The Study Of A Novel Arabidopsis Gene Athspr In Salt Tolerance And Expressional Regulation

Salt stress is a harmful abiotic stress to plants and has an adverse effect on all the physiological processes of plants. However, plants could response to salt stress by the cross talk of different signaling pathways, regulation of different genes expression and protein activation. Under salt stress, the plant can adapt itself to high salinity via toxic ion exclusion, compartmentalization, long-distance transport, the synthesis of chaperone (HSPs, Heat Shock Proteins) and the expression of stress response genes. Among those strategies, molecular chaperones like HSPs are key factors for protein folding under various stresses especially in the heat, drought, salt and heavy metal stresses. HSPs can protect the newly synthetic proteins from denaturalizing to maintain their functions. Although many progresses have been made recently, the specific molecular mechanisms that how HSPs function and the genes related to the process are still covered.In our study, a novel T-DNA insertion mutant was isolated and the related gene was cloned, which encodes a novel protein AtHSPR (Arabidopsis thaliana heat shock protein-related) and may be involved in strigolactones (SLs) signaling pathway. We analyzed the phenotype and salt sensitivity of the Athspr mutant, cis-elements of Athspr promoter, spatio-temporal expression pattern of Athspr gene, salt tolerance of Athspr gene in overexpression(OE), complementation(COM) and RNAi (RNA interference) transgenic plants using comparative morphology, physiological and biochemical techniques, bioinformatics, gene cloning, RT-PCR(Reverse transcription polymerase chain reaction)/QRT-PCR (Quantitative real-time reverse transcription polymerase chain reaction), Western blot and genetic engineering. Furthermore, the transcription levels of the ion transport genes, salt stress response genes and SLs related genes were also analyzed in both Athspr mutant and wild type under salt stress. The results were as follows:1. Under soil growth condition, Athspr mutant showed the reduced organs (leaves, inflorescence stalks and siliques) in size, darker green rosette leaves and delayed flowering time compared with wild-type (WT) plants. The phenotype of young seedlings had no significant difference between Athspr and wild type when growing on normal MS medium. However, under salt stress, the seed germination and growth in seedlings and mature plants of Athspr were markedly inhibited. The salt sensitivity of Athspr mutant was aggravated with the rise of salt concentration. Under 100 mM NaCl, Athspr mutant showed a markedly reduction in seed germination and seedling growth, as well as leaf bleaching compared with WT seedlings. Compared to WT, mature Athspr plants displayed a reduction in survival rate, leaf water content and total chlorophyll content, and an increasing in the plasma membrane damage. When seedlings treated with 200 mM NaCl, the growth suppression of Athspr mutant was more severe and finally leading to death. In addition, the similar morphology of the mutant plants was also induced by LiCl or Na2SO4 treatments However, there was no difference between WT and Athspr seedlinds under osmotic stress.2. Using web database PLACE and Phyre, we analyzed the putative cis-elements of potential Athspr promoter and the structural domain of ATHSPR protein. The resulting putative cis acting regulatory elements in the potential promoter region of Athspr can be grouped into five classes, including stress-responsive elements (hypoxia, heat shock, salt/pathogen, ABA/dehydration, cold and wounding stress), phytohormone-responsive motife (GA, CK, Auxin and ethylene), light-regulated cis-acting elements, tissue-specific elements (leaf and root specific, embryo- and endosperm-specific) and basic transcription elements. Based on analysis above, a 1670-bp promoter containing the key cis-elements from the C24 genomic DNA acts as a part of cloning full Athspr gene. ATHSPR protein contains C1pB protein domains and AAA-ATPase domain. Based on analysis of full-length of Athspr gene, the Athspr gene (5.7 kb) was successfully cloned.3. Spatio-temporal expression pattern of Athspr gene in different organs of wild type was performed by Real Time QRT-PCR and Western blot. The highest expression level of Athspr was observed in open flowers, followed by inflorescence stems, flowers buds, siliques and roots. Finally, Athspr mRNA was largely restricted to express in the leaves and young seedlings.4. The RT-PCR results showed that the expression of Athspr gene in the mutant was interrupted by the T-DNA insertion. While there was no corresponding ATHSPR protein (114 kD) was detected by Western blot.5. The promoter of Athspr in response to salt stress was analyzed in 14-d-old ProAthspr::GUS transgenic seedlings. The results revealed that ProAthspr-promoted GUS was weak under normal conditions, but the ProAthspr-promoted GUS expression was significantly increased after treated with NaCl (150 mM) for 6h and 12h compared to control. Moreover, the expression of Athspr in WT was induced by 150 mM NaCl treatment at transcriptional and translational level. Although the Athspr transcript level in Athspr mutant was much higher than that in WT under salt stress, ATHSPR was not still detected from Athspr mutant.6. The pMDC99-ProAthspr::Athspr and Athspr-RNAi constructs were transferred into Agrobacterium tumefaciens GV3101, and then introduced into A. thaliana(Athspr mutant and wild type C24) plants using the Floral dip method. By PCR and QRT-PCR verification, we screened the stable transgenic plants base on phenotype and salt tolerance. The analysis of Athspr expression level in transgenic plants revealed that the level of Athspr transcripts from high to low was OE, COM, WT and mutant. The phenotype of complementary plants revealed that Athspr could rescue the mutant. Compared with the Athspr mutant, plant height, leaf size, flowering time, silique length and salt tolerance from the pMDC99-ProAthspr::Athspr transgenic plants were obviously enhanced and showed no significant differences compared with WT. Over-expression of Athspr resulted in a increased growth and salt tolerance in contrast with WT. However, RNA interference of Athspr resulted in the retarded growth of transgenic seedlings and reduced the number of rosette leaf and salt tolerance.7. The QRT-PCR results showed that expression of Na+/K+transport gene-related (SOS1/SOS2/SOS3, NHX1, HKT1 and KC05), SL(strigolactone) biosynthesis and signaling genes (MAX1/MAX2/MAX3/MAX4), stress-responsive marker genes (RD29/DREB2A/ERD10) had no difference between WT and Athspr seedlings under normal growth condition, but almost all of the genes selected were induced in WT and Athspr seedlings at various time points after 150 mM NaCl treatment.8. Under 150 mM NaCl, the transcriptional level of SOS1/SOS2/SOS3 and KCO5 in WT was much higher than Athspr seedlings, and the expression of SOS genes in WT was faster than that in Athspr. Among the genes described above, the peak of SOS2/SOS3 in WT seedlings appeared far earlier than that in Athspr. In addition, higher induction of GI and NHX1 genes in Athspr was observed compared to WT.9. QRT-PCR analysis showed that salt stress significantly induced higher expression of MAX1/MAX2/MAX3 in Athspr seedlings than in the wild type. In addition, expression of MAX4 in salt-treated Athspr seedlings clearly decreased in comparison with salt-treated WT seedlings. The decrease was detected at 12h and 24h of NaCl treatments.10. At later stages (8h,12h or 24h) after salt stress, the relative expressions of RD29 and DREB2A in Athspr seedlings were higher than those in WT.The results above suggest that Athspr may act as a possible member of HSP100 family and that the expression and function of Athspr are modulated by salt stress and development signals, which coincide with universal expression in every organ of Arabidopsis. The mutation and lower expression of Athspr or the absence of ATHSPR result in growth retardation, the reduction of organ size and salt sensitivity in Arabidopsis. Hence, Athspr may play essential roles in growth and response to salt stress. Moreover, the transcriptional analysis of various genes indicates that ATHSPR may be functionally involved in the transcriptional activation of SOS and KCO5 genes, and the over-transcriptional repression of GI and MAX genes under salt stress. Finally, the transcriptional regulation of Athspr may be similar to HSPs, that is salt stress signal triggers Athspr expression by transcriptional activating, and the transcriptional expression of Athspr gene is repressed under normal condition or when the synthesis of ATHSPR is enough.