The plant hormone salicylic acid(SA)plays critical roles in plant defense,stress responses,and senescence.Although SA signaling is well understood,the puzzles of its biosynthesis pathways remain to be investigated.Previously,we have characterized an SA 3-hydroxylase(S3H)involved in SA catabolism during leaf senescence and pathogen response.The S3H can convert SA to 2,3-DHBA both in vitro and in vivo,and is inducible by SA and pathogen,thus is a key part of a negative feedback regulation system of SA levels during senescence and pathogen response.The 2,3-DHBA and 2,5-DHBA are both major hydroxylated metabolites of SA in Arabidopsis but the enzyme SA 5-hydroxylase(S5H)responsible for 2,5-DHBA production is missing.To characterize the S5H enzyme and fully understand the functions of SA hydroxylation,we screened the 2-oxoglutarate–Fe(II)oxygenase family genes which S3H belongs to by an enzymatic approach.The candidate gene encoding an enzyme which can convert SA to 2,5-DHBA was chosen for further investigation of its function in vivo.The characterized s5h knockout mutants produce less 2,5-DHBA sugar conjugates,accumulate more SA,2,3-DHBA and their sugar conjugates,and exhibit a precocious senescence phenotype and stronger pathogen resistance.Our research reveals an elegant SA catabolic mechanism by which plants maintain SA homeostasis by converting it to 2,3-DHBA and 2,5-DHBA to prevent SA over-accumulation during leaf senescence and pathogen responses.