Isolation And Functional Analysis Of A Short-day-dependent Lesion Mimic SDL1Gene In Arabidopsis

Lesion mimic mutants spontaneously develop lesions without apparent adversity, injury or pathogens. The lesion formation seems to be similar to the hypersensitive response elicited by inoculation with an avirulent pathogen. In some cases, lesion mimic mutants express cytological and biochemical markers that have been associated with disease-resistant responses, and exhibit local and systemic acquired resistance (SAR).A mutant which develops lesions under short-day conditions(8hr light/16hr dark) was named as short-day-dependent lesion mimic mutant1, sdl1. The sdl1mutant shows no significant differences with the wild-type Col-0under long-day conditions (16hours light/8hours dark), but the mutant plant grows more slowly and is more dwarf than the wild-type plant under short-day conditions. The sdl1mutant spontaneously develops lesions on its leaves without pathogen attack and wrinkles irregularly. After bolting, the acrocarpous plant shows albino stems and wilting siliques and flower buds. But it will not lead to the death of the mutant. Further studies reveal that the lesion phenotype of sdl1could not be suppressed in medium plates, but was able to be obviously attenuated by supplementation of high concentration sucrose in medium.The SDL1gene is identified by map-based cloning. Arabidopsis SDL1gene encodes a421amino acid protein. The mutation in sdll occurs at the last base of the fourth exon of SDL1gene, which directly leads to a substitution of the termination codon TAG for Trp. The translation ends at a premature end, losing265amino acids.In the process of mimic lesion formation, it has been detected in most of the lesion mimic mutants that the pathogen defense-related histochemical reaction and the systemic acquired resistance-related gene expression. Partial cell death has also been observed through the trypan blue staining in the leaves'cells of different growth periods. By detecting the resistance genes expression level, we discovered the constitutive expression of related resistance genes and the accumulation of salicylic acid in the mutants under short-day conditions. It suggests that the lesion mimic formation is related to the accumulation of SA in the mutants, but unrelated to SA signaling pathways by studying of sdl1NahG and sdl1npr1-1double mutants.cDNA sequencing and RT-PCR showed the SDL1mutation in sdl1that changed splicing recognition sequence did not alter its splice site and the transcripts of the mutation SDL1gene in the sdl1mutant decreased markedly. By constructing the expression vector containing the endogenous promoter and35S promoter, we further detect the expression at transcriptional and translational levels. The expression at transcriptional level indicates that mutation at splicing site of sdl1doesn't affect splicing efficiency. The gene is feedback controlled by its own promoter. In addition, the expression at transcriptional and translational levels of SDL1is not light-regulated.SDL1is an unreported new gene, bioinformatics analysis show that SDL1is highly conserved and contains important domains of fumarylacetoacetate hydrolase, suggesting its function as FAH functioning in tyrosine metabolic process. Learning from Tyr metabolism in vivo model, we treat plants with exogenous aromatic amino acids and their metabolic intermediates and end products to further study gene working site. The results show that exogenous high concentrations of Tyr and Trp can inhibit sdl1phenotype under short-day conditions, while there is no effect by adding exogenous Phe. The tyrosine content in sdl1mutant is not changed, illustrating a certain amount of Tyr and Trp seems to remedy the defect. Low concentration of metabolic intermediates homogentisate (HGA) in medium accelerates lesion necrosis phenotype of the sdl1mutants, while high concentrations of HGA in medium strongly inhibit plant growth. Metabolic end product fumaric acid (FA) has no impact to the phenotype of the plants, but the mutation of SDL1gene leads to reduction of FA content in the plants. The results indicate SDL1gene is likely to be working downstream of HGA, upstream of FA. But the phenotype is not due to the HGA accumulation and FA reduction.In summary, a short day-dependent lesion mimic SDL1gene is identified in this study, and its function is preliminary investigated. The findings are of great scientific significance to explore how the light length regulates the plant disease resistance.