specific ZmPEPC1 gene expression.

Targeted knockdown of ribulose-1, 5-bisphosphate carboxylase-oxygenase in rice mesophyll cells

We generated antisense constructs targeting two of the five Rubisco small subunit genes (OsRBCS2 and 4) which account for between 30–40 % of the RBCS transcript abundance in leaf blades. The constructs were driven by a maize phosphoenolpyruvate carboxylase (PEPC) promoter known to have enriched expression in mesophyll cells (MCs). In the resulting lines leaf, Rubisco protein content was reduced by between 30–50 % and CO2 assimilation rate was limited under photorespiratory and non-photorespiratory conditions. A relationship between Rubisco protein content and CO2 assimilation rate was found. This was associated with a significant reduction in dry biomass accumulation and grain yield of between 37–70%. In addition to serving as a resource for reducing Rubisco accumulation in a cell-preferential manner, these lines allow us to characterize gene function and isoform specific suppression on photosynthesis and growth. Our results suggest that the knockdown of multiple genes is required to completely reduce Rubisco accumulation in MCs.

Transgenic maize phosphoenolpyruvate carboxylase alters leaf\u2013atmosphere CO2 and 13CO2 exchanges in Oryza sativa

The engineering process of C4 photosynthesis into C3 plants requires an increased activity of phosphoenolpyruvate carboxylase (PEPC) in the cytosol of leaf mesophyll cells. The literature varies on the physiological effect of transgenic maize (Zea mays) PEPC (ZmPEPC) leaf expression in Oryza sativa (rice). Therefore, to address this issue, leaf–atmosphere CO2 and 13CO2 exchanges were measured, both in the light (at atmospheric O2 partial pressure of 1.84 kPa and at different CO2 levels) and in the dark, in transgenic rice expressing ZmPEPC and wild-type (WT) plants. The in vitro PEPC activity was 25 times higher in the PEPC overexpressing (PEPC-OE) plants (~20% of maize) compared to the negligible activity in WT. In the PEPC-OE plants, the estimated fraction of carboxylation by PEPC (β) was ~6% and leaf net biochemical discrimination against 13CO2left( {Delta_{text{bio}} } right) was ~ 2‰ lower than in WT. However, there were no differences in leaf net CO2 assimilation rates (A) between genotypes, while the leaf dark respiration rates (Rd) over three hours after light–dark transition were enhanced (~ 30%) and with a higher 13C composition left( {delta^{ 1 3} {text{C}}_{text{Rd}} } right) in the PEPC-OE plants compared to WT. These data indicate that ZmPEPC in the PEPC-OE rice plants contributes to leaf carbon metabolism in both the light and in the dark. However, there are some factors, potentially posttranslational regulation and PEP availability, which reduce ZmPEPC activity in vivo.

Transformation and characterization of transgenic rice and Cleome spinosa plants carrying the maize phosphoenolpyruvate carboxylase genomic DNA

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C4 photosynthetic pathway for the initial fixation of atmospheric CO2 into the four-carbon (C4) acid, oxaloacetate. Here, we report that a vector carrying the intact maize C4pepc gene was used to transform C3 monocot japonica rice cultivar Tainung 67 (TNG67) which is an important cultivar in Taiwan, and C3 dicot Cleome spinosa which is closely related to the C4 dicot Cleome gynandra, via an Agrobacterium-mediated method. In total, 29 transgenic rice and 4 transgenic C. spinosa plants were obtained. Genomic PCR and southern blot analyses revealed that the maize pepc and selective antibiotic resistant genes were present in almost all randomly-selected transgenic plants but not in wild-type plants. Reverse transcription-PCR showed that maize pepc mRNA was detected in transgenic plants. Western blot analysis confirmed the presence of 110 kDa maize PEPC protein in transgenic plants but not in wild-type plants. Furthermore, higher PEPC enzyme activities were detected in transgenic rice plants than in transgenic C. spinosa plants. Taken together, we clearly demonstrated monocot maize C4pepc gene is functional in both C3 monocot rice and C3 dicot C. spinosa plants. Two T5 homozygous rice lines, each harboring a single insertion of maize pepc gene, showed 7- to 9.4-fold and 40–54% PEPC enzyme activity as compared to the untransformed rice and maize, respectively. These stable transgenic rice lines will be valuable material for studying the effect of maize PEPC on rice photosynthesis under different conditions.

Physiological characteristics and metabolomics of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene under high temperature stress

In this paper, two transgenic wheat lines, PC27 and PC51, containing the maize PEPC gene and its wild-type (WT) were used as experimental material to study the effects of high temperature on their photosynthetic physiological characteristics and metabolome. The results showed that transgenic wheat lines had higher photosynthetic rate (P n) than WT under non-stress treatment (NT) and high temperature stress treatment (HT), and more significantly under HT. The change trends of F v/F m, Ф PSII, and q P were similar to P n, whereas that of non-photochemical quenching (NPQ) was the opposite. Compared with WT, no differences in chlorophyll content between the transgenic wheat and WT were observed under NT, but two transgenic lines had relatively higher contents than WT under HT. The change trends of Chlorophyll a/b radio, the decreased values of F m, Wk, and Vj, and the activity of the antioxidant enzyme were consistent with the chlorophyll content. Compared with WT, transgenic wheat lines exhibited lower rate of superoxide anion production, H2O2 and malondialdehyde content under HT, and no significant differences were observed under NT. The expression pattern of the ZmPEPC gene and wheat endogenous photosynthesis-related genes were in agreement with that of P n. Compared with WT, about 13 different metabolites including one organic acid, six amino acids, four sugars, and two polyols were identified under NT; 25 different metabolites including six organic acids, 12 amino acids, four sugars, and three polyols were identified under HT. Collectively, our results indicate that ZmPEPC gene can enhance photochemical and antioxidant enzyme activity, upregulate the expression of photosynthesis-related genes, delay degradation of chlorophyll, change contents of proline and other metabolites in wheat, and ultimately improves its heat tolerance.

Drought tolerance and proteomics studies of transgenic wheat containing the maize C4 phosphoenolpyruvate carboxylase (PEPC) gene.

Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.

Overexpression of maize phosphoenolpyruvate carboxylase improves drought tolerance in rice by stabilization the function and structure of thylakoid membrane

Drought impacts severely crop photosynthesis and productivity. Development of transgenic rice overexpressing maize phosphoenolpyruvate carboxylase (PEPC) is a promising strategy for improving crop production under drought stress. However, the molecular mechanisms of protection from PEPC are not yet clear. The objective of this study was: first, to characterize the response of individual photosynthetic components to drought stress; second, to study the physiological and molecular mechanisms underlying the drought tolerance of transgenic rice (cv. Kitaake) over-expressing maize PEPC. Our results showed that PEPC overexpressing improved the ability of transgenic rice to conserve water and pigments during drying as compared to wild type. Despite the fact that drought induced reactive oxygen species and damaged photosystems (especially, PSI) in both lines, higher intercellular CO2 concentration protected the photosynthetic complexes, peptides, and also ultrastructure of thylakoid membranes against the oxidative damage in transgenic rice. In conclusion, although photosynthetic apparatus suffered an inevitable and asymmetric impairment during drought conditions, PEPC effectively alleviated the oxidative damage on photosystems and enhanced the drought tolerance by increasing intercellular CO2 concentration. Our investigation provided critical clues for exploring the feasibility of using C4 photosynthesis to increase the yield of rice under the aggravated global warming.

Morphology and photosynthetic enzyme activity of maize phosphoenolpyruvate carboxylase transgenic rice.

The morphology and photosynthetic enzyme activity were studied in maize phosphoenolpyruvate carboxylase transgenic rice and non-transgenic rice. The results showed that compared with non-transgenic rice, phosphoenolpyruvate carboxylase transgenic rice was taller and had a stronger stalk, wider leaves, and more exuberant root system, with increased photosynthetic enzyme activity and improved yield components. Therefore, given the superiority of this plant type and heterosis, this is a novel breeding strategy for rice for the introduction of C4 photosynthesis genes into high-yielding rice.

Production of marker-free transgenic Jatropha curcas expressing hybrid Bacillus thuringiensis δ-endotoxin Cry1Ab/1Ac for resistance to larvae of tortrix moth (Archips micaceanus).

BackgroundThe potential biofuel plant Jatropha curcas L. is affected by larvae of Archips micaceanus (Walker), a moth of the family Tortricidae. The hybrid Bacillus thuringiensis (Bt) δ-endotoxin protein Cry1Ab/1Ac confers resistance to lepidopteran insects in transgenic rice.ResultsHere, we report the production of a marker-free transgenic line of J. curcas (L10) expressing Cry1Ab/1Ac using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L10 carries a single copy of marker-free T-DNA that contains the Cry1Ab/1Ac gene under the control of a maize phosphoenolpyruvate carboxylase gene promoter (P Pepc :Cry1Ab/1Ac:T Nos ). The P Pepc :Cry1Ab/1Ac:T Nos gene was highly expressed in leaves of L10 plants. Insecticidal bioassays using leaf explants of L10 resulted in 80-100% mortality of larvae of A. micaceanus at 4 days after infestation.ConclusionThe results demonstrate that the hybrid Bt δ-endotoxin protein Cry1Ab/1Ac expressed in Jatropha curcas displays strong insecticidal activity to A. micaceanus. The marker-free transgenic J. curcas line L10 can be used for breeding of insect resistance to A. micaceanus.

The interplay between substrate and allosteric effectors of the photosynthetic C4 isoenzyme of phosphoenolpyruvate carboxylase from monocot and dicot plants

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the irreversible formation of oxaloacetate and Pi from phosphoenolpyruvate (PEP) and bicarbonate. This reaction constitutes the first step in the assimilation pathway of atmospheric CO2 in C4 plants, such as Zea mays, monocot, and Amaranthus hypochondriacus, dicot. The maize and amaranth photosynthetic PEPC isoenzymes (ZmPEPC‐C4 and AhPEPC‐C4 respectively) are activated by glucose‐6‐phosphate (Glc6P) and inhibited by malate and aspartate, but only ZmPEPC‐C4 is regulated by glycine (Gly), which is its main activator under physiological conditions. In this work we have explored the interplay between substrate and allosteric effectors in the two enzymes by initial velocity studies. At pH 7.3, 0.4 mM free Mg2+, and 0.1 mM bicarbonate, we found that: 1) The affinities for PEP and malate of AhPEPC‐C4, measured as Ks and Ki respectively, are higher than those of ZmPEPC‐C4, which result in similar I50 values for malate in both enzymes at physiological PEP concentrations. 2) Glc6P is an atypical activator because, unexpectedly, increases the inhibition by malate in both enzymes. 3) As expected, Gly counteracts the inhibition of ZmPEPC‐C4 by malate, which indicates the physiological relevance of the regulation by Gly of this enzyme. Why the activation by Gly of AhPEPC‐C4 is physiologically unnecessary remains to be explained.Supported by PAPIIT‐UNAM IN216911 grant.

Mutational evidence for a substrate activation site in phosphoenolpyruvate carboxylase

The interaction of phosphoenolpyruvate carboxylase (PEPc) with its substrate PEP is unusual because PEP serves as both substrate (as Mg‐PEP complex) and allosteric activator (as free, uncomplexed PEP). Based on results from mutational analysis and the location of these residues within the same pocket on the surface of maize phosphoenolpyruvate carboxylase, we propose that the residues R753, N755, N942, and I956 contribute to the substrate activation site. Although they are not located in the enzyme's active site, modification of any of these residues via site‐directed mutagenesis significantly reduces the enzyme's affinity for the substrate PEP. Using the program Computed Atlas of Surface Topography of proteins, it was determined that pocket 217 houses these four residues in addition to R232 which, based on previous mutational analysis, is also believed to be involved in substrate activation. Mutations R753E and N755A displayed a 9‐fold increase and a 3‐fold increase, respectively, in the Km. R753E displayed a 3 fold reduction in the Vmax, while N755A only showed a one third reduction. N942D showed a two‐fold increase in Km and roughly a 1/3 reduction in Vmax. Preliminary studies of I956F showed a 52‐fold loss of affinity for substrate. These results suggest that these five residues within pocket 217 are critical to the activation of PEPc by its substrate. This work was supported by the NIH MBRS‐RISE grant GM61331.

Physiological Characteristics of Photosynthesis in Transgenic Wheat with Maize C<SUB>4</SUB>-PEPC Gene under Field Conditions

Transformation and high efficiency expression of key enzymes gene of C4 photosynthetic characteristics are the important approach to improve photosynthetic efficiency and seed yield for C3 crop wheat. Phosphoenolpyruvate carboxylase (PEPC) plays an important role in C4 pathway. The authors have obtained several transgenic wheat lines with maize C4 type PEPC gene. To explore the expression and functional characteristics of PEPC gene in transgenic wheat plants, PCR test, PEPC activity, gas exchange parameters, chlorophyll fluorescence parameters and yield related characteristics of transgenic wheat lines were determined, using untransformed wheat as the control, and the value of diurnal photosynthesis cumulative (VDPC) at the four stages and the correlations between net photosynthesis rate and stomata conductance, intercellular CO2 concentration and transpiration rate were analyzed. The results showed that in the four determination stages, net photosynthetic rate of transgenic wheat obviously increased. For instance VDPC was increased by 29.1% and 23.3% on the 15th day after anthesis. Stomatal conductance and transpiration rate in flag leaves of transgenic wheat obviously increased compared to the control, and intercellular CO2 concentration decreased. Net photosynthetic rate of transgenic wheat enhanced by 23.2%, and variation scope of Fv/Fm, qP, NPQ, and ΦPSII of transgenic wheat was smaller than that of the control from 8:00 to 12:00 on the 15th day after anthesis; the major yield characteristics, such as weight per stem, thousand grain weight, single spike weight and harvest index of transgenic wheat, were significantly higher than those of the control. The results described above indicated that the transgenic wheat expressing maize PEPC gene showed better photosynthetic characteristics than untransformed plant and has the potential to increase wheat yield.

Production of marker-free transgenic rice expressing tissue-specific Bt gene

The hybrid Bacillus thuringiensis (Bt) δ-endotoxin gene Cry1Ab/Ac was used to develop a transgenic Bt rice (Oryza sativa L.) targeting lepidopteran insects of rice. Here, we show the production of a marker-free and tissue-specific expressing transgenic Bt rice line L24 using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L24 carries a single copy of marker-free T-DNA that contains the Cry1Ab/Ac gene driven by a maize phosphoenolpyruvate carboxylase (PEPC) gene promoter. The marker-free T-DNA was integrated into the 3' untranslated region of rice gene Os01g0154500 on the short arm of chromosome 1. Compared to the constitutive and non-specific expression of the P (Actin1):Cry1Ab/Ac:T (Nos) gene in the control Bt rice line T51-1, the P ( Pepc ):Cry1Ab/Ac:T (Nos ) gene was detected only in the leaf and stem tissues of L24. More importantly, compared to high levels of CRY1Ab/Ac proteins accumulated in T51-1 seeds, the CRY1Ab/Ac proteins were not detectable in L24 seeds by Western blot analysis. As demonstrated by insect bioassay, L24 provided similar level of resistance to rice leaffolder (Cnaphalocrocis medinalis) as T51-1. The marker-free transgenic line L24 can be used directly in rice breeding for insect resistance to lepidopteran insects where absence of Bt toxin protein in the seed is highly desirable.

二酸化炭素固定酵素のＸ線構造解析による構造―機能相関の解明

Phosphoenolpyruvate carboxylase (PEPC) and ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco) play pivotal roles in CO2 fixation. Both of the enzymes are involved in photosynthetic efficiency of plants. Here, we report the crystal structures of PEPC and Rubisco; the inactivated Escherichia coli PEPC complexed with L-aspartate, the activated maize PEPC complexed with a sulfate, the Chlamydomonas Rubisco complexed with transition state analogue, and Galdieria Rubisco complexed with a sulfate. X-ray crystallographic analysis and site-directed mutagenesis studies revealed the allosteric regulation and the reaction mechanism of PEPC. The crystallographic studies on Rubisco provided detailed understanding of activity and regulation in Rubisco.

CO2 Exchange and Chlorophyll Fluorescence of Phospho‐enolpyruvate Carboxylase Transgenic Rice Pollen Lines

AbstractTo elucidate the photosynthetic physiological characteristics and the physiological inherited traits of rice (Oryza sativa L.) hybrids and their parents, physiological indices of photosynthetic CO2 exchange and chlorophyll fluorescence parameters were measured in leaves of the maize phosphoenolpyruvate carboxylase (PEPC) transgenic rice as the male parent, sp. japonica rice cv. 9516 as the female parent, and the stable JAAS45 pollen line. The results revealed that the PEPC gene could be stably inherited and transferred from the male parent to the JAAS45 pollen line. Moreover, the JAAS45 pollen line exhibited high levels of PEPC activity, manifesting higher saturated photosynthetic rates, photosynthetic apparent quantum yield (AQY), photochemical efficiency of photosystem II and photochemical and non‐photochemical quenching, which indicated that the JAAS45 pollen line has a high tolerance to photo‐inhibition/photo‐oxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ13C carbon isotope determination and was demonstrated to have a limited photosynthetic C4 microcycle by feeding with exogenous C4 primary products, such as oxaloacetate or malate, or phosphoenolpyruvate. The present study explains the physiological inherited properties of PEPC transgenic rice and provides an expectation for the integration of traditional breeding and biological technology.(Managing editor: Ping He)

Characterization and functional analysis of phosphoenolpyruvate carboxylase kinase genes in rice

Phosphoenolpyruvate carboxylase (PEPC), a key enzyme of primary metabolism of higher plants, is regulated by reversible phosphorylation, which is catalyzed by PEPC kinase (PPCK). Rice has three functional PPCK genes, OsPPCK1, OsPPCK2 and OsPPCK3, all of which have an intron close to the 3' end of the coding region. A novel control mechanism was found for expression of OsPPCK2, namely alternative transcription initiation, and two different transcripts were detected. The four different transcripts of the OsPPCK genes showed different expression patterns. While OsPPCK1 and OsPPCK3 were highly expressed in roots and at low levels in other organs, the two OsPPCK2 transcripts were expressed in all organs. OsPPCK3 was expressed mostly at night, while the long OsPPCK2 transcripts were present in the leaves only in the daytime. Nitrate supplementation of leaves selectively induced expression of both OsPPCK2 transcripts, while phosphate starvation only induced the shorter one. Such diverse expression patterns of OsPPCK genes suggest the importance and variety of strict activity regulation of PEPC in rice. From the correlation between gene expression and the phosphorylation level of PEPC, which was monitored as that of the maize PEPC expressed in transgenic rice plants, it was concluded that the short OsPPCK2 transcripts were expressed in rice leaf mesophyll cells upon nitrogen supplementation and phosphate starvation, whereas OsPPCK3 participated in the nocturnal phosphorylation of PEPC in these cells. Expression of PPCK proteins in rice leaves was detected by immunoblotting using a specific antiserum, and the expression of two different OsPPCK2 proteins derived from alternative transcription initiation was confirmed.

The regulatory role of residues 226–232 in phosphoenolpyruvate carboxylase from maize

The regulatory properties of maize phosphoenolpyruvate carboxylase were significantly altered by site-directed mutagenesis of residues 226 through 232. This conserved sequence element, RTDEIRR, is part of a surface loop at the dimer interface. Mutation of individual residues in this sequence caused various kinetic changes, including desensitization of the enzyme to key allosteric effectors or alteration of the K(0.5 PEP) for the substrate phosphoenolpyruvate. R231A, and especially R232Q, displayed decreased apparent affinity for the activator glucose-6-phosphate. Apparent affinity for the activator glycine was reduced in D228N and R232Q, while the maximum activation caused by glycine was greatly reduced in R226Q and E229A. R226Q and E229A also showed significantly lower sensitivity to the inhibitors malate and aspartate. E229A exhibited a low K(0.5 PEP), while the K(0.5 PEP )of R232Q was significantly higher than that of wild type. Thus these seven residues are critical determinants of the enzyme's kinetic responses to activators, inhibitors and substrate. The present results support an earlier suggestion that Arg 231 contributes to the binding site of the allosteric activator glucose-6-phosphate, and are consistent with other proposals that the substrate phosphoenolpyruvate allosterically activates the enzyme by binding at or near the glucose-6-phosphate site. The results also suggest that the glycine binding site may be contiguous with the glucose-6-phosphate binding site. Glu 229, which extends from this interface region through the interior of the protein and emerges near the aspartate binding site, may provide a physical link for propagating conformational changes between the allosteric activator and inhibitor binding regions.

Photoprotective effects of high level expression of C<sub>4</sub> phosphoenolpyruvate carboxylase in transgenic rice during photoinhibition

With untransformed rice cv. Kitaake as control, the characteristics of carbon assimilation and photoprotection of a transgenic rice line over-expressing maize phosphoenolpyruvate carboxylase (PEPC) were investigated. The PEPC activity in untransformed rice was low, but the activity was stimulated under high irradiance or photoinhibitory condition. PEPC in untransformed rice contributed by about 5-10 % to photosynthesis, as shown by the application of the specific inhibitor 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)propenoate (DCDP). When maize PEPC gene was introduced into rice, transgenic rice expressed high amount of maize PEPC protein and had high PEPC activity. Simultaneously, the activity of carbonic anhydrase (CA) transporting CO2 increased significantly. Thus the photosynthetic capacity increased greatly (50 %) under high CO2 supply. In CO2-free air, CO2 release in the leaf was less. In addition, PEPC transgenic rice was more tolerant to photoinhibition. Treating by NaF, an inhibitor of phosphatase, showed that in transgenic rice more phosphorylated light-harvesting chlorophyll a/b-binding complexes (LHC) moved to photosystem 1 (PS1) protecting thus PS2 from photo-damage. Simultaneously, the introduction of maize PEPC gene could activate or induce activities of the key enzymes scavenging active oxygen, such as superoxide dismutase (SOD) and peroxidase (POD). Hence higher PS2 photochemical efficiency and lower superoxygen anion (O2.-) generation and malonyldiadehyde (MDA) content under photoinhibition could improve protection from photo-oxidation.

The molecular binding interactions of inhibitors and activators of phosphoenolpyruvate carboxylase

We have performed molecular modelling studies of the binding to maize phosphoenolpyruvate carboxylase (PEPC) of phosphoenolpyruvate (PEP) and a number of representative competitive inhibitors. We predict that all these compounds share a common binding mode and that the differences in inhibitory activity of the various inhibitors arise mainly from either increased hydrophobic interactions of cis substituents or small but significant changes in their binding mode arising from steric clashes of trans substituents with the active site. We have also performed molecular modelling studies of glucose-6-phosphate (G6P) and a number of other allosteric activators in their putative allosteric binding site in this enzyme. We predict that these molecules share the same binding mode for their phosphate moiety while some of them engage in a variety of hydrogen bonding interactions with residues from different subunits of the enzyme, and others establish hydrophobic and van der Waals interactions with other regions of the allosteric binding site.

Expression of the Cry1Ab Protein in Genetically Modified Sugarcane for the Control of Diatraea saccharalis (Lepidoptera: Crambidae)

Previously, we genetically modified two Brazilian commercial sugarcane (Saccharum sp.) varieties (SP80-3280 and SP80-1842) for resistance to the sugarcane borer Diatraea saccharalis by co-bombardment transformation with three plasmids: pCIB4421 containing the Bacillus thuringiensis gene cry1Ab regulated by a maize phosphoenol-pyruvate carboxylase promoter, pCIB4426 carrying cry1Ab controlled by a pith promoter, and pHA9 with the neo antibiotic resistance gene. The genetically modified plants were submitted to molecular characterization and insect feeding bioassays. Plants also were screened in the field for insect infestation and had their phenotypic characteristics and quantitative traits evaluated. This study presents evidence that the B. thuringiensis cry1Ab gene can be efficiently expressed in sugarcane plants over an extended growing period, and can confer resistance to the sugarcane borer under field conditions, while not measurably altering numerous material traits of the crop.