| Dehydrins (DHNs; late embryogenesis abundant II family), which are stress-relatedproteins, are important for plant survival under various abiotic and biotic stresses. DHNsaccumulate in desiccation-tolerant seed embryos or in vegetative tissues subjected toenvironmental stresses such as drought, low temperature, and high salinity. To elucidate theregulatory mechanisms of wheat-derived DHNs under these stresses, winter wheat Zhengying1and spring Xinchun9were used as materials to isolate genes and promoters of wzy1-2andwzy2and further study their contribution to stress tolerance. The idiographic experimentresults are following:1. Isolation of gene and upstream regulatory region of wzy1-2and wzy2andbioinformatics analysisBased on the full-length cDNA sequence of wzy1-2and wzy2, wzy1-2and wzy2genomicsequence were isolated. Sequence analysis indicated that the wzy1-2gene is1255-bp long andcontain one103-bp intron, which is located in the nucleotide sequence encoding the S-motifand characterised by a GT-AG border; and the wzy2gene is928-bp long and contains one109-bp intron located in the nucleotide sequence encoding the S-motif and characterized by aGT-AG border.A putative wzy1-2transcription promoter region was obtained by using the PCRamplification method. The product was found to have a length of719-bp, corresponding tothe promoter and5’-untranslated region (5’ UTR). The promoter sequence of wzy1-2wasfound to have a conserved transcription start site (TSS) lying107bp upstream from its startcodon ATG. The upstream sequence of wzy1-2was analysed for putative plant regulatoryelements using the PlantCARE database. Based on this analysis, many putative regulatoryelements were identified in wzy1-2promter, which contains One TATA-box and sixCAAT-boxes. TATA-box and CAAT-box were located-26±7bp, and-562±8bp,-545±7bp,-506±8bp,-459±8bp,-445±8bp and-420±6bp upstream from TSS. The promoter containsseveral potential stress-related cis-acting regulatory elements, including ABA-, dehydration-,low temperature-, methyl jasmonate-, gibberellin-, salicylic acid-, and defence andstress-responsive elements. A putative wzy2transcription promoter region was obtained using the PCR amplificationmethod. The product was found to have a length of633-bp, corresponding to the promoterand5’-untranslated region (5’ UTR). The promoter sequence of wzy2was found to have aconserved TSS lying75bp upstream from its start codon ATG. The upstream sequence ofwzy2was analysed for putative plant regulatory elements using the PlantCARE database.Based on this analysis, many putative regulatory elements were identified in wzy1-2promter,which contains One TATA-box and three CAAT-boxes. TATA-box and CAAT-box werelocated-26±7bp, and-450±6bp,-421±6bp and-414±6bp upstream from TSS. The wzy2promoter was revealed to contain several potential stress-related cis-acting regulatoryelements, including elements responsive to abscisic acid (ABA; ABREs), anoxia (GC motifs),low temperature (LTREs), auxin (TGA elements), methyl jasmonate (MeJA; TGACG motifs)and gibberellin (TATC boxes).2. Transcript accumulation of wzy1-2and wzy2under different stress treatmentsThe quantitative PCR analysis demonstrated that the wzy1-2transcript accumulationoccurred in non-stressed treatment, as well as under osmotic stress, cold, methyl jasmonate(MeJA), abscisic acid (ABA), gibberellin (GA) and salicylic acid (SA) treatments; and wzy2transcript accumulation occurred in response to low temperature, anoxia, indoleacetic acid(IAA), MeJA, ABA and gibberellin (GA) treatments. Both of them are inducible promter.3. YSK2DHN wzy2induction patterns under dehydration, low temperature and ABAstressTogether, transcription of wzy2under20%polyethylene glycol (PEG)6000andexogenous ABA treatments and endogenous ABA content assay results reveal that theYSK2-type DHN wzy2can be induced directly by ABA and low temperature and indirectly bydrought stress.4. Deletion analysis of cis-acting elements of Prowzy1-2and Prowzy2via particlebombardment and transient expression assaysHistochemical analysis of GUS expression demonstrated that wzy1-2promoter activitycould be upregulated by osmotic stress, ABA, GA or SA treatment and wzy2promoteractivity could be upregulated by low temperature, anoxia, ABA and GA treatments. Differentcis-acting elements function in response to various abiotic and biotic stresses such as osmoticstress, cold, anoxia and exogenous application of ABA, GA and SA. Interestingly, wzy2promoter element-driven β-glucuronidase expression was first observed in meristemoidsrather than calli of wheat seeds subjected to anoxia.5. Expression patterns of WZY1-2in wheat leaves under different treatmentsWestern blotting analysis revealed that WZY1-2protein is constitutively expressed under non-stress conditions, and can also be induced by cold, osmotic stress, ABA, MeJA, and SAtreatments. Interestingly, WZY1-2protein was negatively regulated by GA at the translationlevel. A comparative analysis of transcript and protein accumulation showed that wzy1-2transcription and protein accumulation do not occur simultaneously under cold, osmotic stress,ABA, MeJA, and SA treatments; instead, WZY1-2protein accumulation follows transcriptaccumulation with a clear time lag except under cold treatment. WZY1-2proteinaccumulation can thus be induced by cold and other stress-signaling pathways at thetranslation level under cold condition. |
PDF Full Text Request