| Citrus is one of the most important fruit crops worldwide.Seedlessness is a highly desirable trait for citrus consumed as fresh fruit.Male sterility（MS）is one of the main causes of seedlessness in citrus.The mechanism of MS is of major interest to citrus researchers that focus on cultivar improvement.Somatic cybrid of pummelo（Citrus.grandis）（G1+HBP）was generated by protoplast fusion between a cytoplasmic male sterility（CMS）variety of Satsuma mandarin（C.unshiu‘Guoqing No.1’,G1）and a fertile pummelo（C.grandis‘Hirado Buntan’,HBP）.G1+HBP is genetically equivalent to the male mutant of its fertile parent HBP.Therefore,G1+HBP and HBP become a pair of ideal materials for uncovering CMS mechanisms in citrus.In this study,the MS in G1+HBP was characterized by the comparative cytological observation,physiological assays and transcriptome and metabolome analysis in the reproductive organs of G1+HBP and HBP.The results provide basis for investigating the regulation mechanism of MS in G1+HBP.micro RNA（miRNA）is a vital regulator of plant reproductive development.In our previous study for exploration molecular mechanism of MS in G1+HBP,CsmiR399a.1 was identified using small RNA sequencing（s RNA-seq）screen,which might be associated with the reproductive development and MS of G1+HBP.In this study,the function of CsmiR399a.1 was validated in Hongkong kumquat（Fortunella hindsii）,a short-juvenile mini-citrus,and the regulatory mechanisms of CsmiR399a.1 regulating floral organ development and MS were further investigated.The main results are as follows:1.Developmental and physiological of MS phenotype and the associated gene expression analysis in cytoplasmic hybrid G1+HBPMS phenotype of G1+HBP:Compared with HBP,the petals and the stamens of G1+HBP were abnormal,the petals were deformed,smaller not completely enclose the inner stamens and pistil before anthesis.The differentiation of stamen primordium was inhibited and the number of stamens was reduced,and the anther was defective and indehiscent.The cross sections of anthers at nine different developmental stages were observed by paraffin sections.In G1+HBP,the differentiation of anther chamber was inhibited,the taptum was abnomally thickened and degraded,the middle layer was abnormally degraded,and the endothecium abnomally thickened,in which the fiber band and secondary lignification formed abnormally in the later developmental stage of anther,and the PCD（programmed cell death）was defective in septum between two adjacent chambers.These defectives in anther wall leading to the anther indehiscence in G1+HBP.Moreover,the number of microspore mother cell（MMC）was reduced and meiosis was abnormal,leading to the reduction of pollens in G1+HBP.In addition,the pollen wall formation was inhibited in G1+HBP.The gene expression profiles associated with MS in G1+HBP:Different expression genes（DEGs）between G1+HBP and HBP anthers collected from the stage of meiosis to the primexine formation of microspore was analysed by RNA-seq to identify genes associated with the abnormal anther development and pollen sterility of G1+HBP.The up-regulated genes in G1+HBP were mainly enriched in gene ontology（GO）associated with cell acticities（including chromosome behavior,DNA behavior,mitosis,meiosis and PCD）,flower organ and microspore development,jasmonic acid（JA）signal pathway and flavonoid metabolism.The down-regulated genes in G1+HBP mainly enriched in GO terms related to primary metabolism pathways,including those of cabohydrate,amino acid,organic acid and fatty acid.Metabolism and physiological characterizations associated with MS in G1+HBP:The identification of primary and secondary metabolites between G1+HBP and HBP were performed in leaves,flowers and various tissues of floral organ.The primary metabolisms in anthers between G1+HBP and HBP were most significantly different.Compared to HBP,most of amino acids and fatty acids were significantly down-regulated in G1+HBP anthers,and most of organic acids were significantly up-regulated.The content of sugars was down-regulated,and the content of alcohols was up-regulated in G1+HBP anther.Among the 43 kinds of flavonoids identified,39accumulated at significantly different levels in anthers of G1+HBP and HBP.The PCD of anther was determinted using TUNEL.Compared to HBP,the PCD process occured earlier in the anther wall and later in taptum of G1+HBP.The content of reactive oxygen species（ROS）was detected in flowers.The H?O?content in G1+HBP flowers was significantly lower than that in HBP flowers.The activities of three ROS scavenging enzymes（i.e.,POD,SOD and CAT）in G1+HBP flowers were significantly higher than those in HBP at three consecutively developmental stages.ATP content showed no significant difference in flowers during stamen primordia stage and pollen development and mature leaves between G1+HBP and HBP,but was significant higher in G1+HBP flowers from MMC to tetrad stage than in HBP.2.Functional characterization of CsmiR399a.1-CsUBC24 module in floral development and male sterilityFunctional validation of CsmiR399a.1:Downregulation the expression of CsmiR399a.1 using a short tandem target mimic（STTM）of CsmiR399a.1 in the short-juvenile mini-citrus Hongkong kumquat（Fortunella hindsii）led to abnormal floral development,inhibition of anther dehiscence and decreased pollen fertility.In the STTM lines,flower development was abnormal from an early stage.These abnormalities mainly reflected in the petals and stamens,including an irregular flower morphology,fewer petals and smaller petal size that incompletely wrap the inner anthers and stigma,irregular filament arrangement,less and shorter filaments,and fewer anthers.Scanning electron microscopy（SEM）analysis showed that the stomata opening on the abaxial surface of anthers was inhibited and the pollen was collapsed.in CsmiR399a.1-STTM transgenic lines.Transmission electron microscopy（TEM）and I2-KI staining analysis showed that the starch in pollens of CsmiR399a.1-STTM transgenic lines,accumulated insufficiently at early developmental stage and degraded incompletely at the later stage.When grown in inorganic phosphate（Pi）-sufficient conditions,CsmiR399a.1-STTM plants had lower total phosphorus content in their leaves than the wild type（WT）and showed typical symptoms of Pi deficiency,such as inhibited growth of seedling,retarded growth of primary roots,increased number of lateral roots.In CsmiR399a.1-STTM plants,the expression of genes involved in starch metabolism and Pi-homeostasis was significantly different than in the WT.Thus,we conclude that down-regulation of CsmiR399a.1 mimicked Pi deficiency,disturbed starch metabolism,and was responsible for pollen grain collapse in the CsmiR399a.1-STTM transgenic lines.The mechanism of CsmiR399a.1 mediated regulation of flower development and male sterility:We identified CsUBC24,a citrus homolog of Arabidopsis At UBC24（PHO2）,as a target of CsmiR399a.1 using RLM-5′RACE and transient co-expression assays in Nicotiana benthamiana leaves,that was proved to interact with the floral development regulators SEPALLATA family（Cs SEP1.1,Cs SEP1.2 and Cs SEP3）,and the anther dehiscence regulator INDUCER OF CBF EXPRESSION 1（Cs ICE1）by yeast two-hybrid and split firefly luciferase complementation assay.The expression of Cs ICE1 was significantly upregulated and floral meristem identity genes Cs LMI2,which are downstream of SEP3 was significantly decreased in the STTM lines relative to wild type.We hypothesize that CsUBC24 downregulates the Cs SEPs proteins,which disrupts the floral meristem identity regulatory network and leads to developmental abnormalities in flowers.By interacting with Cs ICE1,CsUBC24 disturbs stomate function on the anther surface,which inhibits anther dehiscence.In summary,we dissected the physiological basis of MS in G1+HBP using cytological observation,physiological assays and transcriptome metabolome analysis.The function of CsmiR399a.1 in citrus reproductive development was validated,and the molecular mechanism of CsmiR399-CsUBC24 module in regulating citrus floral development and male sterility was revealed. |
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