Impaired reductive activation of stromal bisphosphatases in tomato leaves following low-temperature exposure at high light

Photosynthesis in domestic tomato (Lycopersicon esculentum L.) is highly sensitive to low temperature, particularly when accompanied by high light. Since previous studies have established that the inhibited plants retain photosynthetic electron transfer and ATP formation competence, we sought to identify specific steps in the photosynthetic carbon reduction pathway that could account for the lost photosynthetic capacity. Measurements of steady-state photosynthetic metabolite pool sizes showed an accumulation of fructose 1,6-bisphosphate and sedohepulose 1,7-bisphosphate following chilling in the light. Measurements of in vivo turnover rates of the metabolite pools accompanied by direct determinations of enzymatic activity showed that the capacity of the stromal bisphosphatases was substantially reduced following chilling in the light and was the cause of the bisphosphate accumulation. The time course of the loss of phosphatase activity closely mimicked that of the inhibition of net photosynthesis, further indicating that impaired phosphatase function is the underlying cause of the sensitivity of photosynthesis in tomato to light and chilling. Fructose 1,6-bisphosphatase extracted from inhibited tomato plants could be fully activated in the presence of dithiothreitol, indicating that chilling in the light disrupts the normal, thioredoxin-dependent, activation pathway of the stromal bisphosphatases. This disruption could involve a change in the redox potential of the functional disulfide on the phosphatases.     

组成型表达的ClpB基因提高番茄植株的耐冷性

用农杆菌介导法将CaMV35S启动子驱动的ClpB cDNA导入番茄,并比较了转基因和未转基因番茄的抗冷能力。当受冷胁迫后,转基因番茄比未转基因番茄表现出较轻的冷胁迫症状,并维持较高的PSII水平。     

Altered expression of pyrophosphate: Fructose-6-phosphate 1-phosphotransferase affects the growth of transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis. To examine the role of PFP in plant growth, we have generated transgenic Arabidopsis plants that either overexpress or repress Arabidopsis PFP sub-unit genes. The overexpressing lines displayed increased PFP activity and slightly faster growth relative to wild type plants, although their photosynthetic activities and the levels of metabolites appeared not to have significantly changed. In contrast, the RNAi lines showed significantly retarded growth in parallel with the reduced PFP activity. Analysis of photosynthetic activity revealed that the growth retardation phenotype of the RNAi lines was accompanied by the reduced rates of CO₂ assimilation. Microarray analysis of our transgenic plants further revealed that the altered expression of AtPFPβ affects the expression of several genes involved in diverse physiological processes. Our current data thus suggest that PFP is important in carbohydrate metabolism and other cellular processes. These authors contributed equally to this study.     

Increase in the activity of fructose-1,6-bisphosphatase in cytosol affects sugar partitioning and increases the lateral shoots in tobacco plants at elevated CO<Subscript>2</Subscript> levels

We generated transgenic tobacco plants with high levels of fructose-1,6-bisphosphatase expressing cyanobacterialfructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol. At ambient CO₂ levels (360 ppm), growth, photosynthetic activity, and fresh weight were unchanged but the sucrose/hexose/starch ratio was slightly altered in the transgenic plants compared with wild-type plants. At elevated CO₂ levels (1200 ppm), lateral shoot, leaf number, and fresh weight were significantly increased in the transgenic plants. Photosynthetic activity was also increased. Hexose accumulated in the upper leaves in the wild-type plants, while sucrose and starch accumulated in the lower leaves and lateral shoots in the transgenic plants. These findings suggest that cytosolic fructose-1,6-bisphosphatase contributes to the efficient conversion of hexose into sucrose, and that the change in carbon partitioning affects photosynthetic capacity and morphogenesis at elevated CO₂ levels.     

Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato

In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T(1) and T(2) plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA(3) (gibberellic acid) treatment. More importantly, GA(3)-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H(2)O(2) in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.     

过表达ZmSKIP基因提高烟草抗低温胁迫的能力

以野生型和过表达ZmSKIP基因烟草为试材, 研究了低温胁迫下过表达ZmSKIP对烟草抗氧化能力的影响。测定了不同低温处理时间下过表达ZmSKIP转基因烟草T3代植株和野生型植株抗氧化酶如超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）活性和丙二醛（MDA）含量以及相对电导率, 结果表明, 低温下, 相对于野生型植株, 转基因烟草具有较高的抗氧化酶活性和较低的相对电导率和MDA含量, 说明过表达ZmSKIP提高了转基因植株的耐低温胁迫能力。     

Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato

A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4 degrees C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.     

过表达LeNLP4转录因子提高番茄抗低温胁迫能力

NAC（NAM-ATAF1,2-CUC2）转录因子在植物胁迫响应中起重要作用。为了探讨三舭丹基因在番茄抗低温胁迫中的功能,分离了番茄LeNLP4转录因子基因,并获得转正义LeNLP4基因番茄植株。荧光定量PCR分析表明,LeNLP4的表达受低温诱导。与野生型植株相比,在4℃胁迫下转基因植株具有较高的生长量和光系统II（PSH）最大光化学效率（Fv／Fm）、过氧化氢（H2O2）和超氧阴离子（O2-）清除速率、抗坏血酸过氧化物酶（APX）和超氧化物歧化酶（SOD）活性,以及较低的丙二醛（MDA）含量和相对电导率（REC）。过表达株系中SICBF1的表达高于野生型。上述结果表明,LeNLP4的过表达提高了转基因番茄抗低温胁迫能力。     

Enhancements in sucrose biosynthesis capacity affect shoot branching in Arabidopsis

We previously demonstrated that transgenic tobacco plants expressing cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol increased the number of lateral shoots and leaves at elevated CO₂ levels. These findings suggest that alterations in carbon partitioning affect the development of shoot branching. In order to elucidate the underlying mechanisms at the molecular level, we generated transgenic Arabidopsis plants overexpressing cyanobacterial fructose-1,6-bisphosphatase-II in the cytosol (AcF). At elevated CO₂ levels, the number of lateral shoots was significantly increased in AcF plants. Sucrose and hexose levels were also higher in AcF plants than in wild-type plants. The expression levels of MAX1, MAX4, YUCCA8, YUCCA9, and BRC1, which are involved in auxin or strigolactone biosynthesis and responses, were lower in AcF plants than in wild-type plants. These results suggest that alterations in sugar partitioning affect hormone metabolism and responses, resulting in enhanced shoot branching.     

Differential effects of chilling-induced photooxidation on the redox regulation of photosynthetic enzymes

Photosynthesis in plant species that are evolutionarily adapted for growth in warm climates is highly sensitive to illumination under cool conditions. Although it is well documented that illumination of these sensitive species under cool conditions results in the photosynthetic production of reactive oxygen molecules, the underlying mechanism for the inhibition of photosynthesis remains uncertain. Determinations of chloroplast fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase activity showed that the light-dependent, reductive activation of these key carbon reduction cycle enzymes was substantially inhibited in tomato (Lycopersicon esculentum) following illumination at 4 degrees C. However, other chloroplast enzymes also dependent on thioredoxin-mediated reductive activation were largely unaffected. We performed equilibrium redox titrations to investigate the thermodynamics of the thiol/disulfide exchange between thioredoxin f and the regulatory sulfhydryl groups of fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, NADP-glyceraldehyde phosphate dehydrogenase, and the chloroplast ATPsynthase. We determined that the redox midpoint potentials for the regulatory sulfhydryl groups of the various enzymes spanned a broad range ( approximately 50 mV at pH 7. 9). The electron-sharing equilibria among thioredoxin f and its target enzymes largely explained the differential effects of photooxidation induced at low temperature on thioredoxin-mediated activation of chloroplast enzymes in tomato. These results not only provide a plausible mechanism for the low-temperature-induced inhibition of photosynthesis in this important group of plants, but also provide a quantitative basis to evaluate the influence of thioredoxin/target enzyme electron-sharing equilibria on the differential activation and deactivation kinetics of thioredoxin-regulated chloroplast enzymes.     

过量表达叶绿体小分子热激蛋白提高番茄的抗寒性

小分子热激蛋白与植物耐寒性提高有相关性,但是没有直接的实验证据能证明小分子热激蛋白的存在增加植物抗寒性.我们克隆了番茄叶绿体(定位)小分子热激蛋白cDNA,并将35SCaMV启动子驱动的番茄叶绿体小分子热激蛋白cDNA植物表达构架导入番茄,测定转基因番茄和未转基因番茄的抗寒性水平.低温处理后,转基因番茄的冷害症状轻于未转基因的番茄;转基因番茄细胞电解质外渗较少、花青素和MDA累积量较低;净光合速率和叶绿体含量高于对照.这些实验结果说明叶绿体小分子热激蛋白的过量表达提高了植物抗寒性.     

Antisense-mediated suppression of tomato zeaxanthin epoxidase alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance

A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated and antisense transgenic tomato plants were produced. Northern, southern, and western blot analyses demonstrated that antisense LeZE was transferred into the tomato genome and the expression of LeZE was inhibited. The ratio of (A+Z)/(V+A+Z) in antisense transgenic plants was maintained at a higher level than in the wild type (WT) plants under high light and chilling stress with low irradiance. The value of non-photochemical quenching (NPQ) in WT and transgenic plants was not affected during the stresses. The oxidizable P700 and the maximal photochemical efficiency of PSII (F_v/F_m) in transgenic plants decreased more slowly at chilling temperature under low irradiance. These results suggested that suppression of LeZE caused zeaxanthin accumulation, which was helpful in alleviating photoinhibition of PSI and PSII in tomato plants under chilling stress.     

Overexpression of bacterial catalase in tomato leaf chloroplasts enhances photo-oxidative stress tolerance

The Escherichia coli gene katE, which is driven by the promoter of the Rubisco small subunit gene of tomato, rbcS3C, was introduced into a tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens‐mediated transformation. Catalase activity in progeny from transgenic plants was approximately three‐fold higher than that in wild‐type plants. Leaf discs from transgenic plants remained green at 24 h after treatment with 1 µm paraquat under moderate light intensity, whereas leaf discs from wild‐type plants showed severe bleaching after the same treatment. Moreover, ion leakage from transgenic leaf discs was significantly less than that from wild‐type leaf discs at 24 h after treatment with 1 µm paraquat and 10 mm H₂O₂, respectively, under moderate light intensity. To evaluate the efficiency of the E. coli catalase to protect the whole transgenic plant from the oxidative stress, transgenic and wild‐type plants were sprayed with 100 µm paraquat and exposed to high light illumination (800 µmol m^?2 s^?1). After 24 h, the leaves of the transgenic plants were less damaged than the leaves of the wild‐type plants. The catalase activity and the photosynthesis activity (indicated by the F_v/F_m ratio) were less affected by paraquat treatment in leaves of transgenic plants, whereas the activities of the chloroplastic ascorbate peroxidase isoenzymes and the ascorbate content decreased in both lines. In addition, the transgenic plants showed increased tolerance to the oxidative damage (decrease of the CO₂ fixation and photosystem II activity and increase of the lipid peroxidation) caused by drought stress or chilling stress (4 °C) under high light intensity (1000 µmol m^?2 s^?1). These results indicate that the expression of the catalase in chloroplasts has a positive effect on the protection of the transgenic plants from the photo‐oxidative stress invoked by paraquat treatment, drought stress and chilling stress.     

Overexpression of Arabidopsis hexokinase in tomato plants inhibits growth, reduces photosynthesis, and induces rapid senescence.

Sugars are key regulatory molecules that affect diverse processes in higher plants. Hexokinase is the first enzyme in hexose metabolism and may be a sugar sensor that mediates sugar regulation. We present evidence that hexokinase is involved in sensing endogenous levels of sugars in photosynthetic tissues and that it participates in the regulation of senescence, photosynthesis, and growth in seedlings as well as in mature plants. Transgenic tomato plants overexpressing the Arabidopsis hexokinase-encoding gene AtHXK1 were produced. Independent transgenic plants carrying single copies of AtHXK1 were characterized by growth inhibition, the degree of which was found to correlate directly to the expression and activity of AtHXK1. Reciprocal grafting experiments suggested that the inhibitory effect occurred when AtHXK1 was expressed in photosynthetic tissues. Accordingly, plants with increased AtHXK1 activity had reduced chlorophyll content in their leaves, reduced photosynthesis rates, and reduced photochemical quantum efficiency of photosystem II reaction centers compared with plants without increased AtHXK1 activity. In addition, the transgenic plants underwent rapid senescence, suggesting that hexokinase is also involved in senescence regulation. Fruit weight, starch content in young fruits, and total soluble solids in mature fruits were also reduced in the transgenic plants. The results indicate that endogenous hexokinase activity is not rate limiting for growth; rather, they support the role of hexokinase as a regulatory enzyme in photosynthetic tissues, in which it regulates photosynthesis, growth, and senescence.     

Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage

Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10 degrees C causes various injuries and greatly decreases fruit set in most cultivars. We have transformed tomato (cv. Moneymaker) with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express codA and synthesize choline oxidase, while accumulating GB in their leaves and reproductive organs up to 0.3 and 1.2 micromol g(-1) fresh weight (FW), respectively. Their chloroplasts contain up to 86% of total leaf GB. Over various developmental phases, from seed germination to fruit production, these GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. During reproduction, they yield, on average, 10-30% more fruit following chilling stress. Endogenous GB contents as low as 0.1 micromol g(-1) FW are apparently sufficient to confer high levels of tolerance in tomato plants, as achieved via transformation with the codA gene. Exogenous application of either GB or H2O2 improves both chilling and oxidative tolerance concomitant with enhanced catalase activity. These moderately increased levels of H2O2 in codA transgenic plants, as a byproduct of choline oxidase-catalyzed GB synthesis, might activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in GB-accumulating codA transgenic plants. Thus, introducing the biosynthetic pathway of GB into tomato through metabolic engineering is an effective strategy for improving chilling tolerance.     

“Sink” regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin-cycle enzymes and an increase of glycolytic enzymes

Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the sink regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO₂. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.Abbreviations Fru 1,6bisP fructose-1,6-bisphosphate - Fru 1,6bisPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc 1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PFK phosphofructokinase - PEP phosphoenolpyruvate - PFP pyrophosphate:fructose-6-phosphate phosphotransferase - PGA glycerate-3-phosphate - PK pyruvate kinase - Pi inorganic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SPS sucrose-phosphate synthase - triose-P triose-phosphates     

Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet: I. Changes in growth, gas exchange, and calvin cycle enzymes

Sugar beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically for 2 weeks in growth chambers with two levels of orthophosphate (Pi) supplied in half strength Hoagland solution. Low-P plants were supplied with 1/20th of the Pi supplied to control plants. With low-P treatment, the acid soluble leaf phosphate and total leaf P decreased by about 88%. Low-P treatment had a much greater effect on leaf area than on photosynthesis. Low-P decreased total leaf area by 76%, dry weight per plant by 60%, and the rate of photosynthesis per area at light saturation by 35%. Low-P treatment significantly decreased the total extractable activity of phosphoglycerate kinase (by 18%) and NADP-glyceraldehyde-3-phosphate dehydrogenase (by 16%), but did not decrease the total activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) and ribulose-5-phosphate kinase. Low-P treatment decreased the initial activities of three rate-limiting Calvin cycle enzymes, but had no effect on the initial activity of RuBPCase. Furthermore, low-P treatment significantly increased the total extractable activities of fructose-1,6-bisphosphatase (by 61%), fructose-1,6-bisphosphate aldolase (by 53%), and transketolase (by 46%). The results suggest that low-P treatment affected photosynthetic rate through an effect on RuBP regeneration rather than through RuBPCase activity and that the changes in Calvin cycle enzymes with low-P resulted in an increased flow of carbon to starch.     

Genetic and physiological characterization of Flaveria linearis plants having a reduced activity of cytosolic fructose-1.6-bisphosphatase

We investigated the genetic control of cytosolic fructose-1,6-bisphosphatase (cytFBPase) activity, and the relationships between sucrose synthesis capacity and photosynthesis, growth, flowering and whole-plant carbon partitioning in Flaveria linearis Lag. F₁; F₂, and selfed lines generated from plants with low or high cytFBPase activity were used. CytFBPase activity was controlled by one gene and inherited co-dominantly, giving three classes of activity (low, intermediate and high). Reversed O₂ sensitivity of photosynthesis, which indicates an end-product limitation on photosynthesis, was controlled by one gene and co-segregated with low cytFBPase activity. A low activity of cytFBPase decreased the growth rate. A recessive day-neutral flowering trait in Flaveria linearis did not co-segregate with cytFBPase activity. Plants with low cytFBPase activity had an increased shoot-to-root ratio, and flowering caused an additional shift in carbon partitioning to shoots only in plants with low cytFBPase activity. These data indicate that altering sucrose synthesis can affect photosynthesis and plant growth and development.