Mechanisms Of Cell Death And Its Resistance In Rice Lesion Mimic Mutant Spl5

Lesion mimics broadly represent a phenotypical class of mutants that spontaneously display cell death.The phenotype of lesion mimics,resembling that of HR,is resulted from spontaneous activating programmed cell death(PCD) pathways.Thus,lesion mimics are a powerful tool to unravel PCD pathways in plants.A lesion mimic mutant in rice,spotted leaf 5,spl5,was identified from a population of japonica rice Norin8 mutagenized byγradiation and spontaneously exhibited small reddish brown spots form seedling stage.The detailed analyses of formation mechanisms of phenotype in spl5 mutant showed that O₂^-accumulation preceded to initiation of cell death and reached its highest level at the early stage of lesion spot development.Activity analyses of ROI-scavenging enzymes SOD and CAT indicate that ROI-scavenging machinery in spl5 mutant was not disrupted,suggesting that misregulating of the ROI metabolism is probably due to enhanced ROI-production machinery.Identification of the resistance to rice bacterial blight showed that spl5 mutant significantly enhanced resistance to Philippines strains of Xanthomonas oryzae pv.Oryzae (Xoo) as compared to wild type,moreover,exhibited a high resistance to PXO145(P8) strain and a resistance to PXO280(P7),PXO87(P9),and PXO124(P10) strains.The analyses of resistance mechanisms showed that activities of POD and PAL increased more rapidly and followed by a higher steady level in spl5 mutant than in wild-type after inoculation,indicating that PCD or HR pathways in spl5 mutant are more sensitively activated than wild-type is. OsPR1 was generally used as a marker gene related with systematic acquired resistance (SAR),while OsAOS was a key controlling enzyme in jasmonate acid(JA) biosynthetic pathway.Expression analyses of resistance-related gene showed that expression of OsAOS and PR genes including OsPR1,OsPR3,and OsPR8 were higher in spl5 mutant than in wild-type.RT-PCR assay indicates that SAR and JA biosynthetic pathway might be involved in the formation of spotted lesions.All of above data support that spl5 mutation enhances resistance to pathogens.To further explore the mechanisms of cell(?)eath in spl5 mutant and interactions between spl5 mutant and pathogens,suspension cell lines from spl5 mutant and wild type were established,respectively,and some key factors affecting the establishment of cell lines were also analyzed.Our data showed that 2,4-D is a key factor to induce calli,and extracellular low POD and excessive H₂O₂ prevented establishment of spl5 cell line to great extents,and shortening subculture period would greatly improve establishment of spl5 cell line.