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Plant stems comprise nodes and internodes that specialize in solute exchange and elongation. However, their boundaries are not well defined, and how these basic units arise remains elusive. In rice with clear nodes and internodes, we found that one subclade of class I knotted1-like homeobox (KNOX1) genes for shoot meristem indeterminacy restricts node differentiation and allows internode formation by repressing YABBY genes for leaf development and genes from another node-specific KNOX1 subclade. YABBYs promote nodal vascular differentiation and limit stem elongation. YABBY and node-specific KNOX1 genes specify the pulvinus, which further elaborates the nodal structure for gravitropism. Notably, this KNOX1 subclade organization is specific to seed plants. We propose that nodes and internodes are distinct domains specified by YABBY-KNOX1 cross-regulation that diverged in early seed plants.
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Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio , Meristema , Oryza , Proteínas de Plantas , Tallos de la Planta , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Tallos de la Planta/anatomía & histología , Tallos de la Planta/genética , Tallos de la Planta/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Meristema/genética , Meristema/crecimiento & desarrollo , Oryza/genética , Oryza/crecimiento & desarrollo , Gravitropismo/genética , Hojas de la Planta/genética , Hojas de la Planta/anatomía & histología , Hojas de la Planta/crecimiento & desarrollo , Genes de PlantasRESUMEN
The stem is one of the major organs in seed plants and is important for plant survival as well as in agriculture. However, due to the lack of clear external landmarks in many species, its developmental and evolutionary processes are understudied compared to other organs. Recent approaches tackling these problems, especially those focused on KNOX1 and BLH transcription factors belonging to the TALE homeodomain superfamily have started unveiling the patterning process of nodes and internodes by connecting previously accumulated knowledge on lateral organ regulators. Fossil records played crucial roles in understanding the evolutionary process of the stem. The aim of this review is to introduce how the stem evolved from ancestorial sporophyte axes and to provide frameworks for future efforts in understanding the developmental process of this elusive but pivotal organ.
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The stem, consisting of nodes and internodes, is the shoot axis, which supports aboveground organs and connects them to roots. In contrast to other organs, developmental processes of the stem remain elusive, especially those initiating nodes and internodes. By introducing an intron into the Cre recombinase gene, we established a heat shock-inducible clonal analysis system in a single binary vector and applied it to the stem in the flag leaf phytomer of rice (Oryza sativa). With detailed characterizations of stem structure and development, we show that cell fate acquisition for each domain of the stem occurs stepwise. Cell fate for a single phytomer was established in the shoot apical meristem (SAM) by one plastochron before leaf initiation. Cells destined for the foot (nonelongating domain at the stem base) also started emerging before leaf initiation. Cell fate acquisition for the node began just before leaf initiation at the flank of the SAM, separating cell lineages for leaves and stems. Subsequently, cell fates for the axillary bud were established in early leaf primordia. Finally, cells committed to the internode emerged from, at most, a few cell tiers of the 12- to 25-cell stage stem epidermis. Thus, internode cell fate is established last during stem development. This study provides the groundwork to unveil underlying molecular mechanisms in stem development and a valuable tool for clonal analysis, which can be applied to various species.
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Oryza , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Diferenciación Celular , Meristema , Hojas de la Planta/metabolismo , Respuesta al Choque Térmico/genética , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
The shoot and root system architectures are fundamental for crop productivity. During the history of artificial selection of domestication and post-domestication breeding, the architecture of rice has significantly changed from its wild ancestor to fulfil requirements in agriculture. We review the recent studies on developmental biology in rice by focusing on components determining rice plant architecture; shoot meristems, leaves, tillers, stems, inflorescences and roots. We also highlight natural variations that affected these structures and were utilized in cultivars. Importantly, many core regulators identified from developmental mutants have been utilized in breeding as weak alleles moderately affecting these architectures. Given a surge of functional genomics and genome editing, the genetic mechanisms underlying the rice plant architecture discussed here will provide a theoretical basis to push breeding further forward not only in rice but also in other crops and their wild relatives.
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Callose is a plant cell wall polysaccharide whose deposition is spatiotemporally regulated in various developmental processes and environmental stress responses. The appearance of callose in premeiotic anthers is a prominent histological hallmark for the onset of meiosis in flowering plants; however, the biological role of callose in meiosis remains unknown. Here, we show that rice (Oryza sativa) GLUCAN SYNTHASE LIKE5 (OsGSL5), a callose synthase, localizes on the plasma membrane of pollen mother cells (PMCs) and is responsible for biogenesis of callose in anther locules through premeiotic and meiotic stages. In Osgsl5 mutant anthers mostly lacking callose deposition, aberrant PMCs accompanied by aggregated, unpaired, or multivalent chromosomes were frequently observed and, furthermore, a considerable number of mutant PMCs had untimely progress into meiosis compared to that of wild-type PMCs. Immunostaining of meiosis-specific protein HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS2 in premeiotic PMCs revealed precocious meiosis entry in Osgsl5 anthers. These findings provide insights into the function of callose in controlling the timing of male meiosis initiation and progression, in addition to roles in microsporogenesis, in flowering plants.
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Meiosis , Oryza , Meiosis/genética , Glucanos/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Oryza/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
In transgenic experiments, we often face fundamental requirements such as overexpressing a certain gene, developing organelle markers, testing promoter activities, introducing large genomic fragments, and combinations of them. To fulfill these multiple requirements in rice, we developed simple binary vectors with or without maize ubiquitin (UBQ) promoter, Gateway cassette and fluorescent proteins. First, we compared stabilities of cauliflower mosaic virus 35S and maize UBQ promoters for constitutive gene expression in transgenic rice. We show that the 35S promoter was frequently silenced after shoot regeneration, whereas maize UBQ promoter achieved stable expression in various young tissues. Binary vectors with Gateway cassettes under the control of the UBQ promoter allowed us to develop stable organelle markers for nuclei, microtubules and P-bodies in rice. The maize UBQ promoter can be easily replaced with any promoters of interest as exemplified by reporters of mitotic cells and provascular bundles. Finally, by introducing two genomic fluorescent reporters, we showed utilities of the Gateway cassette and two selection markers in large DNA fragment transfer and sequential transformations, respectively. Thus, these binary vectors provide useful choices of transgenic experiments in rice.
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Biological resources are the basic infrastructure of bioscience research. Rice (Oryza sativa L.) is a good experimental model for research in cereal crops and monocots and includes important genetic materials used in breeding. The availability of genetic materials, including mutants, is important for rice research. In addition, Oryza species are attractive to researchers for both finding useful genes for breeding and for understanding the mechanism of genome evolution that enables wild plants to adapt to their own habitats. NBRP-RICE contributes to rice research by promoting the usage of genetic materials, especially wild Oryza accessions and mutant lines. Our activity includes collection, preservation and distribution of those materials and the provision of basic information on them, such as morphological and physiological traits and genomic information. In this review paper, we introduce the activities of NBRP-RICE and our database, Oryzabase, which facilitates the access to NBRP-RICE resources and their genomic sequences as well as the current situation of wild Oryza genome sequencing efforts by NBRP-RICE and other institutes.
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BACKGROUND: OryzaGenome ( http://viewer.shigen.info/oryzagenome21detail/index.xhtml ), a feature within Oryzabase ( https://shigen.nig.ac.jp/rice/oryzabase/ ), is a genomic database for wild Oryza species that provides comparative and evolutionary genomics approaches for the rice research community. RESULTS: Here we release OryzaGenome2.1, the first major update of OryzaGenome. The main feature in this version is the inclusion of newly sequenced genotypes and their meta-information, giving a total of 217 accessions of 19 wild Oryza species (O. rufipogon, O. barthii, O. longistaminata, O. meridionalis, O. glumaepatula, O. punctata, O. minuta, O. officinalis, O. rhizomatis, O. eichingeri, O. latifolia, O. alta, O. grandiglumis, O. australiensis, O. brachyantha, O. granulata, O. meyeriana, O. ridleyi, and O. longiglumis). These 19 wild species belong to 9 genome types (AA, BB, CC, BBCC, CCDD, EE, FF, GG, and HHJJ), representing wide genomic diversity in the genus. Using the genotype information, we analyzed the genome diversity of Oryza species. Other features of OryzaGenome facilitate the use of information on single nucleotide polymorphisms (SNPs) between O. sativa and its wild progenitor O. rufipogon in rice research, including breeding as well as basic science. For example, we provide Variant Call Format (VCF) files for genome-wide SNPs of 33 O. rufipogon accessions against the O. sativa reference genome, IRGSP1.0. In addition, we provide a new SNP Effect Table function, allowing users to identify SNPs or small insertion/deletion polymorphisms in the 33 O. rufipogon accessions and to search for the effect of these polymorphisms on protein function if they reside in the coding region (e.g., are missense or nonsense mutations). Furthermore, the SNP Viewer for 446 O. rufipogon accessions was updated by implementing new tracks for possible selective sweep regions and highly mutated regions that were potentially exposed to selective pressures during the process of domestication. CONCLUSION: OryzaGenome2.1 focuses on comparative genomic analysis of diverse wild Oryza accessions collected around the world and on the development of resources to speed up the identification of critical trait-related genes, especially from O. rufipogon. It aims to promote the use of genotype information from wild accessions in rice breeding and potential future crop improvements. Diverse genotypes will be a key resource for evolutionary studies in Oryza, including polyploid biology.
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Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T1 generation. These results show that many Oryza species can be transformed by using modified immature-embryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.
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[This corrects the article DOI: 10.1371/journal.pgen.1007238.].
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Inflorescence architecture is diverse in angiosperms, and is mainly determined by the arrangement of the branches and flowers, known as phyllotaxy. In rice (Oryza sativa), the main inflorescence axis, called the rachis, generates primary branches in a spiral phyllotaxy, and flowers (spikelets) are formed on these branches. Here, we have studied a classical mutant, named verticillate rachis (ri), which produces branches in a partially whorled phyllotaxy. Gene isolation revealed that RI encodes a BELL1-type homeodomain transcription factor, similar to Arabidopsis PENNYWISE/BELLRINGER/REPLUMLESS, and is expressed in the specific regions within the inflorescence and branch meristems where their descendant meristems would soon initiate. Genetic combination of an ri homozygote and a mutant allele of RI-LIKE1 (RIL1) (designated ri ril1/+ plant), a close paralog of RI, enhanced the ri inflorescence phenotype, including the abnormalities in branch phyllotaxy and rachis internode patterning. During early inflorescence development, the timing and arrangement of primary branch meristem (pBM) initiation were disturbed in both ri and ri ril1/+ plants. These findings suggest that RI and RIL1 were involved in regulating the phyllotactic pattern of the pBMs to form normal inflorescences. In addition, both RI and RIL1 seem to be involved in meristem maintenance, because the ri ril1 double-mutant failed to establish or maintain the shoot apical meristem during embryogenesis.
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Inflorescencia/embriología , Inflorescencia/metabolismo , Meristema/embriología , Meristema/metabolismo , Oryza/embriología , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Inflorescencia/genética , Meristema/genética , Oryza/genética , Proteínas de Plantas/genéticaRESUMEN
In plants, transcription factors often act as cell-to-cell trafficking mobile proteins and specify cell fate. Thus, to visualize spatiotemporal expression pattern and localization of transcription factors are essential to understand their functions during development. Several protocols have been developed to observe fluorescent protein. However, plant-specific autofluorescent compounds and various tissue components with different refractive indexes interfere with detection of fluorescent signals of your interest. Furthermore, cell fate specification often occurs in a limited number of cells covered by lateral/layers of organs. To overcome those issues, the plant clearing method, ClearSee, was recently developed for high-resolution imaging inside tissues by making background transparent. In this chapter, we provide three-dimensional imaging of fluorescent-protein-fused transcription factors by two-photon excitation microscopy in Arabidopsis and rice. Complex cell patterning with gene expression could be observed from any direction three-dimensionally. This method could be applicable to visualize any protein of your interest or it can readily be adapted in various other plants.
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Imagenología Tridimensional , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Factores de Transcripción/metabolismo , Urea/metabolismo , Xilitol/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Flores/citología , Flores/metabolismo , Oryza/citología , Oryza/metabolismo , Coloración y Etiquetado , Fijación del TejidoRESUMEN
The 24-nucleotides (nt) phased secondary small interfering RNA (phasiRNA) is a unique class of plant small RNAs abundantly expressed in monocot anthers at early meiosis. Previously, 44 intergenic regions were identified as the loci for longer precursor RNAs of 24-nt phasiRNAs (24-PHASs) in the rice genome. However, the regulatory mechanism that determines spatiotemporal expression of these RNAs has remained elusive. ETERNAL TAPETUM1 (EAT1) is a basic-helix-loop-helix (bHLH) transcription factor indispensable for induction of programmed cell death (PCD) in postmeiotic anther tapetum, the somatic nursery for pollen production. In this study, EAT1-dependent non-cell-autonomous regulation of male meiosis was evidenced from microscopic observation of the eat1 mutant, in which meiosis with aberrantly decondensed chromosomes was retarded but accomplished somehow, eventually resulting in abortive microspores due to an aberrant tapetal PCD. EAT1 protein accumulated in tapetal-cell nuclei at early meiosis and postmeiotic microspore stages. Meiotic EAT1 promoted transcription of 24-PHAS RNAs at 101 loci, and importantly, also activated DICER-LIKE5 (DCL5, previous DCL3b in rice) mRNA transcription that is required for processing of double-stranded 24-PHASs into 24-nt lengths. From the results of the chromatin-immunoprecipitation and transient expression analyses, another tapetum-expressing bHLH protein, TDR INTERACTING PROTEIN2 (TIP2), was suggested to be involved in meiotic small-RNA biogenesis. The transient assay also demonstrated that UNDEVELOPED TAPETUM1 (UDT1)/bHLH164 is a potential interacting partner of both EAT1 and TIP2 during early meiosis. This study indicates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play some important roles in this process. Spatiotemporal expression control of these bHLH proteins is a clue to orchestrate precise meiosis progression and subsequent pollen production non-cell-autonomously.
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Flores/genética , Flores/metabolismo , Oryza/genética , Polen/metabolismo , Factores de Transcripción/fisiología , Diferenciación Celular/genética , Flores/citología , Regulación de la Expresión Génica de las Plantas , Meiosis/genética , Oryza/fisiología , Infertilidad Vegetal/genética , Proteínas de Plantas/fisiología , Polen/genética , ARN de Planta/genéticaRESUMEN
Plant vascular systems in the stem connect roots with aerial organs to move solutes containing minerals, nutrients as well as signaling molecules, and therefore, they play pivotal roles in plant growth and development. However, stem vascular systems, especially in crop species, have been poorly described since they are deeply embedded in the tissue. Here we describe a protocol to utilize micro-computed tomography (micro-CT) scanning to visualize vascular networks in the maize stem. The protocol covers sample fixation and staining with contrasting reagents, data acquisition using micro-CT, reconstructing three-dimensional (3D) models of stem inner structures and extraction of vascular networks from the model. This protocol can be easily applied to various types of species and organs/tissues.
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Monocot stems lack the vascular cambium and instead have characteristic structures in which intercalary meristems generate internodes and veins remain separate and scattered. However, developmental processes of these unique structures have been poorly described. BELL1-like homeobox (BLH) transcription factors (TFs) are known to heterodimerize with KNOTTED1-like homeobox TFs to play crucial roles in shoot meristem maintenance, but their functions are elusive in monocots. We found that maize (Zea mays) BLH12 and BLH14 have redundant but important roles in stem development. BLH12/14 interact with KNOTTED1 (KN1) in vivo and accumulate in overlapping domains in shoot meristems, young stems, and provascular bundles. Similar to kn1 loss-of-function mutants, blh12 blh14 (blh12/14) double mutants fail to maintain axillary meristems. Unique to blh12/14 is an abnormal tassel branching and precocious internode differentiation that results in dwarfism and reduced veins in stems. Micro-computed tomography observation of vascular networks revealed that blh12/14 double mutants had reduced vein number due to fewer intermediate veins in leaves and precocious anastomosis in young stems. Based on these results, we propose two functions of BLH12/14 during stem development: (1) maintaining intercalary meristems that accumulate KN1 and prevent precocious internode differentiation and (2) preventing precocious anastomosis of provascular bundles in young stems to ensure the production of sufficient independent veins.
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Proteínas de Plantas/metabolismo , Zea mays/citología , Zea mays/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Meristema/citología , Meristema/genética , Meristema/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Brotes de la Planta/citología , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Zea mays/genéticaRESUMEN
KNOX genes were initially found as shoot meristem regulators in angiosperms. Recent studies in diverse plant lineages however, have revealed the divergence of KNOX gene function during the evolution of diploid body plans. Using genomic approaches, class I KNOX transcription factors have been shown to regulate multiple hormone pathways including auxin and brassinosteroid as well as many transcription factors that play important roles in plant development. Class I KNOX proteins appear to be activators, whereas class II proteins act as repressors in transcriptional regulation of their target genes.
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Evolución Biológica , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Proteínas de Plantas/genética , Factores de Transcripción/genética , Diploidia , Desarrollo de la Planta , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In flowering plants, knotted1-like homeobox (KNOX) transcription factors play crucial roles in establishment and maintenance of the shoot apical meristem (SAM), from which aerial organs such as leaves, stems, and flowers initiate. We report that a rice (Oryza sativa) KNOX gene Oryza sativa homeobox1 (OSH1) represses the brassinosteroid (BR) phytohormone pathway through activation of BR catabolism genes. Inducible overexpression of OSH1 caused BR insensitivity, whereas loss of function showed a BR-overproduction phenotype. Genome-wide identification of loci bound and regulated by OSH1 revealed hormonal and transcriptional regulation as the major function of OSH1. Among these targets, BR catabolism genes CYP734A2, CYP734A4, and CYP734A6 were rapidly upregulated by OSH1 induction. Furthermore, RNA interference knockdown plants of CYP734A genes arrested growth of the SAM and mimicked some osh1 phenotypes. Thus, we suggest that local control of BR levels by KNOX genes is a key regulatory step in SAM function.
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Brasinoesteroides/metabolismo , Redes Reguladoras de Genes , Genes de Plantas , Meristema/genética , Oryza/genética , Brasinoesteroides/farmacología , Inmunoprecipitación de Cromatina , Secuencia Conservada , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Meristema/efectos de los fármacos , Mutación/genética , Oryza/efectos de los fármacos , Fenotipo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Maintenance of organ separation is one of the essential phenomena for normal plant development. We have identified and analyzed ONION3 (ONI3), which is required for avoiding organ fusions in rice. Loss-of-function mutations of ONI3, which were identified as mutants with ectopic expression of KNOX genes in leaves and morphologically resembling KNOX overexpressors, showed abnormal organ fusions in developing shoots. The mutant seedlings showed fusions between neighboring organs and also within an organ; they stopped growing soon after germination and subsequently died. ONI3 was shown to encode an enzyme that is most similar to Arabidopsis HOTHEAD and is involved in biosynthesis of long-chain fatty acids. Expression analyses showed that ONI3 was specifically expressed in the outermost cell layer in the shoot apex throughout life cycle, and the oni3 mutants had an aberrant outermost cell layer. Our results together with previous studies suggest that long-chain fatty acids are required for avoiding organ fusions and promoting normal shoot development in rice.
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Mutación/genética , Organogénesis , Oryza/crecimiento & desarrollo , Oryza/genética , Proteínas de Plantas/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/genética , Clonación Molecular , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Ácidos Indolacéticos/metabolismo , Organogénesis/genética , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Hojas de la Planta/anatomía & histología , Hojas de la Planta/ultraestructura , Proteínas de Plantas/metabolismo , Raíces de Plantas/anatomía & histología , Análisis de Secuencia de Proteína , Ceras/metabolismoRESUMEN
A plant's surface is covered with epicuticular wax, which protects plants from inappropriate environmental conditions such as drought and pathogen attack. Very-long-chain fatty acids (VLCFAs) are the main component of epicuticular wax on the surface of above-ground organs. Here we show that a fatty acid elongase catalyzing an elongation reaction of VLCFAs is required for shoot development in rice. onion2 (oni2) mutants produced very small shoots in which leaves were fused to each other, and ceased growing after germination. The midrib of oni2 leaf blades was not developed correctly. Molecular cloning showed that ONI2 encodes a fatty acid elongase, which catalyzes the first step of elongation reactions of a carbon chain of VLCFAs, and oni2 had a reduced amount of VLCFAs. Expression analysis showed that ONI2 is specifically expressed in the outermost cell layer of young lateral organs. These results suggest that ONI2 is a layer 1-specific gene required for development of the entire shoot and that VLCFAs play an essential role in normal shoot development in rice.
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Acetiltransferasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Oryza/enzimología , Proteínas de Plantas/metabolismo , Brotes de la Planta/crecimiento & desarrollo , Acetiltransferasas/genética , Alelos , Clonación Molecular , Elongasas de Ácidos Grasos , Ácidos Grasos no Esterificados/metabolismo , Genes de Plantas , Mutación , Especificidad de Órganos , Oryza/genética , Oryza/crecimiento & desarrollo , Fenotipo , Enfermedades de las Plantas/genética , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/genética , Brotes de la Planta/enzimología , Brotes de la Planta/genética , Plantones/enzimología , Plantones/genética , Plantones/crecimiento & desarrolloRESUMEN
Self-maintenance of the shoot apical meristem (SAM), from which aerial organs are formed throughout the life cycle, is crucial in plant development. Class I Knotted1-like homeobox (KNOX) genes restrict cell differentiation and play an indispensable role in maintaining the SAM. However, the mechanism that positively regulates their expression is unknown. Here, we show that expression of a rice (Oryza sativa) KNOX gene, Oryza sativa homeobox1 (OSH1), is positively regulated by direct autoregulation. Interestingly, loss-of-function mutants of OSH1 lose the SAM just after germination but can be rescued to grow until reproductive development when they are regenerated from callus. Double mutants of osh1 and d6, a loss-of-function mutant of OSH15, fail to establish the SAM both in embryogenesis and regeneration. Expression analyses in these mutants reveal that KNOX gene expression is positively regulated by the phytohormone cytokinin and by KNOX genes themselves. We demonstrate that OSH1 directly binds to five KNOX loci, including OSH1 and OSH15, through evolutionarily conserved cis-elements and that the positive autoregulation of OSH1 is indispensable for its own expression and SAM maintenance. Thus, the maintenance of the indeterminate state mediated by positive autoregulation of a KNOX gene is an indispensable mechanism of self-maintenance of the SAM.