RESUMO
MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice (Oryza sativa) Argonaute (AGO) protein, has been reported to function specifically at premeiotic and meiotic stages of germ cell development and is associated with a novel class of germ cell-specific small noncoding RNAs called phased small RNAs (phasiRNAs). MEL1 accumulation is temporally and spatially regulated and is eliminated after meiosis. However, the metabolism and turnover (i.e. the homeostasis) of MEL1 during germ cell development remains unknown. Here, we show that MEL1 is ubiquitinated and subsequently degraded via the proteasome pathway in vivo during late sporogenesis. Abnormal accumulation of MEL1 after meiosis leads to a semi-sterile phenotype. We identified a monocot-specific E3 ligase, XBOS36, a CULLIN RING-box protein, that is responsible for the degradation of MEL1. Ubiquitination at four K residues at the N terminus of MEL1 by XBOS36 induces its degradation. Importantly, inhibition of MEL1 degradation either by XBOS36 knockdown or by MEL1 overexpression prevents the formation of pollen at the microspore stage. Further mechanistic analysis showed that disrupting MEL1 homeostasis in germ cells leads to off-target cleavage of phasiRNA target genes. Our findings thus provide insight into the communication between a monocot-specific E3 ligase and an AGO protein during plant reproductive development.
Assuntos
Oryza/fisiologia , Proteínas de Plantas/metabolismo , Esporos/crescimento & desenvolvimento , Ubiquitina/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Regulação da Expressão Gênica de Plantas , Lisina/metabolismo , Meiose , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA de Plantas/genética , RNA de Plantas/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Esporos/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Plant spermatogenesis is a complex process that directly affects crop breeding. A rapid change in gene abundance occurs at early meiosis prophase, when gene regulation is selective. However, how these genes are regulated remains unknown. Here, we show that rice reproductive phasiRNAs are essential for the elimination of a specific set of RNAs during meiotic prophase I. These phasiRNAs cleave target mRNAs in a regulatory manner such that one phasiRNA can target more than one gene, and/or a single gene can be targeted by more than one phasiRNA to efficiently silence target genes. Our investigation of phasiRNA-knockdown and PHAS-edited transgenic plants demonstrates that phasiRNAs and their nucleotide variations are required for meiosis progression and fertility. This study highlights the importance of reproductive phasiRNAs for the reprogramming of gene expression during meiotic progression and establishes a basis for future studies on the roles of phasiRNAs with a goal of crop improvement.
Assuntos
Regulação da Expressão Gênica de Plantas , Meiose/genética , Oryza/citologia , Oryza/genética , RNA de Plantas/metabolismo , Sequência de Bases , Fertilidade/genética , Gametogênese Vegetal/genética , Modelos Biológicos , Nucleotídeos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Pólen/citologia , Pólen/genética , Clivagem do RNA , RNA de Plantas/genética , Reprodutibilidade dos TestesRESUMO
The intine, the inner layer of the pollen wall, is essential for the normal development and germination of pollen. However, the composition and developmental regulation of the intine in rice (Oryza sativa) remain largely unknown. Here, we identify a microRNA, OsmiR528, which regulates the formation of the pollen intine and thus male fertility in rice. The mir528 knockout mutant aborted pollen development at the late binucleate pollen stage, significantly decreasing the seed-setting rate. We further demonstrated that OsmiR528 affects pollen development by directly targeting the uclacyanin gene OsUCL23 (encoding a member of the plant-specific blue copper protein family of phytocyanins) and regulating intine deposition. OsUCL23 overexpression phenocopied the mir528 mutant. The OsUCL23 protein localized in the prevacuolar compartments (PVCs) and multivesicular bodies (MVBs). We further revealed that OsUCL23 interacts with a member of the proton-dependent oligopeptide transport (POT) family of transporters to regulate various metabolic components, especially flavonoids. We propose a model in which OsmiR528 regulates pollen intine formation by directly targeting OsUCL23 and in which OsUCL23 interacts with the POT protein on the PVCs and MVBs to regulate the production of metabolites during pollen development. The study thus reveals the functions of OsmiR528 and an uclacyanin during pollen development.