| Biotic and abiotic stresses severely affect quantity and quality of crop product. The plant-specific NAC (for NAM, ATAF1,2 and CUC2) transcription factors, which have been found playing important roles in plant growth, development and stress responses, are taken as good genetic resource for the improvement of crop tolerance to environmental stresses. Previous researches about NAC proteins mainly focused on a few model plants such as Arabidopsis and rice. In view of functional difference of orthologues from different species, further investigation on the functions of NAC genes from other plant species will be helpful to understand the common and special molecular mechanisms of plant development and stress responses. Chickpea(Cicer arietinum L.) is the third important legume crop in terms of cultivation area. It is an annual plant with a short life, a small genome size and strong resistance to biotic and abiotic stresses. And chickpea has been suggested as a model plant for investigation of physiological mechanisms of plant development and responses to stresses. However, chickpea is scarcely studied in the molecular biology, especially in china. To build foundation for molecular biology research and to identify the stress responsive genes, we have constructed a drought-related cDNA library using the stress resistance germplasm from Sinkiang. Six expressed sequence tags (EST) of NAC genes, an EST of actin gene and an contig of GPRP (glycine-and proline-rich protein) gene were found in chickpea cDNA library. Since NAC proteins have important physiology function and no NAC gene is cloned in chickpea, we isolated and identified six NAC genes based on these ESTs. Furthermore, expression patterns of these NAC genes were globally investigated in various developmental processes and under diverse stresses. And function of three NAC genes were analyzed using transgenic Arabidopsis. In view of no reference gene for expression analysis in chickpea, we also isolated an actin gene and estimated usability of this gene for normalization purposes. In addition, a GPRP gene also was cloned and characterized functionally. Main results of this study are as follows.CARNAC1～6 (Cicer arietinum NAC gene) genes were obtained using rapid amplification of cDNA end (RACE) technique and their cDNA length were 753,706,927,1108,987 and 921bp, respectively. The putative corresponding proteins were composed of 239,191,285, 339,291 and 307 amino acids, respectively. Genomic DNA sequences of all CarNAC genes contained two location-conserved introns with different length. Multiple sequences alignment revealed that the N-terminus of CARNAC1～6 proteins had conserved NAC domain and the C-terminus represented a more variable region. Based on the DNA gel blot analysis, all CarNAC genes were found to be either single or low copy number in the chickpea genome. All CarNAC:GFP fusion proteins were localized in the nucleus of onion epidemical cells by subcellular localization assay. Moreover, the trans-activation activity CARNAC1～6 proteins were proved to be located in the C-terminal region by trans-activation activity analysis in yeast. These data suggested that CARNAC1～6 were typical NAC genes and the corresponding proteins probably were transcriptional activators in chickpea.Expression patterns of CARNAC～6 genes were globally investigated in various developmental processes and under diverse stress and chemical treatments. All CarNAC genes had different tissue-specific expression profiles except for CarNAC3 and CarNAC4 genes. Expression of CarNAC1,3 genes was strongly induced by leaf age, whereas expression of CarNAC6 gene was significantly inhibited. Meanwhile, CARNAC1～6 showed differential expression patterns with cooperativity during seed development and germination. CARNAC1～6 showed differential expression patterns with cooperativity during seed development and germination, indicating that the physiological functions of CARNAC1～6 genes are different as well as mutually complementary. Transcription of CARNAC1～6 genes was enhanced by drought treatment in different degree at different time points. Moreover, four (CarNAC1, 4-6), two (CarNAC4,5), three (CarNAC1,4,6) and two (CarNAC1,5) NAC genes were significantly induced by high salinity, high temperature (37℃), low temperature (4℃) and mechanical wound, respectively. Additionally, four (CARNAC2～4,6), one (CarNAC4), four (CarNAC1,3,4,6), four (CarNAC1,3-5), five (CarNAC1,3-6), three (CarNAC1,4,6) and four (CarNAC1,3,4,6) NAC genes were significantly up-regulated by abscisic acid (ABA), methyl jasmonate (MeJA), ethephon (Et), salicylic acid (SA), indole-3-acetic acid (IAA), gibberellin (GA3) and H2O2, respectively, whereas two genes (CarNAC3,6) were down-regulated by N-6-benzyl-adenine (6-BA) treatment. These findings suggested that functions of all six CarNAC transcription factors were involved in plant growth and development regulation as well as environmental stress responses. Transgenic Arabidopsis plants expressing CarNAC2 gene showed some abnormalities such as germination delay, shorter stem of seedling, longer root of seedling, dwarfish aerial part, early blossoming and low propagation coefficient. XND1 (xylem NAC domain), an orthologue of CarNAC2 gene in Arabidopsis, which dramatically suppresses secondary wall deposition in the xylary fiber, stunts plant development in transgenic Arabidopsis, suggesting that CarNAC2 gene has physiology function similar to that of XND1 gene.Expression of CarNAC3 gene, like that of other NAP (NAC-like, activated by APETALA3/PISTILLATA) subgroup members including Arabidopsis NAP, NAM-B1 from ancestral wild wheat and soybean GmNAC1, was found to be induced by leaf age using semi-quantitative RT-PCR assay. However, overexpression of NAP, NAM-B1 or GmNAC1 accelerate senescence process of transgenic plants, whereas CarNAC3 gene did not, suggesting there are functional differences between Car NAC3 and other three orthologues in senescence physiology. Transcription of CarNAC3 gene was significantly up-regulated by drought stress and ABA treatment. Furthermore, transgenic Arabidopsis plants expressing Car NAC3 gene showed enhanced tolerance to drought stress and high sensitivity to ABA, indicating that this gene is an ABA-dependent gene response to drought stress. Since overexpression of CarNAC3 gene did not affect plant development but enhanced tolerance to dehydration, it has the potential value for crop drought-resistant genetic engineering.Overexpression of CarNAC6 gene did not alter the phenotype and developmental process of transgenic Arabidopsis plants but enhanced tolerance to osmotic stress in seedlings. Additionally, the transgenic plants overexpressing CarNAC6 gene exhibited the higher ABA sensitivity than wild type plants, indicating that CarNAC6 is an ABA-dependent gene involved in sress response. Among six known stress-related genes, four genes(COR15A, RD22, RD29A, KIN1) were induced by overexpression of CarNAC6 gene in transgenic plants, suggesting that CarNAC6 protein can promote the transcription of these stress-related genes and then enhance tolerance to osmotic stress in transgenic plants. Like, CarNAC3 gene, CarNAC6 gene also has the potential value for crop drought-resistant genetic engineering.CarPRP1 (Cicer arietinum L. glycine- and proline-rich protein) gene containing two introns in genomic sequence encoded a XYPPX-repeat protein of 186 aa and had 3 or 4 copys in chickpea genome. The CarPRP1:GFP fusion protein was localized in cell nulclear and membrane. The transcripts of CarPRP1 appeared in many chickpea organisms including seedling leaves, stems, roots, flowers, developing seeds, and pods, but mostly accumulated in developing seeds and pods. The expression of CarPRP1 gene was not affected by leaf age but obviously fluctuated during seed development and germination. With expanding of seeds, the transcription of CarPRP1 gene was significantly enhanced. With elongation of germs, the expression of CarPRP1 gene gradually decreased in the embryo of germinating seeds. Furthermore, the expression of CarPRP1 gene was significantly induced by various stresses (drought, cold, high salility and mechanical wound) and several chemical treatments (ABA, IAA, GA3 and H2O2). Overexpression of CarPRP1 gene enhanced resistance to high salility and freezing stresses in transgenic plants. Although, the expression difference of six stresss-related genes between transgenic and wild type plants was not observed in normol conditions, their expression levels of transgenic plants were obviously higher than that of wild type plants, which can partially explain why CarPRP1 can enhance tolerance to environmental stresses in transgenic Arabidopsis. Our findings suggest CarPRP1 gene is a potential candidate for crop drought-resistant genetic engineering.CarACT1(Cicer arietinum L. actin) gene, the first actin gene from chickpea, was obtained using RACE technique and its cDNA length was 1418bp. The putative corresponding protein was composed of 377 amino acids. Genomic DNA sequence of CarACT1 gene contained four location-conserved introns with different length. Phylogenetic analysis revealed that all actin genes were conserved in the nucleotide level throughout the whole organic world. Transcript of CarACT1 was extensively detected by semi-quantitative RT-PCR in many organs of chickpea and during diverse developmental processes. Furthermore, expression pattern of CAP2 (Cicer arietinum L. APETALA2) was the same as previous findings using CarACT1 as a presumptive reference gene under drought and salt treatments. Therefore, we presumed that CarACT1 can serve as an internal reference gene for normalization of genes expression in the vegetative tissues of chickpea. The generation of the first reference gene in chickpea is useful to expression analysis through northern blot assay and RT-PCR method. |
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