Function And Expression Pattern Analysis Of PYK10 And BGLU28 In Arabidopsis Thaliana

Glucosinolates are important secondary metabolites in cruciferous plants including model plants Arabidopsis and Brassicales vegetables.The intact glucosinolates do not usually have biological activity.When subjected to external biological stress,such as infestation of insects or pathogens and other forms of mechanical damage,degradation of glucosinolates may occur catalyzed by myrosinase such as TGGs and PEN2.A series of chemical reactions that break the glycosidic bond between S and glucose will produce degradation products with a variety of biological activities.Among them,the degradation product of glucosinolates,isothiocyanates,has anticancer activity for humans and effectively inhibit the proliferation of tumor cells.For plants,isothiocyanates has a defensive effect,that can protect against herbivores and pathogens,and act as a signaling molecule to initiate plant defense responses.Therefore,studies on the degradation of glucosinolates in Arabidopsis have attracted much attention.Studies have shown that in addition to mechanical damage,there is degradation of glucosinolates in intact tissues under sulfur deficiency in Arabidopsis.However,the myrosinase that catalyse glucosinolate degradation in intact tissues remains unclear.Under sulfur deficiency,the expression levels of ?-glycoside hydrolase genes PYK10 and BGLU28 are increased.Combining the bioinformatic analysis of the two genes,we hypothesized that PYK10 and BGLU28 may be involved in the degradation of glucosinolates in Arabidopsis intact tissue under condition of sulfur deficiency.To validate our hypothesis,Arabidopsis pyk10-1 and bglu28 homozygous mutants were used as experimental materials to carry out preliminary functional verification of PYK10 and BGLU28.The spatial expression patterns of PYK10 and BGLU28 were determined by real-time quantitative RT-PCR.The patterns of the two genes response to a variety of different hormones and stresses were systematically analyzed,which laid a foundation for further study of the role of PYK10 and BGLU28 in the metabolism of glucosinolates and its functions in response to to different stresses.The main conclusions of this paper are as follows:1.Tissue localization of PYK10 and BGLU28 in ArabidopsisThe real-time quantitative RT-PCR was used to analyze the tissue localization of PYK10 and BGLU28 in Arabidopsis.The results showed that PYK10 was expressed in young roots,young leaves and mature roots.BGLU28 was expressed in young roots,young leaves,mature roots,inflorescences and siliques.2.Responses of PYK10 and BGLU28 to different hormones and stress in ArabidopsisThe real-time quantitative RT-PCR method was used to analyze the spatial expression patterns of PYK10 and BGLU28 under different hormone treatments and external stress.The results showed that PYK10 and BGLU28 were strongly induced by salicylic acid(SA),methyl jasmonate(Me JA),gibberellin(GA),and 1-amino-1-cyclopropanecarboxylic acid(ethylene of precursor).Sulfur deficiency,Flagelin 22(A bacterial flagellin having a polypeptide of 22 amino acids at the N-terminus,flg22),and UV(ultraviolet)significantly up-regulated PYK10 expression.Sulfur deficiency and Na Cl significantly increased the expression of BGLU28.3.Functional identification of PYK10 and BGLU28 in ArabidopsisBy analyzing the content of glucosinolates and related phenotypes in Arabidopsis pyk10-1 and bglu28,The results indicated that PYK10 is a myrosinase that can participate in the degradation of glucosinolates in intact tissues.And it plays a role in pathogen-mediated pathways in which glucosinolates and auxin are crossed,and positive for the synthesis of auxin which are dependent on indole-3-acetaldehyde oxime(IAOx).BGLU28 may not be a myrosinase,or it is also possible that its activity may be redundant with other myrosinases and therefore no phenotype can be observed.pyk10-1and bglu28 both showed shorter root phenotype,indicating that PYK10 and BGLU28 may play a role in development of the roots.However,there was no significant difference in the expression level of key genes involved in auxin synthesis in both mutants,indicated that the affection in roots is not related with auxin biosynthesis.