SOD 活性增高的拟南芥晚花突变体具有增强的非生物胁迫耐受性

为了研究晚花突变表型和非生物胁迫耐受性之间是否存在相关性, 以遗传背景同为Wassilewskija (Ws-2)的7个拟南芥(Arabidopsis thaliana L.)晚花突变体(CS2235、CS2238、CS2239、CS2240、CS2246、CS2248和CS6208)为材料,通过干旱胁迫、盐胁迫和氧化胁迫实验, 发现晚花突变体的耐旱、耐盐和耐氧化性都较野生型(wildtype, WT)强。对叶片失水速率和超氧化物歧化酶superoxide dismutase, SOD)活性测定结果也表明, 晚花突变体的保水能力和SOD活性均高于野生型。相关性回归分析表明SOD活性与耐氧化性、耐旱性、耐盐性之间呈正相关, 耐旱性和耐盐性随耐氧化性增强而增强。实验结果表明, 这些晚花突变体的表型和非生物胁迫耐受性之间存在相关性。     

水稻OsCatB敲除突变体的构建及耐逆性初步分析

水稻过氧化氢酶B (catalase B, CatB)主要在非光合组织、愈伤组织、根和种子中表达,参与调控活性氧动态水平的平衡和根系的生长。目前, CatB参与非生物胁迫响应的功能在很大程度上是未知的。本研究利用基因编辑技术CRISPR/Cas9获得OsCatB基因敲除的纯合突变体catb,并分析了catb在盐、高温和氧化等胁迫处理下的生理和生化表型。研究结果发现,在盐处理下,突变体catb的生理和生化表型与野生型植株无显著差异;在热胁迫下, catb的过氧化氢酶活性和存活率与野生型植株相比显著降低,而H2O2含量显著升高;在氧化胁迫下, catb的幼苗高度低于野生型。这些结果说明,水稻CatB参与盐胁迫响应的调控不明显,而主要参与了高温和氧化胁迫响应,并正调控水稻的高温和氧化胁迫耐受性。该研究为进一步探究Os CatB参与逆境响应的分子机制和培育抗逆水稻品种提供了一定的理论指导和遗传材料。     

向日葵转录因子HB-12的定位分析及过表达验证

向日葵转录因子HB-12属于HD-Zip I类转录因子家族,在植物逆境胁迫应答中发挥着极其重要的作用。该研究构建HB-12基因瞬时表达载体并进行亚细胞定位分析,表明HB-12基因定位于细胞核。构建HB-12基因植物超表达载体转化野生型烟草获得了转基因植株。进行了Na Cl胁迫对转基因烟草耐盐性检测、生理生化指标测定和胁迫相关基因的表达分析。结果表明,Na Cl胁迫条件下,转基因烟草叶色失绿程度较野生型的轻,分化情况较野生型的好,生长速度和生根率均较野生型的高;转基因烟草叶绿素和脯氨酸含量及POD和SOD活性均高于野生型烟草;与野生型烟草相比,转基因烟草P5CS、POD、Mn SOD和Gu Zn SOD相对表达水平显著提高。这说明,向日葵HB-12在转基因烟草中的过量表达可抑制叶绿素降解酶的活性,降低叶绿素的分解,还可诱导脯氨酸合成酶基因P5CS及抗氧化相关基因POD、Mn SOD和Gu Zn SOD的上调表达,促进脯氨酸的生物合成,增强POD和SOD的活性,提高烟草抵抗盐害的能力。该研究结果将为进一步探讨植物的耐盐机理及改良作物的耐盐性状奠定基础。     

胁迫相关蛋白激酶OsSAPK9调控水稻对铝胁迫的反应

水稻胁迫相关蛋白激酶(OsSAPKs,stress-activated protein kinase genes in rice)在调控水稻非生物胁迫信号传导中起着重要作用。本研究对OsSAPK9在水稻耐铝(Al)胁迫中的功能进行了初步研究。结果表明10 mmol/L Al处理12 h以上,OsSAPK9过表达转基因水稻植株根部Al的吸收量显著低于野生型植株; Al胁迫处理20 d,OsSAPK9过表达转基因植株株高降低百分比显著低于野生型植株。进一步分析发现OsSAPK9过表达转基因水稻植株根部超氧化歧化酶(SOD,Superoxide Dismutase)和过氧化物酶(POD,Peroxidase)的活性高于野生型,而根上部的SOD、POD和过氧化氢酶(CAT,Catalase)的活性则低于野生型植株。上述结果为改良水稻的耐铝性以及进一步揭示OsSAPK9调控水稻耐铝胁迫反应的分子机制提供了信息。     

耐辐射异常球菌dlp基因缺失突变株构建及其生物学功能研究

耐辐射异常球菌(Deinococcus radiodurans,DR)因其具有对非生物胁迫超强的抵抗能力而备受关注。旨在了解该菌亲水蛋白Dlp在细胞耐受非生物胁迫中的作用,采用融合PCR和基因同源重组技术,获得了dlp基因缺失突变株Δdlp,对野生型DR及突变株Δdlp进行非生物胁迫。结果表明,dlp的缺失导致DR细胞对高盐和氧化胁迫敏感。体外检测氧化胁迫条件下Dlp蛋白对苹果酸脱氢酶(MDH)和乳酸脱氢酶(LDH)活性的保护,结果显示,Dlp蛋白的加入能缓解氧化胁迫条件下MDH、LDH酶活性的损失。由此表明,亲水蛋白Dlp增强了耐辐射异常球菌对非生物胁迫的抗性,并能以类似分子伴侣的形式保护胁迫条件下酶的活性。     

NaCl胁迫和外源ABA对枸杞愈伤组织膜脂过氢化及抗氢化酶活性的影响

不同浓度NaCl胁迫下,枸杞愈伤组织生长量下降,质膜相对透性和丙二醛(MDA)含量增大,且二者呈正相关;过氧化酶(POD)和超氧化歧化酶(SOD)活性升高,但对POD同工酶酶带无影响,仅使酶带染色程度加深。加入ABA后,SOD、POD活性升高,说明盐胁迫下ABA可以延缓盐胁迫对膜的伤害,从而提高枸杞的耐盐性。     

拟南芥APX家族基因在植物生长发育与非生物逆境胁迫响应中的作用分析

活性氧造成的氧化胁迫是植物主要非生物逆境胁迫之一。在不利的生长条件下,植物细胞内的各种代谢过程不协调可导致过氧化氢(hydrogen peroxide, H_2O_2)含量增加,从而对细胞造成多种威胁和伤害。抗坏血酸过氧化物酶(ascorbate peroxidase, APX)是植物中清除H_2O_2的一种重要酶,拟南芥(Arabidopsis thaliana) APX家族包括8个成员:APX1～APX6、sAPX和tAPX。本研究以拟南芥野生型和突变体为材料,对拟南芥不同发育时期和不同逆境胁迫下的8种APX基因表达模式进行了分析,同时研究了其相应的缺失突变体对盐、干旱和热胁迫的耐受性。mRNA差异表达模式分析显示:在拟南芥生长的第4～8周,APX1表达量最高,APX2表达量最低,APX4、sAPX和tAPX随着生长发育的时间进程表达量逐渐减少,但APX6表达量不断增加;在非生物胁迫下,APX1、APX2和APX6受热胁迫诱导表达明显,sAPX响应盐胁迫,APX3和APX5对盐、干旱和热胁迫均表现出明显的诱导表达应答。盐和干旱胁迫耐受性分析结果表明:无论是在拟南芥的萌发期还是成熟期,任何一个APX基因缺失均使抗逆性降低;在萌发期,与盐胁迫相比,突变体对干旱胁迫更敏感;在成熟期,与野生型和其他突变体相比,apx1和apx6对盐和干旱胁迫更加不耐受。生理指标检测结果显示:干旱胁迫10 d后,所有突变体植株中的H_2O_2含量均明显高于野生型,其中apx1、sapx和tapx中最高;盐胁迫5 d后,突变体中丙二醛(malondialdehyde,MDA)的含量显著高于野生型;热胁迫2h就会导致apx1、apx2和apx6中H_2O_2和MDA含量大幅增加,其中在apx2中最高。本研究结果表明,拟南芥APX基因家族的8个成员均不同程度地参与植物生长发育及非生物胁迫响应的过程,在不同发育时期或逆境响应过程有特定的一种或几种APX发挥主要作用。     

西伯利亚蓼谷胱甘肽转移酶和半胱氨酸合成酶基因在酿酒酵母中的共表达

谷胱甘肽转移酶和半胱氨酸合成酶在清除活性氧(reactive oxygen species,ROS)中起重要作用。采用0.36 mol.L-1NaHCO3对西伯利亚蓼(Polygonum sibiricum)进行胁迫处理, 荧光定量PCR分析表明这2个基因的表达受盐胁迫强烈诱导。为了分析2个基因是否具有抗盐能力以及其相互协同能力, 从cDNA文库中获得谷胱甘肽转移酶(GST)和半胱氨酸合成酶(CS)2个基因, 分别将GST、CS和GST+CS转入酿酒酵母(Saccharomyces cerevisiae)中, 并分别命名转基因酵母为ty-gst、tycs和ty-gc。在1 mol.L-1 Na2CO3和5 mol.L-1 NaCl胁迫处理下, 转基因酵母(ty-gst、ty-cs和ty-gc)的耐盐能力均明显高于野生型酵母(wy), 而三者之间并无显著差别。在0.4 mol.L-1 NaCl胁迫处理下, 转基因酵母(ty-gst、ty-cs和ty-gc)的抗氧化酶类相关基因SOD1、SOD2、GPX1和GPX3的表达量均低于野生型酵母(对照)(wy), 而CTA1表达量均高于野生型酵母(对照)(wy)。转基因酵母ty-cs在0.4 mol.L-1 NaCl胁迫处理前后其超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)和谷胱甘肽过氧化物酶(glutathione peroxidase, GPX)的活性均表现为最高。     

水稻幼苗叶绿体保护系统对盐胁迫的反应

以耐盐性不同的水稻品种Pokkali(耐盐)和Peta(盐敏感)为材料,研究了叶绿体中保护系统对盐胁迫的响应。结果表明：随着NaCl胁迫时间的增加,供试两品种叶绿体中H2O2和MDA含量增加,耐盐品种Pokkali增加的幅度明显小于盐敏感品种Peta;NaCl胁迫下叶绿体内的SOD活性下降,APX、GR活性和．ASA、GSH含量均为先升后降,耐盐品种Pokkali下降的幅度小于Peta。在200mmol／L NaCl胁迫过程中,Pokkali叶绿体内SOD、APX、GR活性和ASA、GSH含量均高于Peta,说明在NaCl胁迫下耐盐品种叶绿体内清除活性氧的能力强于盐敏感品种。     

外源亚精胺对盐胁迫下黄瓜幼苗体内抗氧化酶活性的影响

以不同耐盐性黄瓜品种“长春密刺”和“津春2号”为材料,采用营养液栽培,研究了外源亚精胺(Spd)对NaCl胁迫下黄瓜幼苗叶片与根系中超氧阴离子(O2-.)产生速率、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性的影响。结果表明,外源Spd对未经盐胁迫处理(对照)黄瓜幼苗体内O2-.产生速率、SOD、CAT和POD活性均无显著性影响;盐胁迫处理提高了O2-.产生速率,SOD、POD和CAT活性都有不同程度的升高;外源Spd处理进一步提高了盐胁迫下SOD、POD和CAT活性,减缓了O2-.产生速率。与耐盐型“长春密刺”品种相比,盐胁迫对盐敏感型“津春2号”影响较大,外源Spd对盐敏感型黄瓜品种盐胁迫伤害的缓解作用较大。表明盐胁迫下外源Spd可缓解盐胁迫对膜的伤害,从而提高黄瓜幼苗的耐盐性。     

The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants’ tolerance to multiple stress conditions.     

Coupled expression of Cu/Zn-superoxide dismutase and catalase in cassava improves tolerance against cold and drought stresses

Recently we reported that the joint expression of cassava Cu/Zn superoxide dismutase (MeCu/ZnSOD) and catalase (MeCAT1) prolonged the shelf life of cassava storage-roots by the stabilization of reactive oxygen species (ROS) homeostasis after harvest. Since oxidative damage is a major feature of plants exposed to environmental stresses, transgenic cassava showing increased expression of the cytosolic MeCu/ZnSOD and the peroxisomal MeCAT1 should have improved resistance against other abiotic stresses. After cold treatment, the transgenic cassava maintained higher SOD and CAT activities and lower malendialdehyde content than those of wild type plants (WT). Detached leaves of transgenic cassava also showed slower transpirational water loss than those of WT. When plants were not watered for 30 d, transgenic lines exhibited a significant increase in water retention ability, accumulated 13% more proline and 12% less malendialdehyde than WT’s, and showed enhanced activity of SOD and CAT. These results imply that manipulation of the antioxidative mechanism allows the development of staple crops with improved tolerance to abiotic stresses.     

Oxidative stress tolerance and longevity in Arabidopsis: the late‐flowering mutant gigantea is tolerant to paraquat

Recent genetic analyses of longevity in animals have revealed that long-lived strains are more tolerant to environmental stresses. To investigate whether extended longevity in Arabidopsis also correlates with an increase in stress tolerance, the response was tested of 11 late-flowering mutants to the superoxide radical-generating herbicide paraquat. A tight correlation between flowering time and paraquat tolerance was found when plants were exposed to low doses of herbicide. Furthermore, the mutant gigantea (gi-3) with the longest delay in flowering time had a high tolerance level to paraquat-induced oxidative stress. All the tested gi alleles had an increased tolerance to paraquat toxicity compared to wild-type, although the actual levels of tolerance differed. In addition, the gi-3 mutant was more tolerant to hydrogen peroxide. These results suggest that the link between longevity and oxidative stress resistance in plants is similar to that found in animals, implying that this phenomenon may be general for all aerobic organisms.     

Effects of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa overexpressing superoxide dismutases

The antioxidant composition and relative water stress tolerance of nodulated alfalfa plants ( Medicago sativa L. ×  Sinorhizobium meliloti 102F78) of the elite genotype N4 and three derived transgenic lines have been studied in detail. These transgenic lines overproduced, respectively, Mn-containing superoxide dismutase (SOD) in the mitochondria of leaves and nodules, MnSOD in the chloroplasts, and FeSOD in the chloroplasts. In general for all lines, water stress caused moderate decreases in MnSOD and FeSOD activities in both leaves and nodules, but had distinct tissue-dependent effects on the activities of the peroxide-scavenging enzymes. During water stress, with a few exceptions, ascorbate peroxidase and catalase activities increased moderately in leaves but decreased in nodules. At mild water stress, transgenic lines showed, on average, 20% higher photosynthetic activity than the parental line, which suggests a superior tolerance of transgenic plants under these conditions. However, the untransformed and the transgenic plants performed similarly during moderate and severe water stress and recovery with respect to important markers of metabolic activity and of oxidative stress in leaves and nodules. We conclude that the base genotype used for transformation and the background SOD isozymic composition may have a profound effect on the relative tolerance of the transgenic lines to abiotic stress.     

Modification of reactive oxygen species scavenging capacity of chloroplasts through plastid transformation

Reactive oxygen species (ROS), including superoxide anions, hydrogen peroxide and hydroxyl radicals are generated through normal biochemical processes, but their production is increased by abiotic stresses. The prospects for enhancing ROS scavenging, and hence stress tolerance, by direct gene expression in a vulnerable cell compartment, the chloroplast, have been explored in tobacco. Several plastid transformants were generated which contained either a Nicotiana mitochondrial superoxide dismutase (MnSOD) or an Escherichia coli glutathione reductase (gor) gene. MnSOD lines had a three-fold increase in MnSOD activity, but interestingly a five to nine-fold increase in total chloroplast SOD activity. Gor transgenic lines had up to 6 times higher GR activity and up to 8 times total glutathione levels compared to wild type tobacco. Photosynthetic capacity of transplastomic plants, as measured by chlorophyll content and variable fluorescence of PSII was equivalent to non-transformed plants. The response of these transplastomic lines to several applied stresses was examined. In a number of cases improved stress tolerance was observed. Examples include enhanced methyl viologen (Paraquat)-induced oxidative stress tolerance in Mn-superoxidase dismutase over-expressing plants, improved heavy metal tolerance in glutathione reductase expressing lines, and improved tolerance to UV-B radiation in both sets of plants.     

Aluminum tolerance in a spermidine synthase-overexpressing transgenic European pear is correlated with the enhanced level of spermidine via alleviating oxidative status

Aluminum (Al) stress is a major cause of poor crop yields, particularly in those countries where acid soil predominate. To verify whether polyamine can confer Al tolerance, in vitro shoots of a transgenic European pear (Pyrus communis L. ‘Ballad’) line #32 overexpressing apple spermidine synthase (MdSPDS1) and the wild type (WT) were subjected to long-term stress for 30 μM AlCl₃. Based on net increment of shoot height (SHI) or fresh weight (FWI) and morphological changes upon the stress, the performance of line #32 was much better than that of WT. Although SPDS expression levels and spermidine (Spd) titers in line #32 were higher than those in WT, firmly due to the transgene (MdSPDS1) expression, no further induction of SPDS expression was observed from the long-term Al stress trial in both lines. While, Spd titers were considerably increased in both lines after the stress. The activities of superoxide dismutase (SOD) or glutathione reductase (GR) and the accumulation of proline or malondialdehyde (MDA) altered upon this stress toward a more favorable status for survival in the transgenic line #32 than in WT. These antioxidant parameters were closely related to Spd titer. Concentrations of calcium (Ca) and some co-factor metals of SOD in line #32 were diversely higher than that in WT after the stress. These evidences indicate that Spd is implicated in elevating of Al stress tolerance of the transgenic line #32 chiefly via ameliorating oxidative status as well as by affecting mineral element balance.     

Linking oxidative and salinity stress tolerance in barley: can root antioxidant enzyme activity be used as a measure of stress tolerance?

Aims

A causal relationship between salinity and oxidative stress tolerance and a suitability of using root antioxidant activity as a biochemical marker for salinity tolerance in barley was investigated.

Methods

Net ion fluxes were measured from the mature zone of excised roots of two barley varieties contrasting in their salinity tolerance using non-invasive MIFE technique in response to acute and prolonged salinity treatment. These changes were correlated with activity of major antioxidant enzymes; ascorbate peroxidase, catalase, and superoxide dismutase.

Results

It was found that genotypic difference in salinity tolerance was largely independent of root integrity, and observed not only for short-term but also long-term NaCl exposures. Higher K⁺ retention ability (and, hence, salinity tolerance) positively correlated with oxidative stress tolerance. At the same time, antioxidant activities were constitutively higher in a sensitive but not tolerant variety, and no correlation was found between SOD activity and salinity tolerance index during large-scale screening.

Conclusion

Although salinity tolerance in barley correlates with its oxidative stress tolerance, higher antioxidant activity at one particular time does not correlate with salinity tolerance and, as such, cannot be used as a biochemical marker in barley screening programs.     

Cadmium-induced changes in antioxidative systems,hydrogen peroxide content,and lipid peroxidation in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

To study the relationship between cadmium (Cd)-induced phytotoxicity and oxidative stress, we grew Cd-sensitive wild-type (WT) and Cd-resistant type (RT) seedlings ofArabidopsis thaliana on MS media containing up to 500 μM CdCl₂. The resistant seedlings showed higher biomasses and lower hydrogen peroxide and lipid peroxidation levels, the latter expressed in terms of malondialdehyde (MDA) production. These results indicate that RT plants experience lower oxidative stress when exposed to Cd. Furthermore, compared with the WT, RT seedlings have significantly higher activities of superoxide dismutase (SOD) and enzymes related to hydrogen peroxide removal, e.g., guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR). These differential responses suggest that such phytotoxicity could be induced by oxidative stress, and that lower accumulations of hydrogen peroxide confer Cd tolerance in seedlings.