| Rice is one of the most important staple food crops in the world,which is also the main food crop in China.Rice feeds more than half of the world's population and almost 65%people of China depend on it,cultivating new rice varieties with higher and more stable yield is conducive to ensuring the national food security.The recent advancements in rice genomics have brought new opportunities and challenges for developing new rice varieties in recent years.Normal leaf color is beneficial to leaf photosynthesis,while some special leaf color can be used as an effective marker for rice material identification.The change of leaf color is directly or indirectly related to the leaf senescence,which has great influence on yield and quality of rice.So far,some genes related to the leaf color have been identified in rice;however,the genetic and molecular mechanisms behind leaf color changing as well as leaf senescence remain largely unknown.In-depth analysis of the biological functions and metabolic pathways of these genes will contribute to the application of leaf color marker traits in rice genetic breeding and have the important theoretical and practical significance in rice anti-aging breeding.In the current study,we isolated two leaf color mutants from a rice(Oiyza sativa L.cv'Zhenong 34')M2 population by mutagenesis with ethylmethane sulfonate(EMS),named brow n mictdrib leaf(bml)and purple leaf(pl).Phenotype observation,map-based cloning and gene expression analysis were used to analyze the function of mutated genes.The results showed that the phenotypes controlled by two leaf color genes could be stably inherited and both of them played an important role in leaf senescence,in which bml and pl were involved in leaf senescence regulation,respectively.Additionally,two genes were also involved in the process responses to biological or abiotic stress in rice to some extent.The main results are the following:(1)Both mutants showed the abnormal leaf color phenotype,early leaf senescence,reduced seed setting rate and 1000-grain weight.Compared to wild type(WT),the bml mutant exhibited the brown rust-colored with early leaf senescence at the heading stage,where at first the midrib of the bml leaf started to brown with lesion-like spots and gradually covered the whole leaf.However,the leaf of pl mutant attained purple at late grain filling stage,which showed that the midrib and left part of the leaf began to turn purple,and then gradually spread to the whole leaf.(2)The bml and pl mutants showed abnormal leaf cell development,chloroplast degeneration or distortion,and significantly decreased the chlorophyll content in leaf.In addition,the chlorophyll a/b ratio of the two mutants decreased significantly during the grain filling stage,and the photosynthetic related genes were down-regulated,indicating that the photosystems in the mutants had been damaged.The bml mutants showed the excessive accumulation of reactive oxygen species(ROS)and up-regulation of senescence-induced genes along with the senescence-related transcription factors.Meanwhile,the contents of superoxide dismutase,catalase and malondialdehyde in both mutants were significantly higher than those in WT,indicating that the degree of membrane damage of the mutants increased with the leaf senescence.The levels of abscisic acid(ABA)and jasmonic acid(JA)were also significantly increased,which might aggravate the leaf senescence of the two mutants.(3)Genetic analysis results showed that both of the bml and pl mutant phenotypes were controlled by a single recessive nucleus gene.The bml gene was positional fine-mapped to a 57.3 kb interval between InDel markers L5IS7 andL5IS11 on the short arm of chromosome 5;within this region,a putative gene LOC_Os05g02020,encoding a kinase APK1A(a chloroplast precursor),was considered as the optimal candidate gene.Detailed DNA sequencing analysis of the candidate gene showed a single base substitution(G to A)in the 5'untranslated region(UTR,+98 bp)of bml mutant.Meanwhile,the pl gene was fine plotted into a region of 36.7 kb on the long arm of chromosome 5,flanked by InDel markers L1S4 and L1S5.Sequencing results showed that a single base(C)was inserted in the third exon(+901 bp)of LOC_Os05g48010,which resulted in the shift mutation of the coding sequence and the premature termination of translation.Functional annotation found that the gene encoded a MYB transcription factor.(4)The results of qRT-PCR analysis for cDNAs in leaf,stem,panicle and root tissues at heading stage showed that OsBML gene was expressed in these tested organs.The gene expressions in leaf,stem,panicle and root tissues of the bml mutant were lower than those in the corresponding part of WT,this might be caused by the substitution of G to A at the 5'-UTR and affected the transcriptional level of bml gene expression in mutant.Organ specific expression profiling of OsPL by qRT-PCR also revealed ubiquitous expression of this gene at different organs of the WT.The highest expression was found in leaf followed on sheath among all the tested tissues in rice plant.(5)For clarifying the biological function of OsBML,artificial microRNA(amiRNA)silencing strategy was used for observing the knockdown influence of OsBML.The calluses of OsBML knockdown lines could not regenerate to new plants with all the calluses showing severe brown,suggesting that OsBML played the important roles during plant regeneration from callus.(6)The bml and WT plants were inoculated with rice bacterial pathogen Xanthomonas oryzae pv.osyzae(Xoo)at seedling stage,booting stage and mature stage,respectively.The results of disease grading showed that the lesion length in all stages of bml plants was shorter than those of WT,indicating that the mutant had stronger resistance to Xoo.The expression of four pathogenesis-related(PR)marker genes(PRIa,PRIb,PR5 and PR10)were significantly up-regulated in mutant bml at booting stage,indicating bml mutant had a positive immune defense response to Xoo.Therefore,OsBML gene negatively regulated the immune response of rice bacterial pathogens.(7)Expression of OsPL gene was persuaded in pl and WT plants after 1 h of high temperature(40?)treatment,which was reduced to background level at 24 h of treatment.This might be related to the OsPL promoter region carrying cis-acting elements of stress response,such as heat shock protein DREs/CRTs(dehydration or drought response elements).The coleoptiles of pl plants grew longer than those of WT at 40 °C after 5 d of treatment,which revealed that the pl mutant might have stronger heat resistance,by that OsPL gene plays a negative role in response to heat stress in rice. |
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