Functional Identification Of BGLU28 And BGLU30 And Their Role In Sulfur Metabolism

The specialized(secondary)metabolic pathway in plants has long been considered as one-way routes to produce bioactive end products with primary metabo lites as precursors.However,endogenous degradation of these"end products"can be observed in plant tissues under nutritional stress.Therefore,there is a matter of general interest whether the secondary metabolites,especially the compounds rich in nitrogen-and/or sulfur-atoms,can be reintegrated into the primary metabolism for reuse under environmental stress.In the study,glucosinolates(GLS),the secondary metabolites widely existing in the order Brassicales,are usually composed of?-D-thioglucosyl,sulfoxime groups and side chain R groups which are derived from different amino acids.GLSs are sulfur rich plant metabolites containing at least two sulfur atoms.When plants suffer sulfur deficiency conditions,the content of GLSs in vivo will be greatly reduced.Previous studies suggested that the decrease of GLSs was due to the hydrolysis by myrosinase(s),and then released sulfur atoms from the structure to form sulfur-containing primary metabolites to compensate the sulfur demand in plant growth.However,it is not clear which myrosinase plays the important role in this process and how sulfur atoms in GLSs reintegrate to primary metabolites.The model plant,Arabidopsis thaliana(hereafter Arabidopsis)was used in our study.The main results are as follows:(1)Based on the analysis of published transcriptome data,BGLU28 and BGLU30 genes may perform the function of myrosinases under sulfur deficiency condition.In order to veri fy the hypothesis,real-time quantitative PCR was used to analyze the transcriptional level of all BGLU family genes in Arabidopsis.The results showed that only the expression of BGLU28 and BGLU30was significantly increased in all BGLU 47 genes.(2)The GLS contents of bglu28,bglu30 and bglu28 bglu30 double knockout mutant grown under different sulfur contents were analyzed.The content of GLSs in bglu28 bglu30 mutant was significantly higher than that in Col-0,bglu28 and bglu30 single mutants,which indicated that the degradation of GLSs in bglu28 bglu30 mutant was significantly inhibited,our experiments showed that BGLU28 and BGLU30 were involved in the degradation of GLSs under sulfur deficiency.(3)Enzyme activity in vitro using various glucosinolates as substrates were determined to verify that at least BGLU28 has the myrosinase activity.(4)The metabolic pathway of glucosinolates was determined by adding deuterium labeled(d ₅)glucosinolates to the culture medium.Glucosinolates were degraded b y myrosinases BGLU28 and BGLU30 to form isothiocyanates(ITCs)and ITCs combine with glutathione(GSH)to form conjugates.In this process,PAD2 were involved in the biosynthesis of GSH.Then,glycine(Gly)and?-glutamine(?-Glu)residues were cleaved from ITC-GSH to form ITC-cysteine(Cys)conjugate,and finally cyclized to form ITC-NH₂ and Raphanus amic acid(RA),which were further degraded by OXP1 to form Cys.(5)By adding ³⁴S labeled glucosinolates in the culture medium,the intermediates in the meta bolic process were further verified,and the fate of sulfur atoms was further determined.(6)Based on the analysis of microarray data,candidate genes that may be involved in the metabolic pathway were obtained,and the functional genes in the metabolic p athway were identified by analyzing the content changes of intermediate products through adding glucosinolates to the culture medium growing candidate mutants.Overall,our findings demonstrate the bidirectional interaction between primary and specialized metabolism,which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.