Cloning of new members of heat shock protein HSP101 gene family in wheat (Triticum aestivum (L.) Moench) inducible by heat, dehydration, and ABA(1)

We have cloned two cDNAs, TaHSP101B and TaHSP101C, encoding two heat stress-inducible members of HSP101/ClpB family in bread wheat (Triticum aestivum (L.) Moench.). Proteins encoded by these cDNAs are highly similar at the primary sequence level and diverged from the previously reported TaHSP101 (designated TaHSP101A) both in the consensus ATP/GTP-binding region II and in the carboxy terminal region. The HSP101 gene was determined to be a single copy gene or a member of a small gene family in hexaploid wheat. Messages encoding HSP101 proteins were inducible by heat stress treatments in both wheat leaves and roots. Accumulation of the TaHSP101C mRNA was less abundant than that of TaHSP101B mRNA. We are showing for the first time that in addition to heat stress, expression of HSP101 mRNAs in wheat leaves was induced by a 2-h dehydration and a treatment with 5x10(-5)M ABA, but not affected by chilling or wounding, indicating that HSP101 proteins may be involved in both heat and drought responses in wheat.     

两个小麦14-3-3基因的特征、亚细胞定位及其对非生物胁迫的响应(英文)

为了探讨14-3-3基因在小麦逆境胁迫应答中的调控作用,利用RACE技术克隆了两个包含完整编码框的14-3-3基因(命名为Ta14R1和Ta14R2),其中Ta14R1 cDNA长999 bp,编码262个氨基酸,而Ta14R2 cDNA长897 bp,编码261个氨基酸。Ta14R1/Ta14R2-GFP融合载体瞬时表达结果显示,Ta14R1和Ta14R2蛋白均定位于细胞质和细胞膜,但不在叶绿体中。荧光定量PCR分析表明,Ta14R1和Ta14R2均在萌发1 d的胚芽鞘中表达量最高;在高温、低温、模拟干旱和ABA处理下,两个基因在小麦的根和叶中都受胁迫诱导而且显著上调表达,推测这两个14-3-3基因通过依赖ABA的非生物胁迫响应途径发挥作用,可能参与了小麦中高温、低温和干旱胁迫的耐受调节过程。     

Synthesis of soluble heat shock proteins in seminal root tissues of some cultivated and wild wheat genotypes

Effect of heat stress on the synthesis of soluble heat shock proteins (HSPs) and the regrowth in seminal roots of three cultivated and three wild wheat genotypes was examined. In regrowth experiments, 2-d-old etiolated seedlings were exposed to 23 (control), 32, 35, 37 and 38 degrees C for 24 h, and 35 and 37 degrees C (24 h) followed by 50 degrees C (1 h). The lengths of the seminal roots generally decreased significantly at the end of 48 and 72 h recovery growth periods at 35, 37 and 38 degrees C temperature treatments compared with control. Genotypic variability was significant level at all temperature treatments for the seminal root length. Also, genotypic differences for the number of seminal roots were determined among the wheat cultivars and between the wild wheat species and the wheat cultivars at all temperature treatments; but genotypic differences among wild wheat species were only detected at 37-->50 degrees C treatment. Acquired thermotolerance for the seminal root length is over 50% at 37-->50 degrees C treatment. The genotypic variability of soluble heat shock proteins in seminal root tissues were analyzed by two-dimensional electrophoresis (2-DE). Total number of low molecular weight (LMW) HSPs was more than intermediate-(IMW) and high- (HMW) HSPs at high temperature treatments. The most of LMW HSPs which were generally of acidic character ranged between 14.2-30.7 kDa. The genotypes had both common (43 HSP spots between at least two genotypes and 23 HSP spots between 37 and 37-->50 degrees C) and genotype-specific (72 HSP spots) LMW HSPs.     

The influence of protonophore high concentration on the structure and functions of wheat root cells

Protonophore induced structural and functional changes in cells of excised roots of wheat seedlings have been investigated. The vector transfer of H+ inside the cells was accompanied by a decrease in energy supply of these cells (suppression of oxygen consumption and heat release), an output of K+ ions to the incubation medium, and by an increase in its pH value. The initial increase in heat release by roots (1 h) apparently reflects the process of dissipation of deltamicro H+ in plasma membrane. Within the first 5-10 min of exposure of 50 microM CCCP, changes in cell ultrastructure were observed that involved activation of Golgi apparatus, secretion of vesicle contents to the vacuole, and swelling of endoplasmic reticulum canals. Following a 2 h treatment with CCCP, structural and functional changes acquired a destructive character, and after 5-6 h of treatment with protonophore a complete desintegration of cell structure occurred demonstrating formations of myelin-like bodies, fragmentation of plasma membrane, and destruction of the nucleus. Thus, the protonophore induced proton excessive transport inside cells is fast and may cause an irreversible cell de-energization followed by serious disruption of ultrastructural organization of cells leading eventually to their death.     

氮磷亏缺对小麦TaIPS基因表达的影响

为了解小麦高效利用土壤磷的分子机理和实现对小麦缺磷的分子诊断,以普通小麦(Triticum aestivum L.)小偃54为材料,克隆了5个受缺磷诱导的IPS基因,同源比较结果显示,小麦IPS基因属于典型的受缺磷条件特异诱导的TPSI1/MT4小基因家族.对小麦根系和地上部的半定量RT-PCR研究结果表明,与全营养处理对照相比,3叶期小麦幼苗经过缺氮、缺磷和氮磷同时缺乏处理8d后,缺磷显著增加了根系中3个TaIPS1(TaIPS1.1、TaIPS1.2和TaIPS1.3)基因和地上部TaIPS1.1基因的表达,中度上调了根系中2个TaIPS2基因(TaIPS2.1和TaIPS2.2)的表达,轻度上调了地上部TaIPS1.2和2个TaIPS2基因的表达.通过比较5个基因在根系和地上部对缺磷的响应,认为TaIPS1.1是一个较理想的用于诊断小麦植株磷素丰缺的基因.缺氮不仅降低了3个TaIPS1基因在根系中的表达,并抑制了IPS基因对缺磷的响应.这一研究结果预示了TaIPS基因对低磷胁迫的响应依赖于植株体内的氮素营养状况.     

小麦NO3-转运蛋白基因TaNRT2.3的克隆和表达分析

用RT-PCR和RACE技术在NO3-诱导处理的小麦(Triticum aestivum L.)根中克隆到一个硝酸根转运蛋白基因的cDNA,命名为TaNRT2.3(GenBank登录号AY053452).序列分析表明,TaNRT2.3全长1 744 bp,其中含有1 521bp的ORF,编码507个氨基酸,具有12个跨膜区,属于MFS超基因家族中的NNP家族.TaNRT2.3与其他植物中已知的NRT2具有很高的同源性.Northern杂交表明:TaNRT2具有在根中表达的组织特异性,而在叶中未检测到.TaNRT2的表达受NO3-诱导,在含NH4 介质中不表达.NO3-在低浓度(5～200μmol/L)和高浓度(2.0 mmol/L)时均起作用.通过研究小麦在0.2 mmol/LNO3-条件下TaNRT2的表达水平及对NO3-的吸收效率,表明TaNRT2在小麦高效吸收NO3-方面起着重要的作用.分根实验表明植物中N循环本身可以作为吸收N的调节信号.     

Wheat and faba bean intercropping changes phenolic acids from roots to rhizosphere

The changed phenolic acids (PAs) allelochemicals exuded by the roots induced by interspecific interactions is related to intercropping alleviates soil-borne disease. However, the presence of PAs in roots and root exudations and their rhizodeposition under intercropping are still unclear. Hydroponic and soil experiments of wheat, faba bean, and wheat intercropped with faba bean were conducted, and the major compositions and contents of PAs in roots, root exudations, and rhizospheric soil were determined. The results showed that ρ-hydroxybenzoic, vanillic, and syringic acids were the major components of PAs in roots, root exudations, and rhizospheric soil in a wheat and faba bean intercropping system. The compositions and percentages of PAs in roots of faba bean were altered when faba bean intercropped with wheat. The total exudation rate of PAs in root exudations was decreased by 30%–60% under the wheat and faba bean intercropping (W//F) system as compared to mono-cropped faba bean (MF). ρ-hydroxybenzoic acid was identified in the root exudation of both MF and mono-cropped wheat (MW), but not detected in the intercropping on 60 days after transplanting. Vanillic acid was only detected in the root exudation of MF on 30 days after transplanting. The rhizodepostion of vanillic and cumaric acid were decreased at both branching and pod setting stages in W//F as compared to MF. In conclusion, interspecific interaction changed the compositions and contents of PAs in faba bean roots and root exudations. W//F constrained vanillic acid exuded by roots and decreased vanillic and coumaric acid rhizodeposition by faba bean, which provides insight into root-soil interactions in the intercropping systems.     

Energy metabolism in wheat roots during modification of cell surface

A study was made of dynamics of wheat production, intensity of respiration and changes in bioelectric characteristics of exised roots. Response reactions of two wheat varieties were compared in the process of adaptive reactions. The varieties differed in bioelectric characteristics of root cells in intact seedlings grown in CaCl2 and EDTA containing media. Different changes of membrane characteristics of root cells were observed: in soft wheat MP and Rin increased, but in hard wheat these decreased after a 5 h incubation of excised root. The rate of heat production was at the same level in both wheat varieties, but oxygen absorption of the root cells was lower in hard wheat compared with soft wheat. The rate of respiration of excised roots was stable in EDTA-containing medium. The obtained data allow to discuss more in detail the role of Ca(2+)-ions in the regulation of cell functions under formation of adaptive processes as the tissue level.     

Wheat germ agglutinin in wheat seedling roots: induction by elicitors and fungi

Summary Treatment of wheat (Triticum aestivum L.) seedlings with elicitors originating from either plant or fungal cell walls induces about a 2-fold increase of wheat germ agglutinin (WGA) in the roots. While the WGA content in roots of healthy plants normally decreases as a function of germination time, a transient accumulation of WGA could be observed in plants challenged with different fungi, including Rhizoctonia solani, Fusarium culmorum, Pythium ultimum and Neurospora crassa. Peak levels in challenged roots were 2 to 5 times as high as in control plants. Most of this induced WGA could be released from the roots by soaking them in a solution of the hapten N-acetylglucosamine. On the basis of the results obtained it is postulated that WGA may be involved in the defence of wheat against fungal attack.     

Effect of lectins from <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> to peroxidase and oxalate oxidase activity regulation in wheat roots

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability of lectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect of lectin in under 10 min in a concentration of 10 μg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H+-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响.结果表明,0.1 mmol/L SNP处理显著缓解了1 50 mmol/L NaCl胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性.进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜H -ATPase和焦磷酸酶活性诱导有关.此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响.应用外源CaSO4和EGTA处理也证实,Ca2 可能在NO诱导的质膜H -ATPase和焦磷酸酶活性的提高过程中起信号作用.另外,分析盐胁迫下小麦幼苗根部Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一.     

The Effect of Local Cooling of Cucumber and Wheat Seedlings on Various Kinds of Stress Resistance of Their Leaves and Roots

Shoots and roots of wheat (Triticum aestivum L., cold-resistant species) and cucumber (Cucumis sativus L., cold-sensitive species) were chilled at 2°C or 10°C, respectively, for 7 h. The changes in cold, heat, and salt resistance in treated leaf and root cells were recorded. Local cooling of the leaf resulted in an increase of its cold and salt tolerance, but its heat tolerance remained unchanged. At the same time, cold tolerance of the root slightly increased as a result of local cooling, but its heat and salt tolerance decreased. Cooling of the shoot did not affect the cold and heat tolerance of root cells but caused a decrease in their salt tolerance. Finally, in the leaf maintained at a normal temperature, there was an increase in all kinds of stress resistance as a result of root cooling. We discuss the possibility of an unspecific change in stress resistance caused by metabolic shifts. These shifts are induced by a signal, which is transmitted inside the plant into plant organs located at a considerable distance from the chilled ones.     

A comparative study on the aluminium- and copper-induced organic acid exudation from wheat roots

It is well established that aluminium (Al) and some heavy metals can elicit organic acid exudation from a range of species. In the present research we found that copper (Cu) can also induce organic acid exudation from the roots of wheat, rye, triticale, maize and soybean. Using intact wheat plants, we made a comparative study of Al- and Cu- induced organic acid exudation. In 5-day-old wheat seedlings, severe Cu stress (40 µ M CuCl₂) mainly induced the exudation of malate and citrate, and Al-tolerant genotypes could release significantly greater amounts of malate than Al-sensitive genotypes. The time course of the exudation of malate and citrate from the roots of 5-day-old seedlings of wheat (cv. Atlas) in 200 µ M AlCl₃ was similar to that in 40 µ M CuCl₂. In older wheat plants (15-day-old), moderate Cu stress (12 µ M CuCl₂) induced the exudation of large amounts of citrate and addition of Al or La sharply reduced Cu-induced citrate exudation, while Cu or La did not affect Al-induced malate efflux. When half of the root system of Atlas wheat was immersed in Al- or Cu-containing solution and the remaining half in Al- or Cu-free solution, organic acids were only exuded into the solution containing Al or Cu. This suggests that no long distance signal transport is involved in organic acid exudation induced by Al or Cu, and that direct contact of Al or Cu with plant roots is a prerequisite for the induction of organic acid exudation. The anion-channel inhibitor niflumic acid (NIF) significantly stimulated the exudation of both citrate and malate from 5-day-old wheat seedlings under severe Al or Cu stress. Our results suggest that Cu-induced organic acid efflux may be a common response, which may play a role in alleviating Cu toxicity in plants.     

Induction of heat sensitivity of wheat roots and its effects on mitochondria, adenosine triphosphate, triglyceride, and total lipid content

Wheat seedlings were subjected to heat shock for 2 min at 45 °C. After heat treatment, the wheat seedlings were incubated at 25 or 35 °C. At 25 °C, but not at 35 °C, the root tips survived the heat shock. Immediately after the heat treatment the free triglyceride content in the treated root tips was higher than in the untreated roots, but the total lipid content was not changed. The ATP content immediately after the heat treatment was variable, but after about 1 h it stabilized at the same level as in the control or at a higher level. After 45 min at 25 °C after heat shock, the endoplasmic reticulum cisternae had expanded, giving rise to small irregular vacuoles. Golgi vesicles were also irregular. Four hours after heat treatment the endoplasmic reticulum and Golgi vesicles again were normal, but mitochondria were irregular with fewer tubules and with adhering membrane curls containing lipids. These membrane curls were not observed 24 h after heat treatment. When incubated at 35 °C after heat shock wheat root meristems died. Some cells in the meristem were still alive 4 h after treatment. They had large vacuoles with membrane whorls and plasmalemmasomes, and in some cases the cells were partly lysed.     

Structure of Syncytia Induced by Heterodera avenae Woll. in Roots of Susceptible and Resistant Wheat (Triticum aestivum L.)

The structure of syncytia induced by Heterodera avenae in roots of susceptible Triticum aestivum cv. ‘Capa’ and resistant wheat AUS 10894 was investigated. In susceptible plants the first syncytia were observed 4 days after placing the seedlings into a substrate containing nematode cysts. They were located on different root levels. In resistant plants syncytia were first found after 14 days, exclusively in the root maturity zone. In both wheats syncytia consisted mostly of stelar parenchymatic cells. The syncytia induced in roots of Triticum aestivum AUS 10894 were characterized by less immediate contact with the host tracheary elements than the syncytia induced in roots of susceptible Triticum aestivum cv. ‘Capa’. In both wheats a stimulation of pericycle cell divisions, giving rise to lateral roots, was found in parts where the syncytia developed.