Characterization and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis thaliana

We have characterized cDNAs for two new dhn/lea/rab (dehydrin, late embryogenesis-abundant, responsive to ABA)-related genes from Arabidopsis thaliana. The two genes were strongly induced in plants exposed to low temperature (4 °C) and were accordingly designated lti45 and lti30 (low temperature-induced). The lti45 gene product contains the conserved serine stretch and three lysine-rich repeats characteristic of DHN/LEA/RAB proteins and is very similar to another low temperature-responsive protein of A. thaliana, COR47 [17]. Both proteins have the same repeat structure and an overall amino acid identity of 64%. This structural similarity of the proteins and the tandem array of the genes suggest that this gene pair arose through a duplication. The other polypeptide, LTI30, consists of several lysine-rich repeats, a structure found in CAP85, a low temperature-and water stress-responsive protein in spinach [41] and similar proteins found in wheat [20].The expression pattern of the five dhn/lea/rab-related genes (cor47, dhnX, lti30, lti45 and rab18) identified so far in A. thaliana, was characterized in plants exposed to low temperature, drought and abscisic acid (ABA). Expression of both lti30 and lti45 was mainly responsive to low temperature similar to cor47. The lti45 and lti30 genes show only a weak response to ABA in contrast to cor47, which is moderately induced by this hormone. The three genes were also induced in severely water-stressed plants although the expression of lti30 and lti45 was rather low. In contrast to these mainly low temperature-induced genes, the expression of rab18 was strongly induced both in water-stressed and ABA-treated plants but was only slightly responsive to cold. The dhnX gene showed a very different expression pattern. It was not induced with any of the treatments tested but exhibited a significant constitutive expression. The low-temperature induction of the genes in the first group, lti30 and lti45, is ABA-independent, deduced from experiments with the ABA-deficient (aba-1) and ABA-insensitive (abi1) mutants of A. thaliana, whereas the induction of rab18 is ABA-mediated. The expression of dhnX was not significantly affected in the ABA mutants.     

Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh.

A cDNA clone corresponding to a novel low-temperature-induced Arabidopsis thaliana gene, named lti140, was employed for studies of the environmental signals and the signal pathways involved in cold-induced gene expression. The single-copy lti140 gene encodes a 140 kDa cold acclimation-related polypeptide. The lti140 mRNA accumulates rapidly in both leaves and roots when plants are subject to low temperature or water stress or are treated with the plant hormone abscisic acid (ABA), but not by heat-shock treatment. The low-temperature induction of lti140 is not mediated by ABA, as shown by normal induction of the lti140 mRNA in both ABA-deficient and ABA-insensitive mutants and after treatment with the ABA biosynthesis inhibitor fluridone. The effects of low temperature and exogenously added ABA are not cumulative suggesting that these two pathways converge. The induction by ABA is abolished in the ABA-insensitive mutant abi-1 indicating that the abi-1 mutation defines a component in the ABA response pathway. Accumulation of the lti140 mRNA in plants exposed to water stress was somewhat reduced by treatment with fluridone and in the ABA-insensitive mutant abi-1 suggesting that the water stress induction of lti140 could be partly mediated by ABA. It is concluded that three separate but converging signal pathways regulate the expression of the lti140 gene.     

Purification of chloroplast elongation factor Tu and cDNA analysis in tobacco: the existence of two chloroplast elongation factor Tu species

We have purified a chloroplast elongation factor Tu (EF-Tu) from tobacco (Nicotiana tabacum) and determined its N-terminal amino acid sequence. Two distinct cDNAs encoding EF-Tu were isolated from a leaf cDNA library of N. sylvestris (the female progenitor of N. tabacum) using an oligonucleotide probe based on the EF-Tu protein sequence. The cDNA sequence and genomic Southern analyses revealed that tobacco chloroplast EF-Tu is encoded by two distinct genes in the nuclear genome of N. sylvestris. We designated the corresponding gene products EF-Tu A and B. The mature polypeptides of EF-Tu A and B are 408 amino acids long and share 95.3% amino acid identity. They show 75–78% amino acid identity with cyanobacterial and chloroplast-encoded EF-Tu species.     

西葫芦NCED基因家族鉴定及其响应干旱胁迫分析

NCED基因家族成员在调节植物响应干旱胁迫中发挥着关键作用,该研究通过生物信息学技术分析NCED在西葫芦基因组中的分布、结构及进化,研究家族成员在不同组织中的表达特异性及其对10%PEG 6000模拟干旱、0.1 mmol·L-1ABA激素和自然干旱胁迫的响应,以解析NCED基因家族的生物学功能。结果表明:(1)从西葫芦全基因组中鉴定出6个NCED家族基因(CpNCED1~6),且6个基因均不含内含子、分别分布于西葫芦的1、10、12、14、19和20号共6条染色体上。(2)理化性质分析发现,CpNCED1~6蛋白长度为569~590 aa,理论分子量在62.64~65.54 kD之间。(3)蛋白保守元件分析显示,除CpNCED3蛋白在遗传进化过程中出现3个基序(motif 12、motif 13和motif 15)的缺失外,其余5个蛋白都有完整的16个motif保守基序,且分布在600个氨基酸以内,同时大部分NCED蛋白序列保守性较高。(4)顺式作用元件分析显示,西葫芦CpNCED1~6基因均含ABRE、W box、MBS、P-box、TCA-element、CGTCA-motif、TGA-element和TGA-box等潜在的干旱胁迫响应元件。(5)qRT-PCR分析表明,CpNCED1~6基因在西葫芦不同组织中的表达具有组织特异性,其中,CpNCED4和CpNCED1在茎中的表达量显著高于其他4个基因,CpNCED2、CpNCED4、CpNCED6在花中的表达显著高于其余3个基因且CpNCED2表达量最高,CpNCED1~6在果实和叶中的表达量均相对较低;与对照组相比,CpNCED1~6受模拟干旱、ABA激素和自然干旱胁迫均上调表达;伴随干旱胁迫的产生,叶片中脱落酸(ABA)含量逐渐升高,暗示CpNCEDs在西葫芦干旱胁迫响应与ABA的生物合成过程中发挥着正向调控作用。研究发现,6个CpNCED1~6基因与西葫芦干旱胁迫响应密切相关,且对西葫芦干旱胁迫的响应以及ABA生物合成具有重要作用,尤其以CpNCED2和CpNCED4基因的作用更为明显。     

cDNA cloning and characterization of a new stress-responsive gene BoRS1 from Brassica oleracea var. acephala

A new stress-responsive gene BoRS1 (GenBank accession No. AY373021 ) was isolated from Brassica oleracea var. acephala by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoRS1 was 2076 bp and contained a 1851 bp open reading frame (ORF) encoding 617 amino acids. Sequence analysis indicated that the deduced BoRS1 shared some identities with LTI65 , RD29A, RD29B and COR78 from Arabidopsis thaliana . Southern blot analysis of genomic DNA indicated that other related genes existed and there were two copies of BoRS1 in the genome of B. oleracea . Northern blot analysis revealed that BoRS1 was up-regulated by cold, mannitol, NaCl and abscisic acid (ABA). Expressional fluctuation of time course with ABA implied a two-step induction process. Tissue-specific expression analysis indicated that BoRS1 was expressed in all the tested plant tissues including leaves, stems and roots. Our studies imply that BoRS1 is a new gene that is responsive to environmental stresses such as low temperature, salinity and osmotic stress.     

Isolation and characterization of a water-stress-inducible cDNA clone from Solanum chacoense

A rich source of valuable genes are wild species. Solanum chacoense Bitter with its extreme resistance to viruses, insects and drought, is a good example.In the present study, a stress gene, designated DS2, has been isolated from S. chacoense. We have shown that the expression of the gene is organ-specific being detected in leaf, stem and stolon, but not in root, tuber or flower. Treatment of detached leaves with abscisic acid (ABA), salicylic acid or methyl jasmonate resulted in only very moderate accumulation of DS2 mRNA. Thus, DS2 represents a very rare type of the water-stress-inducible genes whose signalling pathway is not primarily related to ABA.Based on DNA sequence analysis, DS2 encodes a putative protein starting with 20 amino acids homologous to the ABA- and water-stress-inducible, ripening-related (ASR) proteins of tomato continued by an insert of 155 amino acids structurally similar to certain LEAs (late embryogenesis-abundant proteins) and ending in 88 amino acids homologous again to the ASR sequences and to an unpublished partial cDNA fragment isolated from the root of rice. The N-terminal region of the DS2 protein is hydrophilic with ten 13-mer amino acid motifs and random coil structure. In contrast, the C-terminus predicts an -helix and possesses a bipartite nuclear targeting sequence motif. These data suggest that the function of the DS2 may be the protection of the nuclear DNA from desiccation.     

Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins,a family of putative kinase regulators

We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.     

青杄转录因子PwNAC30及其启动子序列的克隆与表达分析

该研究以青杄(Picea wilsonii)为实验材料,通过PCR从青杄的cDNA文库中克隆得到一个NAC转录因子,命名为PwNAC30。生物信息学分析显示,PwNAC30开放阅读框1 179bp,共编码392个氨基酸,在其N端存在保守的NAM(no apical meristem)结构域,可分为A～E等5个亚结构域。多序列对比和系统进化树分析显示,PwNAC30蛋白与同为云杉属的北美云杉(Picea sitchensis)聚为一类。启动子克隆分析显示,PwNAC30基因启动子上存在脱落酸(ABA)、赤霉素(GA)、茉莉酸甲酯(MeJA)、TC-rich repeats等激素和逆境响应元件,在GA、ABA、MeJA、低温、干旱、盐的处理下,其启动子活性均明显增强。荧光定量PCR分析表明,PwNAC30在球果中的表达量最高,而在花粉和种子中的表达量最低。PwNAC30对于盐、干旱、低温、ABA、MeJA、GA处理均有响应,尤其对盐、干旱、MeJA的响应最为显著。亚细胞定位结果显示,PwNAC30蛋白定位于细胞核与细胞质,主要定位于细胞核中。酵母单杂及双杂结果表明,PwNAC30蛋白的全长和N端没有转录激活活性,而C端有转录激活活性,且PwNAC30自身能形成同源二聚体。研究表明,青杄PwNAC30基因可以作为一个转录因子发挥作用,其转录激活活性在C端,且自身能够形成同源二聚体结构;PwNAC30基因广泛参与了ABA、GA、MeJA等激素的信号通路,并对盐、干旱、低温处理有响应。     

辣椒CaWRKY14转录因子的生物学特性研究

WRKY转录因子是植物响应病原菌胁迫最重要的转录因子之一,且参与抗病反应及信号传导通路的调控。为研究辣椒WRKY基因的生物学特征,以辣椒高抗疫病材料CM334为试材,克隆获得响应疫霉菌诱导的转录因子CaWRKY14。生物信息学分析表明,该基因DNA全长2 530 bp,cDNA全长1 662 bp,含有5个内含子,编码553个氨基酸,含有1个WRKY保守结构域,属于Group Ⅱ(b)。实时荧光定量表达分析表明,CaWRKY14不仅受ABA和疫霉菌胁迫诱导表达,且表达量分别在12 h和24 h时达到峰值,分别是对照的8.54和8.04倍,同时也受高盐、热激和干旱胁迫诱导。利用VIGS技术对CaWRKY14转录因子进行沉默后发现,抗病材料CM334接种疫霉菌后趋于发病。研究表明,CaWRKY14基因在辣椒响应疫霉菌胁迫进程中可能发挥着重要作用。     

Pea dehydrins: identification,characterisation and expression

An antiserum raised against dehydrin from maize (Zea mays) recognised several polypeptides in extracts of pea (Pisum sativum) cotyledons. A cDNA expression library was prepared from mRNA of developing cotyledons, screened with the antiserum and positive clones were purified and characterised. The nucleotide sequence of one such clone, pPsB12, contained an open reading frame which would encode a polypeptide with regions of significant amino acid sequence similarity to dehydrins from other plant species.The deduced amino acid sequence of the pea dehydrin encoded by B12 is 197 amino acids in length, has a high glycine content (25.9%), lacks tryptophan and is highly hydrophilic. The polypeptide has an estimated molecular mass of 20.4 kDa and pI=6.4. An in vitro synthesised product from the clone comigrates with one of the in vivo proteins recognised by the antiserum.A comparison of the pea dehydrin sequence with sequences from other species revealed conserved amino acid regions: an N-terminal DEYGNP and a lysine-rich block (KIKEKLPG), both of which are present in two copies. Unexpectedly, pea dehydrin lacks a stretch of serine residues which is conserved in other dehydrins.B12 mRNA and dehydrin proteins accumulated in dehydration-stressed seedlings, associated with elevated levels of endogenous abscisic acid (ABA). Applied ABA induced expression of dehydrins in unstressed seedlings. Dehydrin expression was rapidly reversed when seedlings were removed from the stress or from treatment with ABA and placed in water.During pea cotyledon development, dehydrin mRNA and proteins accumulated in mid to late embryogenesis. Dehydrin proteins were some of the most actively synthesised at about the time of maximum fresh weight and represent about 2% of protein in mature cotyledons.     

Isolation and characterization of a novel dehydrin gene from Capsella bursa-pastoris

A novel dehydrin gene designated as Cbcor29 was cloned from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE) and genome walker technique. The full-length cDNA of Cbcor29 was 1101 bp long with a 783 bp open reading frame (ORF), encoding a putative protein of 261 amino acids. Like other dehydrin proteins, CbCOR29 contained a high percentage of charged and polar amino acids, in which Cys and Trp amino acids were absent. In addition, the predicted CbCOR29 protein possesses three conserved repeats of the characterized Lys-rich domains (K-segments), and a Ser-rich domain (S-segment) prior to the first Lys-rich domain, which presented a typical SK3 structure of dehydrins. Analysis of Cbcor29 genomic DNA revealed that it contains 2 exons and 1 intron, which is a typical character of dehydrin genes. Subsequent bioinformatic analysis also showed that the sequence of CbCOR29 has high homology with other dehydrin proteins, especially with cor47 from Arabidopsis thaliana. Moreover, semi-quantitative RT-PCR revealed that the expression of Cbcor29 can be induced by exposure to drought, low temperature, NaCl, and exogenous ABA treatment. Our study led to the conclusion that the Cbcor29 gene is a new member of the dehydrin gene family and might exert functions in responsiveness to drought, cold, and salt in Capsella bursa-pastoris. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 1, pp. 52–60. The article was submitted by the authors in English.     

Two genes that encode Ca2+-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana

Two cDNA clones, AATCDPK1 and cATCDPK2, encoding Ca²⁺-dependent, calmodulin-independent protein kinases (CDPK) were cloned from Arabidopsis thaliana and their nucleotide sequences were determined. Northern blot analysis indicated that the mRNAs corresponding to the ATCDPK1 and ATCDPK2 genes are rapidly induced by drought and high-salt stress but not by low-temperature stress or heat stress. Treatment of Arabidopsis plants with exogenous abscisic acid (ABA) had no effect on the induction of ATCDPK1 or ATCDPK2. These findings suggest that a change in the osmotic potential of the environment can serve as a trigger for the induction of ATCDPK1 and ATCDPK2. Putative proteins encoded by ATCDPK1 and ATCDPK2 which contain open reading frames of 1479 and 1488 bp, respectively, are designated ATCDPK1 and ATCDPK2 and show 52% identity at the amino acid sequence level. ATCDPK1 and ATCDPK2 exhibit significant similarity to a soybean CDPK (51 % and 73%, respectively). Both proteins contain a catalytic domain that is typical of serine/threonine protein kinases and a regulatory domain that is homologous to the Ca²⁺-binding sites of calmodulin. Genomic Southern blot analysis suggests the existence of a few additional genes that are related to ATCDPK1 and ATCDPK2 in the Arabidopsis genome. The ATCDPK2 protein expressed in Escherichia coli was found to phosphorylate casein and myelin basic protein preferentially, relative to a histone substrate, and required Ca²⁺ for activation.     

辣椒CaNAC55基因克隆与表达分析

为揭示辣椒NAC转录因子的功能,以高抗疫病辣椒CM334为试验材料,克隆获得CaNAC55基因全长gDNA和cDNA序列。生物信息学分析表明,CaNAC55基因gDNA全长4 164 bp, cDNA完整开放阅读框(ORF)为1 299 bp,基因编码的蛋白由432个氨基酸残基组成;基因序列比对和同源性分析结果表明,CaNAC55与辣椒(XM-016722474)、番茄(XM-004241285)和马铃薯(XM-006361027)的亲缘关系最近,氨基酸相似度分别达到99.87%、93.37%和92.62%。实时荧光定量分析表明,干旱、高盐、热激处理均可诱导CaNAC55基因表达,其中干旱、高盐、热激处理分别在24 h、24 h和12 h时表达量达到峰值,且分别为对照的3.01倍、20.92倍和8.84倍;ABA处理下,CaNAC55基因的相对表达量显著低于对照,说明CaNAC55基因的表达受到ABA的抑制。研究表明,辣椒CaNAC55转录因子对不同逆境胁迫的响应不同,推测辣椒CaNAC55基因可能作为重要的调节因子参与逆境胁迫响应。     

Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts

Using the expression vector gt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112–114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4–12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.     

Abscisic acid causes changes in gene expression involved in the induction of the landform of the liverwortRiccia fluitans L.

The conversion of the submerged form ofRiccia fluitans to the landform either by transfer to a moist solid surface or by treatment with abscisic acid (ABA), is accompanied by the formation of a set of new polypeptides and concomitant down-regulation of other polypeptides. Changes in gene expression were analyzed by twodimensional separations of proteins and differential screening of a cDNA library. One of the landform-specific proteins might depend on the expression of the newly discoveredRic 1 gene. The deduced amino acid sequence of the isolatedRic 1 cDNA clone codes for a protein with a molecular mass of 30.1 kDa. This polypeptide possesses two amino acid sequences which are repeated five times each and it is largely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Under ABA treatment the expression of theRic 1 mRNA had already reached its maximum after 1 h of incubation. Transferring submerged thalli onto an agar surface resulted in a slower induction. TheRic 1 gene product shows homology to an embryo-specific polypeptide of carrot seeds and to the group 3 of late-embryogenesis-abundant (LEA) proteins. Interestingly, ABA treatment improved the desiccation tolerance of the submerged thalli. Additionally, ABA stimulated the synthesis of a protein which is immunologically related to a tonoplast protein. This finding, together with the fact that the ABA-induced landform exhibits an increased activity of several vacuolar enzymes, may indicate a special role of the tonoplast and the vacuole during ABA-induced conversion of the thallus from the submerged to the terrestrial form.Abbreviations ABA abscisic acid - 2-D PAGE two-dimensional polyacrylamide gel electrophoresis - F_o, F_m initial and maximal chlorophylla fluorescence - LEA late embryogenesis abundant - Q _a primary quinone acceptor of PS II We are grateful to the Deutsche Forschungsgemeinschaft for financial support SFB 251, SFB 176 to Prof. O.H. Volk (Institut für Biowissenschaften, Universität Würzburg, Germany) for generous supply ofRiccia fluitans thalli and many useful discussions and to Mrs B. Arbinger and Mrs B. Dierich for skilful technical assistance.