Superoxide dismutase and glutathione reductase overexpression in wheat protoplast: photooxidative stress tolerance and changes in cellular redox state

In previous works, we have established a correlation between antioxidant system response and tolerance to drought, osmotic stress and photooxidative stress of different wheat cultivars with contrasting drought tolerance. In the present work, a protocol to obtain and transform wheat protoplasts was established. Transgenic protoplasts with Manganese Superoxide Dismutase (Mn-SOD) (E.C.: 1.15.1.1) and Glutathione Reductase (GR) (E.C.: 1.6.4.2) overexpression in chloroplasts were obtained, and their responses to photooxidative stress were characterized. Protoplasts with Mn-SOD or GR overexpression, showed different responses and tolerance to photooxidative stress. Protoplasts with Mn-SOD overexpression showed lower levels of oxidative damage, higher level of endogenous hydrogen peroxide and a great induction of total SOD and GR activities during photooxidative treatments. In protoplasts with GR overexpression the oxidative damage provoked by the photooxidative treatment was similar to control protoplasts, the GSH content and GSH/GSH + GSSG ratio were higher than control and Mn-SOD transformed protoplast, and total SOD and GR activities were not induced. Our results suggest that the differential responses and tolerance to photooxidative stress given by Mn-SOD or GR overexpression, also depend on the effects of these enzyme activities over the cellular redox state balance, which modulate the responses to photooxidative stress.     

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.     

A proteomics view on the role of drought-induced senescence and oxidative stress defense in enhanced stem reserves remobilization in wheat

Drought is one of the major factors limiting the yield of wheat (Triticum aestivum L.) particularly during grain filling. Under terminal drought condition, remobilization of pre-stored carbohydrates in wheat stem to grain has a major contribution in yield. To determine the molecular mechanism of stem reserve utilization under drought condition, we compared stem proteome patterns of two contrasting wheat landraces (N49 and N14) under a progressive post-anthesis drought stress, during which period N49 peduncle showed remarkably higher stem reserves remobilization efficiency compared to N14. Out of 830 protein spots reproducibly detected and analyzed on two-dimensional electrophoresis gels, 135 spots showed significant changes in at least one landrace. The highest number of differentially expressed proteins was observed in landrace N49 at 20days after anthesis when active remobilization of dry matter was observed, suggesting a possible involvement of these proteins in effective stem reserve remobilization of N49. The identification of 82 of differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in leaf senescence, oxidative stress defense, signal transduction, metabolisms and photosynthesis which might enable N49 to efficiently remobilized its stem reserves compared to N14. The up-regulation of several senescence-associated proteins and breakdown of photosynthetic proteins in N49 might reflect the fact that N49 increased carbon remobilization from the stem to the grains by enhancing senescence. Furthermore, the up-regulation of several oxidative stress defense proteins in N49 might suggest a more effective protection against oxidative stress during senescence in order to protect stem cells from premature cell death. Our results suggest that wheat plant might response to soil drying by efficiently remobilize assimilates from stem to grain through coordinated gene expression.     

Overexpression of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato

One strategy to increase the level of drought and salinity tolerance is the transfer of genes codifying different types of proteins functionally related to macromolecules protection, such as group 2 of late embryogenesis abundant (LEA) proteins or dehydrins. The TAS14 dehydrin was isolated and characterized in tomato and its expression was induced by osmotic stress (NaCl and mannitol) and abscisic acid (ABA) [Godoy et al., Plant Mol Biol 1994;26:1921-1934], yet its function in drought and salinity tolerance of tomato remains elusive. In this study, transgenic tomato plants overexpressing tas14 gene under the control of the 35SCaMV promoter were generated to assess the function of tas14 gene in drought and salinity tolerance. The plants overexpressing tas14 gene achieved improved long-term drought and salinity tolerance without affecting plant growth under non-stress conditions. A mechanism of osmotic stress tolerance via osmotic potential reduction and solutes accumulation, such as sugars and K(+) is operating in tas14 overexpressing plants in drought conditions. A similar mechanism of osmotic stress tolerance was observed under salinity. Moreover, the overexpression of tas14 gene increased Na(+) accumulation only in adult leaves, whereas in young leaves, the accumulated solutes were K(+) and sugars, suggesting that plants overexpressing tas14 gene are able to distribute the Na(+) accumulation between young and adult leaves over a prolonged period in stressful conditions. Measurement of ABA showed that the action mechanism of tas14 gene is associated with an earlier and greater accumulation of ABA in leaves during short-term periods. A good feature for the application of this gene in improving drought and salt stress tolerance is the fact that its constitutive expression does not affect plant growth under non-stress conditions, and tolerance induced by overexpression of tas14 gene was observed at the different stress degrees applied to the long term.     

核桃JrGRAS2基因响应热胁迫的表达及功能分析

GRAS转录因子在植物响应逆境中起重要作用。为更好的了解核桃（Juglans regia）在逆境胁迫下的适应机制,本研究从‘香玲’核桃转录组中克隆获得一条GRAS基因（命名为JrGRAS2）,对其在不同高温胁迫下的表达进行分析,并将该基因插入酵母表达载体pYES2中构建重组载体pYES2-JrGRAS2,将pYES2-JrGRAS2转入酿酒酵母（Saccharomyces cerevisiae）INVSCI,同时以转化pYES2的重组酵母作为阴性对照,在酵母表达系统中研究该基因的抗热胁迫功能。结果显示,该基因开放读码框（ORF）全长1296bp,拟推导的蛋白分子量为47405.83Da,含有氨基酸数为431,理论等电点为5.66。在热胁迫下,JrGRAS2基因被显著诱导,特别是在36℃胁迫0.5h的茎内,其表达相对于对照被上调了335.5倍。对两种酵母进行热胁迫,发现转JrGRAS2基因酵母表现出较对照更高的生存活性。表明JrGRAS2基因具有响应热胁迫的能力,且能提高酵母的抗性,JrGRAS2基因可作为核桃逆境应答的重要候选基因。     

Over-expression of Arabidopsis thaliana β-carotene hydroxylase (chyB) gene enhances drought tolerance in transgenic tobacco

Carotenoids are essential for photosynthesis and photoprotection in plant life. In order to study the protective role of zeaxanthin under drought stress, we increased the capacity for its accumulation in tobacco by over-expression of Arabidopsis β-carotene hydroxylase chyB gene. This manipulation leads to a 2–4 fold increase of xanthophylls cycle pigments. Under high-light condition, the transformants converted more β-carotene into zeaxanthin compared to the controls. The enhancement of zeaxanthin increased the total antioxidant capacity in lipid phase and made plants more tolerant to drought stress, as shown by less leaf necrosis, reduced lipid peroxidation and enhanced photosynthesis rate. The function of the gene in drought tolerance was explored and discussed. We conclude that genetic manipulation of chyB gene may present a powerful method in the production of drought-tolerant crops.     

Hyponastic leaf growth decreases the photoprotective demand, prevents damage to photosystem II and delays leaf senescence in Salvia broussonetii plants

The influence of leaf angle on the response of plants to high light was studied in Salvia broussonetii, a species endemic of the Canary Islands that shows hyponastic leaf growth. The response of vertical, naturally oriented leaves was compared with that of horizontal, artificially held leaves for 1, 13, 24 and 29 days in terms of photoinhibition [efficiency of photosystem II (PSII)], photoprotection (by the xanthophyll cycle, alpha-tocopherol and beta-carotene) and progression of leaf senescence. Vertical leaves not only showed a decreased photoprotective demand compared with horizontal leaves but also kept the maximum efficiency of PSII (F(v)/F(m) ratio) constant throughout the experiment, thus reflecting the capacity of naturally oriented leaves to avoid photooxidative stress in the field. By contrast, horizontal leaves, which were exposed to higher light intensities, showed a higher photoprotective demand (reflected by a higher de-epoxidation of the xanthophyll cycle, carotenoid losses and increases in alpha-tocopherol), damage to PSII (as indicated by decreases in the F(v)/F(m) ratio) and accelerated leaf senescence, which was associated with cell death after 24 days of high light exposure. It is concluded that hyponastic leaf growth prevents photoinhibition and decreases the photoprotective demand of leaves by reducing the incident light, which helps maintaining leaf vigor and delaying the progression of leaf senescence in S. broussonetii plants. Hyponastic leaf growth is therefore one of the first photoprotection mechanisms activated in this species to avoid the negative impact of high-light stress in the field.     

Differential photosynthetic compensatory mechanisms exist in the immutans mutant of Arabidopsis thaliana

Variegation in the immutans ( im ) mutant of Arabidopsis is induced by a nuclear recessive gene. The white leaf sectors of im contain abnormal plastids lacking pigments and organized lamellae, whereas the green leaf sectors possess normal-appearing chloroplasts. IMMUTANS codes for a thylakoid membrane terminal oxidase that functions as a safety valve to dissipate excess energy. Previous studies have shown that the green sectors of im , regardless of illumination conditions, have anatomical adaptations that are reminiscent of acclimation to high-light stress. It has been suggested that these adaptations provide a means of enhancing photosynthesis to feed the white sectors and maximize plant growth. We have utilized Chl fluorescence imaging to better understand these compensatory mechanisms using, as our experimental material, im leaves with predominantly green ( img ) or predominantly white ( imw ) tissues. The samples were examined under conditions of normal growth or high-light stress (photoinhibition). Steady-state fluorescence quenching revealed that the green sectors of the imw leaves had lower levels of 1 −  q _p than the img leaves, and that this was accompanied by increased electron transport rates. In response to short-term high-light exposure, the green sectors of the imw leaves displayed enhanced non-photochemical quenching (NPQ), which correlated with increased xanthophyll pool sizes and increased amounts of several different Lhcb polypeptides and the PsbS protein. In summary, our data show that, compared with primarily green leaves ( img ), the green sectors of predominantly white leaves ( imw ) have elevated rates of electron transport and an enhanced NPQ capacity. We conclude that, in the absence of IM, green sectors develop morphological and biochemical adaptations that allow them to maximize photosynthesis to feed the white sectors, and to protect against photodamage.     

过表达AtchyB基因提高洋桔梗光胁迫耐受性的研究

类胡萝卜素尤其是叶黄素循环类物质在植物抵抗由强光照引起的非生物胁迫中发挥着重要的作用,为了提高洋桔梗对强光照的抗性,从拟南芥中克隆了类胡萝卜素生物合成途径中参与叶黄素循环关键酶——β-胡萝卜素羟化酶基因(AtchyB),利用农杆菌介导法将其转入洋桔梗中,最终得到遗传转化植株2个株系,研究发现,转基因植株中总类胡萝卜素含量高于对照组,且叶黄素循环池被不同程度地放大。在不同光照强度下,转基因洋桔梗植株对光照耐受性明显强于对照组,且转基因植株生物量也明显提高。表明过表达AtchyB基因使洋桔梗光胁迫耐受性有所增强。     

Overexpression of AtchyB in Eustoma grandiflorum Shinn Enhances its Tolerance to High-Light Via Zeaxanthin Accumulation

Carotenoids are essential components of the photosynthetic apparatus involved in plant photoprotection. To investigate the protective role of zeaxanthin and the xanthophyll cycle under high-light stress, we increased the capacity for their biosynthesis in Eustoma grandiflorum Shinn by overexpression of a gene (AtchyB) from Arabidopsis thaliana encoding ??-carotene hydroxylase (BCH). This enzyme is involved in the conversion of ??-carotene into zeaxanthin and plays an important role in the carotenoid biosynthetic pathway. Not only was the total carotenoid content of the transgenics enhanced (1.046- to?3.141-fold) but zeaxanthin biosynthesis was also faster and the compound was produced in larger quantities in transgenics (up to 3.344-fold) than in controls upon exposure to high-light stress. Additionally, a greater amount of xanthophyll cycle pigments (1.46- to?2.44-fold) was detected in the transgenics. Under high-light stress, untransformed controls showed obvious growth retardation, while transformants were more tolerant. The net addition of biomass in the transformants was more than that of non-transformants under high-light exposure. Furthermore, a new phenomenon was found: high-light stress induced an apparent periodical accumulation of biomass and zeaxanthin in transformants. Our results supplement data from previous research, and indicate that the periodic enhancement of zeaxanthin formation together with the periodic enlargement of the xanthophyll cycle pool contributes to long-term high-light stress protection and prevents plant damage.     

Effects of Shading on the Senescence and Photosynthetic Physiology of the Early-Flowering Rice Mutant FTL10 at Noon

FTL10 is an early-flowering mutant of rice (Oryza sativa L.) with a premature senescent phenotype. Early leaf senescence can cause negative effects on rice yield. Moreover, rice leaves are damaged under high-light conditions, which promote rice senescence. Artificial shading can reduce the amount of light absorbed by rice leaves. The aim of this study was to investigate the effects of shading at noon (11:30–14:00) on the senescence and yield of FTL10. The results showed that shading improved the total antioxidant capacity of rice leaves, reduced the accumulation of reactive oxygen species (ROS) and reduced the expression of genes related to senescence. In the shaded group, the degradation rate of chlorophyll and Rubisco proteins, which are related to photosynthesis, was relatively slow, and the photosynthetic rate was relatively high. Compared with those under the natural growth conditions, the proportion of photosynthetic electron allocated to photorespiration in the shaded group rice leaves was lower, and the proportion allocated to carbon fixation was higher. The yield data showed that the single-spike weight and yield per plant of rice significantly increased after shading. Therefore, our research shows that shading at noon could delay FTL10 senescence and increase yields.