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.     

The alternative pathway in cucumber seedlings under low temperature stress was enhanced by salicylic acid

The alternative pathway is a cyanide-resistant and non-phosphorylatory electron transport pathway in mitochondria of higher plants. Alternative oxidase (AOX) is the terminal oxidase of this pathway. Our present study investigated the effect of exogenous salicylic acid (SA) on alternative pathway in cucumber (Cucumis sativus L.) seedlings under low temperature stress. Results showed that during the process of low temperature stress, the alternative pathway capacity was enhanced as AOX expression increased in SA pretreated seedlings. Compared with seedlings without SA pretreatment, slower decrease of relative water content and lower levels of electrolyte leakage, H₂O₂ and malonyldialdehyde content were detected in SA pretreated seedlings. These results indicated that SA could alleviate the injury caused by low temperature on cucumber seedlings. Since the special protective functions of alternative pathway and AOX in plants, we suggested that the alternative pathway was related to SA-mediated plant resistance to environmental stresses such as low temperature.     

水稻磷脂氢谷胱甘肽过氧化物酶在蛋白质水平上的组织和诱导表达特征

蛋白质是生命活动的主要承担分子,了解蛋白质在有机体中的时空分布对于正确解析蛋白质的功能十分重要．磷脂氢谷胱甘肽过氧化物酶 (PHGPx) 是目前发现的唯一能够直接还原膜上脂类过氧化物的抗氧化酶,在保护生物膜免受过氧化损伤方面有着重要作用．采用Western blot技术,分析了水稻PHGPx (OsPHGPx) 在水稻不同组织以及多种胁迫条件下的蛋白质表达特征．结果表明,OsPHGPx在成熟水稻植株内主要分布于叶组织中,以旗叶中含量最高,而在水稻幼苗中则在茎及叶组织中均检测到较强的杂交信号．OsPHGPx在幼苗中的表达受到H₂O₂和NaCl的强烈诱导,但植物激素对其表达的影响较弱．H₂O₂和NaCl的诱导效果呈现出时间及剂量的相关性,当用0.5 mmol/L H₂O₂处理12 h或用500 mmol/L NaCl处理24 h,此时OsPHGPx表达量达到最大值．对H₂O₂清除剂二甲基硫脲处理的水稻幼苗,外源H₂O₂的再处理并不能诱导OsPHGPx的表达,而NaCl的诱导效果并不受影响,说明H₂O₂可能并不介导NaCl诱导OsPHGPx的表达．这些结果为进一步研究OsPHGPx在水稻中生物学功能奠定了基础．     

NaCl胁迫下交替呼吸途径对烟草悬浮细胞死亡调节作用的研究

盐分胁迫是植物在自然环境中经常遭遇的环境胁迫因素之一,会引起植物代谢紊乱乃至细胞死亡,这严重限制了植物的生长、繁育和生存。交替呼吸途径是植物较之动物独特的线粒体呼吸途径。该研究在烟草悬浮细胞中调查了交替呼吸途径对Na Cl胁迫引起的植物细胞死亡过程的调节作用及相应的内在机制,以及在200 mmol·L~(-1)Na Cl处理的烟草悬浮细胞中研究了交替呼吸途径和细胞死亡发生及H_2O_2之间的关系。结果表明:(1)随着Na Cl处理浓度的增加,烟草悬浮细胞死亡水平逐渐增加,而交替呼吸途径的容量也逐渐上升。(2)与Na Cl处理相似,外源H_2O_2的处理也能导致烟草悬浮细胞死亡水平的增加。200 mmol·L~(-1)Na Cl的胁迫导致明显的细胞死亡发生和H_2O_2产量的显著性增加;而较之200 mmol·L~(-1)Na Cl胁迫下的细胞,用水杨基氧肟酸(交替呼吸途径的抑制剂)预处理后的细胞再置于200 mmol·L~(-1)Na Cl的胁迫下导致更高水平的细胞死亡和H_2O_2的产生。综上表明,高盐胁迫诱导了烟草悬浮细胞的交替呼吸途径的增加,而交替呼吸途径则可能通过抑制活性氧的产生而起到缓解细胞死亡发生的作用。     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced acclimation of photosystem II to excess light is mediated by alternative respiratory pathway and salicylic acid

Acclimation to excess light is required for optimizing plant performance under natural environment. The present work showed that the treatment of Arabidopsis leaves with exogenous H₂O₂ can increase the acclimation of PSII to excess light. Treatments with H₂O₂ also enhanced the capacity of the mitochondrial alternative respiratory pathway and salicylic acid (SA) content. Our work also showed that the lack in alternative oxidase (AOX1a) in AtAOX1a antisense line and the SA deficiency in NahG (salicylate hydroxylase gene) transgenic mutant attenuated the H₂O₂-induced acclimation of PSII to excess light. It indicates that the H₂O₂-induced acclimation of PSII to excess light could be mediated by the alternative respiratory pathway and SA.     

Importance of AOX pathway in optimizing photosynthesis under high light stress: role of pyruvate and malate in activating AOX

The present study shows the importance of alternative oxidase (AOX) pathway in optimizing photosynthesis under high light (HL). The responses of photosynthesis and respiration were monitored as O₂ evolution and O₂ uptake in mesophyll protoplasts of pea pre‐incubated under different light intensities. Under HL (3000 µmol m^?2 s^?1), mesophyll protoplasts showed remarkable decrease in the rates of NaHCO₃‐dependent O₂ evolution (indicator of photosynthetic carbon assimilation), while decrease in the rates of respiratory O₂ uptake were marginal. While the capacity of AOX pathway increased significantly by two fold under HL, the capacity of cytochrome oxidase (COX) pathway decreased by >50% compared with capacities under darkness and normal light (NL). Further, the total cellular levels of pyruvate and malate, which are assimilatory products of active photosynthesis and stimulators of AOX activity, were increased remarkably parallel to the increase in AOX protein under HL. Upon restriction of AOX pathway using salicylhydroxamic acid (SHAM), the observed decrease in NaHCO₃‐dependent O₂ evolution or p‐benzoquinone (BQ)‐dependent O₂ evolution [indicator of photosystem II (PSII) activity] and the increase in total cellular levels of pyruvate and malate were further aggravated/promoted under HL. The significance of raised malate and pyruvate levels in activation of AOX protein/AOX pathway, which in turn play an important role in dissipating excess chloroplastic reducing equivalents and sustenance of photosynthetic carbon assimilation to balance the effects of HL stress on photosynthesis, was depicted as a model.     

Nitrogen deprivation induces cross-tolerance of Poa annua callus to salt stress

Alternative respiration pathway (AP) is an important pathway which can be induced by environment stresses in plants. In the present study, we show a new mechanism involving the AP in nitrogen deprivation-induced tolerance of Poa annua callus to salt stress. The AP capacity markedly increased under a 600 mM NaCl treatment or nitrogen deprivation pretreatment and reached a maximum under the nitrogen deprivation pretreatment combined with the NaCl treatment (–N+NaCl). Malondialdehyde (MDA) and H₂O₂ content and Na⁺/K⁺ ratio significantly increased under the 600 mM NaCl treatment but less under the–N+NaCl treatment. Moreover, both the nitrogen deprivation and the NaCl stress stimulated the plasma membrane (PM) H⁺-ATPase activity and increased pyruvate content. The maximal stimulating effect was found under the–N+NaCl treatment. When the AP capacity was reduced by salicylhydroxamic acid (SHAM, an inhibitor of AP), content of MDA and H₂O₂ and Na⁺/K⁺ ratio dramatically increased, whereas PM H⁺-ATPase activity decreased. Moreover, exogenous application of pyruvate produced a similar effect as the nitrogen deprivation pretreatment. The effects of SHAM on the Poa annua callus were counteracted by catalase (a H₂O₂ scavenger) and diphenylene iodonium (a plasma membrane NADPH oxidase inhibitor). Taken together, our results suggest that the nitrogen deprivation enhanced the capacity of AP by increasing pyruvate content, which in turn prevented the Poa annua callus from salt-induced oxidative damages and Na⁺ over-uptake.     

NaCl-induced heme oxygenase in roots of rice seedlings is mediated through hydrogen peroxide

Recent findings have suggested that H₂O₂ is an important signaling molecule for regulating plant responses to abiotic stress. H₂O₂ plays a critical role in NaCl stress. Heme oxygenase (HO) is known to play a protective role against oxidative stress. In this study, we examined the possible involvement of H₂O₂ in regulating NaCl-promoted HO activity in rice roots. Treatment with NaCl increased HO activity and H₂O₂ content in rice roots. As well, NaCl could induce OsHO1 mRNA expression. NaCl (150 mM) and NaNO₃ (150 mM) were equally effective in inducing HO activity. However, mannitol at the concentration (276 mM) iso-osmotic with 150 mM NaCl had no effect on HO activity. NaCl-promoted HO activity and OsHO1 expression in rice roots was reduced by NADPH oxidase inhibitors i.e. dipehnyleneiodonium and imidazole. Moreover, exogenous application of H₂O₂ enhanced the activity of HO and the mRNA level of OsHO1. Our data suggest that H₂O₂ production plays a positive role in NaCl- induced HO activity by enhancing its mRNA level in rice roots.     

Effect of NaCl stress on H2O2 metabolism in rice leaves

The effect of NaCl stress on H₂O₂ metabolismin detached rice leaves was studied. NaCl (200 mM)treatment did not cause the accumulation ofH₂O₂ and resulted in no increase in lipidperoxidation and membrane leakage of leaf tissues. The activities of peroxidase, ascorbate peroxidase,superoxide dismutase, and glutathione reductase wereobserved to be greater in NaCl-stressed rice leavesthan in control leaves. However, glycolate oxidasewas lower in NaCl-treated rice leaves than in thecontrol leaves. There was no difference in catalaseactivity between NaCl and control treatments. Theseresults suggest that some antioxidant enzymes can beactivated in response to oxidative stress induced byNaCl.     

Pre-treatment with H<Subscript>2</Subscript>O<Subscript>2</Subscript> induces salt tolerance in Barley seedlings

Barley seedlings were pre-treated with 1 and 5 μM H₂O₂ for 2 d and then supplied with water or 150 mM NaCl for 4 and 7 d. Exogenous H₂O₂ alone had no effect on the proline, malondialdehyde (MDA) and H₂O₂ contents, decreased catalase (CAT) activity and had no effect on peroxidase (POX) activity. Three new superoxide dismutase (SOD) isoenzymes appeared in the leaves as a result of 1 μM H₂O₂ treatment. NaCl enhanced CAT and POX activity. SOD activity and isoenzyme patterns were changed due to H₂O₂ pre-treatment, NaCl stress and leaf ageing. In pre-treated seedlings the rate of ¹⁴CO₂ fixation was higher and MDA, H₂O₂ and proline contents were lower in comparison to the seedlings subjected directly to NaCl stress. Cl⁻ content in the leaves 4 and 7 d after NaCl supply increased considerably, but less in pre-treated plants. It was suggested that H₂O₂ metabolism is involved as a signal in the processes of barley salt tolerance.     

Heat shock-mediated H2O2 accumulation and protection against Cd toxicity in rice seedlings

Rice (Oryza sativa L.) seedlings stressed with CdCl₂ (0.5 mM or 50 μM) showed typical Cd toxicity (leaf chlorosis, decrease in chlorophyll content, or increase in H₂O₂ and malondialdehyde contents). Rice seedlings pretreated with heat shock at 45°C (HS) for 2 or 3 h were protected against subsequent Cd stress. Rice seedlings pretreated with HS had similar Cd concentration in leaves caused by CdCl₂ as those non-HS. The content of H₂O₂ increased in leaves 1 h after HS exposure. However, APX and GR activities were higher in HS-treated leaves than their respective control, and it occurred after 2 h of HS treatment. Pretreatment of rice seedlings with H₂O₂ under non-HS conditions resulted in an increase in APX, GR, and CAT activities and protected rice seedlings from subsequent Cd stress. HS-induced H₂O₂ production and protection against subsequent Cd stress can be counteracted by imidazole, an inhibitor of NADPH oxidase complex. Results of the present study suggest that early accumulation of H₂O₂ during HS signals the increase in APX and GR activities, which in turn prevents rice seedlings from Cd-caused oxidative damage.     

An alternative respiratory pathway on Candida krusei: implications on susceptibility profile and oxidative stress

Our aim was to detect the presence of an alternative oxidase (AOX) in Candida krusei clinical strains and its influence on fluconazole susceptibility and in reactive oxygen species (ROS) production. Candida krusei clinical isolates were tested to evaluate the presence of AOX. Debaromyces hansenii 2968 (AOX positive) and Saccharomyces cerevisiae BY4742 (AOX negative) were used as control strains. Measurements of oxygen consumption were performed in the presence of 1?mM KCN, an inhibitor of the classical respiratory chain, and 5?mM salicylhydroxamic acid (SHAM). AOX expression was monitored by Western blotting using an AOX monoclonal antibody. Interactions between fluconazole and SHAM were performed using checkerboard assay. ROS production was evaluated in the presence of SHAM plus fluconazole, H(2) O(2) , menadione, or plumbagin. AOX was present in all C.?krusei tested. The combination of fluconazole with SHAM resulted in an indifferent effect. In the presence of SHAM, the treatment with ROS inductors or fluconazole increased ROS production, except in the AOX-negative strain. An alternative respiratory pathway resistant to cyanide is described for the first time as a characteristic of C.?krusei species. This AOX is unrelated to fluconazole resistance; however, it protects C.?krusei from oxidative stress.     

Differential response of cultivated rice to pathogen challenge and abiotic stresses with reference to cationic peroxidase

Seedlings of cultivated rice variety ADT43 was investigated after challenging with two different abiotic (drought and salinity) and biotic (sheath blight and bacterial leaf blight pathogens) stresses. Salinity and drought stress reduced the growth of seedlings, mainly the higher conditions (100 mM NaCl and 10?days of drought, respectively). Increased level of MDA content was observed in biotic and abiotic-stress treated seedlings. The highest H₂O₂ content was observed under salinity-stressed seedlings and lower level observed under biotic stress. Superoxide dismutase activity showed a gradual decrease in all stress conditions compared to control. Salinity stress resulted in highest activity of catalase compared to biotic stress. The peroxidase activity of the seedlings was found to be increased under salt and drought stress conditions and the activity decreased under biotic stress. Drought stress resulted in induced expression of POC1 gene whereas the biotic stress showed lower expression level. Suppression of the rice peroxidase would have been the mechanism of overcoming the intrinsic defence in rice by these pathogens.     

两种外源化学物影响PEG胁迫对水稻根系的伤害

研究过氧化氢内源消除剂和交替氧化酶专一性抑制剂影响渗透胁迫对水稻根系的伤害。结果表明:PEG 6000胁迫抑制了水稻幼根的生长,降低了相对含水量、增加了H₂O₂含量,并导致细胞死亡。用5 mmol·L^-1二甲基硫脲(过氧化氢内源消除剂,dimethylthiourea,DMTU)预处理水稻幼根能明显降低PEG胁迫下水稻幼根过氧化氢的含量,缓解细胞死亡和相对含水量的降低,但对水稻根的生长影响较小。在PEG胁迫下,用1 mmol·L^-1水杨基氧肟酸(交替氧化酶专一性抑制剂,salicylhydroxamic acid,SHAM)预处理水稻幼根能显著降低水稻幼根的生长和相对含水量,并增加水稻幼根的过氧化氢含量和细胞的死亡程度。这说明DMTU能缓解PEG胁迫对水稻根系伤害,而SHAM加剧了PEG胁迫对水稻根系伤害。     

Expression of a carbonic anhydrase gene is induced by environmental stresses in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Expression of the gene (OsCA1) coding for carbonic anhydrase (CA) in leaves and roots of rice was induced by environmental stresses from salts (NaCl, NaHCO₃ and Na₂CO₃), and osmotic stress (10%, w/v, PEG 6000). CA activity of rice seedlings more than doubled under some of these stresses. Transgenic Arabidopsis over-expressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plants in 1/2 MS medium with 5 mM NaHCO₃, 50 mM NaCl, on 100 mM NaCl. Thus CA expression responds to environmental stresses and is related to stress tolerance in rice.     

NaCl-induced expression of glutathione reductase in roots of rice (Oryza sativa L.) seedlings is mediated through hydrogen peroxide but not abscisic acid

Reactive oxygen species (ROS) play an important role in NaCl stress. Plants tolerant to NaCl stress may evolve certain strategies to remove these ROS, thus reducing their toxic effects. Therefore, the expression patterns of the gene family encoding glutathione reductase (GR, EC 1.6.4.2) were analyzed in roots of etiolated rice (Oryza sativa L.) seedlings in response to NaCl stress. Semi-quantitative RT-PCR was applied to quantify the mRNA levels for one cytosolic (OsGR2) and two chloroplastic (OsGR1 and OsGR3) isoforms of glutathione reductase identified in the rice genome. The expression of OsGR2 and OsGR3 but not OsGR1 was increased in rice roots treated with 150 mM NaCl. The Rab16A is an abscisic acid (ABA)-responsive rice gene. Increasing concentrations of ABA, from 1 to 12 μM, progressively increased the expression of OsRab16A in rice roots. In the present study, the ABA level was judged by the expression of OsRab16A in rice roots. Treatment with 150 mM NaCl induced the expression of OsRab16A, and the expression increased with increasing concentrations of ABA, which suggests that ABA may be involved in this response in rice roots. In fact, exogenous application of ABA enhanced the expression of OsGR2 and OsGR3 in rice roots. On inhibiting ABA accumulation with sodium tungstate (Tu), an inhibitor of ABA biosynthesis, the expression of OsGR2 and OsGR3 was still induced by NaCl; therefore, NaCl-triggered expression of OsGR2 and OsGR3 in rice roots is not mediated by accumulation of ABA. However, NaCl treatment could induce H₂O₂ production in rice roots, and H₂O₂ treatment resulted in enhanced OsGR2 and OsGR3 induction. On inhibiting the NaCl-induced accumulation of H₂O₂ with diphenylene iodonium, the expression of OsGR2 and OsGR3 was also suppressed. Moreover, the increase in H₂O₂ level was prior to the induction of OsGR2 and OsGR3 in NaCl-treated rice roots. Thus, H₂O₂, but not ABA, is involved in regulation of OsGR2 and OsGR3 expression in NaCl-treated rice roots.     

Co-expression of the Suaeda salsa SsNHX1 and Arabidopsis AVP1 confer greater salt tolerance to transgenic rice than the single SsNHX1

Transgenic rice plants co-expressing the Suaeda salsa SsNHX1 (vacuolar membrane Na⁺/H⁺ antiporter) and Arabidopsis AVP1 (vacuolar H⁺-PPase) showed enhanced salt tolerance during 3 d of 300 mM NaCl treatment under outdoor growth conditions. These transgenic rice seedlings also grew better on MS medium containing 150 mM NaCl compared to SsNHX1-transformed lines and non-transformed controls. Measurements on isolated vacuolar membrane vesicles derived from the salt stressed SsNHX1+AVP1-transgenic plants demonstrated that the vesicles had increased V-PPase hydrolytic activity in comparison with the Ss-transgenics and non-transgenics. Moreover the V-PPase activity was closely related to the development period of the SA-transgenic seedlings and markedly higher in 3-week-old seedlings than in 5-week-old seedlings. Statistic analysis indicated that the SA-transgenic rice plants contained relatively more ions with higher K⁺/Na⁺ ratio in their shoots compared to the SsNHX1-transformed lines upon salt treatment. Furthermore, these SA-transformants also exhibited relatively higher level of photosynthesis and root proton exportation capacity whereas reduced H₂O₂ generation in the same plants. In general, these results supported the hypothesis that simultaneous expression of the SsNHX1 and AVP1 conferred greater performance to the transgenic plants than that of the single SsNHX1.Feng-Yun Zhao and Xue-Jie Zhang contributed equally to this work     

Alternative pathway is involved in the tolerance of highland barley to the low-nitrogen stress by maintaining the cellular redox homeostasis

Key message

Alternative pathway (AP) is involved in the tolerance of highland barley seedlings to the low-nitrogen stress by dissipating excessive reducing equivalents generated by photosynthesis and maintaining the cellular redox homeostasis.

Abstract

Low nitrogen (N) is a major limiting factor for plant growth and crop productivity. In this study, we investigated the roles of the alternative pathway (AP) in the tolerance of two barley seedlings, highland barley (Kunlun12) and barley (Ganpi6), to low-N stress. The results showed that the chlorophyll content and the fresh weight decreased more in Ganpi6 than those in Kunlun12 under low-N stress, suggesting that Kunlun12 has higher tolerance to low-N stress than Ganpi6. AP capacity was markedly induced by low-N stress; and it was higher in Kunlun12 than in Ganpi6. Comparatively, the cytochrome pathway capacity was not affected under all conditions. Western-blot analysis showed that the protein level of the alternative oxidase (AOX) increased under low-N stress in Kunlun12 but not in Ganpi6. Under low-N stress, the NAD(P)H content and the NAD(P)H to NAD(P)⁺+NAD(P)H ratio in Ganpi6 increased more than those in Kunlun12. Furthermore, photosynthetic parameters (Fv/Fm, qP, ETR and Yield) decreased markedly and qN increased, indicating photoinhibition occurred in both barley seedlings, especially in Ganpi6. When AP was inhibited by salicylhydroxamic acid (SHAM), the NAD(P)H content and the NAD(P)H to NAD(P)⁺+NAD(P)H ratio dramatically increased under all conditions, resulting in the marked accumulation of H₂O₂ and malondialdehyde in leaves of both barley seedlings. Meanwhile, the malate–oxaloacetate shuttle activity and the photosynthetic efficiency were further inhibited. Taken together, AP is involved in the tolerance of highland barley seedlings to low-N stress by dissipating excess reducing equivalents and maintaining the cellular redox homeostasis.