Enhanced tolerance of transgenic sweetpotato plants that express both CuZnSOD and APX in chloroplasts to methyl viologen-mediated oxidative stress and chilling

Oxidative stress is one of the major factors causing injury to plants exposed to environmental stress. Transgenic sweetpotato [Ipomoea batatas (L.) Lam. cv. Yulmi] plants with an enhanced tolerance to multiple environmental stresses were developed by expressing the genes of both CuZn superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) under the control of an oxidative stress-inducible SWPA2 promoter in the chloroplasts of sweetpotato plants (referred to as SSA plants). SSA plants were successfully generated by the particle bombardment method and confirmed by PCR analysis. When leaf discs of SSA plants were subjected to 5 μM methyl viologen (MV), they showed approximately 45% less damage than non-transformed (NT) plants. When 200 μM MV was sprayed onto the whole plants, SSA plants showed a significant reduction in visible damage compared to leaves of NT plants, which were almost destroyed. The expression of the introduced CuZnSOD and APX genes in leaves of SSA plants following MV treatment was significantly induced, thereby reflecting increased levels of SOD and APX in the chloroplasts. APX activity in chloroplast fractions isolated from SSA plants was approximately 15-fold higher than that in their counterparts from NT plants. SSA plants treated with a chilling stress consisting of 4°C for 24 h exhibited an attenuated decrease in photosynthetic activity (Fv/Fm) relative to NT plants; furthermore, after 12 h of recovery following chilling, the Fv/Fm of SSA plants almost fully recovered to the initial levels, whereas NT plants remained at a lower level of Fv/Fm activity. These results suggest that SSA plants would be a useful plant crop for commercial cultivation under unfavorable growth conditions. In addition, the manipulation of the antioxidative mechanism in chloroplasts can be applied to the development of various other transgenic crops with an increased tolerance to multiple environmental stresses.     

Paclobutrazol pre-treatment enhanced flooding tolerance of sweet potato

The objective of this experiment was to study changes of antioxidants and antioxidative enzymes in the flooding-stressed sweet potato leaf, as affected by paclobutrazol (PBZ) treatment at 24 h prior to flooding. Sweet potato 'Taoyuan 2' were treated with 0 and 0.5 mg/plant of PBZ, afterwards subjected to non-flooding and flooding-stress conditions for 0, 1, 3, and 5 d, followed by a 2 d drainage period. The study was conducted as a factorial experiment in completely randomized blocks with three replications maintained within a screen house. Plants with various antioxidative systems responded differently to flooding stress according to the duration of the flooding period and subsequent drainage period. The increased levels of antioxidants and antioxidative enzymes observed on different days of flooding afforded the sweet potato leaf with improved flooding tolerance. Glutathione reductase activity in the leaf was significantly enhanced over 5 d continuous flooding followed by a drainage period, in comparison with non-flooding conditions. Under non-flooding conditions, antioxidative system of leaf was regulated and elevated by PBZ pre-treatment. PBZ treatment may enable sweet potato 'Taoyuan 2' to maintain the balance between the formation and the detoxification of activated oxygen species. Our results also show that under flooding-stress conditions, the level of 'Taoyuan 2' antioxidative system is linked to PBZ treatment. Pre-treating with PBZ may increase levels of various components of antioxidative systems after exposure to different durations of flooding and drainage, thus inducing flooding tolerance. PBZ exhibited the important function of enhancing the restoration of leaf oxidative damage under flooding stress after the pre-application of 0.5 mg/plant. These findings may have greater significance for farming in frequently flooded areas.     

Differential responses of antioxidative system to chilling and drought in four rice cultivars differing in sensitivity

Responses of antioxidative defense systems to chilling and drought stresses were comparatively studied in four cultivars of rice (Oryza sativa L.) differing in sensitivity, two of them (Xiangnuo no. 1 and Zimanuo) are tolerant to chilling but sensitive to drought and the other two (Xiangzhongxian no. 2 and IR50) are tolerant to drought but sensitive to chilling. The seedlings of rice were transferred into growth chamber for 5 d at 8 degrees C as chilling treatment, or at 28 degrees C as control, or at 28 degrees C but cultured in 23% PEG-6000 solution as drought stress treatment. Under drought stress the elevated levels of electrolyte leakage, contents of H(2)O(2) and total thiobarbituric acid-reacting substances (TBARS) in Xiangzhongxian no. 2 and IR50 are lower than those in Xiangnuo no. 1 and Zimanuo. On the contrary, Xiangnuo no. 1 and Zimanuo have much lower level of electrolyte leakage, H(2)O(2) and TBARS than Xiangzhongxian no. 2 and IR50 under chilling stress. Activities of antioxidant enzymes (superoxide dismutase (SOD), catalase, and ascorbate-peroxidase (APX)) and contents of antioxidants (ascorbaic acid and reduced glutathione) were measured during the stress treatments. All of them were enhanced greatly until 3 d after drought stress in the two drought-tolerant cultivars, or after chilling stress in the two chilling-tolerant cultivars. They all were decreased at 5 d after stress treatments. On the other hand, activities of antioxidant enzymes and contents of antioxidants were decreased greatly in the drought-sensitive cultivars after drought stress, or in the chilling-sensitive cultivars after chilling stress. The results indicated that tolerance to drought or chilling in rice is well associated with the enhanced capacity of antioxidative system under drought or chilling condition, and that the sensitivity of rice to drought or chilling is linear correlated to the decreased capacity of antioxidative system.     

茄子种质资源的耐冷性鉴定

采用低温胁迫下幼苗叶片冷害指数、种子活力指数、叶片电导百分率为指标,对34份不同类型的茄子种质资源进行耐冷性鉴定.结果表明,活力指数与冷害指数、电导百分率均呈极显著负相关,电导百分率与冷害指数呈极显著正相关.通过耐冷隶属函数值和聚类分析,初步筛选出11份耐冷性较强的材料.     

低温胁迫对嫁接西瓜耐冷性和活性氧清除系统的影响

研究了西瓜实生苗和以黑籽南瓜、超丰F1为砧木的嫁接苗的耐冷性及活性氧清除系统的差异．结果表明,低温胁迫下,嫁接苗的耐冷性明显高于实生苗,表现为以黑籽南瓜为砧木的嫁接苗的耐冷性>以超丰F1为砧木的嫁接苗>实生苗,此外嫁接苗和实生苗均表现为叶片中叶绿素含量下降,丙二醛(MDA)含量上升,非酶促抗氧化剂抗坏血酸(AsA)、谷胱甘肽(GSH)含量和抗氧化酶超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(AsA-POD)、脱氢抗坏血酸还原酶(DR)活性下降,说明低温逆境降低了植物体防御活性氧有关的酶促和非酶促保护系统能力,提高了体内自由基浓度,加剧了膜脂过氧化．嫁接苗的活性氧清除能力均高于自根苗,且嫁接苗中耐冷性越强的活性氧清除能力越高,说明西瓜嫁接后耐冷性的提高是与植物体内活性氧清除系统中抗氧化剂含量和抗氧化酶活性提高有关。     

Coordination of light,circadian clock with temperature: The potential mechanisms regulating chilling tolerance in rice

Rice (Oryza sativa L.) is a major staple food crop for over half of the world's population. As a crop species originated from the subtropics, rice production is hampered by chilling stress. The genetic mechanisms of rice responses to chilling stress have attracted much attention, focusing on chilling‐related gene mining and functional analyses. Plants have evolved sophisticated regulatory systems to respond to chilling stress in coordination with light signaling pathway and internal circadian clock. However, in rice, information about light‐signaling pathways and circadian clock regulation and their roles in chilling tolerance remains elusive. Further investigation into the regulatory network of chilling tolerance in rice is needed, as knowledge of the interaction between temperature, light, and circadian clock dynamics is limited. Here, based on phenotypic analysis of transgenic and mutant rice lines, we delineate the relevant genes with important regulatory roles in chilling tolerance. In addition, we discuss the potential coordination mechanism among temperature, light, and circadian clock in regulating chilling response and tolerance of rice, and provide perspectives for the ongoing chilling signaling network research in rice.     

Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance

Fructans are water-soluble fructose oligomers and polymers thatare based on sucrose, and have been implicated in protectingplants against water stress. Rice (Oryza sativa L.) is highlysensitive to chilling temperatures, and is not able to synthesizefructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose1-fructosyltransferase, encoded by wft2, or sucrose:fructan6-fructosyltransferase, encoded by wft1, were introduced intorice plants, and rice transformants that accumulate fructanswere successfully obtained. The mature leaf blades of transgenicrice lines with wft2 or wft1 accumulated 16.2 mg g^–1 FWof oligo- and polysaccharides mainly composed of inulin oligomersof more than DP7, and 3.7 mg g^–1 FW of oligo- and polysaccharides,mainly composed of phlein oligomers of more than DP15, respectively.The transgenic rice seedlings with wft2 accumulated significantlyhigher concentrations of oligo- and polysaccharides than non-transgenicrice seedlings, and exhibited enhanced chilling tolerance. Theoligo- and polysaccharide concentrations of seedlings expressingwft1 were obviously lower than those of lines expressing wft2,and no correlation between oligo- and polysaccharide concentrationsand chilling tolerance was detected in wft1-expressing ricelines. The results suggest that transgenic rice lines expressingwheat-derived fructosyltransferase genes accumulated large amountsof fructans in mature leaf blades and exhibited enhanced chillingtolerance at the seedling stage. This is the first report owingthat fructan accumulation enhanced tolerance to non-freezinglow temperatures. Key words: Chilling tolerance, fructan, fructosyltransferase, Oryza sativa, transgenic plant     

The inheritance of chilling tolerance in tomato (Lycopersicon spp.)

During the past 25 years, chilling tolerance of the cultivated (chilling-sensitive) tomato Lycopersicon esculentum and its wild, chilling-tolerant relatives L. peruvianum and L. hirsutum (and, less intensively studied, L. chilense) has been the object of several investigations. The final aim of these studies can be seen in the increase in chilling tolerance of the cultivated genotypes. In this review, we will focus on low-temperature effects on photosynthesis and the inheritance of these traits to the offspring of various breeding attempts. While crossing L. peruvianum (male symbol) to L. esculentum (female symbol) so far has brought the most detailed insight with respect to physiological questions, for practical purposes, e.g., the readily cross ability, crossing programmes with L. hirsutum as pollen donor at present seem to be a promising way to achieve higher chilling-tolerant genotypes of the cultivated tomato. This perspective is due to the progress that has been made with respect to the genetic basis of chilling tolerance of Lycopersicon spp. over the past five years.     

Inhibition of glutathione synthesis decreases chilling tolerance in Chorispora bungeana callus

The possible roles of reduced glutathione (GSH) in chilling tolerance were studied in callus generated from a representative alpine plant, Chorispora bungeana Fisch. & C.A. Mey (C. bungeana). The callus grew well under low-temperature and chilling treatment led only to slight injury, as indicated by a low level of ion leakage (IL). Malondialdehyde measurements also were not elevated, however GSH was. Exogenously application of l-buthionine-(S R)-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase (γ-ECS), arrested the GSH accumulation induced by chilling and resulted in a significant decrease in cell growth and an increase in IL and malondialdehyde. These results implied that C. bungeana is a plant with a strong low-temperature tolerance mechanism, and the tolerance of C. bungeana may be associated with GSH accumulation. Under chilling treatment, the proportion of unsaturated fatty acid in the plasma membrane (PM) increased significantly in callus of C. bungeana mainly due to increases in C18:2 and C18:3, the membrane fluidity (indicated by DPH fluorescent polarization) however was maintained. High PM H⁺-ATPase activities were also induced by chilling. Exogenously application of BSO blocked the effects of chilling treatments on the changes of fatty acids and PM H⁺-ATPase activities, reducing the PM membrane fluidity. On the other hand, simultaneous application of GSH and BSO to callus under chilling treatments reversed the effects of BSO on the changes of fatty acids, PM fluidity and PM H⁺-ATPase activities. These results suggested that GSH induced by low-temperature treatments may confer chilling tolerance to C. bungeana, probably by increasing unsaturated fatty acid compositions and maintaining PM fluidity and high enzymatic activity.     

Low temperature acclimation to chilling tolerance in rice roots

Rice seedlings (Oryza sativa L.) were subjected to low temperature pretreatment (LT-PT; 10°C) for various length of time (1, 2, 4, 6, 12, 18, 24 h) followed by a 48-h chilling temperature stress (2°C). Chilling temperature tolerance of rice roots was improved with increasing duration of LT-PT, but LT-PT longer than 12 h gave no additional improvement. Alcohol dehydrogenase activity and ethanol concentration in the roots were increased with increasing duration of LT-PT up to 12 h. Chilling tolerance was also improved by exogenously applied ethanol. These results suggest that LT-PT may increase chilling tolerance in rice roots owing to ethanol accumulation in the roots and LT-PT acclimation to chilling temperature may occur within 12 h.     

Exogenously applied paclobutrazol modulates growth in salt-stressed wheat plants

Effect of paclobutrazol (PBZ) treatment on salinity tolerance of wheat (Triticum aestivum) was investigated on a salt-tolerant (Karchia-65) and salt-sensitive (Ghods) cultivars. Salinity significantly reduced the investigated growth parameters such as plant height, length and area of sixth leaf, root length, fresh and dry weight of shoot, roots and sixth leaf, water content (WC) of plant and seeds weight in the both cultivars. The negative effect of salinity in Ghods cultivar was more than Karchia cultivar. However, PBZ treatment reduced the growth in both cultivars, the differences in plant growth among various levels of NaCl decreased in PBZ-treated plants. Salt stress resulted in high accumulation of Na⁺ in the sixth leaf and roots in both cultivars, particularly in Ghods cultivar. Against Karchia cultivar, salt stress decreased the storage of K⁺, P and N in sixth leaf and roots in Ghods cultivar. In the both cultivars, PBZ treatment enhanced the K⁺, P and N contents in sixth leaf and roots by increasing salinity. Although PBZ treatment decreased the growth of plants, it improved the weight of seeds against stress damage. PBZ treatment reduced the accumulation of harmful Na⁺ ion in plant tissues while increased the K⁺, P and N contents. These observations suggest that PBZ treatment may increase tolerance by diminishing ionic imbalance caused by salt stress.     

Growth promotion of ivy (Hedera helix L.) by paclobutrazol

Paclobutrazol was applied to juvenile ivy plants in an attempt to induce the mature form by the inhibition of gibberellin biosynthesis. Contrary to expectation, shoot elongation and adventitious root formation on aerial shoots were promoted.     

Antioxidants and antioxidative enzymes in wild-type and transgenic Lycopersicon genotypes of different chilling tolerance

The Mehler–Ascorbate–Peroxidase cycle is a protection system against reactive oxygen species (ROS) occurring during over-excitation of the photosynthetic apparatus. In the cultivated tomato, Lycopersicon esculentum, long-term chilling under moderate light leads to oxidation of the Calvin cycle key enzyme, ribulose-1,5-bisphosphate carboxylase (rubisco), presumably by generation of ROS. In contrast, high-altitude lines of the wild tomato species L. peruvianum were tolerant against the same chilling stress. In the present study, we analysed leaf contents of antioxidants (ascorbate, glutathione) and activities of enzymes of the Mehler–Ascorbate–Peroxidase cycle in the two Lycopersicon species. While antioxidant levels and activities of chloroplast superoxide dismutase (SOD) and ascorbate peroxidase (APX), both inducible by chilling stress, were similar in chilling-tolerant and chilling-sensitive genotypes, chilled L. esculentum showed lower glutathione reductase (GR) activities than high-altitude L. peruvianum. We constructed transgenic plants overexpressing an Escherichia coli GR in the chloroplast (approximately 60-fold of the wild-type (WT) activity). However, these plants resembled identical chilling sensitivity of the photosynthetic apparatus as WT plants as measured after a photoinhibition treatment and by the effect of long-term chilling on rubisco activity. We conclude that the Mehler–Ascorbate–Peroxidase cycle is not the limiting factor for the sensitivity of the photosynthetic apparatus of L. esculentum towards long-term chilling under moderate light. We suggest that a possible cause for the higher chilling tolerance of L. peruvianum is prevention of ROS formation by better conversion of light energy to photochemistry at suboptimal temperatures.     

Changes in activities of antioxidant enzymes and their gene expression during leaf development of sweetpotato

To understand the functions of antioxidant enzymes during leaf development in sweetpotato, we investigated the activities of several antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX) and catalase (CAT). Significant increases were observed in the activities of SOD, POX and APX during the late stage of leaf development, whereas CAT activity increased during the early developmental stage. By RT-PCR analysis, various POX and APX genes showed differential expression patterns during leaf development. Four POX genes swpa3, swpa4, swpa6, swpb4 and one APX gene swAPX1 exhibited high levels of gene expression during the senescence stage of leaf development, but two POX genes, swpa1 and swpa7 were preferentially expressed at both the mature green and the late senescence stages of leaf development. These results indicate that hydrogen peroxide (H₂O₂)-related antioxidant enzymes are differentially regulated in the process of leaf development of sweetpotato.     

Galactolipase activity and free fatty acid levels in chloroplasts of domestic and wild tomatoes with different chilling tolerance

In order to establish differences in the chilling sensitivity of domestic and wild Lycopersicon species, galactolipase (EC 3.1.1.26) activity, free fatty acid (FFA) level and Hill reaction activity were measured in chloroplasts isolated from control and cold treated leaves of L. esculentum Mill., cv. Norton, L. hirsutum Humb. and Bonpl., L. peruvianum var. glandulosum Mill. Galactolipase activity was higher in chloroplasts from Lycopersicon species with high chilling sensitivity than in chloroplasts of more chilling-resistant ones. A similar relationship was observed for FFA level in chloroplasts from both cold-stored and control leaves. Decrease in Hill reaction activity due to cold stress was greater in chloroplasts of more chilling-sensitive species. The changes are accompanied by a decline of photochemical activity. Considering the changes in the three parameters noted above, an increasing order of chilling tolerance was established: L. esculentum < L. hirsutum (700 m) < L. hirsutum (3100 m) < L. peruvianum (3400 m). It is suggested that measurements of galactolipase activity and FFA may be useful in an evaluation of differences in resistance to chilling injury of closely related species.     

Structures of clusters in sweetpotato amylopectin

Sweetpotato amylopectin was subjected to partial hydrolysis by α-amylase from Bacillus amyloliquefaciens to release the clusters. Clusters were then fractionated and precipitated by methanol and structurally characterized by gel-permeation chromatography and high-performance anion-exchange chromatography. An initial stage of α-amylolysis on the amylopectin isolated mostly domains but also clusters. A second stage of α-amylolysis on the domains and clusters further isolated their respective clusters and sub-clusters. All the domains, clusters and sub-clusters were sequentially subjected to phosphorolysis and β-amylolysis to obtain their internal part. The degree of polymerization of the clusters in the form of φ,β-limit dextrins were from 58 to 86. Each domain contained 2–8 clusters. Two types of clusters were structurally identified. Type A clusters were larger and contained about 12 chains per cluster with higher degree of branching (DB), whereas those of type B were smaller and contained about eight chains per cluster with lower DB.