Disruption of LEAF LESION MIMIC 4 affects ABA synthesis and ROS accumulation in rice

Lesion mimic mutants(LMMs) are advantageous materials for studying programmed cell death(PCD).Although some rice LMM genes have been cloned, the diversity of functions of these genes indicates that the mechanism of cell death regulation in LMMs needs further study. In this study, we identified a rice light-dependent leaf lesion mimic mutant 4(llm4) that showed abnormal chloroplast structure, photoinhibition, reduced photosynthetic protein levels, massive accumulation of reactive oxygen species(ROS), and PCD. Map-based cloning and complementation testing revealed that LLM4 encodes zeaxanthin epoxidase(ZEP), an enzyme involved in the xanthophyll cycle, which functions in plant photoprotection,ROS scavenging, and carotenoid and abscisic acid(ABA) biosynthesis. The ABA content was decreased,and the contents of 24 carotenoids differed between the llm4 mutant and the wild type(WT). The llm4mutant showed reduced dormancy and greater sensitive to ABA than the WT. We concluded that the mutation of LLM4 resulted in the failure of xanthophyll cycle, in turn causing ROS accumulation. The excessive ROS accumulation damaged chloroplast structure and induced PCD, leading eventually to the formation of lesion mimics.

Deciphering Rice Lesion Mimic Mutants to Understand Molecular Network Governing Plant Immunity and Growth

Plant lesion mimic mutants(lmms)generally possess autoimmunity and hypersensitive response(HR)-like cell death in the absence of biotic or abiotic stress.They have attracted much attention because they are useful tools for deciphering the interaction between defense signaling and growth.Recent studies have identified more than 30 lmms involved in the plant immune response and cell death in rice.Genes underlying these lmms,coding for diverse types of proteins,mainly regulate transcription,protein translation and modification,vesicular trafficking and catalyzation of metabolism.Here,we presented an overview of the most recent advances on the study of lmms in rice and proposed a perspective on potential utilization of LMM genes in agriculture.

Identification and Genetic Mapping of a Lesion Mimic Mutant in Rice

A lesion mimic stripe mutant, designated as Ims1 （lesion mimic stripe 1）, was obtained from the M2 progeny of a ^60Co y-radiation treated japonica rice variety Jiahua 1. The Ims1 mutant displayed propagation type lesions across the whole growth and developmental stages. Physiology and histochemistry analysis showed that the mutant exhibited a phenotype of white stripe when grown under high temperature （30 ℃）, and the lesion mimic caused by programmed cell death under low temperature （20 ℃）. The genetic analysis indicated that this lesion-mimic phenotype is controlled by a single locus recessive nuclear gene. Furthermore, by using simple sequence repeat markers and an F2 segregating population derived from two crosses of Ims1 ×93-11 and Ims1 ×Pei＇ai 64S, the Imsl gene was mapped between markers Indel1 and MM0112-4 with a physical distance of 400 kb on chromosome 6 in rice.

Characterization and Proteomic Analysis of Novel Rice Lesion Mimic Mutant with Enhanced Disease Resistance

Lesion mmic mutants(LMMs)are plants that spontaneously form lesions without pathogeninfection or external stimulus and exhibit resistance to pathogens.Here,a rice LMM was created by ethylmethane sulfonate mutagenesis,named as hpil(hydrogen peroxide induced lesion).Diaminobenzidineand trypan blue staining showed that large amounts of H_(2)O_(2) were produced and cell death was occurredat and around the parts of lesion mimic in the rice leaves.The phenotype of hpil is controlled by a singlerecessive gene,localized at a 2 Mb interval on chromosome 2.The data suggested that hpil is a novelLMM with enhanced bacterial and fungal disease resistance,and multiple pathogenesis-related proteins(PRs)were up-regulated.The proteomes of leaves at three positions(different degrees of lesion mimicseverity)were characterized in hpil compared with its wild type plant.Differentially expressed proteinswere detected by two dimensional difference gel electrophoresis and 274 proteins were identified byMALDITOF/TOFTM.These proteins were related to metabolic process,cellular process and response tostimulus,with mostly down-regulated in hpil leaves.Many of these proteins were related to the Calvincycle,photosynthetic electron transport chain,glycolysis/gluconeogenesis and phosphonates pathways.Some resistance-related proteins including 14-3-3 proteins,OsPR10 and antioxidases such asperoxidase,superoxide dismutase and ascorbate peroxidase were up-regulated in leaves with lesionmimic.These results provide the foundation for cloning of the target gene and shed light on themechanism involved in autaimmunity of rice.

Characterization and mapping of a novel light-dependent lesion mimic mutant lmm6 in rice(Oryza sativa L.)

A novel rice lesion mimic mutant (LMM) was isolated from an ethane methyl sulfonate (EMS)-induced 02428 mutant bank. The mutant, tentatively designated as lmm6, develops necrotic lesions in the whole growth period along with changes in several important agronomic traits. We found that the initiation of the lesions was induced by light and cel death occurred in lmm6 accompanied with accumulation of reactive oxygen species (ROS). The lower chlorophyl content, soluble protein content and superoxide dismutase (SOD) activity, the higher malondialdehyde (MDA) content were detected in lmm6 than in the wild type (WT). Moreover, the observation by transmission electronic microscope (TEM) demonstrated that some organel es were damaged and the stroma lamel a of chloroplast was irregular and loose in mesophyl cel of lmm6. In addition, lmm6 was more resistant than WT to rice blast fungus Magnaporthe grisea infection, which was consistent with increased expression of four genes involved in the defense-related reaction. Genetic analysis showed that mutant trait of lmm6 is inherited as a monogenic recessive nuclear gene located on the long arm of chromosome 6. Using simple sequence repeat (SSR) markers, the target gene was ifnal y delimited to an interval of 80.8 kb between markers MM2359 and MM2370, containing 7 annotated genes. Taken together, our results provide the information to identify a new gene involved in rice lesion mimic, which wil be helpful in clarifying the mechanism of cel death and disease resistance in rice.

Deterioration of Antioxidant Competence in Barley Lesion Mimic Mutant 194

A barley mutant,194,was observed to exhibit a leaf spot phenotype over the whole course of its growing period.In this study,the phenotype and antioxidant competence were studied in the lesion mimic mutant 194.Plant height was slightly higher in mutant 194 than in the wild type(WT).In addition,leaf spot per plant in mutant 194 was significantly higher than in WT.Antioxidant competence,as indicated by reactive oxygen species(ROS)accumulation,antioxidant enzyme activity,and the expression of antioxidant enzyme-encoding genes was also assessed in mutant 194.Compared to the WT,mutant 194 displayed a relatively higher accumulation of ROS,accompanied by lower activities of some antioxidant enzymes and downregulation of antioxidant enzyme-encoding genes.This demonstrated reduced antioxidant competence in mutant 194.The results suggested that this lower antioxidant competence of mutant 194 could lead to the accumulation of excessive ROS.This excess of ROS could induce programmed cell death and has the potential to promote disease resistance in mutant 194.

The gene encoding flavonol synthase contributes to lesion mimic in wheat

Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection,and is characterized by a hypersensitive-response(HR)that closely linked to plant disease resistance.Despite this,only a few lesion mimic genes have been identified in wheat.In this investigation,a lesion mimic wheat mutant named je0297 was discovered,showing no alteration in yield components when compared to the wild type(WT).Segregation ratio analysis of the F_(2)individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297.Using Bulked segregant analysis(BSA)and exome capture sequencing,we mapped the lesion mimic gene designated as lm6 to chromosome 6BL.Further gene fine mapping using 3315 F_(2)individuals delimited the lm6 within a 1.18 Mb region.Within this region,we identified 16 high-confidence genes,with only two displaying mutations in je0297.Notably,one of the two genes,responsible for encoding flavonol synthase,exhibited altered expression levels.Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6.Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis,including flavone and flavonol biosynthesis,isoflavonoid biosynthesis,and flavonoid biosynthesis pathways.Furthermore,more than 30 pathogen infection-related(PR)genes exhibited upregulation in the mutant.Corresponding to this expression pattern,the flavonoid content in je0297 showed a significant decrease in the 4^(th)leaf,accompanied by a notable accumulation of reactive oxygen,which likely contributed to the development of lesion mimic in the mutant.This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.

Characterization and mapping of a lesion mimic mutant in rice (Oryza sativa L.)

A rice initiation-type lesion mimic mutant (imi) was identified, which was isolated from an indica rice Zhongxian 3037 through γ radiation mutagenesis. Trypan blue staining and sterile culture revealed that the mutant spontaneously developed lesions on the leaves in a develop mentally regulated and light-dependent manner. Genetic analysis indicated that the lesion mimic trait was controlled by a single resessive locus. Using public molecular markers and an F2 population derived from lmi and 93-11, we mapped the lmi locus to the short arm of chromosome 8, nearby the centromere, between two SSR markers RM547 and RM331. The genetic distance was 1.2 and 3.2 Cm, respectively. Then according to the public rice genomic sequence between the two SSR markers, lmi was further finely tagged by three CAPS markers: C4135-8, C4135-9 and C4135-10. And lmi locus was a co-segregated with marker C4135-10, providing a starting point for imi gene cloning.

A combination of leaf rust resistance gene Lr34 and lesion mimic gene lm significantly enhances adult plant resistance to Puccinia triticina in wheat

Leaf rust caused by Puccinia triticina is an economically-important disease in wheat worldwide.A combination of different types of resistance genes may significantly enhance rust resistance under rust-favorable conditions.To investigate the interactions between the rust resistance gene Lr34 and the lesion mimic gene lm on 1BL in Ning 7840,a segregating F8-10 population of 180 recombinant inbred lines was developed from Ning 7840/Chokwang and evaluated for both lesion mimic expression and leaf rust response at the adult plant stage in a greenhouse.A major quantitative trait locus(QTL),derived from Sumai 3,was co-localized with Lr34 on chromosome 7D and explained 41.5% of phenotypic variations for rust severity and 22.1% for leaf tip necrosis(LTN).The presence of Lr34 was confirmed by Lr34-specific markers cssfr1 and cssfr2 in Ning 7840 and Sumai 3.Unlike Lr34,lm conditioned a spontaneous lesion mimic phenotype and had a significant effect on reducing uredinial size,and a smaller effect on severity.Additive effects were observed between lm and Lr34 for severity and LTN,and an epistatic effect was observed for infection type.Single marker analysis also identified several other QTL with minor effects on severity,infection type,or LTN.

Radiological Mimics of Popliteal Cysts: An Algorithmic Approach Using US and MRI to Identify the Potentially Malignant Lesions: Case Series

Background: Popliteal cysts are common and present as asymptomatic lumps in the medial popliteal fossa. Some have complex internal characteristics such as septa and loose-bodies. However, not all are popliteal cysts and can be aggressive. These lesions need to be differentiated by the absence of the communicating neck with the joint on ultrasound. Presence of Doppler flow of non-communicating cysts requires further evaluation on MRI, prior to performing a biopsy. Using a case series, we propose an algorithmic approach that is simple and will help identify the malignant lesions and institute appropriate management. Case-Presentation: Popliteal Cyst: On ultrasound: characteristic neck communicating with knee joint. Synovial Sarcoma: Gadolinium enhancement, with areas of low-, iso- and hyper-intense signal to fat on T2. Synovial-Osteochondromatosis: Non-mineralized: T1-low/intermediate intensity;T2-high intensity. Mineralized type: low intensity on T1 & T2. Thrombosed Popliteal Aneurysm: Lamellated appearance-high/low signal intensity on T2. Myxoid-Liposarcomas: Inhomogeneous appearance;homogenous with gadolinium. Usually require a biopsy for diagnosis. Conclusion: The cystic lesions in the medial aspect of the popliteal fossa can be misdiagnosed. Our article reiterates the importance of the communicating neck that separates popliteal cysts from other mimics. We have proposed an algorithm to identify these mimics.

水稻类病变突变体lms1的表型鉴定与抗病分子机制分析

类病变突变体是研究植物细胞死亡和抗病分子机制的重要遗传材料。通过辐射诱变粳稻品种武运粳7号,获得一个少见的抗病性下降的类病变突变体lms1(lesion mimic and disease susceptible mutant 1)。与野生型相比,该突变体叶片自发出现红褐色斑点,其株高、穗长、每穗粒数以及单株产量降低,但千粒重增加;对水稻白叶枯病的抗性显著下降,组织染色表明突变体的叶片中存在明显的细胞死亡以及活性氧的过量积累。遗传分析表明,lms1突变体的表型受单隐性核基因控制,利用图位克隆技术将lms1基因精细定位于水稻9号染色体Indel7和Indel8两个分子标记间,物理距离为62 kb。定位区间内候选基因的PCR扩增测序结果表明,其中一个编码泛素羧基末端水解酶的OsLMP1(Lesion Mimic Phenotype 1)基因第1个外显子中插入了一段长度为654 bp的序列,导致其蛋白翻译提前终止。利用蛋白组学技术分析了lms1突变体与野生型对照的蛋白积累水平,共鉴定到19个差异蛋白(7个上调、12个下调),主要参与氧化还原、叶绿素合成、光合作用等代谢途径。上述结果为进一步研究OsLMP1基因的功能及其调控细胞程序性死亡与抗病性的分子机制提供借鉴。

小麦类病斑突变体lmpa1的早衰特性研究

本研究以小麦品系KD527及其经EMS诱变创制出的类病斑型早衰突变体lmpa1和两者的杂交F1、F2群体为材料,分析其类病斑发生动态、农艺及产量性状,并进行相关光合指标测定与组织化学染色鉴定,以期分析lmpa1发生类病斑早衰的生理生化机制.结果表明,KD527能正常完成生长成熟过程,而lmpa1在生长前期表现正常,挑旗后叶片受光诱导而发生类病斑反应,出现黄褐色类病斑,且随生育期延长,类病斑逐渐蔓延至叶鞘、茎秆和穗部,引发整株早衰直至干枯死亡,导致其产量降低;类病斑发生期间,lmpa1叶片净光合速率、气孔导度和蒸腾速率显著低于KD527(P<0.01),胞间CO_(2)浓度显著高于KD527(P<0.01);总叶绿素量含量明显低于KD527,且二者之间的差异随类病斑发生程度的增加而增大;类病斑发生初期,lmpa1的叶片过氧化氢(H_(2)O_(2))含量及过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性均较KD527高,随着类病斑扩散,其CAT、SOD、POD活性急剧下降,H2O2含量急剧增加;组织化学染色分析表明类病斑发生部位出现细胞死亡.本结论为类病斑早衰基因Lmpa1调控小麦早衰发生的生理生化机制研究奠定了理论基础.

水稻颖壳类病斑突变体glmm1的鉴定与基因定位

【目的】鉴定水稻颖壳类病斑突变体,并进行基因定位,为基因克隆及其分子机制研究奠定基础。【方法】对野生型材料LR005和经EMS诱变得到的颖壳类病斑突变体glmm1(glume lesion mimics mutant 1)进行农艺性状分析、扫描电镜分析、DAB染色和全硅含量测定。glmm1与广亲和材料L422杂交获得的F2群体用于遗传分析,利用图位克隆和BSA-seq方法进行基因定位。【结果】突变体glmm1在抽穗10 d后颖壳和叶片逐渐出现褐色斑点,成熟后颖壳完全呈现褐色。与野生型相比,突变体的株高、穗长、每穗总粒数、结实率和千粒重等都极显著降低。DAB染色表明glmm1颖壳和叶片的活性氧含量增多;扫描电镜显示突变体颖壳和叶片表面硅质细胞皱缩。遗传分析结果表明,突变体glmm1的颖壳类病斑表型受到一对隐性基因控制。利用glmm1与L422的F2分离群体,通过图位克隆和BSA-seq等策略将glmm1定位在水稻第2染色体上68 kb的区间内。该区间内有10个候选基因。序列分析发现该区间仅有一个SNP位点,位于基因Lsi1(LOC_Os02g51110)的第5个外显子上,导致第238位氨基酸由异亮氨酸变为苏氨酸。对颖壳和叶片全硅含量的测定表明,glmm1突变体中硅的积累减少,说明GLMM1可能是Lsi1的等位突变。【结论】GLMM1是Lsi1新的等位突变基因,该突变造成植株硅含量的降低和活性氧的积累,致使颖壳和叶片产生褐色类病斑。

小麦类病斑突变体lm452的生理和遗传特性分析

在小麦品种西农1376与克旱21构建的近等基因系中发现一个自然突变的类病斑家系lm452。本研究对lm452的类病斑发生进程、生理生化特性、农艺性状、遗传分离规律等进行了研究。结果表明,类病斑最先发生于第一叶,颜色由白色渐变为黄褐色,呈条纹斑块状;类病斑数量随着植株生长发育进程逐渐增加,可蔓延至叶鞘。类病斑的发生受温度和光照影响,遮光可以避免或减轻类病斑的发生;低温和强光可加重类病斑发生。生理生化分析表明类病斑的形成伴随着超氧化物产生、可溶性蛋白质含量降低和细胞活性降低;突变体lm452的千粒重在田间和温室环境条件下均较表型正常姊妹系g451极显著降低。遗传分析表明lm452的类病斑性状受单个隐性核基因控制。上述结果为lm452类病斑基因的克隆和分子调控网络机制解析奠定了基础。

植物类病变形成机制与信号传导途径

植物类病变是指植物在无显著非生物胁迫、物理机械损伤或者外部致病因素的作用下,其叶片、叶鞘或整株上自发地出现坏死斑点的现象。这种局部细胞死亡的现象类似于某些致病菌感染的结果,其症状与植物超敏反应(Hypersensitive response, HR)的表征极为相似,是一种程序性细胞死亡(Programmed cell death, PCD)。许多类病变突变体组成性表达病程相关基因,并增强对某种或多种病原微生物的抗性,因而类病变突变体可作为研究植物防御反应和PCD的理想材料。本文对植物类病变突变体的来源、命名、类型、遗传方式、抗性特点、基因克隆、形成机制以及信号传导途径进行了总结,并展望了植物类病变突变体未来的研究方向,以期为进一步探索PCD调控机制和防御反应作用机理提供理论依据。

水稻类病变突变体lrd40的抗病性与细胞学分析

由中花11经γ射线诱变获得的水稻类病变突变体lrd40,是一个对环境不敏感的全生育期表达的突变体。遗传分析表明,lrd40 的类病变性状为1个隐性位点控制,与已报道的类病变突变体lrd38、lrd39、lrd42 是非等位的。经多点接种试验,lrd40 对水稻白叶枯病2个菌系PXO99A和DY89031都表现抗病。lrd40 与野生型杂交F2 接种结果证实,lrd40 的抗白叶枯病与类病变的表现是一致的。锥虫蓝(trypan blue)染色观察到lrd40 的类病变发生可能是一个程序性细胞死亡过程,经二氨基联苯胺(diamino benzidine,DAB)染色检测到lrd40 的类病变表达时,叶片上产生H2 O2 沉淀。细胞学观察结果说明,lrd40 的类病变发生类似于植物防卫反应的早期特征,如细胞死亡和过敏反应导致的氧迸发(oxidative burst)。

一个水稻类病变黄叶突变体的鉴定和精细定位

从15 000多个水稻转基因株系中发现了一个类病变黄叶突变体。该突变体最显著的特征为叶片由下而上依次黄化,同时出现类似病原体感染的病斑。根据突变体表型,将该突变体命名为syl1(spotted and yellow leaves 1)。遗传学分析表明,该突变性状受1对隐性核基因控制。PCR检测和潮霉素抗性分析显示,该突变表型不是由T-DNA插入引起的。为了克隆该基因,将此突变体和籼稻品种龙特甫杂交获得F2和F3定位群体,利用网上公布的SSR标记首先将突变基因定位在第12染色体短臂端分子标记RM7619附近。随后利用日本晴和9311序列的差异,在该区域新设计了9对InDel分子标记,进一步将syl1基因定位在BAC克隆OSJNBa0002O20内分子标记WL32与WL35之间约102kb的区域。为进一步克隆syl1基因奠定了基础。

水稻类病变突变体C23的遗传分析与基因的精细定位

【目的】对水稻类病变突变体C23进行遗传分析,并对其突变基因进行分子标记定位。【方法】通过EMS化学诱变获得类病变突变体。对突变体的形态特征、主要农艺性状进行观察,同时进行组织化学分析,对突变性状进行遗传分析,并以C23/浙辐802F2作群体,应用分子标记进行基因定位。【结果】突变体植株三叶期开始在叶片上出现近似圆形淡黄色斑点,随着植株的生长,斑点数量增多,面积逐渐扩大并连成中心枯黄色,边缘黄褐色的病斑,至成熟时病斑几乎遍布整个植株,该类病变发生可能属于程序性细胞死亡过程。与野生型相比,突变体C23的成熟期株高降低,有效分蘖数和每穗着粒数减少,千粒重和结实率降低。遗传分析表明,C23的突变性状由1对隐性核基因控制,该基因位于水稻第12染色体着丝粒附近,SSR标记RM101和InDel标记Ch12-112之间,与这2个标记的遗传距离分别为0.36和0.73cM,并与该区段内的4个InDel标记Ch12-91、Ch12-92、Ch12-95和Ch12-104共分离。【结论】认为C23的突变基因位点与已鉴定的spl1位点接近,但其植株类病变表型与spl1突变体表型有较大差异。

水稻类病斑突变体spl34的鉴定与基因精细定位

利用化学诱变剂EMS处理籼型水稻恢复系"缙恢10号",从其后代中筛选到1个遗传稳定的类病斑突变体spl34。该突变体于分蘖后期在下部叶片的叶鞘上开始出现褐色的类病斑,随后沿着中脉扩散至整个叶片,成熟期扩散至整个植株。相比于野生型,该突变体的株高显著变矮,穗长显著变短,穗粒数、结实率和千粒重极显著降低。遮光试验和组织化学分析表明,突变体类病斑的形成受光诱导,在类病斑形成部位发生大量过氧化氢沉积和细胞程序性死亡。荧光显微镜观察发现,在紫外光照射下突变体产生的荧光较野生型弱。与野生型相比,突变体spl34的H_2O_2和O_2^-含量较高,而CAT、POD和T-SOD等保护酶的活性显著降低;稻瘟病抗性无明显差异或略显降低。遗传分析表明,突变体spl34的表型受1对隐性核基因控制。基因定位结果表明,该基因定位于第4染色体的LR49和LR52两个分子标记之间,物理距离为200 kb。测序分析发现该区间内的候选基因LOC_Os04g56480的第3449位碱基发生突变(G3449T),导致色氨酸替换为半胱氨酸。qRT-PCR结果表明该基因在突变体内表达量降低,而部分病程相关基因的表达量则升高。

水稻类病斑突变体lmm4的鉴定及其基因定位

在EMS诱变的日本晴突变体库中,筛选到一个类病斑突变体。该突变体类病斑出现于分蘖始期,而后慢慢扩散至叶鞘、茎秆,最后至整个植株,属于扩散型类病斑突变体。相比野生型,突变体的lmm4的株高明显降低,有效穗数减少,穗长缩短,结实率、每穗实粒数、千粒重均显著降低。遮光处理实验表明,突变体lmm4类病斑的产生受自然光诱导。突变体lmm4的光合色素含量和净光合速率明显低于野生型。电子显微镜观察发现突变体类病斑部位叶肉细胞中的叶绿体结构发育异常。台盼蓝染色实验表明突变体类病斑部有大量的死亡细胞。二氨基联苯胺染色实验表明类病斑部位有过氧化氢沉积。稻瘟病抗性鉴定结果表明,与野生型相比,突变体lmm4对C7和G1等稻瘟病菌生理小种的抗性明显增强。实时PCR分析证明在突变体lmm4中防卫反应相关基因POC1、PAL、PBZ1、PR1的表达量显著提高。遗传分析表明突变体表型符合单隐性核基因控制的遗传规律。lmm4定位在第5染色体着丝粒附近Ind-4和Ind-6之间大约235kb的区域内。