Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat

Expression of the acidic dehydrin gene wcor410 was found to be associated with the development of freezing tolerance in several Gramineae species. This gene is part of a family of three homologous members, wcor410, wcor410b, and wcor410c, that have been mapped to the long arms of the homologous group 6 chromosomes of hexaploid wheat. To gain insight into the function of this gene family, antibodies were raised against the WCOR410 protein and affinity purified to eliminate cross-reactivity with the WCS120 dehydrin-like protein of wheat. Protein gel blot analyses showed that the accumulation of WCOR410 proteins correlates well with the capacity of each cultivar to cold acclimate and develop freezing tolerance. Immunoelectron microscope analyses revealed that these proteins accumulate in the vicinity of the plasma membrane of cells in the sensitive vascular transition area where freeze-induced dehydration is likely to be more severe. Biochemical fractionation experiments indicated that WCOR410 is a peripheral protein and not an integral membrane protein. These results provide direct evidence that a subtype of the dehydrin family accumulates near the plasma membrane. The properties, abundance, and localization of these proteins suggest that they are involved in the cryoprotection of the plasma membrane against freezing or dehydration stress. We propose that WCOR410 plays a role in preventing the destabilization of the plasma membrane that occurs during dehydrative conditions.     

Overexpression of the acidic dehydrin WCOR410 improves freezing tolerance in transgenic strawberry leaves

Progress in freezing tolerance (FT) improvement through plant breeding approaches has met with little success in the last 50 years. Engineering plants for greater FT through plant transformation is one possible way to reduce the damage caused by freezing. Here, we report an improvement of the selection procedure and the transfer of the wheat Wcor410a acidic dehydrin gene in strawberry. The encoded protein has previously been shown to be associated with the plasma membrane, and its level of accumulation has been correlated with the degree of FT in different wheat genotypes. The WCOR410 protein was expressed in transgenic strawberry at a level comparable with that in cold-acclimated wheat. Freezing tests showed that cold-acclimated transgenic strawberry leaves had a 5 degrees C improvement of FT over wild-type or transformed leaves not expressing the WCOR410 protein. However, no difference in FT was found between the different plants under non-acclimated conditions, suggesting that the WCOR410 protein needs to be activated by another factor induced during cold acclimation. These data demonstrate that the WCOR410 protein prevents membrane injury and greatly improves FT in leaves of transgenic strawberry. A better understanding of the limiting factors allowing its activation may open up the way for engineering FT in different plant organs, and may find applications for the cryopreservation of human tissues and organs.     

干旱胁迫下耐旱性小麦在不同生育期脱水素的表达分析

以2种耐旱性不同的盆栽小麦陕合6号(干旱耐受型)和郑引1号(干旱敏感型)为材料,分别在其苗期、分蘖期、拔节期、开花期对土壤实施不同程度的自然干旱胁迫和复水处理,采用SDS-PAGE和Western blotting技术研究其叶片脱水素的表达规律,探究小麦整个生长期脱水素的表达与干旱胁迫的关系.结果表明:2种小麦的脱水素均仅在干旱胁迫时表达,其中45 kD和37 kD的脱水素在2种小麦的4个发育期的叶片中均有表达,28 kD的脱水素仅在特定发育时期表达.在干旱耐受型小麦(陕合6号)中,脱水素在胁迫初期少量表达,随着胁迫程度加剧表达量急剧增加,在重度干旱胁迫下达到峰值,复水后小麦叶片中脱水素含量迅速下降;在干旱敏感型小麦(郑引1号)中,脱水素在胁迫初期大量表达,中度胁迫表达量小幅度回落,到复水1 d达到峰值,此后随着复水时间增加小麦叶片中脱水素的量逐渐下降.研究表明,小麦叶片脱水素表达与干旱胁迫程度和生育期迫密切相关,不同耐旱型小麦材料中叶片脱水素表达的差异与品种之间的干旱耐受能力密切相关.     

Characterization of an abiotic stress-inducible dehydrin gene, OsDhn1, in rice (Oryza sativa L.)

A full-length 1.1 kb cDNA, designated Oryza sativa Dehydrin 1 (OsDhn1), was isolated from the seed coat of rice. The deduced protein is hydrophilic and has three K-type and one S-type motifs (SK3-type), indicating that OsDhn1 belongs to the acidic dehydrin family, which includes wheat WCOR410 and Arabidopsis COR47. Expression of OsDhn1 was strongly induced by low temperature as well as by drought. Induction of OsDhn1 by cold stress was clearcut in the roots of seedlings and the epidermis of palea and lemma. OsDhn1 was also up-regulated in UBI::CBF1/DREB1b transgenic plants indicating that it is regulated by the CBF/DREB stress signaling pathway.     

Differential accumulation of a 24-kd dehydrin protein in wheat seedlings correlates with drought stress tolerance at grain filling

The Pacific Northwest (PNW), an important region for wheat production in the USA, is often subject to water deficits during sowing and grain filling. These deficits reduce the quality and yield of the crop. As a consequence, an important objective of breeding programs in the region is improving the genetic adaptation of wheat cultivars to drought stress. One response to dehydrative stresses is the accumulation of proteins called dehydrins, which are believed to protect membranes and macromolecules against denaturation. We characterized dehydrin accumulation in seedlings during drought stress and its correlation with stress tolerance during grain filling in seven wheat cultivars, 'Connie', 'Gene', 'TAM105', 'Rod', 'Hiller', 'Rhode', and 'Stephens'. A 24-kd dehydrin accumulated in seedlings under stress, but not in irrigated control plants. Connie, TAM105, and Gene started to accumulate dehydrins at the fourth day of stress, while the other cultivars showed dehydrins after twelve days of stress. This differential accumulation in seedlings was associated with stress tolerance at grain filling, characterized by a lower reduction in yield and in the rate of decrease in leaf water potential per day of stress. Connie, TAM105, Gene and Rod where the most tolerant cultivars. The results indicate that expression of this 24-kd dehydrin might serve as a rapid and non-destructive screening technique at the seedling stage. Even though the results are promising, selection experiments using a population segregating for stress tolerance are needed to test more conclusively whether this dehydrin can serve as a genetic marker for cultivars with tolerance to drought stress.     

水分胁迫下内生真菌球毛壳ND35对冬小麦苗期生长和抗旱性的影响

为明确不同水分条件下内生真菌对冬小麦苗期生长和抗旱性的影响,以抗旱型小麦品种山农16和水分敏感型小麦品种山农22为材料,利用荧光定量PCR技术检测小麦干旱诱导基因脱水素wzy2的表达量来了解冬小麦在干旱胁迫下相关基因的表达差异,通过测定相关生理指标与酶活性来判断小麦发育及其在干旱胁迫下的生理响应状况。结果表明,与正常水分ND35组相比,接种球毛壳菌(Chaetomium globosum)ND35的干旱处理组小麦的根冠比、总蛋白含量、脯氨酸含量及丙二醛含量等指标显著提高,小麦叶片含水量和可溶性糖含量有所降低。在干旱处理组中,球毛壳菌ND35可以显著提高小麦山农16的根长和山农22的株高,接种球毛壳ND35的山农16脯氨酸含量、可溶性糖含量、过氧化氢酶活性比对照组均显著提高,丙二醛含量比对照组降低9.0%,但差异不显著;山农22脯氨酸含量和过氧化氢酶活性比对照组显著提高,丙二醛含量和可溶性糖含量比对照组有所降低,但可溶性糖含量差异不显著;相对定量检测数据显示,接种球毛壳ND35后,两种小麦脱水素wzy2基因的表达量较对照组均能够显著提高。综合分析说明内生真菌球毛壳ND35可以促进冬小麦苗期根系和植株发育,小麦提前进入三叶期,增强小麦避旱性,同时提高小麦根系活力,增强小麦耐旱性;提高个体细胞内水分、糖分、脯氨酸含量,降低丙二醛的氧化性损伤,增强过氧化氢酶活性,从而提高两种冬小麦对干旱胁迫的耐受能力;球毛壳ND35促进小麦干旱诱导相关基因wzy2的表达量,进而提高抗旱相关蛋白的表达,从而提高两种冬小麦耐脱水性和对干旱胁迫的适应性。     

Low temperature promotes intron retention in two <Emphasis Type="Italic">e-cor</Emphasis> genes of durum wheat

Following the screening of a suppression subtractive library developed from durum wheat plants exposed to low temperature for 6 h, two early cold-regulated (e-cor) genes have been isolated. These genes, coding putatively for a ribokinase (7H8) and a C3H2C3 RING-finger protein (6G2), were characterized by the stress-induced retention of a subset of introns in the mature mRNA. This feature was dependent on cold for 7H8 and on cold and dehydration for 6G2. When other genes, such as the stress-related gene WCOR410c, coding for a dehydrin (one intron), or a gene coding for a putative ATP binding cassette transporter (16 introns) were analyzed, no cold-dependent intron retention was observed. Cold-induced intron retention was not observed in mutants defective in the chloroplast development; nevertheless treatment with cycloheximide in the absence of cold was able to promote intron retention for the 7H8 e-cor gene. These results suggest that the cold-induced intron retention reflects the response of the spliceosoma to specific environmental signals transduced to the splicing protein factors through a chloroplast-dependent pathway. Notably, when the 7H8 Arabidopsis orthologous gene was analyzed, no stress induction in terms of mRNA abundance and no cold-dependent intron retention was detected. Otherwise, 6G2 Arabidopsis homologous sequences sharing the same genomic structure of the durum wheat 6G2 showed a similar intron retention event although not strictly dependent on stress.Electronic Supplementary Material Supplementary material is available for this article at     

Generation and analysis of drought stressed subtracted expressed sequence tags from safflower (Carthamus tinctorius L.)

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of?~1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78?%) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22?%) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance are discussed in our study.     

Preliminary screening for water stress tolerance and genetic diversity in wheat (Triticum aestivum L.) cultivars from Sudan

The present study was carried out to screen 12 Sudanese wheat (Triticum aestivum L.) cultivars for their response to water stress at early germination stages and to characterize sources that could be used in breeding programs to develop wheat cultivars with better adaptation to water stress. The effect of osmotic stress on the early growing stages was evaluated, in vitro, using five concentrations of Polyethylene glycol. Genetic diversity was studied using 24 allele specific simple sequence repeats (SSR) markers associated with drought tolerance in wheat. The presence of the drought genes and their chromosomal location was also investigated by isolating and sequencing the dehydration responsive element binding protein (dreb1). Results of the in vitro screening among the cultivars showed significant differences in the root length, shoot length and root/shoot ratio. The 24 drought specific SSR markers used revealed 50 alleles, with an average of 2.0 alleles per locus. Of these, 60% were polymorphic with polymorphism information content (PIC) ranging from 0.16 to 0.89. A dendrogram based on the similarity values generated from the SSR data revealed three major clusters. Of the five specific primers for dreb1 genes, only primer P25F/PR produced amplification products with the expected fragment sizes. Sequencing and BLAST results of the cloned fragments excised from the gels showed 99% homology to the dreb1 gene on chromosome 3A.