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.     

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.     

干旱条件下外源蔗糖对高表达玉米C4型pepc水稻种子萌发的影响

为揭示外源蔗糖参与干旱胁迫下高表达转玉米C₄ 型磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase, PEPC)基因(C₄ pepc)水稻(简称：PC)种子萌发的生理机制,该研究以 PC及其未转基因野生型受体‘Kitaake’(简称：WT)的种子为材料,研究外施不同浓度蔗糖联合模拟干旱(10% PEG 6000)处理下,其种子发芽参数、总可溶性糖及可溶性蛋白含量、蔗糖非发酵1 (sucrose nonfermenting 1, SNF1)相关蛋白激酶(SNF1 related protein kinase 1s, SnRK1s)基因以及PEPC基因表达等参数的变化。结果表明：(1)PEG 6000模拟干旱处理均显著抑制两材料发芽,但明显促进胚根的生长;外施蔗糖则呈现浓度效应,高浓度蔗糖(>150 mmol·L^-1)进一步加剧了干旱对发芽的抑制效应,而低浓度(<30 mmol·L^-1)则可缓解干旱的抑制,但与WT(<30 mmol·L^-1)相比,促进PC水稻萌发的外施蔗糖浓度(<6 mmol·L^-1)更低,且各处理的发芽表现与其α 淀粉酶活性的动态表现一致。(2)与WT相比,外施3 mmol·L^-1蔗糖联合干旱处理下,显著提高了PC种子的发芽率,且伴随PC内源蔗糖含量、总可溶糖和可溶性蛋白含量显著增加;且外施3 mmol·L^-1蔗糖使PC中内源C₃ pepc基因表达下调,而外源导入C₄ pepc基因表达显著增加。(3)与WT相比,干旱处理下外施3 mmol·L^-1蔗糖,PC的糖信号相关基因SnRKs亚家族基因(包括SnRK1s：OsK1a OsK24 OsK35和SnRK2s:SAPK6)的表达也显著增加。研究发现,外施低浓度蔗糖通过上调PC水稻种子中可溶性糖和可溶性蛋白含量,增强SnRK1s亚家族基因和外源C₄ pepc基因的表达,提高了α 淀粉酶活性,从而缓解了干旱胁迫对PC种子萌发的抑制。     

低浓度NaHSO3促进田间水稻的光合磷酸化和光合作用

用1～2 mmol/L NaHSO3喷施于水稻(Oryza sativa L.)叶面可以提高叶片的光合速率,并能持续3 d以上.在此条件下,光下叶片中的ATP含量明显增高,叶片的叶绿素毫秒延迟荧光加强,反映与光合磷酸化活力有关的跨类囊体膜质子梯度增加.乳熟期喷施2次1 mmol/L NaHSO3后,水稻产量提高约10%.研究表明NaHSO3的主要作用和PMS(phenazine methosulfate)促进光合速率的原因可能类似,都是增加了ATP的供应.与此同时,观察到低浓度NaHSO3可促进水稻中反映循环电子传递的叶绿素荧光在作用光关闭后的短时上升.以上现象表明低浓度NaHSO3的促进作用很可能是通过促进围绕PSⅠ的循环电子传递及其耦联的光合磷酸化而促进水稻光合作用的.     

低浓度NaHSO3促进田间水稻的光合磷酸化和光合作用

用１～２ｍｍｏｌ／Ｌ ＮａＨＳＯ３喷施于水稻（Ｏｒｙｚａ ａｔｉｖａ Ｌ．）叶面可以提高叶片的光合速率,并能持续３ｄ以上。在此条件下,光下叶片中的ＡＴＰ含量明显增高,叶片的叶绿素毫秒延迟荧光加强,反映与光合磷酸化活力有关的跨类囊体膜质子梯度增加。乳熟期喷施２次１ｍｍｏｌ／Ｌ ＮａＨＳＯ３后,水稻产量提高约１０％。研究表明ＮａＨＳＯ３的主要作用和ＰＭＳ（ｐｈｅｎａｚｉｎｅ ｍｅｔｈｏｓｕｌｆａｔｅ）     

光/暗对高粱叶片磷酸烯醇式丙酮酸羧化酶基因表达的调节

高粱幼苗黄化叶片经照光转绿后,其PEP-Case活性提高4～15倍,mRNA含量提高了1.03倍,并测定出PEPCase mRNA的分子量为3.4kb。以等量的总RNA及mRNA进行体外翻译,发现转绿后PEPCase专一性翻译活性提高了51％～53％。这表明光照可以在转录水平上调节PEP-Case的基因表达。     

Photosynthetic Characteristics of Transgenic Rice Plants Overexpressing Maize Phosphoenopyruvate Carboxylase

转PEPC基因水稻的光合生理特性 迟伟¹焦德茂^1* 黄雪清¹ 李霞¹ 匡廷云² Ku S..B.MAURICE ³     

Photosynthesis and the influence of CO2-enrichment on δ13C values in a C3 halophyte

Abstract Shifts in ?¹³C of the graminaceous C₃ halophyte Puccinellia nuttalliana (Schultes) Hitch. can be induced by salinization. To investigate this phenomenon, three approaches were taken: assay of carboxylases, CO₂-enrichment studies, and gas exchange analysis. Although ribulose-1,5-bisphosphate carboxylase activity decreased with salinity, phosphoenolpyruvate carboxylase activity did not increase and its levels were not atypical of C₃ plants. When plants were grown at four NaCl concentrations under atmospheres of 310 and 1300 cm³ m^?3 CO₂, the CO₂-enrichment enhanced the effects of salinity on ?¹³C. This is consistent with a biophysical explanation for salt-induced shifts in ?¹³C, whereby there is a steepening of the CO₂ diffusion gradient into the leaf. Gas exchange analysis indicated that intercellular CO₂ concentrations were depressed in the leaves of salt-affected plants. This resulted from a greatly decreased stomatal conductance coupled with only small effects on intrinsic photosynthetic capacity. Water-use efficiency was enhanced.     

低温对玉米叶片PEP羧化酶及其调节特性的影响

低温贮存期间,玉米叶片PEP羧化酶活性随贮存时间的延长而明显降低,对效应剂Gly的敏感性也减弱。多羟基醇(甘油和山梨醇)以及PEP羧化酶的正效应剂G-6-P在与Gly和甘油同时作用时,对PEP羧化酶在低温贮存期间的活性和对Gly的敏感性均有保护效应,且对两者的保护程度相一致,表明低温贮存期间PEP羧化酶对效应剂敏感性减弱与其低温失活有直接关系。     

On the significance of C3—C4 intermediate photosynthesis to the evolution of C4 photosynthesis

Abstract Evidence is drawn from previous studies to argue that C₃—C₄ intermediate plants are evolutionary intermediates, evolving from fully-expressed C₃ plants towards fully-expressed C₄ plants. On the basis of this conclusion, C₃—C₄ intermediates are examined to elucidate possible patterns that have been followed during the evolution of C₄ photosynthesis. An hypothesis is proposed that the initial step in C₄-evolution was the development of bundle-sheath metabolism that reduced apparent photorespiration by an efficient recycling of CO₂ using RuBP carboxylase. The CO₂-recycling mechanism appears to involve the differential compartmentation of glycine decarboxylase between mesophyll and bundle-sheath cells, such that most of the activity is in the bundlesheath cells. Subsequently, elevated phosphoenolpyruvate (PEP) carboxylase activities are proposed to have evolved as a means of enhancing the recycling of photorespired CO₂. As the activity of PEP carboxylase increased to higher values, other enzymes in the C₄-pathway are proposed to have increased in activity to facilitate the processing of the products of C₄-assimilation and provide PEP substrate to PEP carboxylase with greater efficiency. Initially, such a ‘C₄-cycle’ would not have been differentially compartmentalized between mesophyll and bundlesheath cells as is typical of fully-expressed C₄ plants. Such metabolism would have limited benefit in terms of concentrating CO₂ at RuBP carboxylase and, therefore, also be of little benefit for improving water- and nitrogen-use efficiencies. However, the development of such a limited C₄-cycle would have represented a preadaptation capable of evolving into the leaf biochemistry typical of fully-expressed C₄ plants. Thus, during the initial stages of C₄-evolution it is proposed that improvements in photorespiratory CO₂-loss and their influence on increasing the rate of net CO₂ assimilation per unit leaf area represented the evolutionary ‘driving-force’. Improved resourceuse efficiency resulting from an efficient CO₂-concentrating mechanism is proposed as the driving force during the later stages.     

C4 photosynthesis at low temperatures

Abstract. C₄ plants grown in optimum conditions are, by comparison to C₃, capable of higher maximum dry-matter yields and greater efficiencies of water and nitrogen use, yet they are rare outside the subtropics. Both latitudinal and altitudinal limits of C₄ distributions correlate most closely with a mean minimum temperature of 8-10°C during the period of active growth. The possibility that the C₄ process is inherently incapable of functioning at low temperatures is examined. The reversible effects of chilling on the quantum efficiency of C₄ photosynthesis and the functioning of the individual steps in the C₄ cycle are examined. Chilling also produces an irreversible loss of capacity to assimilate CO₂ which is directly proportional to the light received during chilling. It is suggested that the reversible reduction in capacity to assimilate CO₂ and the lack of an alternative pathway for the utilization of lightgenerated reducing power may make C₄ species more prone to chilling-dependent photoinhibition. Laboratory studies and limited field observations suggest that this damage would be most likely to occur during photosynthetic induction at the temperatures and light levels encountered on clear, cool mornings during the spring and early summer in cool climates. Even those C₄ species occurring naturally in cool climates do not appear fully capable of tolerating these conditions; indeed their growth patterns suggest that they may be adapted by avoiding 'rather than enduring' such conditions.     

Altered night-time CO2 concentration affects the growth, physiology and biochemistry of soybean

Soybean plants (Glycine max (L.) Merr. c.v. Williams) were grown in CO₂ controlled, natural-light growth chambers under one of four atmospheric CO₂ concentrations ([CO₂]): (1) 250 μmol mol^–1 24 h d^–1[250/250]; (2) 1000 μmol mol^–1 24 h d^–1[1000/1000]; (3) 250 μmol mol^–1 during daylight hours and 1000 μmol mol^–1 during night-time hours [250/1000] or (4) 1000 μmol mol^–1 during daylight hours and 250 μmol mol^–1 during night-time hours [1000/250]. During the vegetative growth phase few physiological differences were observed between plants exposed to a constant 24 h [CO₂] (250/250 and 1000/1000) and those that were switched to a higher or lower [CO₂] at night (250/1000 and 1000/250), suggesting that the primary physiological responses of plants to growth in elevated [CO₂] is apparently a response to daytime [CO₂] only. However, by the end of the reproductive growth phase, major differences were observed. Plants grown in the 1000/250 regime, when compared with those in the 1000/1000 regime, had significantly more leaf area and leaf mass, 27% more total plant dry mass, but only 18% of the fruit mass. After 12 weeks of growth these plants also had 19% higher respiration rates and 32% lower photosynthetic rates than the 1000/1000 plants. As a result the ratio of carbon gain to carbon loss was reduced significantly in the plants exposed to the reduced night-time [CO₂]. Plants grown in the opposite switching environment, 250/1000 versus 250/250, showed no major differences in biomass accumulation or allocation with the exception of a significant increase in the amount of leaf mass per unit area. Physiologically, those plants exposed to elevated night-time [CO₂] had 21% lower respiration rates, 14% lower photosynthetic rates and a significant increase in the ratio of carbon gain to carbon loss, again when compared with the 250/250 plants. Biochemical differences also were found. Ribulose-1,5-bisphosphate carboxylase/ oxygenase concentrations decreased in the 250/ 1000 treatment compared with the 250/250 plants, and phosphoenolpyruvate carboxylase activity decreased in the 1000/250 compared with the 1000/1000 plants. Glucose, fructose and to a lesser extent sucrose concentrations also were reduced in the 1000/250 treatment compared with the 1000/1000 plants. These results indicate that experimental protocols that do not maintain elevated CO₂ levels 24 h d^–1 can have significant effects on plant biomass, carbon allocation and physiology, at least for fast-growing annual crop plants. Furthermore, the results suggest some plant processes other than photosynthesis are sensitive to [CO₂] and under ecologically relevant conditions, such as high night-time [CO₂], whole plant carbon balance can be affected.     

Comparative ecophysiology of C3 and C4 plants

Abstract. In this review we relate the physiological significance of C₄ photosynthesis to plant performance in nature. We begin with an examination of the physiological consequences of the C₄ pathway on photosynthesis, then discuss the ecophysiological performance of C₄ plants in contrasting environments. We then compare the performance of C₃ and C₄ plants when they occur together in similar habitats, and finally discuss the distribution of C₄ photosynthesis with respect to the physical environment, phylogeny, and life form.     

RuBPCO kinetics and the mechanism of CO2 entry in C3 plants

Abstract. The CO₂ partial pressure in the chloroplasts of intact photosynthetic C₃ leaves is thought to be less than the intercellular CO₂ partial pressure. The intercellular CO₂ partial pressure can be calculated from CO₂ and H₂O gas exchange measurements, whereas the CO₂ partial pressure in the chloroplasts is unknown. The conductance of CO₂ from the intercellular space to the chloroplast stroma and the CO₂ partial pressure in the chloroplast stroma can be calculated if the properties of photosynthetic gas exchange are compared with the kinetics of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPCO). A discrepancy between gas exchange and RuBPCO kinetics can be attributed to a deviation of CO₂ partial pressure in the chloroplast stroma from that calculated in the intercellular space. This paper is concerned with the following: estimation of the kinetic constants of RuBPCO and their comparison with the CO₂ compensation concentration; their comparison with differential uptake of ¹⁴CO₂ and ¹²CO₂; and their comparison with O₂ dependence of net CO₂ uptake of photosynthetic leaves. Discrepancy between RuBPCO kinetics and gas exchange was found at a temperature of 12.5 °C, a photosynthetic photon flux density (PPFD) of 550 μmol quanta m^?2 s^?1, and an ambient CO₂ partial pressure of 40 Pa. Consistency between RuBPCO kinetics and gas exchange was found if CO₂ partial pressure was decreased, temperature incresed and PPFD decreased. The results suggest that a discrepancy between RuBPCO kinetics and gas exchange is due to a diffusion resistance for CO₂ across the chloroplast envelope which decreases with increasing temperature. At low CO₂ partial pressure, the diffusion resistance appears to be counterbalanced by active CO₂ (or HCO₃) transport with high affinity and low maximum velocity. At low PPFD, CO₂ partial pressure in the chloroplast stroma appears to be in equilibrium with that in the intercellular space due to low CO₂ flux.     

脱落酸和己糖激酶抑制剂对高表达C4-PEPC转基因稻苗耐旱性的影响

为了研究高表达转玉米C₄-磷酸烯醇式丙酮酸羧化酶（phosphoenolpyruvate carboxylase,PEPC）基因水稻（PC）的耐旱性机制,本研究以PC和未转基因野生型原种kitaake为材料,分别在光照和黑暗预处理24 h,其中光照处理中光强为600 μmol·m^-2·s^-1,预处理后稻苗再在15％聚乙二醇-6000模拟干旱胁迫下,同时使用药理学的方法,通过加入脱落酸和己糖激酶的专一性抑制剂100 μmol·L^-1去甲二氢愈创木酸和10 mmol·L^-1葡萄糖胺,观察两种水稻4~5叶期稻苗耐旱表现。结果发现,与WT水稻相比,在PEG-6000处理后,经过光预处理的PC水稻叶片相对含水量下降较少,始终比WT的高,而且丙二醛含量则较少,脯氨酸诱导增加,表现耐旱;而经过暗预处理后PC植株显著降低这个优势,表明光预处理有利于PC耐旱性的表现;黑暗预处理均显著下调了2供试材料植株叶片中可溶性糖的含量,而对可溶性蛋白的含量影响不显著;而光预处理后PC水稻叶片内可溶性糖含量比WT增加,而在黑暗预处理则PC的显著低于WT的,其中葡萄糖胺对光预处理下PC的可溶性糖含量的下调作用最显著;暗预处理逆转或消除了NO,H₂O₂和钙离子含量变化趋势,这些变化与暗预处理减少了两材料叶片蔗糖和葡萄糖含量变化同步;光暗预处理对两材料的PEPC酶活性的差异影响不大,表明外源玉米C₄-PEPC在水稻中是组成型表达。可见PC叶片可部分通过糖组分,参与内源糖介导ABA和HXK信号途径,缓解干旱处理对叶片的伤害,稳定光合能力。     

Photosynthesis, immediate export and carbon partitioning in source leaves of C3, C3-C4 intermediate, and C4Panicum and Flaveria species at ambient and elevated CO2 levels

Immediate export in leaves of C₃‐C₄ intermediates were compared with their C₃ and C₄ relatives within the Panicum and Flaveria genera. At 35 Pa CO₂, photosynthesis and export were highest in C₄ species in each genera. Within the Panicum, photosynthesis and export in ‘type I’ C₃‐C₄ intermediates were greater than those in C₃ species. However, ‘type I’ C₃‐C₄ intermediates exported a similar proportion of newly fixed ¹⁴C as did C₄ species. Within the Flaveria, ‘type II’ C₃‐C₄ intermediate species had the lowest export rather than the C₃ species. At ambient CO₂, immediate export was strongly correlated with photosynthesis. However, at 90 Pa CO₂, when photosynthesis and immediate export increased in all C₃ and C₃‐C₄ intermediate species, proportionally less C was exported in all photosynthetic types than that at ambient CO₂. All species accumulated starch and sugars at both CO₂ levels. There was no correlation between immediate export and the pattern of ¹⁴C‐labelling into sugars and starch among the photosynthetic types within each genus. However, during CO₂ enrichment, C₄Panicum species accumulated sugars above the level of sugars and starch normally made at ambient CO₂, whereas the C₄Flaveria species accumulated only additional starch.     

浓度NO对高表达转玉米C4型pepc水稻光合的促进

为了揭示C₃植物中C₄ pepc高表达带来的生理差异与其高光合效率的关系。本文以高表达的转玉米C₄ pepc光合基因水稻（PC）及原种Kitaake(WT）为材料,通过水培在孕穗期通过根吸入的方法,进行不同浓度的NO供体、NO合成抑制剂以及相关影响信号分子的试剂单独和联合过夜处理12 h,选取倒二叶研究NO对供试材料净光合速率（P_n）,气孔导度(G_s)和胞间CO₂浓度(C_i)的影响。结果表明：WT和PC在200 μmol·L^-1 SNP（Sodium nitroprusside）和1 mmol·L^-1 L-Arg(L-Arginine)处理下,P_n分别增加20.8%、10.7%,差异显著（p<0.05）;随SNP和L-Arg浓度的增加,其表现不同程度的抑制,与PC相比,WT的P_n抑制更显著（p<0.05）,而G_s和C_i的变化则相反（p<0.05）;进一步结合200 μmol·L^-1 SNP和1 mmol·L^-1 L-Arg与SA处理,结果与高浓度的NO供体处理类似;在联合6 mmol·L^-1 Ca²⁺螯合剂EGTA处理下,与PC相比,WT的P_n抑制达到极显著水平（p<0.01）,C_i的变化则相反（p<0.05）。相关分析结果表明：PC的P_n的高低与G_s的相关性小于WT,PC与WT决定系数分别为0.654 9、0.773 5;而与C_i的相关性则更大些,PC与WT决定系数分别为0.466 5、0.419 6,显示PC可能有不同的调节方式,尤其在低浓度的NO,PC可在Ca²⁺参与下调节气孔的开放,在气孔关闭的条件下,仍能维持一定的P_n。