RESUMEN
Thermospermine suppresses auxin-inducible xylem differentiation, whereas its structural isomer, spermine, is involved in stress responses in angiosperms. The thermospermine synthase, ACAULIS5 (ACL5), is conserved from algae to land plants, but its physiological functions remain elusive in non-vascular plants. Here, we focused on MpACL5, a gene in the liverwort Marchantia polymorpha, that rescued the dwarf phenotype of the acl5 mutant in Arabidopsis. In the Mpacl5 mutants generated by genome editing, severe growth retardation was observed in the vegetative organ, thallus, and the sexual reproductive organ, gametangiophore. The mutant gametangiophores exhibited remarkable morphological defects such as short stalks, fasciation and indeterminate growth. Two gametangiophores fused together, and new gametangiophores were often initiated from the old ones. Furthermore, Mpacl5 showed altered responses to heat and salt stresses. Given the absence of spermine in bryophytes, these results suggest that thermospermine has a dual primordial function in organ development and stress responses in M. polymorpha. The stress response function may have eventually been assigned to spermine during land plant evolution.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Marchantia , Espermina/análogos & derivados , Reguladores del Crecimiento de las Plantas , Proteínas de Arabidopsis/genética , Marchantia/genética , Arabidopsis/genética , PlantasRESUMEN
Ethylene is a gaseous phytohormone involved in various physiological processes, including fruit ripening, senescence, root hair development and stress responses. Recent genomics studies have suggested that most homologous genes of ethylene biosynthesis and signaling are conserved from algae to angiosperms, whereas the function and biosynthesis of ethylene remain unknown in basal plants. Here, we examined the physiological effects of ethylene, an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) and an inhibitor of ethylene perception, silver thiosulfate (STS), in a basal land plant, Marchantia polymorpha. M. polymorpha plants biosynthesized ethylene, and treatment with high concentrations of ACC slightly promoted ethylene production. ACC remarkably suppressed the growth of thalli (vegetative organs) and rhizoids (root-hair-like cells), whereas exogenous ethylene slightly promoted thallus growth. STS suppressed thallus growth and induced ectopic rhizoid formation on the dorsal surface of thalli. Thus, ACC and ethylene have different effects on the vegetative growth of M. polymorpha. We generated single and double mutants of ACC synthase-like (ACSL) genes, MpACSL1 and MpACSL2. The mutants did not show obvious defects in thallus growth, ACC content and ethylene production, indicating that MpACSL genes are not essential for the vegetative growth and biosynthesis of ACC and ethylene. Gene expression analysis suggested the involvement of MpACSL1 and MpACSL2 in stress responses. Collectively, our results imply ethylene-independent function of ACC and the absence of ACC-mediated ethylene biosynthesis in M. polymorpha.
Asunto(s)
Aminoácidos Cíclicos/metabolismo , Etilenos/metabolismo , Marchantia/metabolismo , Aminoácidos Cíclicos/farmacología , Etilenos/biosíntesis , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Marchantia/efectos de los fármacos , Marchantia/genética , Marchantia/crecimiento & desarrollo , Mutación , Compuestos Organofosforados/farmacología , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tiosulfatos/farmacologíaRESUMEN
Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying ß-glucuronidase (GUS) genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.
Asunto(s)
Agrobacterium tumefaciens/fisiología , Marchantia/genética , Plantas Modificadas Genéticamente/genética , Transducción Genética/métodos , Transformación Genética , Marchantia/microbiologíaRESUMEN
Tip growth is driven by turgor pressure and mediated by the polarized accumulation of cellular materials. How a single polarized growth site is established and maintained is unclear. Here, we analyzed the function of NIMA-related protein kinase 1 (MpNEK1) in the liverwort Marchantia polymorpha In the wild type, rhizoid cells differentiate from the ventral epidermis and elongate through tip growth to form hair-like protrusions. In Mpnek1 knockout mutants, rhizoids underwent frequent changes in growth direction, resulting in a twisted and/or spiral morphology. The functional MpNEK1-Citrine protein fusion localized to microtubule foci in the apical growing region of rhizoids. Mpnek1 knockouts exhibited increases in both microtubule density and bundling in the apical dome of rhizoids. Treatment with the microtubule-stabilizing drug taxol phenocopied the Mpnek1 knockout. These results suggest that MpNEK1 directs tip growth in rhizoids through microtubule organization. Furthermore, MpNEK1 expression rescued ectopic outgrowth of epidermal cells in the Arabidopsis thaliana nek6 mutant, strongly supporting an evolutionarily conserved NEK-dependent mechanism of directional growth. It is possible that such a mechanism contributed to the evolution of the early rooting system in land plants.
Asunto(s)
Marchantia , Quinasas Relacionadas con NIMA/fisiología , Rizoma/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Secuencia Conservada , Embryophyta , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Marchantia/genética , Marchantia/crecimiento & desarrollo , Quinasa 1 Relacionada con NIMA/genética , Quinasas Relacionadas con NIMA/genética , Desarrollo de la Planta/genética , Plantas Modificadas Genéticamente , Rizoma/genéticaRESUMEN
KEY MESSAGE: We found that mutations in a Ca2+-permeable mechanosensitive channel MCA1, an ethylene-regulated microtubule-associated protein WDL5, and a versatile co-receptor BAK1 affect root growth response to mechanical stress. Plant root tips exposed to mechanical impedance show a temporal reduction in the elongation growth. The process involves a transient Ca2+ increase in the cytoplasm followed by ethylene signaling. To dissect the molecular mechanisms underlying this response, we examined the root growth of a series of Arabidopsis mutants with potentially altered response to mechanical stress after transfer from vertical to horizontal plates that were covered by dialysis membrane as an impedance. Among the plant hormone-response mutants tested, the ethylene-insensitive mutant ein3 was confirmed to show no growth reduction after the transfer. The root growth reduction was attenuated in a mutant of MCA1 encoding a Ca2+-permeable mechanosensitive channel and that of WDL5 encoding an ethylene-regulated microtubule-associated protein. We also found that the growth reduction was enhanced in a mutant of BAK1 encoding a co-receptor that pairs with numerous leucine-rich repeat receptor kinases to modulate growth and immunity. These results suggest the root growth reduction in response to mechanical stress involves ethylene-mediated microtubule reorganization and also transmembrane receptor-mediated signal transduction.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Arabidopsis/genética , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , Gravitropismo/fisiología , Proteínas de la Membrana/genética , Meristema/crecimiento & desarrollo , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Mutación , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Estrés FisiológicoRESUMEN
In Arabidopsis, spermine is produced in most tissues and has been implicated in stress response, while its structural isomer thermospermine is only in xylem precursor cells. Studies on acaulis5 (acl5), a mutant defective in the biosynthesis of thermospermine, have revealed that thermospermine plays a repressive role in xylem development through enhancement of mRNA translation of the SAC51 family. In contrast, the pao5 mutant defective in the degradation of thermospermine has high levels of thermospermine and shows increased salt tolerance, suggesting a role of thermospermine in salt stress response. Here we compared acl5 with a mutant of spermine synthase, spms, in terms of abiotic stress tolerance and found that acl5 was much more sensitive to sodium than the wild-type and spms. A double-mutant of acl5 and sac51-d, which suppresses the excessive xylem phenotype of acl5, recovered normal sensitivity, while a quadruple T-DNA insertion mutant of the SAC51 family, which has an increased thermospermine level but shows excessive xylem development, showed increased salt sensitivity, unlike pao5. Together with the result that the salt tolerance of both wild-type and acl5 seedlings was improved by long-term treatment with thermospermine, we suggest a correlation of the salt tolerance with reduced xylem development rather than with the thermospermine level. We further found that the mutants containing high thermospermine levels showed increased tolerance to drought and heat stress, suggesting another role of thermospermine that may be common with that of spermine and secondary to that in restricting excess xylem development associated with salt hypersensitivity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Espermina/análogos & derivados , Xilema/metabolismo , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Espermina/metabolismoRESUMEN
Plants generally possess a strong ability to regenerate organs; for example, in tissue culture, shoots can regenerate from callus, a clump of actively proliferating, undifferentiated cells. Processing of pre-mRNA and ribosomal RNAs is important for callus formation and shoot regeneration. However, our knowledge of the roles of RNA quality control via the nonsense-mediated mRNA decay (NMD) pathway in shoot regeneration is limited. Here, we examined the shoot regeneration phenotypes of the low-beta-amylase1 (lba1)/upstream frame shift1-1 (upf1-1) and upf3-1 mutants, in which the core NMD components UPF1 and UPF3 are defective. These mutants formed callus from hypocotyl explants normally, but this callus behaved abnormally during shoot regeneration: the mutant callus generated numerous adventitious root structures instead of adventitious shoots in an auxin-dependent manner. Quantitative RT-PCR and microarray analyses showed that the upf mutations had widespread effects during culture on shoot-induction medium. In particular, the expression patterns of early auxin response genes, including those encoding AUXIN/INDOLE ACETIC ACID (AUX/IAA) family members, were significantly affected in the upf mutants. Also, the upregulation of shoot apical meristem-related transcription factor genes, such as CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, was inhibited in the mutants. Taken together, these results indicate that NMD-mediated transcriptomic regulation modulates the auxin response in plants and thus plays crucial roles in the early stages of shoot regeneration.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Degradación de ARNm Mediada por Codón sin Sentido , Reguladores del Crecimiento de las Plantas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Hipocótilo/genética , Hipocótilo/fisiología , Ácidos Indolacéticos/metabolismo , Meristema/genética , Meristema/fisiología , Mutación , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/fisiología , Transducción de SeñalRESUMEN
Mechanical sensing is one of the most fundamental processes for sessile plants to survive and grow. The response is known to involve calcium elevation in the cell. Arabidopsis seedlings grown horizontally on agar plates covered with a dialysis membrane show a 2-fold reduction in root growth compared with those grown vertically, a response to mechanical stress generated due to gravitropism of the root. To understand the molecular mechanism of how plant roots sense and respond to mechanical stimuli, we screened chemical libraries for compounds that affect the horizontal root growth in this experimental system and found that, while having no effect on root gravitropism, omeprazole known as a proton pump inhibitor significantly enhanced the mechanical stress-induced root growth reduction especially in lower pH media. In contrast, omeprazole reversed neither the alleviation of the mechanical stress-induced growth reduction caused by calcium depletion nor the insensitivity to the mechanical stress in the ethylene signaling mutant ein2. Together with the finding that omeprazole increased expression of touch-induced genes and ETHYLENE RESPONSE FACTOR1, our results suggest that the target of omeprazole mediates ethylene signaling in the root growth response to mechanical stress.
Asunto(s)
Omeprazol/farmacología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Estrés Mecánico , Arabidopsis/efectos de los fármacos , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Gravitropismo/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Microtubules are dynamic filaments, the assembly and disassembly of which are under precise control of various associated proteins, including motor proteins and regulatory enzymes. In Arabidopsis thaliana, two such proteins are the ARMADILLO-REPEAT KINESIN 1 (ARK1), which promotes microtubule disassembly, and the NIMA-RELATED KINASE 6 (NEK6), which has a role in organizing microtubule arrays. Previous yeast two-hybrid and in vitro pull-down assays determined that NEK6 can interact with ARK1 through the latter protein's Armadillo-repeat (ARM) cargo domain. To explore the function of the ARM domain, we generated fluorescent reporter fusion proteins to ARK1 lacking the ARM domain (ARK1ΔARM-GFP) and to the ARM domain alone (ARM-GFP). Both of these constructs strongly associated with the growing plus ends of microtubules, but only ARK1ΔARM-GFP was capable of inducing microtubule catastrophe and rescuing the ark1-1 root hair phenotype. These results indicate that neither the ARM domain nor NEK6's putative interaction with it is required for ARK1 to induce microtubule catastrophe. In further exploration of the ARK1-NEK6 relationship, we demonstrated that, despite evidence that NEK6 can phosphorylate ARK1 in vitro, the in vivo distribution and function of ARK1 were not affected by the loss of NEK6, and vice versa. Moreover, NEK6 and ARK1 were found to have overlapping but non-identical distribution on microtubules, and hormone treatments known to affect NEK6 activity did not stimulate interaction. These findings suggest that ARK1 and NEK6 function independently in microtubule dynamics and cell morphogenesis. Despite the results of this functional analysis, we found that overexpression of the ARM domain led to complete loss of NEK6 transcription, suggesting that the ARM domain might have a regulatory role in NEK6 expression.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Quinasas Relacionadas con NIMA/metabolismo , Aminoácidos Cíclicos/farmacología , Arabidopsis/citología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Giberelinas/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Cinesinas/genética , Microtúbulos/genética , Mutación , Fosforilación , Plantas Modificadas Genéticamente , Dominios y Motivos de Interacción de ProteínasRESUMEN
The plant hormone auxin is a key morphogenetic signal that controls many aspects of plant growth and development. Cellular auxin levels are coordinately regulated by multiple processes, including auxin biosynthesis and the polar transport and metabolic pathways. The auxin concentration gradient determines plant organ positioning and growth responses to environmental cues. Auxin transport systems play crucial roles in the spatiotemporal regulation of the auxin gradient. This auxin gradient has been analyzed using SCF-type E3 ubiquitin-ligase complex-based auxin biosensors in synthetic auxin-responsive reporter lines. However, the contributions of auxin biosynthesis and metabolism to the auxin gradient have been largely elusive. Additionally, the available information on subcellular auxin localization is still limited. Here we designed fluorescently labeled auxin analogs that remain active for auxin transport but are inactive for auxin signaling and metabolism. Fluorescent auxin analogs enable the selective visualization of the distribution of auxin by the auxin transport system. Together with auxin biosynthesis inhibitors and an auxin biosensor, these analogs indicated a substantial contribution of local auxin biosynthesis to the formation of auxin maxima at the root apex. Moreover, fluorescent auxin analogs mainly localized to the endoplasmic reticulum in cultured cells and roots, implying the presence of a subcellular auxin gradient in the cells. Our work not only provides a useful tool for the plant chemical biology field but also demonstrates a new strategy for imaging the distribution of small-molecule hormones.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Ácidos Indolacéticos/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Transporte Biológico , Fluorescencia , Ácidos Indolacéticos/química , Meristema/citología , Meristema/metabolismo , Raíces de Plantas/metabolismo , Transducción de Señal , Fracciones Subcelulares/metabolismoRESUMEN
The acaulis5 (acl5) mutant of Arabidopsis thaliana is defective in the biosynthesis of thermospermine and shows a dwarf phenotype associated with excess xylem differentiation. SAC51 was identified from a dominant suppressor of acl5, sac51-d, and encodes a basic helix-loop-helix protein. The sac51-d mutant has a premature termination codon in an upstream open reading frame (uORF) that is conserved among all four members of the SAC51 family, SAC51 and SACL1-SACL3 This suggests that thermospermine cancels the inhibitory effect of the uORF in main ORF translation. Another suppressor, sac57-d, has a mutation in the conserved uORF of SACL3 To define further the function of the SAC51 family in the thermospermine response, we analyzed T-DNA insertion mutants of each gene. Although sacl1-1 may not be a null allele, the quadruple mutant showed a semi-dwarf phenotype but with an increased level of thermospermine and decreased sensitivity to exogenous thermospermine that normally represses xylem differentiation. The sac51-1 sacl3-1 double mutant was also insensitive to thermospermine. These results suggest that SAC51 and SACL3 play a key role in thermospermine-dependent negative control of thermospermine biosynthesis and xylem differentiation. Using 5' leader-GUS (ß-glucuronidase) fusion constructs, however, we detected a significant enhancement of the GUS activity by thermospermine only in SAC51 and SACL1 constructs. Furthermore, while acl5-1 sac51-1 showed the acl5 dwarf phenotype, acl5-1 sacl3-1 exhibited an extremely tiny-plant phenotype. These results suggest a complex regulatory network for the thermospermine response in which SAC51 and SACL3 function in parallel pathways.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Espermina/análogos & derivados , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/efectos de los fármacos , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Modelos Biológicos , Mutagénesis Insercional , Sistemas de Lectura Abierta/genética , Fenotipo , Plantones/citología , Plantones/efectos de los fármacos , Plantones/genética , Plantones/fisiología , Alineación de Secuencia , Espermina/metabolismo , Espermina/farmacología , Xilema/citología , Xilema/efectos de los fármacos , Xilema/genética , Xilema/fisiologíaRESUMEN
Microtubules are highly dynamic structures that control the spatiotemporal pattern of cell growth and division. Microtubule dynamics are regulated by reversible protein phosphorylation involving both protein kinases and phosphatases. Never in mitosis A (NIMA)-related kinases (NEKs) are a family of serine/threonine kinases that regulate microtubule-related mitotic events in fungi and animal cells (e.g. centrosome separation and spindle formation). Although plants contain multiple members of the NEK family, their functions remain elusive. Recent studies revealed that NEK6 of Arabidopsis thaliana regulates cell expansion and morphogenesis through ß-tubulin phosphorylation and microtubule destabilization. In addition, plant NEK members participate in organ development and stress responses. The present phylogenetic analysis indicates that plant NEK genes are diverged from a single NEK6-like gene, which may share a common ancestor with other kinases involved in the control of microtubule organization. On the contrary, another mitotic kinase, polo-like kinase, might have been lost during the evolution of land plants. We propose that plant NEK members have acquired novel functions to regulate cell growth, microtubule organization, and stress responses.
Asunto(s)
Evolución Molecular , Microtúbulos/metabolismo , Plantas/clasificación , Plantas/enzimología , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Fosforilación , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Proteínas Quinasas/genéticaRESUMEN
Morphological response of cells to environment involves concerted rearrangements of microtubules and actin microfilaments. A mutant of WAVE-DAMPENED2-LIKE5 (WDL5), which encodes an ethylene-regulated microtubule-associated protein belonging to the WVD2/WDL family in Arabidopsis thaliana, shows attenuation in the temporal root growth reduction in response to mechanical stress. We found that a T-DNA knockout of WDL6, the closest homolog of WDL5, oppositely shows an enhancement of the response. To know the functional relationship between WDL5 and WDL6, we attempted to generate the double mutant by crosses but failed in isolation. Close examination of gametophytes in plants that are homozygous for one and heterozygous for the other revealed that these plants produce pollen grains with a reduced rate of germination and tube growth. Reciprocal cross experiments of these plants with the wild type confirmed that the double mutation is not inherited paternally. These results suggest a critical and cooperative function of WDL5 and WDL6 in pollen tube growth.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Tubo Polínico/metabolismo , Polen/metabolismo , Mutación/genética , GerminaciónRESUMEN
NimA-related kinase 6 (NEK6) has been implicated in microtubule regulation to suppress the ectopic outgrowth of epidermal cells; however, its molecular functions remain to be elucidated. Here, we analyze the function of NEK6 and other members of the NEK family with regard to epidermal cell expansion and cortical microtubule organization. The functional NEK6-green fluorescent protein fusion localizes to cortical microtubules, predominantly in particles that exhibit dynamic movement along microtubules. The kinase-dead mutant of NEK6 (ibo1-1) exhibits a disturbance of the cortical microtubule array at the site of ectopic protrusions in epidermal cells. Pharmacological studies with microtubule inhibitors and quantitative analysis of microtubule dynamics indicate excessive stabilization of cortical microtubules in ibo1/nek6 mutants. In addition, NEK6 directly binds to microtubules in vitro and phosphorylates ß-tubulin. NEK6 interacts and co-localizes with NEK4 and NEK5 in a transient expression assay. The ibo1-3 mutation markedly reduces the interaction between NEK6 and NEK4 and increases the interaction between NEK6 and NEK5. NEK4 and NEK5 are required for the ibo1/nek6 ectopic outgrowth phenotype in epidermal cells. These results demonstrate that NEK6 homodimerizes and forms heterodimers with NEK4 and NEK5 to regulate cortical microtubule organization possibly through the phosphorylation of ß-tubulins.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Microtúbulos/ultraestructura , Epidermis de la Planta/citología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/citología , Proteínas de Arabidopsis/genética , Benzamidas/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Mutación , Quinasas Relacionadas con NIMA , Paclitaxel/farmacología , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Thermospermine, a structural isomer of spermine, is produced through the action of ACAULIS5 (ACL5) and suppresses xylem differentiation in Arabidopsis thaliana. To elucidate the molecular basis of the function of thermospermine, we screened chemical libraries for compounds that can modulate xylem differentiation in the acl5 mutant, which is deficient in thermospermine and shows a severe dwarf phenotype associated with excessive proliferation of xylem vessels. We found that the isooctyl ester of a synthetic auxin, 2,4-D, remarkably enhanced xylem vessel differentiation in acl5 seedlings. 2,4-D, 2,4-D analogs and IAA analogs, including 4-chloro IAA (4-Cl-IAA) and IAA ethyl ester, also enhanced xylem vessel formation, while IAA alone had little or no obvious effect on xylem differentiation. These effects of auxin analogs were observed only in the acl5 mutant but not in the wild type, and were suppressed by the anti-auxin, p-chlorophenoxyisobutyric acid (PCIB) and α-(phenyl ethyl-2-one)-IAA (PEO-IAA), and also by thermospermine. Furthermore, the suppressor of acaulis51-d (sac51-d) mutation, which causes SAC51 overexpression in the absence of thermospermine and suppresses the dwarf phenotype of acl5, also suppressed the effect of auxin analogs in acl5. These results suggest that the auxin signaling that promotes xylem differentiation is normally limited by SAC51-mediated thermospermine signaling but can be continually stimulated by exogenous auxin analogs in the absence of thermospermine. The opposite action between thermospermine and auxin may fine-tune the timing and spatial pattern of xylem differentiation.
Asunto(s)
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Espermina/análogos & derivados , Xilema/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Espermina/metabolismo , Xilema/genética , Xilema/crecimiento & desarrolloRESUMEN
A NIMA-related protein kinase, MpNEK1, directs tip growth of rhizoids through microtubule depolymerization in a liverwort Marchantia polymorpha. The Mpnek1 knockouts were shown to develop curly and spiral rhizoids due to the fluctuated direction of growth. Still, physiological roles and mechanisms of MpNEK1-dependent rhizoid tip growth remain to be clarified. Here, we developed novel culture methods to further study rhizoid growth of M. polymorpha, in which plants were grown on vertical plates. We applied the established methods to investigate MpNEK1 function in rhizoid growth. Rhizoids of the wild-type and Mpnek1 plants grew toward the gravity. The aerial rhizoids were longer in Mpnek1 than in the wild type. When the rhizoids were grown on the surface of a cellophane sheet, rhizoid length was comparable between the wild type and Mpnek1, whereas Mpnek1 developed more rhizoids compared to the wild type. We also applied gellan gum, which is more transparent than agar, to analyze rhizoids grown in the medium. Rhizoids of Mpnek1 displayed defect on entering into the solid medium. These results suggest that Mpnek1 rhizoids have the deficiency in invasive tip growth. Thus, stable directional growth is important for rhizoids to get into the soil to anchor plant body and to adsorb water and nutrients. Collectively, our newly designed growth systems are valuable for analyzing rhizoid growth.
RESUMEN
In Arabidopsis thaliana, the ACL5 gene encodes thermospermine synthase and its mutant, acl5, exhibits a dwarf phenotype with excessive xylem formation. Studies of suppressor mutants of acl5 reveal the involvement of thermospermine in enhancing mRNA translation of the SAC51 gene family. We show here that a mutant, sac59, which partially suppresses the acl5 phenotype, has a point mutation in JMJ22 encoding a D6-class Jumonji C protein (JMJD6). A T-DNA insertion allele, jmj22-2, also partially suppressed the acl5 phenotype while mutants of its closest two homologs JMJ21 and JMJ20 had no such effects, suggesting a unique role for JMJ22 in plant development. We found that mRNAs of the SAC51 family are more stabilized in acl5 jmj22-2 than in acl5.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Xilema/genética , Xilema/metabolismo , Fenotipo , MutaciónRESUMEN
In higher plants, many extracellular proteins are involved in developmental processes, including cell-cell signaling and cell wall construction. Xylogen is an extracellular arabinogalactan protein (AGP) isolated from Zinnia elegans xylogenic culture medium, which promotes xylem cell differentiation. Xylogen has a unique structure, containing a non-specific lipid transfer protein (nsLTP) domain and AGP domains. We searched for xylogen-type genes in the genomes of land plants, including Arabidopsis thaliana, to further our knowledge of xylogen-type genes as functional extracellular proteins in plants. We found that many xylogen-type genes, including 13 Arabidopsis genes, comprise a gene family in land plants, including Populus trichocarpa, Vitis vinifera, Lotus japonicus, Oryza sativa, Selaginella moellendorffii and Physcomitrella patens. The genes shared an N-terminal signal peptide sequence, a distinct nsLTP domain, one or more AGP domains and a glycosylphosphatidylinositol (GPI)-anchored sequence. We analyzed transgenic plants harboring promoter::GUS (ß-glucuronidase) constructs to test expression of the 13 Arabidopsis xylogen-type genes, and detected a diversity of gene family members with related expression patterns. AtXYP2 was the best candidate as the Arabidopsis counterpart of the Zinnia xylogen gene. We observed two distinct expression patterns for several genes, with some anther specific and others preferentially expressed in the endodermis/pericycle. We conclude that xylogen-type genes, which may have diverse functions, form a novel chimeric AGP gene family with a distinct nsLTP domain.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Perfilación de la Expresión Génica , Familia de Multigenes , Proteoglicanos/genética , Arabidopsis/metabolismo , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Glicosilfosfatidilinositoles/metabolismo , Filogenia , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Señales de Clasificación de ProteínaRESUMEN
MicroRNAs (miRNAs) are sequence-specific negative regulators of gene expression generated by DICER-LIKE1 (DCL1) with the assistance of a double-stranded RNA-binding protein, HYPONASTIC LEAVES1 (HYL1), in Arabidopsis. To achieve a better understanding of miRNA biogenesis, we isolated hyl1 suppressors. Our genetic screening identified a novel semidominant mutation in DCL1 (dcl1-13), which causes an amino acid substitution of Glu-395 with Lys in the ATPase/DExH-box RNA helicase domain. This mutation confers significant restoration from the developmental abnormality and a reduction in the level of miRNA in the loss-of-function mutant of HYL1. However, the dcl1-13 single mutant, exhibiting a decreased number of leaves, showed a slight decrease in miRNA accumulation. Thus, the effect of the dcl1-13 mutation is HYL1 dependent: it promotes miRNA processing in the absence of HYL1, but conversely, impairs it in the presence of HYL1. Our results suggest significant roles of the helicase domain of DCL1, which remain unclear to date, possibly in relation with HYL1.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ARN/genética , Ribonucleasa III/genética , Sustitución de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , MicroARNs/metabolismo , Modelos Moleculares , Mutación , Fenotipo , ARN de Planta/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/química , Ribonucleasa III/metabolismoRESUMEN
Inductive cell-cell interactions are essential for controlling cell fate determination in both plants and animals; however, the chemical basis of inductive signals in plants remains little understood. A proteoglycan-like factor named xylogen mediates local and inductive cell-cell interactions required for xylem differentiation in Zinnia cells cultured in vitro. Here we describe the purification of xylogen and cloning of its complementary DNA, and present evidence for its role in planta. The polypeptide backbone of xylogen is a hybrid-type molecule with properties of both arabinogalactan proteins and nonspecific lipid-transfer proteins. Xylogen predominantly accumulates in the meristem, procambium and xylem. In the xylem, xylogen has a polar localization in the cell walls of differentiating tracheary elements. Double knockouts of Arabidopsis lacking both genes that encode xylogen proteins show defects in vascular development: discontinuous veins, improperly interconnected vessel elements and simplified venation. Our results suggest that the polar secretion of xylogen draws neighbouring cells into the pathway of vascular differentiation to direct continuous vascular development, thereby identifying a molecule that mediates an inductive cell-cell interaction involved in plant tissue differentiation.