RESUMEN
Abscission is the shedding of plant organs in response to developmental and environmental cues. Abscission involves cell separation between two neighboring cell types, residuum cells (RECs) and secession cells (SECs) in the floral abscission zone (AZ) in Arabidopsis thaliana. However, the regulatory mechanisms behind the spatial determination that governs cell separation are largely unknown. The class I KNOTTED-like homeobox (KNOX) transcription factor BREVIPEDICELLUS (BP) negatively regulates AZ cell size and number in Arabidopsis. To identify new players participating in abscission, we performed a genetic screen by activation tagging a weak complementation line of bp-3. We identified the mutant ebp1 (enhancer of BP1) displaying delayed floral organ abscission. The ebp1 mutant showed a concaved surface in SECs and abnormally stacked cells on the top of RECs, in contrast to the precisely separated surface in the wild-type. Molecular and histological analyses revealed that the transcriptional programming during cell differentiation in the AZ is compromised in ebp1. The SECs of ebp1 have acquired REC-like properties, including cuticle formation and superoxide production. We show that SEPARATION AFFECTING RNA-BINDING PROTEIN1 (SARP1) is upregulated in ebp1 and plays a role in the establishment of the cell separation layer during floral organ abscission in Arabidopsis.
RESUMEN
In flowering plants, double fertilization between male and female gametophytes, which are separated by distance, largely depends on the unique pattern of the male gametophyte (pollen): two non-motile sperm cells suspended within a tube-producing vegetative cell. A morphological screen to elucidate the genetic control governing the strategic patterning of pollen has led to the isolation of a sticky generative cell (sgc) mutant that dehisces abnormal pollen with the generative cell immobilized at the pollen wall. Analyses revealed that the sgc mutation is specifically detrimental to pollen development, causing ectopic callose deposition that impedes the timely internalization and differentiation of the generative cell. We found that the SGC gene encodes the highly conserved domain of unknown function 707 (DUF707) gene that is broadly expressed but is germline specific during pollen development. Additionally, transgenic plants co-expressing fluorescently fused SGC protein and known organelle markers showed that SGC localizes in the endoplasmic reticulum, Golgi apparatus and vacuoles in pollen. A yeast two-hybrid screen with an SGC bait identified a thaumatin-like protein that we named GCTLP1, some homologs of which bind and/or digest ß-1,3-glucans, the main constituent of callose. GCTLP1 is expressed in a germline-specific manner and colocalizes with SGC during pollen development, indicating that GCTLP1 is a putative SGC interactor. Collectively, our results show that SGC suppresses callose deposition in the nascent generative cell, thereby allowing the generative cell to fully internalize into the vegetative cell and correctly differentiate as the germline progenitor, with the potential involvement of the GCTLP1 protein, during pollen development in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glucanos/metabolismo , Polen/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Glucanos/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Polen/fisiologíaRESUMEN
Sexual reproduction in flowering plants relies on the production of haploid gametophytes that consist of germline and supporting cells. During male gametophyte development, the asymmetric mitotic division of an undetermined unicellular microspore segregates these two cell lineages. To explore genetic regulation underlying this process, we screened for pollen cell patterning mutants and isolated the heterozygous myb81-1 mutant that sheds ~50% abnormal pollen. Typically, myb81-1 microspores fail to undergo pollen mitosis I (PMI) and arrest at polarized stage with a single central vacuole. Although most myb81-1 microspores degenerate without division, a small fraction divides at later stages and fails to acquire correct cell fates. The myb81-1 allele is transmitted normally through the female, but rarely through pollen. We show that myb81-1 phenotypes result from impaired function of the GAMYB transcription factor MYB81. The MYB81 promoter shows microspore-specific activity and a MYB81-RFP fusion protein is only expressed in a narrow window prior to PMI. Ectopic expression of MYB81 driven by various promoters can severely impair vegetative or reproductive development, reflecting the strict microspore-specific control of MYB81. Our data demonstrate that MYB81 has a key role in the developmental progression of microspores, enabling formation of the two male cell lineages that are essential for sexual reproduction in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Factores Generales de Transcripción/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Linaje de la Célula , Haploidia , Mitosis , Fenotipo , Polen/genética , Polen/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores Generales de Transcripción/genéticaRESUMEN
Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA-protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Arabidopsis/embriología , Proteínas de Arabidopsis/genética , División Celular/genética , Nucléolo Celular/metabolismo , Mutación , Proteínas Nucleares/genética , Óvulo Vegetal/embriología , Óvulo Vegetal/genética , Precursores del ARN/genética , Estabilidad del ARN , ARN Ribosómico/genética , Proteínas de Unión al ARN/genéticaRESUMEN
In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin-celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6-1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)-dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6-1 during male meiosis and pollen mitosis I using fluorescent MT-markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6-1 mutants as a valuable tool for the investigation of augmin-dependent MT nucleation and dynamics in plant cells.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Microtúbulos/metabolismo , Óvulo Vegetal/crecimiento & desarrollo , Polen/genética , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Meiosis/fisiología , Mitosis/fisiología , Polen/fisiología , Reproducción/genética , Reproducción/fisiologíaRESUMEN
Angiosperm reproduction is characterized by alternate diploid sporophytic and haploid gametophytic generations. Gametogenesis shares similarities with that of animals except for the formation of the gametophyte, whereby haploid cells undergo several rounds of postmeiotic mitosis to form gametes and the accessory cells required for successful reproduction. The mechanisms regulating gametophyte development in angiosperms are incompletely understood. Here, we show that the nucleoporin Nup88-homolog MOS7 (Modifier of Snc1,7) plays a crucial role in mitosis during both male and female gametophyte formation in Arabidopsis thaliana. Using a mutagenesis screen, we identify the mos7-5 mutant allele, which causes ovule and pollen abortion in MOS7/mos7-5 heterozygous plants, and preglobular stage embryonic lethality in homozygous mos7-5 seeds. During interphase, we show that MOS7 is localized to the nuclear membrane but, like many nucleoporins, is associated with the spindle apparatus during mitosis. We detect interactions between MOS7 and several nucleoporins known to control spindle dynamics, and find that in pollen from MOS7/mos7-5 heterozygotes, abortion is accompanied by a failure of spindle formation, cell fate specification, and phragmoplast activity. Most intriguingly, we show that following gamete formation by MOS7/mos7-5 heterozygous spores, inheritance of either the MOS7 or the mos7-5 allele by a given gamete does not correlate with its respective survival or abortion. Instead, we suggest a model whereby MOS7, which is highly expressed in the Pollen- and Megaspore Mother Cells, enacts a dosage-limiting effect on the gametes to enable their progression through subsequent mitoses.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/embriología , Células Germinativas/crecimiento & desarrollo , Mitosis/fisiología , Semillas/crecimiento & desarrollo , Alelos , Arabidopsis/genética , Microtúbulos/fisiología , MutaciónRESUMEN
The THO/TREX complex mediates transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and has a role in small interfering RNA-dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, which encodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only were the levels of siRNAs reduced in tho2 mutants, but also those of mature miRNAs. As a consequence, a feedback mechanism is triggered, increasing the amount of miRNA precursors, and finally causing accumulation of miRNA-targeted mRNAs. Yeast two-hybrid experiments and confocal microscopy showed that THO2 does not appear to interact with any of the known miRNA biogenesis components, but rather with the splicing machinery, implying an indirect role of THO2 in small RNA biogenesis. Using an RNA immunoprecipitation approach, we found that THO2 interacts with miRNA precursors, and that tho2 mutants fail to recruit such precursors into the miRNA-processing complex, explaining the reduction in miRNA production in this mutant background. We also detected alterations in the splicing pattern of genes encoding serine/arginine-rich proteins in tho2 mutants, supporting a previously unappreciated role of the THO/TREX complex in alternative splicing.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , MicroARNs/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Plantas Modificadas Genéticamente , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.
Asunto(s)
Oryza/metabolismo , Polen/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
Pollen germination may occur via the so-called germination pores or directly through the pollen wall at the site of contact with the stigma. In this study, we addressed what processes take place during pollen hydration (i.e. before tube emergence), in a species with extra-poral pollen germination, Arabidopsis thaliana. A T-DNA mutant population was screened by segregation distortion analysis. Histological and electron microscopy techniques were applied to examine the wild-type and mutant phenotypes. Within 1 h of the start of pollen hydration, an intine-like structure consisting of cellulose, callose and at least partly de-esterified pectin was formed at the pollen wall. Subsequently, this 'germination plaque' gradually extended and opened up to provide passage for the cytoplasm into the emerging pollen tube. BURSTING POLLEN (BUP) was identified as a gene essential for the correct organization of this plaque and the tip of the pollen tube. BUP encodes a novel Golgi-located glycosyltransferase related to the glycosyltransferase 4 (GT4) subfamily which is conserved throughout the plant kingdom. Extra-poral pollen germination involves the development of a germination plaque and BUP defines the correct plastic-elastic properties of this plaque and the pollen tube tip by affecting pectin synthesis or delivery.
Asunto(s)
Arabidopsis/genética , Pectinas/metabolismo , Arabidopsis/crecimiento & desarrollo , Citoplasma/metabolismo , Glucanos/metabolismo , Mutagénesis Insercional , Polen/genética , Polen/crecimiento & desarrollo , Tubo Polínico/genética , Tubo Polínico/crecimiento & desarrollo , PolinizaciónRESUMEN
The phospholipase A(2) (PLA(2)) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA(2)s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA(2)s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA(2) paralogs (PLA(2)-ß, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA(2) using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA(2) inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA(2)s that are conserved in other organisms.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Germinación , Fosfolipasas A2/metabolismo , Polen/crecimiento & desarrollo , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Retículo Endoplásmico/enzimología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Aparato de Golgi/enzimología , Lisofosfolípidos/metabolismo , Mutación , Fosfolipasas A2/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Polen/genética , Polen/ultraestructura , Interferencia de ARN , ARN de Planta/genéticaRESUMEN
Flowering plants possess a unique reproductive strategy, involving double fertilization by twin sperm cells. Unlike animal germ lines, the male germ cell lineage in plants only forms after meiosis and involves asymmetric division of haploid microspores, to produce a large, non-germline vegetative cell and a germ cell that undergoes one further division to produce the twin sperm cells. Although this switch in cell cycle control is critical for sperm cell production and delivery, the underlying molecular mechanisms are unknown. Here we identify a novel F-box protein of Arabidopsis thaliana, designated FBL17 (F-box-like 17), that enables this switch by targeting the degradation of cyclin-dependent kinase A;1 inhibitors specifically in male germ cells. We show that FBL17 is transiently expressed in the male germ line after asymmetric division and forms an SKP1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex (SCF(FBL17)) that targets the cyclin-dependent kinase inhibitors KRP6 and KRP7 for proteasome-dependent degradation. Accordingly, the loss of FBL17 function leads to the stabilization of KRP6 and inhibition of germ cell cycle progression. Our results identify SCF(FBL17) as an essential male germ cell proliferation complex that promotes twin sperm cell production and double fertilization in flowering plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Ciclo Celular/fisiología , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas F-Box/metabolismo , Polen/citología , Arabidopsis/embriología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , División Celular/genética , Proliferación Celular , Proteínas F-Box/genética , Regulación de la Expresión Génica de las Plantas , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismoRESUMEN
KEY MESSAGE: Two pollen-preferential thaumatin-like proteins show both common and distinctive expression profiles. Precocious expression of one of them drastically disturbs timely deposition and dissolution of callose during microsporogenesis, leading to microspore death. Thaumatin-like proteins (TLPs), members of the pathogenesis-related protein family 5 (PR-5), are involved in plant defenses against biotic and abiotic stresses through antifungal activity and enhanced tolerance. Accordingly, studies on TLPs have focused on their responses to various pathogens and stresses and on engineering agronomically valuable crops that can be cultivated in suboptimal environments. On the other hand, the role of TLP members in plant development and their genetic regulation remains largely unexplored. Recently, we reported that the generative cell internalization after pollen mitosis I, an essential pollen patterning step for the nonmotile sperm cell delivery through a pollen tube, depends on STICKY GENERATIVE CELL which suppresses callose deposition in the nascent generative cell and interacts with a germline cell preferential GCTLP1 in Arabidopsis. Here, we additionally identified GCTLP2 which is similarly expressed in the germline cells. We generated various transgenic lines and examined their expressions and phenotypes to elucidate GCTLP functions during pollen development. Expression profiles suggest two GCTLP proteins may have common but also distinctive roles during pollen development. Importantly, ectopic expression analyses show that precocious expression of GCTLP2 severely disturbs the timely deposition and degradation of callose during microsporogenesis which is essential to produce viable microspores. Therefore, our study broadens the knowledge of TLP function and callose regulation for successful pollen development in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Glucanos , Infertilidad Vegetal , Polen , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Glucanos/metabolismo , Polen/genética , Polen/crecimiento & desarrollo , Polen/metabolismo , Infertilidad Vegetal/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Expresión Génica Ectópica , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Gametogénesis en la Planta/genética , Plantas Modificadas GenéticamenteRESUMEN
In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.
Asunto(s)
Oryza , Oryza/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Germinación/genética , Polen/metabolismo , Tubo Polínico/genética , Calmodulina/genética , Calmodulina/metabolismo , Fosfotransferasas , Nucleótidos Cíclicos/metabolismoRESUMEN
The conserved Fused kinase plays vital but divergent roles in many organisms from Hedgehog signalling in Drosophila to polarization and chemotaxis in Dictyostelium. Previously we have shown that Arabidopsis Fused kinase termed TWO-IN-ONE (TIO) is essential for cytokinesis in both sporophytic and gametophytic cell types. Here using in vivo imaging of GFP-tagged microtubules in dividing microspores we show that TIO is required for expansion of the phragmoplast. We identify the phragmoplast-associated kinesins, PAKRP1/Kinesin-12A and PAKRP1L/Kinesin-12B, as TIO-interacting proteins and determine TIO-Kinesin-12 interaction domains and their requirement in male gametophytic cytokinesis. Our results support the role of TIO as a functional protein kinase that interacts with Kinesin-12 subfamily members mainly through the C-terminal ARM repeat domain, but with a contribution from the N-terminal kinase domain. The interaction of TIO with Kinesin proteins and the functional requirement of their interaction domains support the operation of a Fused kinase signalling module in phragmoplast expansion that depends upon conserved structural features in diverse Fused kinases.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Citocinesis , Microtúbulos/metabolismo , Secuencias de Aminoácidos , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas Fluorescentes Verdes , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/genética , Modelos Biológicos , Mutación , Fenotipo , Polen/citología , Polen/enzimología , Polen/genética , Polen/fisiología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
Cellular patterning and differentiation in plants depend on the balance of asymmetric and symmetric divisions. Patterning of the male gametophyte (pollen grains) in flowering plants requires asymmetric division of the microspore followed by a symmetric division of the germ cell to produce three highly differentiated cells: a single vegetative cell and two sperm cells. In Arabidopsis sidecar pollen (scp) mutants a proportion of microspores first divide symmetrically, and then go on to produce 'four-celled' pollen with an extra vegetative cell; however, details of the timing and origin of phenotypic defects in scp and the identity of the SCP gene have remained obscure. Comparative analysis of the original hypomorphic scp-1 allele and a T-DNA-induced null allele, scp-2, revealed that in the absence of SCP, microspores undergo normal nuclear positioning, but show delayed entry into mitosis, increased cell expansion and alterations in the orientation of nuclear division. We identified the SCP gene to encode a male gametophyte-specific LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES 2-like (LBD/ASL) protein that is expressed in microspore nuclei in a tightly regulated phase-specific manner. Therefore, our study demonstrates that the correct patterning of male gametophyte depends on the activity of a nuclear LBD/ASL family protein that is essential for the correct timing and orientation of asymmetric microspore division.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Mitosis , Polen/citología , Arabidopsis/citología , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Clonación Molecular , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Mutagénesis Insercional , Mutación , Fenotipo , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Polen/genéticaRESUMEN
The haploid microspore division during pollen development in flowering plants is an intrinsically asymmetric division which establishes the male germline for sexual reproduction. Arabidopsis gem1 mutants lack the male germline as a result of disturbed microspore polarity, division asymmetry, and cytokinesis and represent loss-of-function mutants in MOR1/GEM1, a plant orthologue of the conserved MAP215/Dis1 microtubule associated protein (MAP) family. This provides genetic evidence for the role of MAP215/Dis1 in the organization of gametophytic microtubule arrays, but it has remained unknown how microtubule arrays are affected in gem1 mutant microspores. Here, novel male gametophytic microtubule-reporter Nicotiana tabacum plants were constructed, expressing a green fluorescent protein-alpha-TUBULIN fusion protein (GFP-TUA6) under the control of a microspore-specific promoter. These plants allow effective visualization of all major male gametophytic microtubule arrays and provide useful tools to study the regulation of microtubule arrays by MAPs and other effectors. Depletion of TMBP200, a tobacco homologue of MOR1/GEM1 in gametophytic microtubule-reporter plants using microspore-targeted RNA interference, induced defects in microspore polarity, division asymmetry and cytokinesis that were associated with striking defects in phragmoplast position, orientation, and structure. Our observations further reveal a requirement for TMBP200 in gametophytic spindle organization and a novel role in spindle position and orientation in polarized microspores. These results provide direct evidence for the function of MAP215/Dis1 family protein TMBP200 in the organization of microtubule arrays critical for male germline formation in plants.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Polen/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Proteínas de Plantas/genética , Polen/genética , Polen/metabolismo , Especificidad de la Especie , Nicotiana/genética , Nicotiana/crecimiento & desarrolloRESUMEN
Gene duplication is important for gene family evolution, allowing for functional divergence and innovation. In flowering plants, duplicated genes are widely observed, and functional redundancy of closely related duplicates has been reported, but few cases of functional divergence of close duplicates have been described. Here, we show that the Arabidopsis AtKIN14a and AtKIN14b genes encoding highly similar kinesins are two of the most closely related Arabidopsis paralogs, which were formed by a duplication event that occurred after the split of Arabidopsis and poplar. In addition, AtKIN14a and AtKIN14b exhibit varying degrees of coding sequence divergence. Further genetic studies of plants carrying atkin14a and/or atkin14b mutations indicate that, although these two genes have similar functions, there is clear evidence for functional divergence. Although both genes are important for male and female meiosis, AtKIN14a plays a more critical role in male meiosis than AtKIN14b. Moreover, either one of these two genes is necessary and sufficient for gametophyte development, indicating that they are redundant for this function. Therefore, AtKIN14a and AtKIN14b together play important roles in controlling plant reproductive development. Our results suggest that the AtKIN14a and AtKIN14b genes have retained similar functions in gametophyte development and female meiosis, but have evolved partially distinct functions in male meiosis, with AtKIN14a playing a more substantive role.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Células Germinativas/crecimiento & desarrollo , Meiosis/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Evolución Molecular , Flores/citología , Flores/genética , Flores/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Células Germinativas/metabolismo , Cinesinas , Meiosis/fisiología , Mutación , Filogenia , Infertilidad Vegetal/genéticaRESUMEN
The FUSED (FU) Ser/Thr protein kinase family has a key role in the hedgehog signaling pathway known to control cell proliferation and patterning in fruit flies and humans . The genomes of Arabidopsis thaliana and rice each encode a single Fu ortholog, but their role is unknown. Here, we show that cytokinesis-defective mutants, which we named two-in-one (tio), result from mutations in Arabidopsis Fu. Phenotypic analysis of tio mutants reveals an essential role for TIO in conventional modes of cytokinesis in plant meristems and during male gametogenesis. TIO also has a key role in nonconventional modes of cytokinesis (cellularization) during female gametogenesis. We demonstrate that TIO is tightly localized to the midline of the nascent phragmoplast and remains associated with the expanding phragmoplast ring. These data reveal the evolution of a divergent role for the Fu kinase family as an essential phragmoplast-associated protein that functions in different cell type-specific modes of cytokinesis in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Citocinesis/genética , Gametogénesis/genética , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal/genética , Secuencia de Bases , Western Blotting , Cromosomas Artificiales Bacterianos , Citocinesis/fisiología , Componentes del Gen , Datos de Secuencia Molecular , Mutación/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Understanding late pollen development, including the maturation and pollination process, is a key component in maintaining crop yields. Transcriptome data obtained through microarray or RNA-seq technologies can provide useful insight into those developmental processes. Six series of microarray data from a public transcriptome database, the Gene Expression Omnibus of the National Center for Biotechnology Information, are related to anther and pollen development. RESULTS: We performed a systematic and functional study across the rice genome of genes that are preferentially expressed in the late stages of pollen development, including maturation and germination. By comparing the transcriptomes of sporophytes and male gametes over time, we identified 627 late pollen-preferred genes that are conserved among japonica and indica rice cultivars. Functional classification analysis with a MapMan tool kit revealed a significant association between cell wall organization/metabolism and mature pollen grains. Comparative analysis of rice and Arabidopsis demonstrated that genes involved in cell wall modifications and the metabolism of major carbohydrates are unique to rice. We used the GUS reporter system to monitor the expression of eight of those genes. In addition, we evaluated the significance of our candidate genes, using T-DNA insertional mutant population and the CRISPR/Cas9 system. Mutants from T-DNA insertion and CRISPR/Cas9 systems of a rice gene encoding glycerophosphoryl diester phosphodiesterase are defective in their male gamete transfer. CONCLUSION: Through the global analyses of the late pollen-preferred genes from rice, we found several biological features of these genes. First, biological process related to cell wall organization and modification is over-represented in these genes to support rapid tube growth. Second, comparative analysis of late pollen preferred genes between rice and Arabidopsis provide a significant insight on the evolutional disparateness in cell wall biogenesis and storage reserves of pollen. In addition, these candidates might be useful targets for future examinations of late pollen development, and will be a valuable resource for accelerating the understanding of molecular mechanisms for pollen maturation and germination processes in rice.
RESUMEN
Development of reproductive tissue and control of cell division are common challenges to all sexually reproducing eukaryotes. The Arabidopsis thaliana TSO1 gene is involved in both these processes. Mild tso1 mutant alleles influence only ovule development, whereas strong alleles have an effect on all floral tissues and cause cell division defects. The tso1 mutants described so far carry point mutations in a conserved cysteine-rich domain, the CRC domain, but the reason for the range of phenotypes observed is poorly understood. In the present study, the tesmin/TSO1-like CXC (TCX) proteins are characterized at the biochemical, genomic, transcriptomic, and functional level to address this question. It is shown that the CRC domain binds zinc, offering an explanation for the severity of tso1 alleles where cysteine residues are affected. In addition, the phylogenetic and expression analysis of the TCX genes suggested an overlap in function between AtTSO1 and the related gene AtTCX2. Their expression ratios indicated that pollen, in addition to ovules, would be sensitive to loss of TSO1 function. This was confirmed by analysis of novel tso1 T-DNA insertion alleles where the development of both pollen and ovules was affected.