Responses of Rubisco and Rubisco activase in cucumber seedlings to low temperature and weak light

Using 'Jinyou 3' cucumber seedlings as test materials, this paper studied their photosynthetic rate (P(n)), Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase (RCA) activities, and gene expression of Rubisco and RCA under optimal temperature and weak light (WL: 25 degrees C/18 degrees C, 100 micromol x m(-2) x s(-1)), suboptimal temperature and weak light (ST+WL: 18 degrees C/12 degrees C, 100 micromol x m(-2) x s(-1)), and low temperature and weak light (LT+WL: 10 degress C/5 degrees C, 100 micromol x m(-2) x s(-1)). Comparing with the control (25 degrees C/18 degrees C, 400 micromol x m(-2) x s(-1)), treatments WL, ST+WL, and LT+WL all led to a remarkable decrease in leaf area and dry matter mass. At initial stage, the P(n), Rubisco activity, rbcL and rbcS expression, RCA activity, and CsRCA expression in the three treatments declined by a big margin; 5-7 days later, these parameters tended to be less changed in treatment WL, ascended slowly in treatment ST+WL, and decreased continuously in treatment LT+WL. These results suggested that the photosynthetic apparatus of test cucumber seedlings could gradually adapt to weak light or suboptimal temperature and weak light. The Rubisco and RCA activities and the gene expression of Rubisco and RCA showed the similar responses to low temperature and weak light as the P(n), suggesting that the decline in Rubisco and RCA activities and gene expression in cucumber seedlings under low temperature and weak light could be the important reason leading to the decrease of P(n).     

低温弱光对黄瓜幼苗Rubisco与Rubisco活化酶的影响

以‘津优3号'黄瓜幼苗为试材,研究弱光(100 μmol·m-2·s-1)下适温(WL:25℃/18℃)、亚适温(ST+WL:18℃/12℃)和低温(LT+WL:10℃/5℃)对黄瓜幼苗光合速率(Pn)、核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)、Rubisco活化酶(RCA)活性及其基因表达量的影响.结果表明:与对照(25℃/18℃,400 μmol·m-2·s-1)相比,WL、ST+WL和LT+WL处理的单株叶面积和干物质量均明显减小.处理初期,Pn、Rubisco活性及其大亚基基因(rbcL)、小亚基基因(rbcS)表达、RCA活性与基因(CsRCA)表达量大幅度降低,5～7 d后,WL处理趋于平稳,ST+WL处理缓慢回升,而LT+WL处理持续下降,表明黄瓜光合机构对适温弱光和亚适温弱光环境有逐步适应机制.Rubisco和RCA活性及其基因表达对低温弱光的响应与Pn基本一致,表明低温弱光下RCA和Rubisco活性及其基因表达量下降是黄瓜幼苗Pn降低的重要原因.     

Overexpression in tobacco of a tomato GMPase gene improves tolerance to both low and high temperature stress by enhancing antioxidation capacity

GDP-mannose pyrophosphorylase (GMPase: EC 2.7.7.22) plays a crucial role in the synthesis of l-ascorbate (AsA) and the consequent detoxification of reactive oxygen species (ROS). Herein, a GMPase (accession ID DQ449030) was identified and cloned from tomato. The full-length cDNA sequence of this gene contains 1,498 bp nucleotides encoding a putative protein with 361 amino acid residues of approximate molecular weight 43 kDa. Northern blot analysis revealed that the GMPase was expressed in all examined tomato tissues, but its expression level was up-regulated in tomato plants subjected to abnormal temperatures. We then overexpressed this tomato GMPase in tobacco plants and observed that the activity of GMPase and the content of AsA were significantly increased by two- to fourfold in the leaves of transgenic tobacco plants. The effect of this gene overexpression was superimposed by the treatments of high or low temperature in tobacco, since the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves were significantly higher in transgenic plants than those of WT, while the contents of H₂O₂ and O₂ ^−· were reduced. Meanwhile, relative electric conductivity increased less in transgenic plants than that in WT, and the net photosynthetic rate (P _n) and the maximal photochemical efficiency of PSII (F _v/F _m) of transgenic plants were notably higher than those of WT under temperature stresses. In conclusion, the overexpression of GMPase increased the content of AsA, thereby leading to the increase in tolerance to temperature stress in transgenic plants.     

Induction of systemic stress tolerance by brassinosteroid in Cucumis sativus

? Brassinosteroids (BRs) are a new class of plant hormones that are essential for plant growth and development. Here, the involvement of BRs in plant systemic tolerance to biotic and abiotic stresses was studied. ? The effects of 24-epibrassinolide (EBR) on plant stress tolerance were studied through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation, the analysis of gene expression using quantitative real-time PCR and the measurement of hydrogen peroxide (H?O?) production using a spectrophotometric assay or confocal laser scanning microscopy. ? Treatment of primary leaves with EBR induced systemic tolerance to photooxidative stress in untreated upper and lower leaves. This was accompanied by the systemic accumulation of H?O? and the systemic induction of genes associated with stress responses. Foliar treatment of EBR also enhanced root resistance to Fusarium wilt pathogen. Pharmacological study showed that EBR-induced systemic tolerance was dependent on local and systemic H?O? accumulation. The expression of BR biosynthetic genes was repressed in EBR-treated leaves, but elevated significantly in untreated systemic leaves. Further analysis indicated that EBR-induced systemic induction of BR biosynthetic genes was mediated by systemically elevated H?O?. ? These results strongly argue that local EBR treatment can activate the continuous production of H?O?, and the autopropagative nature of the reactive oxygen species signal, in turn, mediates EBR-induced systemic tolerance.     

温度对黄瓜幼苗光合生理弱光耐受性的影响

以不耐弱光的津研2号和较耐弱光的戴多星黄瓜(Cucumis sativus L.)为试材,在人工气候室内研究适温25 ℃/18 ℃（昼/夜）、亚适温15 ℃/9 ℃和低温9 ℃/7 ℃对弱光(75～85 μmol·m^-2·s^-1)耐受性的影响.结果表明：弱光下黄瓜叶片的SPAD、净光合速率（P_n）、气孔导度（G_s）、蒸腾速率（T_r）、水分利用效率（WUE）、实际光化学效率（Φ_PSⅡ）、光化学猝灭（q_P）等指标下降,下降程度随温度的降低而加剧,而超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性上升.逆境解除后的恢复过程中,光合和荧光参数逐渐恢复,荧光参数恢复速度快于气体交换参数.弱光下温度越低对黄瓜幼苗叶片光合机构造成的伤害越重,低温降低了叶片对弱光的耐受性.在低温、弱光处理过程中,津研2号P_n、Φ_PSⅡ、q_P等下降程度较戴多星明显,而在随后的恢复过程中其回升速度较戴多星迟缓,表明弱光下戴多星对低温的耐受性强于津研2号.     

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.     

Overexpression of transketolase gene promotes chilling tolerance by increasing the activities of photosynthetic enzymes,alleviating oxidative damage and stabilizing cell structure in Cucumis sativus L.

Despite being a key enzyme of Cavin cycle, transketolase (TK) is believed to be related to abiotic resistance in higher plants. However, how TK affects chilling tolerance still remains largely unknown. Here, we describe the effect of overexpression of the Cucumis sativa TK gene (CsTK) on growth, photosynthesis, ROS metabolism and cell ultrastructure under chilling stress. Low temperature led to a decrease of the photosynthetic rate (Pn), the stomatal conductance (Gs), the actual photochemical efficiency (ΦPSII) and the sucrose content, whereas there was an increase of the intercellular CO₂ concentration (Ci) and MDA content. These changes were alleviated in the CsTK plants after 5 days of chilling stress, however, inhibition of CsTK showed the opposite results. Furthermore, transgenic plants with overexpression of CsTK showed higher increase in leaf area and dry matter, higher activity of the enzymes and higher increase in the contents of metabolism substance involved in Calvin cycle and reactive oxygen scavenging system as well as lower ^?OH and H₂O₂ content, superoxide anion production rate compared with the control cucumber plants under chilling stress. At the end of the chilling stress, compared to wild‐type (WT) which exhibited dramatically destroyed cell ultrastructure, expanded chloroplast, broken cell and chloroplast membranes as well as the disappeared grana lamella, the CsTK sense plants showed a more complete cell ultrastructure, whereas, the damage of the cell ultrastructure was aggravated in CsTK antisense plants. Taken together, these results imply that CsTK promoted chilling tolerance in cucumber plants mainly through increasing the capacity to assimilate carbon, alleviating oxidative damage and stabilizing cell structure.     

Relationship between the heat tolerance of photosynthesis and the thermal stability of rubisco activase in plants from contrasting thermal environments

Inhibition of net photosynthesis (Pn) by moderate heat stress has been attributed to an inability of Rubisco activase to maintain Rubisco in an active form. To examine this proposal, the temperature response of Pn, Rubisco activation, chlorophyll fluorescence, and the activities of Rubisco and Rubisco activase were examined in species from contrasting environments. The temperature optimum of Rubisco activation was 10 degrees C higher in the creosote bush (Larrea tridentata) compared with the Antarctic hairgrass (Deschampsia antarctica), resembling the temperature response of Pn. Pn increased markedly with increasing internal CO(2) concentration in Antarctic hairgrass and creosote bush plants subjected to moderate heat stress even under nonphotorespiratory conditions. Nonphotochemical quenching of chlorophyll fluorescence, the effective quantum yield of photochemical energy conversion (DeltaF/F(m)') and the maximum yield of PSII (F(v)/F(m)) were more sensitive to temperature in Antarctic hairgrass and two other species endemic to cold regions (i.e. Lysipomia pumila and spinach [Spinacea oleracea]) compared with creosote bush and three species (i.e. jojoba [Simmondsia chinensis], tobacco [Nicotiana tabacum], and cotton [Gossypium hirsutum]) from warm regions. The temperature response of activity and the rate of catalytic inactivation of Rubisco from creosote bush and Antarctic hairgrass were similar, whereas the optimum for ATP hydrolysis and Rubisco activation by recombinant creosote bush, cotton, and tobacco activase was 8 degrees C to 10 degrees C higher than for Antarctic hairgrass and spinach activase. These results support a role for activase in limiting photosynthesis at high temperature.     

Influence of salicylic acid on rubisco and rubisco activase in tobacco plant grown under sodium chloride in vitro

The present study was designed to evaluate the influence of salicylic acid (SA) on the growth of salt stress (sodium chloride) induced in tobacco plants. In addition, quantification of rubisco and rubisco activase contents of the plants was also determined in treatments with the control, 10⁻⁴ mM SA, 50 mM NaCl, 100 mM NaCl, 150 mM NaCl, SA + 50 mM NaCl, SA + 100 mM NaCl and SA + 150 mM NaCl, respectively after in vitro culture for 5 weeks. The growth of the tobacco plant decreased in 50 mM and 100 mM NaCl when not treated with SA. However, the growth was accelerated by SA, and the growth retardation caused by NaCl was improved by SA. The content of rubisco was improved by SA only in plants treated with 50 mM NaCl, and the activity of rubisco was increased by SA resulting in the decreased effect of NaCl, but only in 50 mM NaCl treated plants. The content of rubisco activase decreased due to NaCl, and SA did not improve the effect caused by NaCl. The activity of rubisco activase was increased by SA resulting in decreased activity caused by NaCl, but increased effect by SA was not recovered to the level of NaCl untreated plants. The activity of rubisco and rubisco activase, which decreased due to denaturing agents, did not demonstrate significant improvement when compared to the control.     

Species-dependent variation in the interaction of substrate-bound ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and rubisco activase

Purified spinach (Spinacea oleracea L.) and barley (Hordeum vulgare L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase supported 50 to 100% activation of substrate-bound Rubisco from spinach, barley, wheat (Triticum aestivum L.), soybean (Glycine max L.), pea (Pisum sativum L.), Arabidopsis thaliana, maize (Zea mays L.), and Chlamydomonas reinhardtii but supported only 10 to 35% activation of Rubisco from three Solanaceae species, tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida L.), and tomato (Lycopersicon esculentum L.). Conversely, purified tobacco and petunia Rubisco activase catalyzed 75 to 100% activation of substrate-bound Rubisco from the three Solanacee species but only 10 to 25% activation of substrate-bound Rubisco from the other species. Thus, the interaction between substrate-bound Rubisco and Rubisco activase is species dependent. The species dependence observed is consistent with phylogenetic relationships previously derived from plant morphological characteristics and from nucleotide and amino acid sequence comparisons of the two Rubisco subunits. Species dependence in the Rubisco-Rubisco activase interaction and the absence of major anomalies in the deduced amino acid sequence of tobacco Rubisco activase compared to sequences in non-Solanaceae species suggest that Rubisco and Rubisco activase may have coevolved such that amino acid changes that have arisen by evolutionary divergence in one of these enzymes through spontaneous mutation or selection pressure have led to compensatory changes in the other enzyme.     

Two residues of rubisco activase involved in recognition of the Rubisco substrate

Rubisco activase is an AAA(+) protein, a superfamily with members that use a "Sensor 2" domain for substrate recognition. To determine whether the analogous domain of activase is involved in recognition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), two chimeric activases were constructed, interchanging a Sensor 2-containing region between activases from spinach and tobacco. Spinach chimeric activase was a poor activator of both spinach and tobacco Rubisco. In contrast, tobacco chimeric activase activated spinach Rubisco far better than tobacco Rubisco, similar to spinach activase. A point mutation, K311D, in the Sensor 2 domain of the tobacco chimeric activase abolished its ability to better activate spinach Rubisco. The opposite mutation, D311K, in wild type tobacco activase produced an enzyme that activated both spinach and tobacco Rubisco, whereas a second mutation, D311K/L314V, shifted the activation preference toward spinach Rubisco. The involvement of these two residues in substrate selectivity was confirmed by introducing the analogous single and double mutations in cotton activase. The ability of the two tobacco activase mutants to activate wild type and mutant Chlamydomonas Rubiscos was also examined. Tobacco D311K activase readily activated wild type and P89R but not D94K Rubisco, whereas the tobacco L314V activase only activated D94K Rubisco. The tobacco activase double mutant D311K/L314V activated wild type Chlamydomonas Rubisco better than either the P89R or D94K Rubisco mutants, mimicking activation by spinach activase. The results identified a substrate recognition region in activase in which two residues may directly interact with two residues in Rubisco.     

Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.     

Dependence of ovary growth on ovule development in Cucumis sativus

Partition and sorption characteristics of the cytokinin N-(phenylmethyl)-lH-purine-6-amine (N⁶-benzyladenine, BA) in two model systems were studied at 25°C. Apparent partition coefficients (K^pH) for BA in an n -octanol:water (buffer) system were independent of BA concentration but decreased with increasing buffer concentration (ionic strength). A plot of K^pH values versus pH (2 to 12) was a bell-shaped curve. At pH 2 or 12, K^pH values were low (less than 2). As pH approached neutrality, K^pH values increased approximately 20-fold. Estimates of pK₁(3.3) and pK₂ (10.7) values for BA were obtained from these data. The accuracy of this approach to determining pK values was verified with 2-(1-naphthyl)acetic acid (NAA). Correction of K^pH values for dissociation effects on BA yielded partition coefficients (K) whose overall mean was 37. Sorption of BA by cuticles enzymatically isolated from abaxial and adaxial surfaces of mature rubber plant ( Ficus elastica Roxb. cv. decora) leaves was initially rapid, however equilibrium was not reached by 432 h. At pH 2, sorption was low (approximately 5 μmol kg−¹), whereas at pH 6 sorption increased approximately 11-fold. Sorption by abaxial cuticular membranes (CM) and dewaxed CM (DCM) was slightly higher than corresponding adaxial CM and DCM. Using natural logarithm values, K^pH data were linearly-correlated with CM (r = 0.98) and DCM (r = 0.99) sorption data.     

Overexpression of the PtSOS2 gene improves tolerance to salt stress in transgenic poplar plants

In higher plants, the salt overly sensitive (SOS) signalling pathway plays a crucial role in maintaining ion homoeostasis and conferring salt tolerance under salinity condition. Previously, we functionally characterized the conserved SOS pathway in the woody plant Populus trichocarpa. In this study, we demonstrate that overexpression of the constitutively active form of PtSOS2 (PtSOS2TD), one of the key components of this pathway, significantly increased salt tolerance in aspen hybrid clone Shanxin Yang (Populus davidiana × Populus bolleana). Compared to the wild‐type control, transgenic plants constitutively expressing PtSOS2TD exhibited more vigorous growth and produced greater biomass in the presence of high concentrations of NaCl. The improved salt tolerance was associated with a decreased Na⁺ accumulation in the leaves of transgenic plants. Further analyses revealed that plasma membrane Na⁺/H⁺ exchange activity and Na⁺ efflux in transgenic plants were significantly higher than those in the wild‐type plants. Moreover, transgenic plants showed improved capacity in scavenging reactive oxygen species (ROS) generated by salt stress. Taken together, our results suggest that PtSOS2 could serve as an ideal target gene to genetically engineer salt‐tolerant trees.