Elevated cytokinin content in ipt transgenic creeping bentgrass promotes drought tolerance through regulating metabolite accumulation

Increased endogenous plant cytokinin (CK) content through transformation with an adenine isopentyl transferase (ipt) gene has been associated with improved plant drought tolerance. The objective of this study is to determine metabolic changes associated with elevated CK production in ipt transgenic creeping bentgrass (Agrostis stolonifera L.) with improved drought tolerance. Null transformants (NTs) and plants transformed with ipt controlled by a stress- or senescence-activated promoter (SAG12-ipt) were exposed to well-watered conditions or drought stress by withholding irrigation in an environmental growth chamber. Physiological analysis confirmed that the SAG12-ipt line (S41) had improved drought tolerance compared with the NT plants. Specific metabolite changes over the course of drought stress and differential accumulation of metabolites in SAG12-ipt plants compared with NT plants at the same level of leaf relative water content (47% RWC) were identified using gas chromatography-mass spectroscopy. The metabolite profiling analysis detected 45 metabolites differentially accumulated in response to ipt expression or drought stress, which included amino acids, carbohydrates, organic acids, and organic alcohols. The enhanced drought tolerance of SAG12-ipt plants was associated with the maintenance of accumulation of several metabolites, particularly amino acids (proline, γ-aminobutyric acid, alanine, and glycine) carbohydrates (sucrose, fructose, maltose, and ribose), and organic acids that are mainly involved in the citric acid cycle. The accumulation of these metabolites could contribute to improved drought tolerance due to their roles in the stress response pathways such as stress signalling, osmotic adjustment, and respiration for energy production.     

Effect of abiotic stresses on the activity of antioxidative enzymes and contents of phytohormones in wild type and AtCKX2 transgenic tobacco plants

The responses of antioxidant enzymes (AOE) ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in soluble protein extracts from leaves and roots of tobacco (Nicotiana tabacum L. cv. Samsun NN) plants to the drought stress, salinity and enhanced zinc concentration were investigated. The studied tobacco included wild-type (WT) and transgenic plants (AtCKX2) harbouring the cytokinin oxidase/dehydrogenase gene under control of 35S promoter from Arabidopsis thaliana (AtCKX2). The transgenic plants exhibited highly enhanced CKX activity and decreased contents of cytokinins and abscisic acid in both leaves and roots, altered phenotype, retarded growth, and postponed senescence onset. Under control conditions, the AtCKX2 plants exhibited noticeably higher activity of GR in leaves and APX and SOD in roots. CAT activity in leaves always decreased upon stresses in WT while increased in AtCKX2 plants. On the contrary, the SOD activity was enhanced in WT but declined in AtCKX2 leaves. In roots, the APX activity prevailingly increased in WT while mainly decreased in AtCKX2 in response to the stresses. Both WT and AtCKX2 leaves as well as roots exhibited elevated abscisic acid content and increased CKX activity under all stresses while endogenous CKs and IAA contents were not much affected by stress treatments in either WT or transgenic plants.     

Involvement of Lipid Peroxidation in Water Stress-Promoted Senescence of Detached Rice Leaves

Role of lipid peroxidation and antioxidative enzymes (catalase, peroxidase, superoxide dismutase, ascorbate peroxidase and glutathione reductase) in water stress-promoted senescence of detached rice leaves was investigated. The senescence was followed by measuring the decrease in protein content. Increased lipid peroxidation was closely correlated with senescence in water stressed leaves. Decrease in superoxide dismutase activity was evident 8 h after beginning of water stress. However, decreased catalase, peroxidase, and ascorbate peroxidase activity was observed only when senescence was observed. Glutathione reductase was not affected by water stress. Free radical scavengers retarded water stress-enhanced senescence.     

Effects of P(SAG12)-IPT gene expression on development and senescence in transgenic lettuce

An ipt gene under control of the senescence-specific SAG12 promoter from Arabidopsis (P(SAG12)-IPT) significantly delayed developmental and postharvest leaf senescence in mature heads of transgenic lettuce (Lactuca sativa L. cv Evola) homozygous for the transgene. Apart from retardation of leaf senescence, mature, 60-d-old plants exhibited normal morphology with no significant differences in head diameter or fresh weight of leaves and roots. Induction of senescence by nitrogen starvation rapidly reduced total nitrogen, nitrate, and growth of transgenic and azygous (control) plants, but chlorophyll was retained in the lower (outer) leaves of transgenic plants. Harvested P(SAG12)-IPT heads also retained chlorophyll in their lower leaves. During later development (bolting and preflowering) of transgenic plants, the decrease in chlorophyll, total protein, and Rubisco content in leaves was abolished, resulting in a uniform distribution of these components throughout the plants. Homozygous P(SAG12)-IPT lettuce plants showed a slight delay in bolting (4-6 d), a severe delay in flowering (4-8 weeks), and premature senescence of their upper leaves. These changes correlated with significantly elevated concentrations of cytokinin and hexoses in the upper leaves of transgenic plants during later stages of development, implicating a relationship between cytokinin and hexose concentrations in senescence.     

转PSAG12-ipt基因水稻延衰性能的初步研究

研究了叶片衰老抑制基因PSAG1 2 ipt经基因枪导入籼稻不育系中A后 ,转化植株农艺性状和生理特性的变化。结果显示 ,在水稻生育期 ,转化植株相对于对照而言 ,叶绿素、蛋白质含量和SOD(超氧物歧化酶 )活性下降以及MDA(丙二醛 )相对含量上升趋势较缓 ,表现在农艺性状上是单株有效穗数、千粒重明显高于对照。说明叶片衰老抑制基因PSAG1 2 ipt确实具有延缓叶片衰老的功能 ,延长了水稻叶片利用光能的时间 ,使之积累了大量的光合产物 ,增加了产量     

转基因抗早衰棉的获得

利用花粉管通道技术将含有抑制衰老嵌合基因PCSAG12-ipt的pBG121质粒转入早衰型陆地棉品种中棉所10号中,通过对T1代植株进行卡那霉素田间筛选、PCR检测及GUS组织化学染色,获得了12株转基因棉花。在棉花发育进入衰老时期,对转基因植株进行叶绿素和细胞分裂素含量的测定及形态观察,结果表明转基因植株的衰老得到延迟。     

Effects of SAG12-ipt expression on cytokinin production,growth and senescence of creeping bentgrass (<Emphasis Type="Italic">Agrostis stolonifera</Emphasis> L.) under heat stress

The study was conducted to determine the effects of expression of a transgene encoding adenine isopentenyl transferase (ipt), which controls cytokinin synthesis, on growth and leaf senescence of creeping bentgrass (Agrostis stolonifera L.), subjected to heat stress. Creeping bentgrass (cv. Penncross) was transformed with ipt ligated to a senescence-activated promoter (SAG12). Eight SAG12-ipt transgenic lines exhibiting desirable turf quality and a transgenic control line (transformed with the empty vector) were evaluated for morphological and physiological changes under normal growth temperature (20°C) and after 14 days of heat stress (35°C) in growth chambers. Six of the SAG12-ipt lines developed more tillers than the control line during establishment under normal growth temperature of 20°C. Following 14 days of heat stress, four of the SAG12-ipt lines had increased 65–83% of roots and for all six SAG12-ipt lines root elongation continued, whereas root production ceased and total root length decreased for the control line. Root isopentenyl adenine (iPA) content increased 2.5–3.5 times in five of the SAG12-ipt lines, whereas in the control line iPA decreased 20% after 14 days at 35°C. Total zeatin riboside (ZR) content was maintained at the original level or increased in five of the SAG12-ipt lines, whereas in the control line ZR decreased under heat stress. Our results suggest expression of SAG12-ipt in creeping bentgrass stimulated tiller formation and root production, and delayed leaf senescence under heat stress, suggesting a role for cytokinins in regulating cool-season grass tolerance to heat stress.     

Arabidopsis LOS5/ABA3 overexpression in transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) results in enhanced drought tolerance

Drought is a major environmental stress factor that affects growth and development of plants. Abscisic acid (ABA), osmotically active compounds, and synthesis of specific proteins, such as proteins that scavenge oxygen radicals, are crucial for plants to adapt to water deficit. LOS5/ABA3 (LOS5) encodes molybdenum-cofactor sulfurase, which is a key regulator of ABA biosynthesis. We overexpressed LOS5 in tobacco using Agrobacterium-mediated transformation. Detached leaves of LOS5-overexpressing seedlings showed lower transpirational water loss than that of nontransgenic seedlings in the same period under normal conditions. When subjected to water-deficit stress, transgenic plants showed less wilting, maintained higher water content and better cellular membrane integrity, accumulated higher quantities of ABA and proline, and exhibited higher activities of antioxidant enzymes, i.e., superoxide dismutase, catalase, peroxidase and ascorbate peroxidase, as compared with control plants. Furthermore, LOS5-overexpressing plants treated with 30% polyethylene glycol showed similar performance in cellular membrane protection, ABA and proline accumulation, and activities of catalase and peroxidase to those under drought stress. Thus, overexpression of LOS5 in transgenic tobacco can enhance drought tolerance.     

蒺藜苜蓿MtSAG113基因的转化及表达特征分析

Senescence Associated Gene 113(SAG113)基因属于PP2Cc超家族,该基因的研究主要集中在植物衰老领域.为分析蒺藜苜蓿(Medicago truncatula)MtSAG113基因的表达特征,探究MtSAG113基因的功能.该基因从蒺藜苜蓿中克隆得到,以烟草(Nicotiana tab...     

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

A gene encoding an acyl hydrolase is involved in leaf senescence in Arabidopsis

SAG101, a leaf senescence-associated gene, was cloned from an Arabidopsis leaf senescence enhancer trap line and functionally characterized. Reporter gene and RNA gel blot analyses revealed that SAG101 was not expressed until the onset of senescence in leaves. A recombinant SAG101 fusion protein overexpressed in Escherichia coli displayed acyl hydrolase activity. Antisense RNA interference in transgenic plants delayed the onset of leaf senescence for approximately 4 days. Chemically induced overexpression of SAG101 caused precocious senescence in both attached and detached leaves of transgenic Arabidopsis plants. These data suggest that SAG101 plays a significant role in leaf senescence.     

Effect of drought on biomass, protein content, lipid peroxidation and antioxidant enzymes in two sesame cultivars

The effects of drought on growth, protein content, lipid peroxidation, superoxide dismutase (SOD), peroxidase (POX), catalase (CAT) and polyphenol oxidase (PPO) were studied in leaves and roots of Sesamum indicum L. cvs. Darab 14 and Yekta. Four weeks after sowing, plants were grown under soil moisture corresponding to 100, 75, 50 and 25 % field capacity for next four weeks. Fresh and dry masses, and total protein content in leaves and roots decreased obviously under drought. However, several new proteins appeared and content of some proteins was affected. Measurement of malondialdehyde content in leaves and roots showed that lipid peroxidation was lower in Yekta than in Darab 14. Severe stress increased SOD, POX, CAT and PPO activities in leaves and roots, especially in Yekta. According to the present study Yekta is more resistant to drought than Darab 14.     

Antioxidant protection during ageing and senescence in chloroplasts of tobacco with modulated life span

We studied changes in antioxidant protection during ageing and senescence in chloroplasts of tobacco (Nicotiana tabacum L., cv. Wisconsin) with introduced SAG(12) promoter fused with ipt gene for cytokinin synthesis (transgenic plants with increased levels of cytokinins, SAG) or without it (control). Old leaves of SAG plants as well as their chloroplasts maintained higher physiological parameters compared to controls; accordingly, we concluded that their ageing was diverted due to increased cytokinin content. The chloroplast antioxidant protection did not decrease as well. Although antioxidant protection usually decreased in whole leaves of senescing control plants, ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) activity, which maintained the high redox state of ascorbate, increased in chloroplasts of old control leaves.     

Some physiological responses of chickpea cultivars to arbuscular mycorrhiza under drought stress

A factorial experiment based on RCB design with three replicates was conducted to investigate changes in some physiological responses of two chickpea (Cicer arietinum L.) cultivars (Pirouz from Desi type and ILC482 from Kabuli type) to arbuscular mycorrhiza (Glomus etunicatum Becker and Gerdman) under different irrigation treatments. The experiment was carried out in the greenhouse of the Agricultural Faculty of Kurdistan University from April to August 2009. The results showed that leaf chlorophyll content of chickpea cultivars was significantly increased by arbuscular mycorrhiza (AM) under both well and limited irrigation conditions. Proline accumulation in chickpea leaves under moderate and severe drought stresses was significantly stronger than that under optimum irrigation. Inoculation of chickpea with mycorrhizal fungi caused an increase in the activities of polyphenol oxidase and peroxidase, but a decrease in the activity of catalase. Comparisons among different irrigation levels showed that chickpea plants under drought stress had the most active lipid peroxidation. Non-AM plants showed stronger lipid peroxidation under moderate and severe water stresses than AM plants. Lipid peroxidation was more active in Pirouz leaves than in ILC₄₈₂ leaves. It seems that Kabuli-type cultivar responded better to mycorrhizal symbiosis under drought stress than Desitype cultivar.     

Antioxidative protection and proteolytic activity in tolerant and sensitive wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) varieties subjected to long-term field drought

Field drought studies were performed in order to assess oxidative stress, proteolytic activity and yield loss under natural stress conditions. Flag leaves of two drought-tolerant (Yantar and Zlatitsa) and two drought-sensitive (Miziya and Dobrudjanka) winter wheat varieties were analyzed. Stress intensity was assessed by relative electrolyte leakage and proline accumulation. Senescence progression was followed by loss of chlorophyll and protein. Lipid peroxidation, H₂O₂ content, activities of superoxide dismutase (SOD), catalase (CAT), and non-specific peroxidase (GPX) isoforms, as well as proteolytic activities were analyzed from heading throughout grain filling. Weakening of membrane integrity and oxidative damage to lipids were more pronounced in the sensitive varieties under field drought. The activities of Fe- and Cu/Zn SOD isoforms decreased in the controls, but remained high in drought-treated plants. The activities of MnSOD isoforms and CAT were enhanced towards grain filling, especially in the sensitive varieties under drought. GPX activities were rised under drought but progressively diminished. Accelerated senescence under field drought was linked to higher proteolytic activity with variety specific differences in the protease response, but without a clear correlation to drought resistance or sensitivity. Field drought led to higher oxidative stress more pronounced for drought sensitive varieties, especially during the grain filling period.     

Role of Antioxidant Systems in Wheat Genotypes Tolerance to Water Stress

The role of plant antioxidant systems in stress tolerance was studied in leaves of three contrasting wheat genotypes. Drought imposed at two different stages after anthesis resulted in an increase in H2O2 accumulation and lipid peroxidation and decrease in ascorbic acid content. Antioxidant enzymes like superoxide dismutase, ascorbate peroxidase and catalase significantly increased under water stress. Drought tolerant genotype C 306 which had highest ascorbate peroxidase and catalase activity and ascorbic acid content also showed lowest H2O2 accumulation and lipid peroxidation (malondialdehyde content) under water stress in comparison to susceptible genotype HD 2329 which showed lowest antioxidant enzyme activity and ascorbic acid content and highest H2O2 content and lipid peroxidation. HD 2285 which is tolerant to high temperature during grain filling period showed intermediate behaviour. Superoxide dismutase activity, however, did not show significant differences among the genotypes under irrigated as well as water stress condition. It seems that H2O2 scavenging systems as represented by ascorbate peroxidase and catalase are more important in imparting tolerance against drought induced oxidative stress than superoxide dismutase alone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Drought induced programmed cell death and associated changes in antioxidants, proteases, and lipid peroxidation in wheat leaves

Two wheat (Triticum aestivum L.) genotypes with varying degree of drought tolerance were used to analyze programmed cell death (PCD) and related biochemical changes under drought stress. Drought induced PCD in leaves, as evident by internucleosomal nDNA fragmentation, was observed in sensitive genotype Nesser. Drought tolerant genotype (FD-83) showed higher peroxidase, superoxide dismutase, and catalase activities and ascorbate content under drought stress compared to sensitive genotype. Total phenolic content increased whereas lipid peroxidation remained un-changed under drought in FD-83. In contrast, drought enhanced the proteases and ascorbate peroxidase activities and lipid peroxidation (MDA content) in Nesser.     

Senescence-associated vacuoles with intense proteolytic activity develop in leaves of Arabidopsis and soybean

Vacuolar compartments associated with leaf senescence and the subcellular localization of the senescence-specific cysteine-protease SAG12 (senescence-associated gene 12) were studied using specific fluorescent markers, the expression of reporter genes, and the analysis of high-pressure frozen/freeze-substituted samples. Senescence-associated vacuoles (SAVs) with intense proteolytic activity develop in the peripheral cytoplasm of mesophyll and guard cells in Arabidopsis and soybean. The vacuolar identity of these compartments was confirmed by immunolabeling with specific antibody markers. SAVs and the central vacuole differ in their acidity and tonoplast composition: SAVs are more acidic than the central vacuole and, whereas the tonoplast of central vacuoles is highly enriched in gamma-TIP (tonoplast intrinsic protein), the tonoplast of SAVs lacks this aquaporin. The expression of a SAG12-GFP fusion protein in transgenic Arabidopsis plants shows that SAG12 localizes to SAVs. The analysis of Pro(SAG12):GUS transgenic plants indicates that SAG12 expression in senescing leaves is restricted to SAV-containing cells, for example, mesophyll and guard cells. A homozygous sag12 Arabidopsis mutant develops SAVs and does not show any visually detectable phenotypical alteration during senescence, indicating that SAG12 is not required either for SAV formation or for progression of visual symptoms of senescence. The presence of two types of vacuoles in senescing leaves could provide different lytic compartments for the dismantling of specific cellular components. The possible origin and functions of SAVs during leaf senescence are discussed.