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

To 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 trans- ferred 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/photooxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ^13C 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.     

Photosynthetic characteristics and effect of ATP in transgenic rice with phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase and pyruvate orthophosphate dikinase genes

In the untransformed rice (WT) and transgenic rice with the PEPC and PPDK genes (CK) we determined activities of C₄ photosynthetic enzymes, photosynthetic response to irradiance and temperature, the metabolic index of active oxygen, and the yield component factors. The activities of C₄ photosynthetic enzymes in WT were very low, while those of corresponding enzymes in CK were highly observable. Moreover, after adenosine triphosphate (ATP) treatment, and under high irradiance and high temperature, the net photosynthetic rate of CK increased by 17 and 12 %, respectively, as compared to that achieved without ATP treatment. The resistance of CK against photo-oxidation was enhanced under these conditions, and CK yield increased by 15 %. ATP treatment enhanced the photosynthetic productivity of CK, thereby proving that ATP is the key factor in enhancing the photosynthetic capacity of transgenic rice with C₄ gene. Our new technical approach can be used in breeding rice with high photosynthetic efficiency and high grain yield.     

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.     

Photoprotective effects of high level expression of C<Subscript>4</Subscript> phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate 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.     

Physiological and metabolic enzymes activity changes in transgenic rice plants with increased phosphoenolpyruvate carboxylase activity during the flowering stage

To compare the differences in physiology and metabolism between phosphoenolpyruvate carboxylase (PEPC) transgenic rice and its control, untransformed wild rice, dry matter accumulation, soluble sugar, starch and protein contents and enzyme activities were determined in different plant parts during flowering. Results revealed that PEPC transgenic rice had higher dry weights for leaf, stem and sheath as well as panicle than the untransformed wild rice did, with the largest increase in the panicle. Soluble sugar and protein content in the grains of PEPC transgenic rice were significantly enhanced while starch content changed less. PEPC transgenic rice exhibited high levels of PEPC activity, manifesting in high net photosynthetic rates during flowering. Moreover, transgenic rice with high PEPC expression levels also had elevated levels of the enzymes such as sucrose-p-synthase and sucrose synthase, which may confer a higher capacity to assimilate CO₂ into sucrose. Little increase in grain starch content was observed in transgenic plants due to the stable activities of starch synthase and Q enzyme. However, the PEPC transgenic rice plant induced the activities of nitrate reductase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, glutamine synthetase, and asparagine synthase to high levels, as compared with the untransformed rice plant. PEPC activity was correlated with protein content in grains and the enzymes of nitrogen metabolism, suggesting that high PEPC activity in transgenic rice might be able to redirect carbon and nitrogen flow by regulating some enzymes related to carbon or nitrogen metabolisms. These results may help to understand how the C₃ plants possessing a C₄-like photosynthesis pathway worked by expression of PEPC.     

Physiological and photosynthetic characteristics of indica Hang2 expressing the sugarcane PEPC gene

Phosphoenolpyruvate carboxylase (PEPC) is known to play a key role in the initial fixation of CO₂ in C4 photosynthesis. The PEPC gene from sugarcane (a C4 plant) was introduced into indica rice (Hang2), a process mediated by Agrobacterium tumefaciens. Integration patterns and copy numbers of the gene was confirmed by DNA blot analysis. RT-PCR and western blotting results showed that the PEPC gene was expressed at both the mRNA and protein levels in the transgenic lines. Real-time PCR results indicated that expression of the sugarcane PEPC gene occurred mostly in green tissues and changed under high temperature and drought stress. All transgenic lines showed higher PEPC enzyme activities compared to the untransformed controls, with the highest activity (11.1 times higher than the controls) being observed in the transgenic line, T34. The transgenic lines also exhibited higher photosynthetic rates. The highest photosynthetic rate was observed in the transgenic line, T54 (22.3 μmol m^?2 s^?1; 24.6 % higher than that in non-transgenic plants) under high-temperature conditions. Furthermore, the filled grain and total grain numbers for transgenic lines were higher than those for non-transgenic plants, but the grain filling (%) and 1,000-grain weights of all transgenic lines remained unchanged. We concluded that over-expression of the PEPC gene from sugarcane in indica rice (Hang2) resulted in higher PEPC enzyme activities and higher photosynthesis rates under high-temperature conditions.     

Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes

The photosynthetic characteristics of four transgenic rice lines over-expressing rice NADP-malic enzyme (ME), and maize phosphoenolpyruvate carboxylase (PC), pyruvate,orthophosphate dikinase (PK), and PC+PK (CK) were investigated using outdoor-grown plants. Relative to untransformed wild-type (WT) rice, PC transgenic rice exhibited high PC activity (25-fold increase) and enhanced activity of carbonic anhydrase (more than two-fold increase), while the activity of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) and its kinetic property were not significantly altered. The PC transgenic plants also showed a higher light intensity for saturation of photosynthesis, higher photosynthetic CO₂ uptake rate and carboxylation efficiency, and slightly reduced CO₂ compensation point. In addition, chlorophyll a fluorescence analysis indicates that PC transgenic plants are more tolerant to photo-oxidative stress, due to a higher capacity to quench excess light energy via photochemical and non-photochemical means. Furthermore, PC and CK transgenic rice produced 22–24% more grains than WT plants. Taken together, these results suggest that expression of maize C₄ photosynthesis enzymes in rice, a C₃ plant, can improve its photosynthetic capacity with enhanced tolerance to photo-oxidation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Over-expression of phosphoenolpyruvate carboxylase cDNA from C4 millet (Seteria italica) increase rice photosynthesis and yield under upland condition but not in wetland fields

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the initial fixation of CO₂ in C₄ plants. Under the control of the rice Rubisco small subunit promoter, cDNA of a C₄ SiPPC gene cloned from Seteria italica was introduced into Japonica rice by Agrobacterium-mediated transformation. Integration of the gene was confirmed by PCR analysis. RT-PCR showed expression of the gene at the RNA level in transgenic plants, and enzyme activity measurements confirmed the increase in PEPC protein. The transformants showed improvements in both photosynthesis rate and yield only under upland field cultivation. The possible function of PEPC in rice stress tolerance is discussed.     

Direct and selective small-molecule inhibition of photosynthetic PEP carboxylase: New approach to combat C4 weeds in arable crops

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of C₄ photosynthesis. Besides, non-photosynthetic isoforms of PEPC are found in bacteria and all types of plants, although not in animals or fungi. A single residue in the allosteric feedback inhibitor site of PEPC was shown to adjust the affinity of the photosynthetic and non-photosynthetic isoforms for feedback inhibition by metabolites of the C₄ pathway. Here, we applied computational screening and biochemical analyses to identify molecules that selectively inhibit C₄ PEPC, but have no effect on the activity of non-photosynthetic PEPCs. We found two types of selective inhibitors, catechins and quinoxalines. Binding constants in the lower μM range and a strong preference for C₄ PEPC qualify the quinoxaline compounds as potential selective herbicides to combat C₄ weeds.     

PEPC-mediated carbon fixation in transmitting tract cells reflects style–pollen tube interactions

Styles nurture and guide pollen tubes to the ovules. The styles of Nicotiana tabacum, a C₃ plant, contain a concentric strand of transmitting tract cells replete with well-developed chloroplasts. It is shown that the chloroplasts have normal ultrastructure and electron transport ability. However, they were found to be devoid of Rubisco, the key enzyme responsible for carbon fixation in C₃ plants. Nevertheless, non-invasive fluorescence techniques showed a light-driven photosynthetic flux. Carbon fixation via phosphoenol pyruvate carboxylase (PEPC) into malate was demonstrated, the latter accumulating during stylar development. Characterization of stylar PEPC in vitro and in vivo revealed apparent K_m values consistent with bicarbonate as a rate limiting factor for photosynthetic flux. Presumably, in the closed confines of the intact style, respired CO₂ is the source of carbonate. Enhanced photosynthetic flux was detected following pollination, suggesting utilization of the additional respired bicarbonate and underlining metabolic interactions between the style and the elongating pollen tube.     

Expression of a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> protoporphyrinogen oxidase gene in rice plants reduces sensitivity to peroxidizing herbicides

Protoporphyrinogen oxidase (Protox) in the porphyrin pathway is the target site of the peroxidizing herbicides such as carfentrazone-ethyl and oxyfluorfen. In an attempt to develop herbicide-resistant plants, transgenic rice plants were generated via expression of herbicide-insensitive Bacillus subtilis Protox gene fused to the transit sequence for targeting to the plastid using Agrobacterium-mediated gene transformation. Homozygous transgenic rice lines of T₃ generation selected by hygromycin resistance test were examined if they are resistant to the herbicides carfentrazone-ethyl and oxyfluorfen. The homozygous transgenic lines had single copy insertion of B. subtilis Protox gene into their genomes and express its mRNA. Compared to wild-type rice, the transgenic lines were less susceptible to the herbicides when examined with respect to growth, electrolyte leakage, chlorophyll loss and lipid peroxidation. The in vitro Protox activities in transgenic lines were about 56 % higher than those in wild-type rice. With 10 µM concentration of the herbicides in the enzyme assays, Protox activities in transgenic lines were similar to those in non-inhibited wild-type rice. Less amount of protoporphyrin IX was accumulated in transgenic lines than in wild-type rice upon the treatment of the herbicides at 10 µM concentration. Our results indicated that expression of B. subtilis Protox gene was stably transmitted into T₃ rice plants and reduced their sensitivity to carfentrazone-ethyl and oxyfluorfen.This work was supported by Ministry of Agriculture and Forestry of Korea and Agricultural Plant Stress Research Center (grant No. R11-2001-09203000-0) funded by Korea Science and Engineering Foundation.     

Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure,regulation and genetic engineering

Three to four families of nuclear genes encode different isoforms of phosphoenolpyruvate (PEP) carboxylase (PEPC): C₄-specific, C₃ or etiolated, CAM and root forms. C₄ leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C₄-dicot Flaveria trinervia. Selective expression of ppc in only C₄-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C₄-PEPC pinpoint the phosphorylatable serine near the N-terminus, C₄-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO₂, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO₃ ^--dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C₄ ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C₄ PEPC and the transgenic tobacco plants expressed both C₃ and C₄ isoforms. Site-directed mutagenesis of ppc indicates the importance of His¹³⁸, His⁵⁷⁹ and Arg⁵⁸⁷ in catalysis and/or substrate-binding by the E. coli enzyme, Ser⁸ in the regulation of sorghum PEPC. Important areas for further research on C₄ PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.Abbreviations OAA oxalacetate - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC-protein kinase - PPDK pyruvate, orthophosphate dikinase - Rubisco ribulose 1,5-bis-phosphate carboxylase/oxygenase - CAM Crassulacean acid metabolism     

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.     

Iron supplementation promotes in vitro shoot induction and multiplication of <Emphasis Type="Italic">Baptisia australis</Emphasis>

C₄ plants can efficiently accumulate CO₂ in leaves and thus reduce wasteful oxygen fixation by the RuBisCO enzyme. Three C₄ enzymes, namely carbonic anhydrase (CA), phosphoenol pyruvate (PEPC) and pyruvate orthophosphate dikinase (PPDK), were over expressed in Oryza sativa L. ssp. indica var. Khitish under the control of green tissue specific promoters PD_54o, PEPC and PPDK, respectively. Integration of these genes was confirmed by Southern hybridization. The relative expression of PEPC, CA and PPDK were, respectively, 6.75, 6.57 and 3.6-fold higher in transgenic plants compared to wild type plants (control). Photosynthetic efficiency of the transgenic plants increased significantly along with a 12?% increase in grain yield compared to wild type plants. Compared to control plants, transgenic plants also showed phenotypic changes such as increased leaf blade size, root biomass, and plant height and anatomical changes such as greater leaf vein number, bundle sheath cells, and bulliform cells. Our findings indicate that the combined over expression of these three enzymes is an efficient strategy for incorporating beneficial physiological and anatomical features that will enable subsequent yield enhancement in C₃ rice plants.     

Physiological characteristics of the primitive CO<Subscript>2</Subscript> concentrating mechanism in PEPC transgenic rice

The relationship between carbon assimilation and high-level expression of the maize PEPC in PEPC transgenic rice was studied by comparison to that in the untransformed rice, japonica kitaake. Stomatal conductance and photosynthetic rates in PEPC transgenic rice were higher than those of untransformed rice, but the increase of stomatal conductance had no statistical correlation with that of photosynthetic rate. Under high levels of light intensity, the protein contents of PEPC and CA were increased significantly. Therefore the photosynthetic capacity was increased greatly (50%) with atmospheric CO2 supply. While CO2 release in leaf was reduced and the compensation point was lowered correspondingly under CO2 free conditions. Treatment of the rice with the PEPC-specific inhibitor DCDP showed that overexpression of PEPC and enhancement of carbon assimilation were related to the stability of Fv/Fm. Labeling with 14CO2 for 20 s showed more 14C was distributed to C4 primary photosynthate asperate in PEPC transgenic rice, suggesting that there exists a limiting C4 photosynthetic mechanism in leaves. These results suggest that the primitive CO2 concentrating mechanism found in rice could be reproduced through metabolic engineering, and shed light on the physiological basis for transgenic breeding with high photosynthetic efficiency.     

Promotion of photosynthesis in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene by nitric oxide donors

We determined the effects of exogenous nitric oxide on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C₄ pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC). Seedlings were subjected to treatments with NO donors, an NO scavenger, phospholipase inhibitors, a Ca²⁺ chelator, a Ca²⁺ channel inhibitor, and a hydrogen peroxide (H₂O₂) inhibitor, individually and in various combinations. The NO donors significantly increased the net photosynthetic rate (P_N) of PC and wild-type (WT), especially that of PC. Treatment with an NO scavenger did inhibit the P_N of rice plants. The treatments with phospholipase inhibitors and a Ca²⁺ chelator decreased the P_N of WT and PC, and photosynthesis was more strongly inhibited in WT than in PC. Further analyses showed that the NO donors increased endogenous levels of NO and PLD activity, but decreased endogenous levels of Ca²⁺ both WT and PC. However, there was a greater increase in NO in WT and a greater increase in PLD activity and Ca²⁺ level in PC. The NO donors also increased both PEPC activity and pepc gene expression in PC. PEPC activity can be increased by SNP alone. But the expression of its encoding gene in PC might be regulated by SNP, together with PA and Ca²⁺.     

Field Performance of Transgenic indica Hybrid Rice with Improved Cooking and Eating Quality by Down-regulation of Wx Gene Expression

High amylose content (AC) in rice endosperm is correlated with poor grain quality, particularly in indica hybrid rice. We have generated several homozygous transgenic parent lines of indica hybrid rice carrying an antisense Waxy (Wx) gene and demonstrated that the AC in seeds of these lines decreased dramatically. Two transgenic maintainer lines (L25B and L18B), derived from one of the key maintainer parents of an indica hybrid rice in China, Long-te-fu B (LTF-B), were selected and the antisense Wx gene was subsequently introgressed into the male-sterile counterpart, LTF-A, with the aim to generate improved indica hybrids. The indica hybrids derived from the selected transgenic male-sterile lines and restorer lines were tested for quality and agronomic performance under normal field conditions. Our results demonstrated that the reduction of AC in the homozygous transgenic maintainer lines stably passed down in five successive generations and the improved quality was also found in their relevant transgenic hybrids produced. The other two key characters of rice cooking and eating quality, the gel consistence (GC) and gelatinization temperature (GT), were also improved in the grains of both the transgenic maintainer lines and their relevant hybrids. In addition, no change was observed for most of the agronomic characters of the transgenic maintainer lines and the relevant transgenic hybrids. Although the grain weight of the transgenic line was reduced, the grain yield of the homozygous transgenic parent lines and the transgenic hybrids was similar when compared with that of the wild-type controls. These results suggest that transgenic approaches are an effective way to obtain rice lines with both improved qualities and high yield, especially for indica hybrid rice.     

Promotive Effect of Low Concentrations of NaHSO3 on Photophosphorylation and Photosynthesis in Phosphoenolpyruvate Carboxylase Transgenic Rice Leaves

Spraying a 1-2 mmol/L solution of NaHSO3 on the leaves of wild-type rice (Oryza sativa L.)Kitaake (WT), phosphoenolpyruvate carboxylase (PEPC) transgenic (PC) rice and PEPC phosphate dikinase (PPDK) transgenic rice (PC PK), in which the germplasm was transformed with wild-type Kitaake as the gene receptor, resulted in an enhancement of the net photosynthetic rate by 23.0%, 28.8%, and 34.4%,respectively, for more than 3 d. It was also observed that NaHSO3 application caused an increase in the ATP content in leaves. Spraying PMS (a cofactor catalysing the photophosphorylation cycle) and NaHSO3 separately or together on leaves resulted in an increase in photosynthesis with all treatments. There was no additional effect on photosynthetic rate when the mixture was applied, suggesting that the mechanism by which NaHSO3 promotes photosynthesis is similar to the mechanism by which PMS acts and that both of compounds enhanced the supply of ATP. After spraying a solution of NaHSO3 on leaves, compared with the WT Kitaake rice, a greater enhancement of net photosynthetic rate was observed in PEPC transgenic (PC) and PEPC PPDK transgenic (PC PK) rice, with the greatest increase being observed in the latter group. Therefore ATP supply may become the limiting factor that concentrates CO2 in rice leaves transformed with an exogenous PEPC gene and exogenous PEPC PPDK genes.     

Promotive Effect of Low Concentrations of NaHSO3 on Photophosphorylation and Photosynthesis in Phosphoenolpyruvate Carboxylase Transgenic Rice Leaves

Spraying a 1-2 mmol/L solution of NaHSO3 on the leaves of wild-type rice (Oryza sativa L.)Kitaake (WT), phosphoenolpyruvate carboxylase (PEPC) transgenic (PC) rice and PEPC phosphate dikinase(PPDK) transgenic rice (PC PK), in which the germplasm was transformed with wild-type Kitaake as the gene receptor, resulted in an enhancement of the net photosynthetic rate by 23.0%, 28.8%, and 34.4%,respectively, for more than 3 d. It was also observed that NaHSO3 application caused an increase in the ATP content in leaves. Spraying PMS (a cofactor catalysing the photophosphorylation cycle) and NaHSO3 separately or together on leaves resulted in an increase in photosynthesis with all treatments. There was no additional effect on photosynthetic rate when the mixture was applied, suggesting that the mechanism by which NaHSO3 promotes photosynthesis is similar to the mechanism by which PMS acts and that both of compounds enhanced the supply of ATE After spraying a solution of NaHSO3 on leaves, compared with the WT Kitaake rice, a greater enhancement of net photosynthetic rate was observed in PEPC transgenic(PC) and PEPC PPDK transgenic (PC PK) rice, with the greatest increase being observed in the latter group. Therefore ATP supply may become the limiting factor that concentrates CO2 in rice leaves transformed with an exogenous PEPC gene and exogenous PEPC PPDK genes.