Transcriptional Regulation Of Salicylic Acid Hydroxylase Gene S5H In Arabidopsis Thaliana

Salicylic acid(SA)is an important phenolic plant hormone which plays essential roles in physiological and biochemical processes.It is critical for plants to modulate hormone homeostasis under different conditions.The SA homeostasis is regulated by a fine balance between biosynthesis and metabolism.We previously identified that SA hydroxylation is an important modification and is regarded as the major detoxification pathway to maintain the SA homeostasis in plants.We have previously characterized the SA 5-hydrxoylase(S5H)which can convert SA to 2,5-DHBA in vivo and in vitro.In addition,S5H is induced by SA treatment.So far,the transcriptional regulation network of SA 5-hydroxylase(S5H)is still unknown.To study the regulation network of SA hydroxylation maintaining the SA homeostasis,we studied the regulatory mechanisms of S5H gene at transcriptional levels.Taking advantage of yeast-one hybrid(Y1H)system,we screened the regulators which binding to the promoter region of S5H.The candidate regulators were further investigated by genetic,physiological and morphological approaches.The main conclusions of this study are as following:(1)The 598 bp promoter sequence of S5H was identified to have a complete promoter function.Using the 2.4 Kb promoter region of S5H as bait in yeast-one hybrid by AbA~r reporter,we found that the S5H promoter sequence is likely being recognized by endogenous yeast transcription factors.In order to screening a shorter promoter sequence of S5H with a full function,the promoter truncations including three different length regions of S5H promoter(598 bp,1232 bp and 1843 bp).The three different length region of S5H promoter was 598 bp,1232 bp,1843 bp were ligated fused with the GUS as a reporter gene to test the promoter activities for constructing binary expression vectors.Tobacco transient expression in tobacco and stable the three expression frames were transformed in wild-type Arabidopsis showed that the three different length promoter fragments were all show GUS activities and were all induced by SA,Thus the shorter promoter region(598 bp)of S5H promoter was choosen as a bait for Y1H screening.(2)Six transcription factors were identified to be the candidate regulators of the S5H gene.Y1H cDNA library constructed using the high-quality RNA from senescence and Pst DC3000 treated leaves was used for the Y1H screening.15 transcription factors were initially identified by Y1H and later were rescreened by Y1H and Dual-Luciferase Reporter Assay System using the 598 bp promoter.The transcription factors including WRKY1,AXR2,PH,C2H2,HMG and WRKY18 were found to directly bind to the S5H promoter.(3)The characterization of T-DNA insertion mutants and overexpression lines of WRKY1 showed that WRKY1 is positively regulator of leaf senescence.We identified the T-DNA insertion lines of all the candidate transcription factors.Phenotype analysis revealed that wrky18 and axr2 showed a dark-induced premature senescence whereas wrky1 and nac6 displayed delay-senescence phenotype.The WRKY1 was chosen as a target gene for detailed study.The over-expression plants were generated and found to promote leaf senescence.(4)The expression analysis of SA metabolim related genes in the wrky1,WRKY1-OE and wild-type.The expression of S5H,S3H,SAG13,PR1,ICS1,ICS2 and SGR1 were quantified in the wrky1,WRKY1-OE and wild-type using the qRT-PCR assyas.The expression of SA metabolim related genes ICS1,ICS2,S5H and S3H were significantly increased in the WRKY1 overexpression plants but decrased in the wrky1 mutant.The senescence related genes such as PR1,S3H and SAG13 expression were also increased in the WRKY1-OE but were reduced in the wrky1 mutant,which are consistent with premature leaf senescence phenotype of WRKY1 overexpression lines and delayed leaf senescence phenotype.(5)The metabolites analysis of free SA,total SA and 2,5-DHBA in the WT,wrky1 and WRKY1-OE.The metabolites of SA and 2,5-DHBA were increased in the WRKY1overexpression plants but were decreased in the wrky1 mutant compared with that of the wild-type.We speculated that the WRKY1 may influence the expression of ICS1,ICS2,and S5H and affecting the contents of SA and 2,5-DHBA which contribute to an accelerated leaf senescence phenotype.In summary,we identified six of the 15 transcription factors were confirmed to bind to S5H promoter.The WRKY1 gene was further proved to be associated with the biosynthesis of SA and 2,5-DHBA and has essential role in leaf senscence.Our research provides new insights to understand the regulation of S5H gene in SA homeostasis.