A novel role for cyanide in the control of cucumber (Cucumis sativus L.) seedlings response to environmental stress

The effects of potassium cyanide (KCN) pretreatment on the response of cucumber (Cucumis sativus L.) plants to salt, polyethylene glycol (PEG) and cold stress were investigated in the present study. Here, we found that KCN pretreatment improved cucumber seedlings tolerance to stress conditions with maximum efficiency at a concentration of 20 µM. The results showed that pretreatment with 20 µM KCN alleviated stress‐induced oxidative damage in plant cells and clearly induced the activity of alternative oxidase (AOX) and the ethylene production. Furthermore, the structures of thylakoids and mitochondria in the KCN‐pretreated seedlings were less damaged by the stress conditions, which maintained higher total chlorophyll content, photosynthetic rate and photosystem II (PSII) proteins levels than the control. Importantly, the addition of the AOX inhibitor salicylhydroxamic acid (1 mm ; SHAM) decreased plant resistance to environmental stress and even compromised the cyanide (CN)‐enhanced stress tolerance. Therefore, our findings provide a novel role of CN in plant against environmental stress and indicate that the CN‐enhanced AOX might contribute to the reactive oxygen species (ROS) scavenging and the protection of photosystem by maintaining energy charge homoeostasis from chloroplast to mitochondria.     

Enhancement of stress tolerance in cucumber seedlings by proanthocyanidins

Proanthocyanidins (PAs) are the main products of the flavonoid biosynthetic pathway in many plants. However, their biological function during environmental stresses in plants is rarely reported. In the present study, the effects of pretreatment with PAs on the response of cucumber (Cucumis sativus L.) seedlings to high irradiance (HI), polyethylene glycol (PEG), and cold stress were investigated. The PAs pretreament alleviated stress-induced oxidative damage in plant cells and increased the activity of alternative oxidase (AOX) and content of abscisic acid (ABA). Furthermore, PAs-pretreated seedlings suffered less damage by the stress conditions, maintained higher content of chlorophyll a+b and AOX proteins in comparison with the control. Therefore, our findings suggest that PAs might contribute to plant tolerance to environmental stresses.     

Endogenous salicylic acid accumulation is required for chilling tolerance in cucumber (Cucumis sativus L.) seedlings

Salicylic acid (SA) is an important plant hormone, and its exogenous application can induce tolerance to multiple environmental stresses in plants. In this study, we examine the potential involvement of endogenous SA in response to chilling in cucumber (Cucumis sativus L.) seedlings. A low temperature of 8 °C induces a moderate increase in endogenous SA levels. Chilling stimulates the enzymatic activities and the expression of genes for phenylalanine ammonia-lyase (PAL) and benzoic acid-2-hydroxylase rather than isochorismate synthase. This indicates that the PAL enzymatic pathway contributes to chilling-induced SA production. Cucumber seedlings pretreated with SA biosynthesis inhibitors accumulate less endogenous SA and suffer more from chilling damage. The expression of cold-responsive genes is also repressed by SA inhibitors. The reduction in stress tolerance and in gene expression can be restored by the exogenous application of SA, confirming the critical roles of SA in chilling responses in cucumber seedlings. Furthermore, the inhibition of SA biosynthesis under chilling stress results in a prolonged and enhanced hydrogen peroxide (H₂O₂) accumulation. The application of exogenous SA and the chemical scavenger of H₂O₂ reduces the excess H₂O₂ and alleviates chilling injury. In contrast, the protective effects of SA are negated by foliar spraying with high concentrations of H₂O₂ and an inhibitor of the antioxidant enzyme. These results suggest that endogenous SA is required in response to chilling stress in cucumber seedlings, by modulating the expression of cold-responsive genes and the precise induction of cellular H₂O₂ levels.     

Overexpression of wheat alternative oxidase gene Waox1a alters respiration capacity and response to reactive oxygen species under low temperature in transgenic Arabidopsis

Under low temperature conditions, the cytochrome pathway of respiration is repressed and reactive oxygen species (ROS) are produced in plants. Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for the cyanide-insensitive and salicylhydroxamic acid-sensitive respiration. To study functions of wheat AOX genes under low temperature, we produced transgenic Arabidopsis by introducing Waox1a expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis thaliana. The enhancement of endogenous AOX1a expression via low temperature stress was delayed in the transgenic Arabidopsis. Recovery of the total respiration activity under low temperature occurred more rapidly in the transgenic plants than in the wild-type plants due to a constitutively increased alternative pathway capacity. Levels of ROS decreased in the transgenic plants under low temperature stress. These results support the hypothesis that AOX alleviates oxidative stress when the cytochrome pathway of respiration is inhibited under abiotic stress conditions.     

Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X

Background  

Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1.     

Effects of light on cyanide‐resistant respiration and alternative oxidase function in Arabidopsis seedlings

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)‐resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light‐induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light‐response of AOX1a gene. When aox1a mutant seedlings were grown under a high‐light (HL) condition, photobleaching was more evident in the mutant than the wild‐type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing‐equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing‐equivalents in the mutant during de‐etiolation might be the main reasons.     

Overexpression of the rubisco activase gene improves growth and low temperature and weak light tolerance in Cucumis sativus

Rubisco activase (RCA) is an important enzyme that can catalyze the carboxylation and oxygenation activities of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), which is involved in the photosynthetic carbon reduction cycle. Here, we studied the effects of changes in RCA activity on photosynthesis, growth and development, as well as the low temperature and weak light tolerance of RCA overexpressing transgenic cucumber (Cucumis sativus) plants. CsRCA overexpression increased the plant height, leaf area and dry matter, and decreased the root/top ratio in transgenic cucumber plants compared with the wild‐type (WT) plants. Low temperature and low light stress led to decreases in the CsRCA expression and protein levels, the photosynthetic rate (Pn) and the stomatal conductance (Gs), but an increase in the intercellular CO₂ (Ci) concentration in cucumber leaves. The actual photochemical efficiency and maximal photochemical efficiency of photosystem II in cucumber seedlings also declined, but the initial fluorescence increased during low temperature and weak light stress. Transgenic plants showed a lower decrease in the CsRCA expression level and actual and maximal photochemical efficiencies, as well as increases in the Ci and initial fluorescence relative to the WT plants. Low temperature and low light stress resulted in a significant increase in the malondialdehyde (MDA) content; however, this increase was reduced in transgenic plants compared with that in WT plants. Thus, the overexpression of CsRCA may promote the growth and low temperature and low light tolerance of cucumber plants in solar greenhouses.     

Biochemical study of the effect of stress conditions on the mandelonitrile‐associated salicylic acid biosynthesis in peach

• Salicylic acid (SA) plays a central role in plant responses to environmental stresses. In a recent study, we suggested a third pathway for SA biosynthesis from mandelonitrile (MD) in peach plants. This pathway is an alternative to the phenylalanine ammonia‐lyase pathway and links SA biosynthesis and cyanogenesis. In the present work, using biochemical approaches, we studied the effect of salt stress and Plum pox virus (PPV) infection on this proposed SA biosynthetic pathway from MD.
• Peach plants were submitted to salt stress and Plum pox virus (PPV) infection. We studied the levels of SA and its intermediates/precursors (phenylalanine, MD, amygdalin and benzoic acid) in in vitro shoots. Moreover, in peach seedlings, we analysed the content of H₂O₂‐related enzymes, SA and the stress‐related hormones abscisic acid and jasmonic acid.
• We showed that the contribution of this SA biosynthetic pathway from MD to the total SA pool does not seem to be important under the stress conditions assayed. Nevertheless, MD treatment not only affected the SA content, but also had a pleiotropic effect on abscisic acid and jasmonic acid levels. Furthermore, MD modulates the antioxidative metabolism via SA‐dependent or ‐independent redox‐related signalling pathways.
• Even though the proposed SA biosynthetic pathway seems to be functional under stress conditions, MD, and hence cyanogenic glycosides, may be operating more broadly than by influencing SA pathways and signalling. Thus, the physiological function of the proposed SA biosynthetic pathway remains to be elucidated.

     

不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较

比较了不同低温(14℃和4℃)胁迫对烟草(Nicotiana rusticaL．)愈伤组织抗氰交替途径诱导和交替氧化酶表达的影响。结果显示,不同低温胁迫处理能显著诱导烟草愈伤组织交替途径容量和实际运行的增加,且都呈现出基本相同的变化模式：在胁迫的初期(1—3d)持续增加,在3d时达到最高,而后下降到一个相对恒定的水平。但交替途径容量增加的幅度与温度下降的程度密切相关,而交替途径实际运行量的诱导程度在不同低温胁迫下的差异却很小。表明交替途径容量和实际运行对低温胁迫的响应是不同的。免疫印迹分析结果表明：低温胁迫明显诱导了交替氧化酶总蛋白的增加,且其随低温胁迫进程的变化与交替途径容量的变化基本一致;而对交替氧化酶单体与二聚体在低温胁迫下的含量变化检测结果则显示,烟草愈伤组织中交替氧化酶主要以二聚体形式存在,且这一存在形式并不随低温胁迫程度的加深而发生改变。两种形式的交替氧化酶蛋白含量都能被低温胁迫诱导增加,但其单体水平在两种不同的低温胁迫下并无明显差别,而4℃低温胁迫诱导的二聚体交替氧化酶蛋白含量明显高于14℃。表明不同程度低温对抗氰交替途径发生的不同影响主要是由于对交替氧化酶蛋白二聚体形式的不同诱导程度所致;而高活性的交替氧化酶单体形式则不因低温胁迫程度的加重而被明显诱导升高,使得抗氰交替途径的运行程度在两种不同的低温胁迫处理条件下无显著差异。     

不同低温胁迫对烟草愈伤组织抗氰交替途径诱导和交替氧化酶表达影响的比较

比较了不同低温（14℃和4℃）胁迫对烟草（Nicotiana rustica L.）愈伤组织抗氰交替途径诱导和交替氧化酶表达的影响。结果显示,不同低温胁迫处理能显著诱导烟草愈伤组织交替途径容量和实际运行的增加,且都呈现出基本相同的变化模式：在胁迫的初期（1～3 d）持续增加,在3 d时达到最高,而后下降到一个相对恒定的水平。但交替途径容量增加的幅度与温度下降的程度密切相关,而交替途径实际运行量的诱导程度在不同低温胁迫下的差异却很小。表明交替途径容量和实际运行对低温胁迫的响应是不同的。免疫印迹分析结果表明：低温胁迫明显诱导了交替氧化酶总蛋白的增加,且其随低温胁迫进程的变化与交替途径容量的变化基本一致;而对交替氧化酶单体与二聚体在低温胁迫下的含量变化检测结果则显示,烟草愈伤组织中交替氧化酶主要以二聚体形式存在,且这一存在形式并不随低温胁迫程度的加深而发生改变。两种形式的交替氧化酶蛋白含量都能被低温胁迫诱导增加,但其单体水平在两种不同的低温胁迫下并无明显差别,而4℃低温胁迫诱导的二聚体交替氧化酶蛋白含量明显高于14℃。表明不同程度低温对抗氰交替途径发生的不同影响主要是由于对交替氧化酶蛋白二聚体形式的不同诱导程度所致;而高活性的交替氧化酶单体形式则不因低温胁迫程度的加重而被明显诱导升高,使得抗氰交替途径的运行程度在两种不同的低温胁迫处理条件下无显著差异。     

Identification of Down-regulated Genes Modulated by an Alternative Oxidase Pathway Under Cold Stress Conditions in Watermelon Plants

This study investigated the cold responses of watermelon plants modulated by the alternative oxidase (AOX) pathway. Salicylhydroxamic acid was used in watermelon (Citrullus lanatus) plants in order to chemically inhibit the AOX pathway. After exposure to cold temperature (10°C) for 24 h, the SHAM-treated plants showed more serious cold-induced damage compared to non-treated plants. To investigate how the AOX pathway modulated the watermelon plants’ responses to cold, a polymerase chain reaction (PCR)-based suppression subtractive hybridization technique was used to isolate genes differently expressed between cold-stressed leaves of SHAM-treated and non-treated watermelon germplasm “IVSM No. 9”. After sequencing 400 randomly chosen cDNA clones, 314 uniquely expressed sequence tags (ESTs) were obtained. A total of 114 ESTs encoded unknown proteins or had no similarity in the database. Identified genes with significant protein homology mainly participated in stress and defense responses, metabolism and energetic processes, regulation of gene expression, signal transduction, protein fate and synthesis, cellular transport and others. These results indicated that watermelon plants could not respond appropriately to cold stress and suffer greater damage with an inhibited AOX pathway, thus, the AOX pathway had an important role in defining the plants cold responses.     

The flexible interrelation between AOX respiratory pathway and photosynthesis in rice leaves.

Alternative respiratory pathway was investigated in rice seedlings grown under total darkness, light/dark cycle, or continuous light. The capacity of the alternative pathway was relatively higher in leaves that had longer light exposure. An analysis of rice AOX1 multigene family revealed that AOX1c, but not AOX1a and AOX1b, had a light-independent expression. The alternative oxidase (AOX) inhibitor, salicylhydroxamic acid (SHAM, 1mM), inhibited nearly 68% of the capacity of the alternative pathway in leaves grown under different light conditions. The plants grown under different light periods were treated with SHAM and then were exposed to illumination for 4h. The transition from dark to 4h of light stimulated the capacity of alternative pathway in etiolated rice seedlings and in those grown under light/dark cycle, whereas the capacity of the alternative pathway was constant in seedlings grown under continuous light with additional 4h of illumination. Etiolated leaves did not show any CO(2) fixation after 4h of illumination, and the increase in chlorophyll content was delayed by the SHAM pretreatment. When seedlings grown under light/dark cycle were moved from dark and exposed to 4h of light, increases in chlorophyll content and CO(2) fixation rate were reduced by SHAM. Although these parameters were stable in plants grown under continuous light, SHAM decreased CO(2) fixation rate but not the chlorophyll content. These results indicate that the role and regulation of AOX in light are determined by the developmental stage of plant photosynthetic apparatus.     

Regulation of alternative oxidase 1 in Chlamydomonas reinhardtii during sulfur starvation

The mitochondrial respiratory chain in plants, some protists and many fungi consists of the ATP-coupling cyanide-sensitive cytochrome pathway and the cyanide-resistant alternative respiratory pathway. The alternative pathway is mediated by alternative oxidase (AOX). Although AOX has been proposed to play essential roles in nutrient stress tolerance of plants and protists, the effects of sulfur (S) deprivation, on AOX are largely unknown. The unicellular green alga Chlamydomonas reinhardtii reacts to S limitation conditions with the induced expression of many genes. In this work, we demonstrated that exposure of C. reinhardtii to S deprivation results in the up-regulation of AOX1 expression and an increased AOX1 protein. Furthermore, S-deprived C. reinhardtii cells display the enhanced AOX1 capacity. Moreover, nitrate assimilation regulatory protein (NIT2) is involved in the control of the AOX1 gene expression in the absence of S. Together, the results clearly indicate that AOX1 relates to S limitation stress responses and is regulated in a NIT2-dependent manner, probably together with yet-unknown regulatory factor(s).