Molecular Dissection Of Arabidopsis RNA Helicase Athelps In Regulation Of Salt Stress Tolerance

Plants are sessile organisms capable of adapting to various environmental conditions,such as drought, cold, and high salt content in soil. When they encounter these stressfulconditions, the plant cells reprogram their cellular biochemical processes to defense it bytriggering a network of signaling events, during which phytohormone plays necessary roles.The RNA helicase is a kind of enzyme existed widely in all living organisms, whichcould catalyze double-stranded RNA to unwind using energy from ATP hydrolysis. And RNAhelicases convert RNA with advanced structure that makes it inactive to active functionalRNA, and is treated as the most effective RNA molecular chaperone. The function of RNAhelicase involves in RNA transcription, translation initiation, pre-mRNA splicing, mRNAdecay, protein translation, rRNA processing, assembly of ribosome and nuclear mRNA export,and so on. RNA helicase plays important roles in gene expression and regulation.The best known members in the RNA helicase family is the DEAD-box RNA helicases.Since the first cold induced DEAD-box helicase FL25A was studied in Arabidopsis thaliana,series of the DEAD-box helicase members were identified to function in abiotic stress, suchas LOS4, STSR1/2and AtRH9/25. But SKI2family is rarely studied in plants, and therelationship between SKI2family and abiotic stress is rarely addressed.Using biological information and gene-chip analysis, we identified the members of SKI2family in Arabidopsis. And we confirmed AtHelps as the homologous gene of Ski2p in yeastand Ski2w in human according to the evolutionary relationships. Then the biological functionof AtHelps was analyzed by the method of reverse genetics, and main results are as follow:(1) Identification and characterization of AtHelpsThe RNA helicase AtHelps was the member of Arabidopsis SKI2family, with closestphylogenetic relationship with ScSki2p and HsSki2w (53%and56%in core region aminoacid sequence), and contains a conserved DEVH-box motif.AtHelps expressed highest in root tissue and the mature seed; and the transcripts reducedobviously at the second day of germination process, and slowly returned to normal level after then. The transcription of AtHelps was inhibited by high salt and osmotic stress.Promoter activity of AtHelps was detected in all stages of Arabidopsis, but the intensitywas different: transcriptional activity was mainly found in the vascular tissue, and graduallydisappeared as the tissue turned older; Transcriptional activity in root was high all the time. Inthe root, transcriptional activity was mainly detected in pericycle, especially in theparenchyma cells of phloem. AtHelps might play an important role in loading and unloadingnecessary material to the xylem. High salt and drought stress repressed the transcriptionalactivity of AtHelps at seedling stage.The protein of AtHelps localized in cytoplasm by transient transformation assay withonion epidermal cells and protoplast of homozygous lines observation. However, it does notlocalized in chloroplast.(2) The responses to salt stress of AtHelps in germination and postgermination phasewere depended on different ABI genesWe got mutants by RNA interference technology and overexpression lines for furtherphenotypic analysis. The mutant germinated faster but the overexpression lines were slowerin200mM NaCl. The mutant also showed higher resistance to salt in cotyledon-greeningstage and final survival rate. The results of KCl and LiCl showed AtHelps was mainly inresponse to sodium toxicity.Different kinds of ABA synthesis inhibitor reduced the difference-germinationphenotype of three genotypes in salt stress. It proved AtHelps response to salt stress wasdependent on the phytohormone ABA.The expression of ABA synthesis key genes, ABA2, NCED3, AAO3and degradation keygene CYP707A3were detected by qRT-PCR. And synthesis key genes were reduced anddegradation key gene was induced in mutant. While the overall effect was to make the ABAcontent decrease in mutant; then the ABA content was messured and the results was in accordwith the qRT-PCR results. The difference of ABA content among genotypes was one ofreasons that caused the difference-germination.The sensitivity to ABA was diverse among three genotypes. Western Blot showed lowerABI3and ABI5protein levels in the mutant, and the phenotype caused by decreased proteinlevel can be reduced by proteasome inhibitors. In post-germination growth phase, the reduced transcripts of ABI4was the main reasonof phenotype, which directly enhanced the transcription of AtHKT1;1. AtHKT1;1can achievethe optimal allocation of sodium, which reduced active oxygen damage caused by sodium.(3) AtHelps participated in regulating the stabilization of mRNA in cytosol.AtHelps regulated the stability for some specific mRNAs, such as CYP707A3andAtHKT1;1. The active form was stabilized by protecting the mRNA from turnover, whichfurther played roles in resisting salt stress in mutant.