Investigating the mechanisms of isochorismate synthase:An approach to improve salicylic acid synthesis and increase resistance to Fusarium head blight in wheat

Salicylic acid(SA),a vital endogenous hormone,plays a crucial role in plant growth and the response to abiotic and biotic stress.Isochorismate synthase(ICS)and phenylalanine ammonia lyase(PAL)are critical rate-limiting enzymes for SA synthesis.Fusarium head blight(FHB)seriously threatens the safety of wheat production,but increasing the content of SA can enhance FHB resistance.However,the pathway of SA synthesis and regulation in wheat remains unknown.In this study,three wheat ICS(TaICSA,TaICSB,and TaICSD)were identified,and their functions were validated in vitro for isomerizing chorismate to isochorismate.The mutation of one or two homoeoalleles of TaICSA,TaICSB,and TaICSD in the wheat variety‘Cadenza’reduced SA levels under ultraviolet treatment and Fusarium graminearum infection,further enhancing sensitivity to FHB.Overexpression of TaICSA can significantly enhance SA levels and resistance to FHB.To further study SA synthesis pathways in wheat and avoid interference with pathogenicity related genes,the leaves of wild-type Cadenza and different TaICS mutant lines were subjected to ultraviolet treatment for transcriptomic analysis.The results showed that 37 PALs might be involved in endogenous SA synthesis,and 82 WRKY and MYB family transcription factors may regulate the expression of ICS and PAL.These results were further confirmed by RT-PCR.In conclusion,this study expands our knowledge of SA biosynthesis and identifies TaICSA,as well as several additional candidate genes that encode transcription factors for regulating endogenous SA levels,as part of an efficient strategy for enhancing FHB resistance in wheat.

PBS3 and EPS1 Complete Salicylic Acid Biosynthesis from Isochorismate in Arabidopsis

Salicylic acid(SA)is an important phytohormone mediating both local and systemic defense responses in plants.Despite over half a century of research,how plants biosynthesize SA remains unresolved.In Arabidop-sis,a major part of SA is derived from isochorismate,a key intermediate produced by the isochorismate syn-thase,which is reminiscent of SA biosynthesis in bacteria.Whereas bacteria employ an isochorismate pyru-vate lyase(IPL)that catalyzes the turnover of isochorismate to pyruvate and SA,plants do not contain an IPL ortholog and generate SA from isochorismate through an unknown mechanism.Combining genetic and biochemical approaches,we delineated the SA biosynthetic pathway downstream of isochorismate in Ara-bidopsis.We found that PBS3,a GH3 acyl adenylase-family enzyme important for SA accumulation,catalyzes ATP-and Mg2+-dependent conjugation of L-glutamate primarily to the 8-carboxyl of isochorismate and yields the key SA biosynthetic intermediate,isochorismoyl-glutamate A.Moreover,we discovered that EPS1,a BAHD acyltransferase-family protein with a previously implicated role in SA accumulation upon pathogen attack,harbors a noncanonical active site and an unprecedented isochorismoyl-glutamate A pyruvoyl-glutamate lyase activity that produces SA from the isochorismoyl-glutamate A substrate.Together,PBS3 and EPS1 form a two-step metabolic pathway to produce SA from isochorismate in Arabidopsis,which is distinct from how SA is biosynthesized in bacteria.This study closes a major knowledge gap in plant SA meta-bolism and would help develop new strategies for engineering disease resistance in crop plants.

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase （AalCS1） gene from Artemisia annua

Isochorismate synthase(ICS) is a crucial enzyme in the salicylic acid(SA) synthesis pathway. The full-length complementary DNA(cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site(MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction(qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography(HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.