Chloroplast protein synthesis elongation factor, EF-Tu, reduces thermal aggregation of rubisco activase

Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize. The recombinant precursor of this protein, pre-EF-Tu, has been found to exhibit chaperone activity and protect heat-labile proteins, such as citrate synthase and malate dehydrogenase, from thermal aggregation. Chloroplast EF-Tu is highly conserved and it is possible that the chaperone activity of this protein is not species-specific. In this study, we investigated the effect of native wheat pre-EF-Tu on thermal aggregation of rubisco activase. Additionally, we investigated the effect of native and recombinant maize pre-EF-Tu on activase aggregation. Activase was chosen because it displays an exceptional sensitivity to thermal aggregation and constrains photosynthesis at high temperature. The native precursors of both wheat and maize EF-Tu displayed chaperone activity, as shown by the capacity of both proteins to reduce thermal aggregation of rubisco activase in vitro. Similarly, the recombinant maize pre-EF-Tu protected activase from thermal aggregation. This is the first report on chaperone activity of native pre-EF-Tu and the first evidence for thermal protection of a photosynthetic enzyme by this putative chaperone. The results are consistent with the hypothesis that chloroplast EF-Tu plays a functional role in heat tolerance by acting as a molecular chaperone.     

Analysis of transgenic wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) harboring a maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) gene for plastid EF-Tu: segregation pattern,expression and effects of the transgene

We previously reported that transgenic wheat (Triticum aestivum L.) carrying a maize (Zea mays L.) gene (Zmeftu1) for chloroplast protein synthesis elongation factor, EF-Tu, displays reduced thermal aggregation of leaf proteins, reduced injury to photosynthetic membranes (thylakoids), and enhanced rate of CO₂ fixation following exposure to heat stress (18 h at 45°C) [Fu et al. in Plant Mol Biol 68:277–288, 2008]. In the current study, we investigated the segregation pattern and expression of the transgene Zmeftu1 and determined the grain yield of transgenic plants after exposure to a brief heat stress (18 h at 45°C). We also assessed thermal aggregation of soluble leaf proteins in transgenic plants, testing the hypothesis that increased levels of EF-Tu will lead to a non-specific protection of leaf proteins against thermal aggregation. The transgenic wheat displayed a single-gene pattern of segregation of Zmeftu1. Zmeftu1 was expressed, and the transgenic plants synthesized and accumulated three anti-EF-Tu cross-reacting polypeptides of similar molecular mass but different pI, suggesting the possibility of posttranslational modification of this protein. The transgenic plants also showed better grain yield after exposure to heat stress compared with their non-transgenic counterparts. Soluble leaf proteins of various molecular masses displayed lower thermal aggregation in transgenic than in non-transgenic wheat. The results suggest that overexpression of chloroplast EF-Tu can be beneficial to wheat tolerance to heat stress. Moreover, the results also support the hypothesis that EF-Tu contributes to heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation in a non-specific manner.     

Thermal stress evaluation of suspension cell cultures in winter wheat

Summary The objectives of this study were to compare thermotolerance in whole plants vs. suspension cell cultures of winter wheat, and to evaluate the synthesis of heat shock proteins in relation to genotypic differences in thermotolerance in suspension cells. Whole plant genetic differences in the development of heat tolerance were identified for three wheat genotypes (ND 7532, KS 75210 and TAM 101). Suspension cell cultures of these genotypes were used to evaluatein vitro response to heat stress. Viability tests by triphenyl tetrazolium chloride (TTC) and by fluorescein diacetate (FD) were utilized to determine the relationship of cellular response to heat stress (37°C/24 h, 50°C/1h). KS 75210 and ND 7532 are relatively heat susceptible. TAM 101 is heat tolerant. Both tests at the cellular level were similar to the whole plant response. Thus, cellular selection for enhancing heat tolerance seems feasible. Heat shock protein (HSP) synthesis of two genotypes, ND 7532 and TAM 101 were determined for suspension cultured cells. In suspension cultures, HSPs of molecular weight 16 and 17 kD were found to be synthesized at higher levels in the heat tolerant genotype (TAM 101) than the susceptible genotype (ND 7532), both at 34° and 37°C treatments for 2 hours and 5 hours. HSP 22 kD was synthesized more at 34°C for TAM 101 than ND 7532, but not at 37°C; whereas, HSP 33 kD was synthesized at 37°C at similar abundance for both genotypes, but not at 34°C.These results indicated that there is a differential expression of HSP genes in wheat suspension cells at different temperature stress durations and between heat tolerant and heat susceptible genotypes. It appears that the levels of synthesis of HSPs 16 and 17 kD are correlated with genotypic differences in thermal tolerance at the cellular level in two genotypes of wheat.     

WCS120 protein family and proteins soluble upon boiling in cold-acclimated winter wheat

The amount of proteins soluble upon boiling (especially WCS120 proteins) and the ability to develop frost tolerance (FT) after cold acclimation was studied in two frost-tolerant winter wheat cultivars, Mironovskaya 808 and Bezostaya 1. Protein gel blot analysis, mass spectrometry (MS) and image analysis of two-dimensional gel electrophoresis (2-DE) gels were used to identify and/or quantify the differences in protein patterns before (non-acclimated, NA) and after 3 weeks of cold acclimation (CA) of the wheats, when FT increased from -4 degrees C (lethal temperature (LT(50)), for both cultivars) to -18.6 degrees C in Bezostaya 1 and -20.8 degrees C in Mironovskaya 808. Only WCS120 protein was visible in NA leaves while all five WCS120 proteins were induced in the CA leaves. Mironovskaya 808 had higher accumulation of three members of WCS120 proteins (WCS120, WCS66 and WCS40) than Bezostaya 1. MS analysis of total sample of proteins soluble upon boiling showed seven COR proteins in the CA samples and only three COR proteins in the NA samples of cultivar Mironovskaya 808 (MIR). In conclusion, the level of the accumulation of WCS120, WCS66 and WCS40 distinguished our two frost-tolerant winter wheat cultivars. Moreover, the differences of CA and NA samples of the MIR were shown by liquid chromatography (LC)-tandem mass spectrometry (MS/MS).     

Exogenous Gamma-aminobutyric Acid Coordinates Active Oxygen and Amino Acid Homeostasis to Enhance Heat Tolerance in Wheat Seedlings

Heat stress has detrimental impacts on wheat growth and yield formation. Conferring heat tolerance through applying plant growth regulators is a feasible strategy to reduce loss. Gamma aminobutyric acid (GABA) is a four-carbon non-proteinogenic amino acid existing in organisms and accumulates in response to stress. In this study, spring wheat Liaochun17 and winter wheat Jingdu 40 were used to investigate the function of exogenous GABA on the heat tolerance of wheat seedlings. Data displayed that GABA treatment not only reduced the production of reactive active oxygen (ROS), but also improved the scavenging capacity of diphenyl picryl phenyl hydrazine active oxygen under heat stress, thus alleviating the accumulation of malondialdehyde and the damage of cell membrane. In addition, analysis of protein and amino acids revealed that GABA effectively promoted the accumulation of soluble protein and coordinated amino acid homeostasis. Summarily, our current findings revealed that GABA strengthened the resistance of wheat seedling to heat stress by maintaining the metabolism balance of ROS and amino acids.

     

高山植物圆锥南芥的光合系统耐热性及其修复机制(英文)

高温胁迫包括极端高温和中高温,严重影响了植物的一系列生理活动,尤其是光合作用,而植物应对极端高温和中高温胁迫具有不同的策略。高山植物因长期生长于相对寒冷的环境中,相比而言应缺少对高温胁迫的适应机制。本文以圆锥南芥作为一种高山模式植物来探索其在中高温下是否表现出耐热能力,如果具有耐热能力,那么在光合方面与拟南芥存在怎样的差异。研究发现,圆锥南芥在中高温处理后具有更高的光化学效率及快速可逆的恢复过程,表现出了较强的耐热能力。两物种的F0没有明显的差异,而圆锥南芥在热处理后及恢复过程中具有更高的Fm,促进其快速光合修复。在热处理后,非光化学能量耗散快速瞬时上升,及时保护光系统II免受光损伤和热伤害,另外,HSP101蛋白迅速诱导可能启动了光化学修复。最后,圆锥南芥在严重高温处理后具有更高的存活率再次验证了它在中高温下的耐热能力。结果表明,圆锥南芥具有更耐热的光合系统以及有效的光合修复机制来耐受中高温胁迫。     

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.     

An overview of heat stress in tomato (Solanum lycopersicum L.)

Heat stress has been defined as the rise of temperature for a period of time higher than a threshold level, thereby permanently affecting the plant growth and development. Day or night temperature is considered as the major limiting factor for plant growth. Earlier studies reported that night temperature is an important factor in the heat reaction of the plants. Tomato cultivars capable of setting viable fruits under night temperatures above 21 °C are considered as heat-tolerant cultivars. The development of breeding objectives is generally summarized in four points: (a) cultivars with higher yield, (b) disease resistant varieties in the 1970s, (c) long shelf-life in 1980s, and (d) nutritive and taste quality during 1990s. Some unique varieties like the dwarf “Micro-Tom”, and the first transgenic tomato (FlavrSavr) were developed through breeding; they were distributed late in the 1980s.High temperature significantly affects seed, pollen viability and root expansion. Researchers have employed different parameters to evaluate the tolerance to heat stress, including membrane thermo stability, floral characteristics (Stigma exertion and antheridia cone splitting), flower number, and fruit yield per plant. Reports on pollen viability and fruit set/plant under heat stress by comparing the pollen growth and tube development in heat-treated and non-heat-stressed conditions are available in literature. The electrical conductivity (EC) have been used to evaluate the tolerance of some tomato cultivars in vitro under heat stress conditions as an indication of cell damage due to electrolyte leakage; they classified the cultivars into three groups: (a) heat tolerant, (b) moderately heat tolerant, and (c) heat sensitive.It is important to determine the range in genetic diversity for heat tolerance in tomatoes. Heat stress experiments under field conditions offer breeders information to identify the potentially heat tolerant germplasm.     

小麦产量与品质对灌浆不同阶段高温胁迫的响应

利用人工环境控制室对盆栽冬小麦 (品种 :‘济南 17’和‘鲁麦 2 1’) (Triticumaestivumcv.‘Ji′nan17’and‘Lumai2 1’) 分别在子粒灌浆前期、中期和后期进行了 2 5℃ / 35℃ (夜 /昼 ) 的高温胁迫处理, 以生长在 2 0℃ / 30℃ (夜 /昼 ) 环境中的小麦为对照, 研究了灌浆期不同阶段高温胁迫对小麦产量和品质的影响。结果发现 :1) 子粒蛋白质积累速率在高温处理期间显著提高 (p<0.0 5 ), 但高值持续期缩短, 并最终造成植株氮素积累量减少, 氮素收获指数降低 (p <0.0 5 ) 。 2 ) 小麦蛋白质的组成和品质对不同灌浆阶段的响应存在显著差异, 前期高温胁迫导致麦谷蛋白 /醇溶蛋白的比值以及麦谷蛋白大聚合体 (GMP) 含量增加, 标志蛋白质和淀粉品质的湿面筋含量升高、沉降值增加、膨胀势和高峰粘度等指标也显著提高 ;灌浆中期高温却导致上述指标降低 ;灌浆后期高温在造成粒重减小、产量降低和淀粉品质下降的同时, 却有利于蛋白质含量的提高。 3) 小麦淀粉积累的形成与蛋白质品质的形成是两个既相互联系又相互独立的过程, 高温条件下子粒蛋白质含量的升高是淀粉积累量减少造成的。     

Nitrogen Modulates the Effects of Heat,Drought, and Combined Stresses on Photosynthesis,Antioxidant Capacity,Cell Osmoregulation,and Grain Yield in Winter Wheat

Longer and more intense heat and drought stresses will occur in terrestrial ecosystems in the future. Although the effects of individual heat or drought stress on wheat plants have been largely explored, the regulatory effect of nitrogen (N) on winter wheat under heat, drought, and combined stresses and whether N alleviates damage to wheat plants caused by these stresses remain unclear. Therefore, the objective of the present study was to investigate the growth, photosynthesis, antioxidant enzyme and N metabolism-related enzyme activity, cell membrane system, osmoregulatory substance, and yield responses to heat, drought, and combined stresses in wheat plants and to clarify the regulatory effects of N on the growth, physiological and biochemical characteristics, and yield of wheat plants under stress conditions. The results showed that wheat plant exposure to individual heat or drought stress reduced photosynthesis and N metabolism-related enzyme activities and increased antioxidant enzyme activities, electrolyte leakage (EL), and the contents of MDA (malondialdehyde) and O₂^? (superoxide anion). The above parameters showed typical superposition effects under combined stress. Under individual heat or drought stress, wheat plants treated with a medium (N₂) or high (N₃) N supply maintained higher photosynthesis and N metabolism-related enzyme activities than did those treated with a low N supply (N₁). Enhanced heat and drought tolerance in wheat plants under an appropriate N supply may be attributed to improved antioxidant capacity, as exemplified by increased activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione reductase (GR) and ascorbate peroxidase (APX), and to enhanced osmoregulation capacity, as signified by increased contents of soluble sugar (SS), soluble protein (SP), and proline (Pro). Variable importance in projection (VIP) analysis indicated that efficient SOD, POD, CAT, and GR activities and an increased Pro content had superior potential to alleviate heat, drought, and combined stress stresses in wheat plants, and the improvements in growth and grain yield in wheat plants further confirmed the oxidative stress alleviation and stress tolerance enhancement. However, positive effects of N on wheat growth and grain yield under combined stress were usually observed under a low N supply. These results may facilitate future research on the effects of N fertilizer on the stress resistance of winter wheat.

Graphical Abstract

     

Costs and benefits of hydroxamic acids-related resistance in winter wheat against the bird cherry-oat aphid, Rhopalosiphum padi L.

Estimations of infestation by the bird cherry-oat aphid (Rhopalosiphum padi) as well as measurements of grain yield in 26 Hungarian winter wheat cultivars under field conditions were correlated with the concentration of hydroxamic acids (Hx) in seedlings of those cultivars. The significant inverse relationship between infestation ratings and Hx levels in wheat showed that Hx, despite their decreased accumulation at later plant phenological stages, may be able to confer resistance against aphid infestation in the field. Since no significant relationship was found between grain yield and Hx levels in plants it is suggested that Hx accumulation does not impose a cost to the plant in terms of yield. These findings support earlier claims stressing the potential of Hx as breeding targets for aphid resistance in wheat.     

Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.

The protein synthesis elongation factor, EF-Tu, is a protein that carries aminoacyl-tRNA to the A-site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tolerance, and it has been hypothesized that EF-Tu confers heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation and inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate synthase and malate dehydrogenase. The recombinant pre-EF-Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF-Tu. The purified protein was capable of binding GDP (indicative of protein activity) and was stable at 45 degrees C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre-EF-Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre-EF-Tu protein. The results of this study support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation.     

Low temperature-wheat-fungal interactions: A carbohydrate connection

Winter annual and perennial crop species grown in the northern boreal ecosystem must survive periods of protracted snow cover and low temperatures during the winter. In deep snow regions, plants are susceptible to winter stresses caused by both snow molds and low temperatures. Therefore, high levels of tolerance to freezing and snow molds are requisite for crops adapted to these regions. Accumulation of soluble carbohydrates in winter wheat during the autumn is linked to both hardening and resistance to attack by snow molds. Snow mold-resistant cultivars accumulate higher levels of carbohydrate and metabolize them at slower rates than susceptible cultivars. The quantity and quality of carbohydrates, particularly fructans, remaining in the spring after snow mold attack appear important for survival of winter wheat. However, the total accumulation of carbohydrates is dependent on the stage of development of the winter cereal plant at the beginning of the winter. Recent research findings have shown that sugars are pivotal metabolic activators of the sugar-sensing enzyme, hexokinase, which initiates signal transduction and activation of numerous metabolic genes including host defense genes. Thus, an understanding of the metabolism of soluble carbohydrates, particularly fructans, during plant growth, hardening, and snow mold infection, is essential to the elucidation of survival mechanisms in plants subjected to these winter stresses.     

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.     

Cadmium uptake and bioaccumulation in selected cultivars of durum wheat and flax as affected by soil type

Accumulation of cadmium (Cd) in crop plants is of great concern due to the potential for food chain contamination through the soil-root interface. Although Cd uptake varies considerably with plant species, the processes which determine the accumulation of Cd in plant tissues are affected by soil factors. The influence of soil type on Cd uptake by durum wheat (Triticum turgidum var. durum L.) and flax (Linum usitatissimum L.) was studied in a pot experiment under environmentally controlled growth chamber conditions. Four cultivars/lines of durum wheat (Kyle, Sceptre, DT 627, and DT 637) and three cultivars/lines of flax (Flanders, AC Emerson, and YSED 2) were grown in two Saskatchewan soils: an Orthic Gray Luvisol (low background Cd concentration; total/ABDTPA extractable Cd: 0.12/0.03 mg kg^-1, respectively) and a Dark Brown Chernozem (relatively high background Cd concentration; total/ABDTPA Cd: 0.34/0.17 mg kg^-1 respectively). Plant roots, stems, newly developed heads, and grain/seeds were analyzed for Cd concentration at three stages of plant growth: two and seven weeks after germination, and at plant maturity. The results showed that Cd bioaccumulation and distribution within the plants were strongly affected by both soil type and plant cultivar/line. The Cd concentration in roots leaves and stems varied at different stages of plant growth. However, all cultivars of both plant species grown in the Chernozemic soil accumulated more Cd in grain/seeds than plants grown in the Orthic Gray Luvisol soil. The different Cd accumulation pattern also corresponded to the levels of ABDTPA extractable and metal-organic complex bound soil Cd found in both soils. Large differences were found in grain Cd among the durum wheat cultivars grown in the same soil type, suggesting the importance of rhizosphere processes in Cd bioaccumulation and/or Cd transport processes within the plant. Distribution of Cd in parts of mature plants showed that durum grain contained up to 21 and 36% of the total amount of Cd taken up by the plants for the Orthic Gray Luvisol and Chernozemic soils, respectively. These results indicate the importance of studying Cd speciation, bioaccumulation and cycling in the environment for the management of agricultural soils and crops.