RESUMO
The conservation of GOLVEN (GLV)/ROOT MERISTEM GROWTH FACTOR (RGF) peptide encoding genes across plant genomes capable of forming roots or root-like structures underscores their potential significance in the terrestrial adaptation of plants. This study investigates the function and role of GOLVEN peptide-coding genes in Medicago truncatula. Five out of fifteen GLV/RGF genes were notably upregulated during nodule organogenesis and were differentially responsive to nitrogen deficiency and auxin treatment. Specifically, the expression of MtGLV9 and MtGLV10 at nodule initiation sites was contingent upon the NODULE INCEPTION transcription factor. Overexpression of these five nodule-induced GLV genes in hairy roots of M. truncatula and application of their synthetic peptide analogues led to a decrease in nodule count by 25-50%. Uniquely, the GOLVEN10 peptide altered the positioning of the first formed lateral root and nodule on the primary root axis, an observation we term 'noduletaxis'; this decreased the length of the lateral organ formation zone on roots. Histological section of roots treated with synthetic GOLVEN10 peptide revealed an increased cell number within the root cortical cell layers without a corresponding increase in cell length, leading to an elongation of the root likely introducing a spatiotemporal delay in organ formation. At the transcription level, the GOLVEN10 peptide suppressed expression of microtubule-related genes and exerted its effects by changing expression of a large subset of Auxin responsive genes. These findings advance our understanding of the molecular mechanisms by which GOLVEN peptides modulate root morphology, nodule ontogeny, and interactions with key transcriptional pathways.
Assuntos
Regulação da Expressão Gênica de Plantas , Medicago truncatula , Proteínas de Plantas , Raízes de Plantas , Nódulos Radiculares de Plantas , Medicago truncatula/genética , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/metabolismo , Medicago truncatula/efeitos dos fármacos , Medicago truncatula/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/efeitos dos fármacos , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Nodulação/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/efeitos dos fármacos , Peptídeos/metabolismo , Peptídeos/genéticaRESUMO
Leguminous plants provide carbon to symbiotic rhizobia in root nodules to fuel the energy-consuming process of nitrogen fixation. The carbon investment pattern from the acquired sources is crucial for shaping the growth regime of the host plants. The autoregulation of nodulation (AON) signaling pathway tightly regulates the number of nodules that form. AON disruption leads to excessive nodule formation and stunted shoot growth. However, the physiological role of AON in adjusting the carbon investment pattern is unknown. Here, we show that AON plays an important role in sustaining shoot water availability, which is essential for promoting carbon investment in shoot growth in Lotus japonicus. We found that AON-defective mutants exhibit substantial accumulation of nonstructural carbohydrates, such as sucrose. Consistent with this metabolic signature, resilience against water-deficit stress was enhanced in the shoots of the AON-defective mutants. Furthermore, the water uptake ability was attenuated in the AON-defective mutants, likely due to the increased ratio of nodulation zone, which is covered with hydrophobic surfaces, on the roots. These results increase our physiological understanding of legume-rhizobia symbiosis by revealing a trade-off between root nodule formation and shoot water availability.
Assuntos
Lotus , Brotos de Planta , Nódulos Radiculares de Plantas , Água , Lotus/genética , Lotus/metabolismo , Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Água/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Brotos de Planta/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/genética , Nodulação , Mutação/genética , Simbiose/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Carbono/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Fixação de NitrogênioRESUMO
Legumes attract symbiotic bacteria and create de novo root organs called nodules. Nodule development consists of bacterial infection of root epidermis and subsequent primordium formation in root cortex, steps that need to be spatiotemporally coordinated. The Lotus japonicus mutant "daphne " has uncoupled symbiotic events in epidermis and cortex, in that it promotes excessive bacterial infection in epidermis but does not produce nodule primordia in cortex. Therefore, daphne should be useful for exploring unknown signals that coordinate these events across tissues. Here, we conducted time-course RNA sequencing using daphne after rhizobial infection. We noticed that IAA carboxyl methyltransferase 1 (IAMT1) , which encodes the enzyme that converts auxin (IAA) into its methyl ester (MeIAA), is transiently induced in wild-type roots at early stages of infection but shows different expression dynamics in daphne. IAMT1 serves an important function in shoot development of Arabidopsis, a nonsymbiotic plant, but the function of IAMT1 in roots has not been reported. Phylogenetic tree analysis suggests a gene duplication of IAMT1 in the legume lineage, and we found that one of the two IAMT1s (named IAMT1a) was induced in roots by epidermal infection. IAMT1a knockdown inhibited nodule development in cortex; however, it had no effect on epidermal infection. The amount of root MeIAA increased with rhizobial infection. Application of MeIAA, but not IAA , significantly induced expression of the symbiotic gene NIN in the absence of rhizobial infection. Our results provide evidence for the role of auxin methylation in an early stage of root nodule development.
Assuntos
Duplicação Gênica , Ácidos Indolacéticos/metabolismo , Lotus/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Genes de Plantas , Lotus/genética , Lotus/crescimento & desenvolvimento , Metilação , Mutação , Filogenia , TranscriptomaRESUMO
Legume plants develop two types of root postembryonic organs, lateral roots and symbiotic nodules, using shared regulatory components. The module composed by the microRNA390, the Trans-Acting SIRNA3 (TAS3) RNA and the Auxin Response Factors (ARF)2, ARF3, and ARF4 (miR390/TAS3/ARFs) mediates the control of both lateral roots and symbiotic nodules in legumes. Here, a transcriptomic approach identified a member of the Lateral Organ Boundaries Domain (LBD) family of transcription factors in Medicago truncatula, designated MtLBD17/29a, which is regulated by the miR390/TAS3/ARFs module. ChIP-PCR experiments evidenced that MtARF2 binds to an Auxin Response Element present in the MtLBD17/29a promoter. MtLBD17/29a is expressed in root meristems, lateral root primordia, and noninfected cells of symbiotic nodules. Knockdown of MtLBD17/29a reduced the length of primary and lateral roots and enhanced lateral root formation, whereas overexpression of MtLBD17/29a produced the opposite phenotype. Interestingly, both knockdown and overexpression of MtLBD17/29a reduced nodule number and infection events and impaired the induction of the symbiotic genes Nodulation Signaling Pathway (NSP) 1 and 2. Our results demonstrate that MtLBD17/29a is regulated by the miR390/TAS3/ARFs module and a direct target of MtARF2, revealing a new lateral root regulatory hub recruited by legumes to act in the root nodule symbiotic program.
Assuntos
Medicago truncatula , Proteínas de Plantas , Nodulação , Raízes de Plantas , Fatores de Transcrição , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Ácidos Indolacéticos/metabolismo , Medicago truncatula/genética , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/microbiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Nodulação/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Simbiose/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia.
Assuntos
Glycine max/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Transporte Biológico , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Glycine max/genética , Glycine max/metabolismoRESUMO
Bradyrhizobia are the principal symbiotic partner of the leguminous plant and take active part in biological nitrogen-fixation. The present investigation explores the underlying competition among different strains during colonization in host roots. Six distinct GFP and RFP-tagged Bradyrhizobium strains were engineered to track them inside the peanut roots either independently or in combination. The Bradyrhizobium strains require different time-spans ranging from 4 to 21 days post-infection (dpi) for successful colonization which further varies in presence of another strain. While most of the individual strains enhanced the shoot and root dry weight, number of nodules, and nitrogen fixation capabilities of the host plants, no significant enhancement of plant growth and nodulation efficiency was observed when they were allowed to colonize in combinations. However, if among the combinations one strains is SEMIA 6144, the co-infection results in higher growth and nodulation efficiency of the hosts. From the competition experiments it has been found that Bradyrhizobium japonicum SEMIA 6144 was found to be the most dominant strain for effective nodulation in peanut. The extent of biofilm and exopolysaccharide (EPS) production by these isolates, individually or in combinations, were envisaged to correlate whether these parameters have any impact on the symbiotic association. But the extent of colonization, growth-promotion and nitrogen-fixation ability drastically lowered when a strain present together with other Bradyrhizobium strain. Therefore, it is imperative to understand the interaction between two co-inoculating Bradyrhizobium species for nodulation followed by plant growth promotion to develop suitable consortia for enhancing BNF in peanut and possibly for other legumes.
Assuntos
Arachis , Biofilmes , Bradyrhizobium , Fixação de Nitrogênio , Nodulação , Raízes de Plantas , Nódulos Radiculares de Plantas , Simbiose , Arachis/microbiologia , Arachis/crescimento & desenvolvimento , Bradyrhizobium/crescimento & desenvolvimento , Bradyrhizobium/fisiologia , Raízes de Plantas/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologia , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Polissacarídeos Bacterianos/metabolismo , Interações Microbianas , Desenvolvimento VegetalRESUMO
Nodule Inception (NIN) is one of the most important root nodule symbiotic genes as it is required for both infection and nodule organogenesis in legumes. Unlike most legumes with a sole NIN gene, there are four putative orthologous NIN genes in soybean (Glycine max). Whether and how these NIN genes contribute to soybean-rhizobia symbiotic interaction remain unknown. In this study, we found that all four GmNIN genes are induced by rhizobia and that conserved CE and CYC binding motifs in their promoter regions are required for their expression in the nodule formation process. By generation of multiplex Gmnin mutants, we found that the Gmnin1a nin2a nin2b triple mutant and Gmnin1a nin1b nin2a nin2b quadruple mutant displayed similar defects in rhizobia infection and root nodule formation, Gmnin2a nin2b produced fewer nodules but displayed a hyper infection phenotype compared to wild type (WT), while the Gmnin1a nin1b double mutant nodulated similar to WT. Overexpression of GmNIN1a, GmNIN1b, GmNIN2a, and GmNIN2b reduced nodule numbers after rhizobia inoculation, with GmNIN1b overexpression having the weakest effect. In addition, overexpression of GmNIN1a, GmNIN2a, or GmNIN2b, but not GmNIN1b, produced malformed pseudo-nodule-like structures without rhizobia inoculation. In conclusion, GmNIN1a, GmNIN2a, and GmNIN2b play functionally redundant yet complicated roles in soybean nodulation. GmNIN1b, although expressed at a comparable level with the other homologs, plays a minor role in root nodule symbiosis. Our work provides insight into the understanding of the asymmetrically redundant function of GmNIN genes in soybean.
Assuntos
Glycine max/crescimento & desenvolvimento , Glycine max/genética , Glycine max/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/metabolismo , Simbiose/genética , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Produtos Agrícolas/microbiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Rhizobium , Nódulos Radiculares de Plantas/microbiologia , Glycine max/microbiologiaRESUMO
Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.
Assuntos
Alquil e Aril Transferases/metabolismo , Citocininas/genética , Citocininas/metabolismo , Medicago truncatula/genética , Medicago truncatula/fisiologia , Nodulação/genética , Nódulos Radiculares de Plantas/metabolismo , Simbiose/genética , Alquil e Aril Transferases/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fixação de Nitrogênio/genética , Fixação de Nitrogênio/fisiologia , Organogênese/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Sinorhizobium meliloti/fisiologia , Simbiose/fisiologiaRESUMO
Leguminous plants establish endosymbiotic associations with rhizobia and form root nodules in which the rhizobia fix atmospheric nitrogen. The host plant and intracellular rhizobia strictly control this symbiotic nitrogen fixation. We recently reported a Lotus japonicus Fix- mutant, apn1 (aspartic peptidase nodule-induced 1), that impairs symbiotic nitrogen fixation. APN1 encodes a nodule-specific aspartic peptidase involved in the Fix- phenotype in a rhizobial strain-specific manner. This host-strain specificity implies that some molecular interactions between host plant APN1 and rhizobial factors are required, although the biological function of APN1 in nodules and the mechanisms governing the interactions are unknown. To clarify how rhizobial factors are involved in strain-specific nitrogen fixation, we explored transposon mutants of Mesorhizobium loti strain TONO, which normally form Fix- nodules on apn1 roots, and identified TONO mutants that formed Fix+ nodules on apn1 The identified causal gene encodes an autotransporter, part of a protein secretion system of Gram-negative bacteria. Expression of the autotransporter gene in M. loti strain MAFF3030399, which normally forms Fix+ nodules on apn1 roots, resulted in Fix- nodules. The autotransporter of TONO functions to secrete a part of its own protein (a passenger domain) into extracellular spaces, and the recombinant APN1 protein cleaved the passenger protein in vitro. The M. loti autotransporter showed the activity to induce the genes involved in nodule senescence in a dose-dependent manner. Therefore, we conclude that the nodule-specific aspartic peptidase, APN1, suppresses negative effects of the rhizobial autotransporter in order to maintain effective symbiotic nitrogen fixation in root nodules.
Assuntos
Lotus/metabolismo , Fixação de Nitrogênio/fisiologia , Rhizobium/metabolismo , Simbiose/fisiologia , Sistemas de Secreção Tipo V/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Bacterianos/genética , Bactérias Gram-Negativas , Mesorhizobium/genética , Mesorhizobium/metabolismo , Modelos Moleculares , Fixação de Nitrogênio/genética , Fenótipo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Conformação Proteica , Domínios Proteicos , Rhizobium/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Simbiose/genética , Transcriptoma , Sistemas de Secreção Tipo V/química , Sistemas de Secreção Tipo V/genéticaRESUMO
Legume and rhizobium species can establish a nitrogen-fixing nodule symbiosis. Previous studies have shown that several transcription factors that play a role in (lateral) root development are also involved in nodule development. Chromatin remodeling factors, like transcription factors, are key players in regulating gene expression. However, studies have not investigated whether chromatin remodeling genes that are essential for root development are also involved in nodule development. Here, we studied the role of Medicago (Medicago truncatula) histone deacetylases (MtHDTs) in nodule development. Arabidopsis (Arabidopsis thaliana) orthologs of HDTs have been shown to play a role in root development. MtHDT expression is induced in nodule primordia and is maintained in the nodule meristem and infection zone. Conditional, nodule-specific knockdown of MtHDT expression by RNAi blocks nodule primordium development. A few nodules may still form, but their nodule meristems are smaller, and rhizobial colonization of the cells derived from the meristem is markedly reduced. Although the HDTs are expressed during nodule and root development, transcriptome analyses indicate that HDTs control the development of each organ in a different manner. During nodule development, the MtHDTs positively regulate 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 (MtHMGR1). Decreased expression of MtHMGR1 is sufficient to explain the inhibition of primordium formation.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Histona Desacetilases/metabolismo , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Histona Desacetilases/genética , Medicago truncatula/metabolismo , Morfogênese/genética , Morfogênese/fisiologia , Desenvolvimento Vegetal/genética , Nódulos Radiculares de Plantas/metabolismo , Simbiose/genética , Simbiose/fisiologiaRESUMO
Nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) form symbioses with plant roots and these are established by precise regulation of symbiont accommodation within host plant cells. In model legumes such as Lotus japonicus and Medicago truncatula, rhizobia enter into roots through an intracellular invasion system that depends on the formation of a root-hair infection thread (IT). While IT-mediated intracellular rhizobia invasion is thought to be the most evolutionarily derived invasion system, some studies have indicated that a basal intercellular invasion system can replace it when some nodulation-related factors are genetically modified. In addition, intracellular rhizobia accommodation is suggested to have a similar mechanism as AMF accommodation. Nevertheless, our understanding of the underlying genetic mechanisms is incomplete. Here we identify a L. japonicus nodulation-deficient mutant, with a mutation in the LACK OF SYMBIONT ACCOMMODATION (LAN) gene, in which root-hair IT formation is strongly reduced, but intercellular rhizobial invasion eventually results in functional nodule formation. LjLAN encodes a protein that is homologous to Arabidopsis MEDIATOR 2/29/32 possibly acting as a subunit of a Mediator complex, a multiprotein complex required for gene transcription. We also show that LjLAN acts in parallel with a signaling pathway including LjCYCLOPS. In addition, the lan mutation drastically reduces the colonization levels of AMF. Taken together, our data provide a new factor that has a common role in symbiont accommodation process during root nodule and AM symbiosis.
Assuntos
Lotus/genética , Medicago truncatula/genética , Micorrizas/crescimento & desenvolvimento , Simbiose/genética , Regulação da Expressão Gênica de Plantas/genética , Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/microbiologia , Mutação , Micorrizas/genética , Proteínas de Plantas/genética , Nodulação/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Rhizobium/genética , Rhizobium/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologiaRESUMO
The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5.
Assuntos
Lipopolissacarídeos/genética , Lotus/genética , Nodulação/genética , Simbiose/genética , Citoplasma/enzimologia , Fabaceae/genética , Fabaceae/crescimento & desenvolvimento , Fabaceae/microbiologia , Regulação da Expressão Gênica de Plantas/genética , Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Fosfotransferases/genética , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Rhizobium/genética , Rhizobium/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/microbiologia , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Nicotiana/microbiologiaRESUMO
Soybean nodulation is a highly controlled process that involves complex gene regulation at both transcriptional and post-transcriptional levels. In the present study, we profiled gene expression changes, alternative splicing events, and DNA methylation patterns during nodule formation, development, and senescence. The transcriptome data uncovered key transcription patterns of nodule development that included 9669 core genes and 7302 stage-specific genes. Alternative splicing analysis uncovered a total of 2323 genes that undergo alternative splicing events in at least one nodule developmental stage, with activation of exon skipping and repression of intron retention being the most common splicing events in nodules compared to roots. Approximately 40% of the differentially spliced genes were also differentially expressed at the same nodule developmental stage, implying a substantial association between gene expression and alternative splicing. Genome-wide-DNA methylation analysis revealed dynamic changes in nodule methylomes that were specific to each nodule stage, occurred in a sequence-specific manner, and impacted the expression of 1864 genes. An attractive hypothesis raised by our data is that increased DNA methylation may contribute to the efficiency of alternative splicing. Together, our results provide intriguing insights into the associations between gene expression, alternative splicing, and DNA methylation that may shape transcriptome complexity and proteome specificity in developing soybean nodules.
Assuntos
Processamento Alternativo , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Nodulação , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Metilação de DNA/genética , Metilação de DNA/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas/genética , Genes de Plantas/fisiologia , Nodulação/genética , Nodulação/fisiologia , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Glycine max/crescimento & desenvolvimento , Glycine max/metabolismoRESUMO
Legumes control their nodule numbers through the autoregulation of nodulation (AON). Rhizobia infection stimulates the production of root-derived CLE peptide hormones that are translocated to the shoot where they regulate a new signal. We used soybean to demonstrate that this shoot-derived signal is miR2111, which is transported via phloem to the root where it targets transcripts of Too Much Love (TML), a negative regulator of nodulation. Shoot perception of rhizobia-induced CLE peptides suppresses miR2111 expression, resulting in TML accumulation in roots and subsequent inhibition of nodule organogenesis. Feeding synthetic mature miR2111 via the petiole increased nodule numbers per plant. Likewise, elevating miR2111 availability by over-expression promoted nodulation, while target mimicry of TML induced the opposite effect on nodule development in wild-type plants and alleviated the supernodulating and stunted root growth phenotypes of AON-defective mutants. Additionally, in non-nodulating wild-type plants, ectopic expression of miR2111 significantly enhanced lateral root emergence with a decrease in lateral root length and average root diameter. In contrast, hairy roots constitutively expressing the target mimic construct exhibited reduced lateral root density. Overall, these findings demonstrate that miR2111 is both the critical shoot-to-root factor that positively regulates root nodule development and also acts to shape root system architecture.
Assuntos
Glycine max/crescimento & desenvolvimento , Glycine max/genética , MicroRNAs/metabolismo , Família Multigênica , Brotos de Planta/genética , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Modelos Biológicos , Fenótipo , Floema/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Plant cell infection is tightly controlled by cell surface receptor-like kinases (RLKs). Like other RLKs, the Medicago truncatula entry receptor LYK3 laterally segregates into membrane nanodomains in a stimulus-dependent manner. Although nanodomain localization arises as a generic feature of plant membrane proteins, the molecular mechanisms underlying such dynamic transitions and their functional relevance have remained poorly understood. Here we demonstrate that actin and the flotillin protein FLOT4 form the primary and indispensable core of a specific nanodomain. Infection-dependent induction of the remorin protein and secondary molecular scaffold SYMREM1 results in subsequent recruitment of ligand-activated LYK3 and its stabilization within these membrane subcompartments. Reciprocally, the majority of this LYK3 receptor pool is destabilized at the plasma membrane and undergoes rapid endocytosis in symrem1 mutants on rhizobial inoculation, resulting in premature abortion of host cell infections. These data reveal that receptor recruitment into nanodomains is indispensable for their function during host cell infection.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Medicago truncatula/microbiologia , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Receptores de Superfície Celular/química , Nódulos Radiculares de Plantas/microbiologia , Sinorhizobium meliloti/fisiologia , Simbiose , Proteínas de Transporte/genética , Regulação da Expressão Gênica de Plantas , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/metabolismo , Mutação , Fosfoproteínas/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Rhizobium , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismoRESUMO
Auxin is essential for root development, and its regulatory action is exerted at different steps from perception of the hormone up to transcriptional regulation of target genes. In legume plants there is an overlap between the developmental programs governing lateral root and N2-fixing nodule organogenesis, the latter induced as the result of the symbiotic interaction with rhizobia. Here we report the characterization of a member of the L. japonicus TIR1/AFB auxin receptor family, LjAFB6. A preferential expression of the LjAFB6 gene in the aerial portion of L. japonicus plants was observed. Significant regulation of the expression was not observed during the symbiotic interaction with Mesorhizobium loti and the nodule organogenesis process. In roots, the LjAFB6 expression was induced in response to nitrate supply and was mainly localized in the meristematic regions of both primary and lateral roots. The phenotypic analyses conducted on two independent null mutants indicated a specialized role in the control of primary and lateral root elongation processes in response to auxin, whereas no involvement in the nodulation process was found. We also report the involvement of LjAFB6 in the hypocotyl elongation process and in the control of the expression profile of an auxin-responsive gene.
Assuntos
Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Lotus/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , Genes de Plantas , Lotus/crescimento & desenvolvimento , Lotus/metabolismo , Organogênese Vegetal , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismoRESUMO
Carbonic anhydrase (CA) plays a vital role in photosynthetic tissues of higher plants, whereas its non-photosynthetic role in the symbiotic root nodule was rarely characterized. In this study, 13 CA genes were identified in the model legume Lotus japonicus by comparison with Arabidopsis CA genes. Using qPCR and promoter-reporter fusion methods, three previously identified nodule-enhanced CA genes (LjαCA2, LjαCA6, and LjßCA1) have been further characterized, which exhibit different spatiotemporal expression patterns during nodule development. LjαCA2 was expressed in the central infection zone of the mature nodule, including both infected and uninfected cells. LjαCA6 was restricted to the vascular bundle of the root and nodule. As for LjßCA1, it was expressed in most cell types of nodule primordia but only in peripheral cortical cells and uninfected cells of the mature nodule. Using CRISPR/Cas9 technology, the knockout of LjßCA1 or both LjαCA2 and its homolog, LjαCA1, did not result in abnormal symbiotic phenotype compared with the wild-type plants, suggesting that LjßCA1 or LjαCA1/2 are not essential for the nitrogen fixation under normal symbiotic conditions. Nevertheless, the nodule-enhanced expression patterns and the diverse distributions in different types of cells imply their potential functions during root nodule symbiosis, such as CO2 fixation, N assimilation, and pH regulation, which await further investigations.
Assuntos
Anidrases Carbônicas/metabolismo , Regulação da Expressão Gênica de Plantas , Lotus/enzimologia , Fixação de Nitrogênio , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/enzimologia , Simbiose , Anidrases Carbônicas/genética , Lotus/genética , Lotus/crescimento & desenvolvimento , Fenótipo , Proteínas de Plantas/genética , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/crescimento & desenvolvimentoRESUMO
During the legume-rhizobium symbiotic interaction, rhizobial invasion of legumes is primarily mediated by a plant-made tubular invagination called an infection thread (IT). Here, we identify a gene in Lotus japonicus encoding a Leu-rich repeat receptor-like kinase (LRR-RLK), RINRK1 (Rhizobial Infection Receptor-like Kinase1), that is induced by Nod factors (NFs) and is involved in IT formation but not nodule organogenesis. A paralog, RINRK2, plays a relatively minor role in infection. RINRK1 is required for full induction of early infection genes, including Nodule Inception (NIN), encoding an essential nodulation transcription factor. RINRK1 displayed an infection-specific expression pattern, and NIN bound to the RINRK1 promoter, inducing its expression. RINRK1 was found to be an atypical kinase localized to the plasma membrane and did not require kinase activity for rhizobial infection. We propose RINRK1 is an infection-specific RLK, which may specifically coordinate output from NF signaling or perceive an unknown signal required for rhizobial infection.
Assuntos
Lotus/enzimologia , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Lotus/crescimento & desenvolvimento , Lotus/microbiologia , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/microbiologiaRESUMO
Legume-rhizobia symbiosis is a time-limited process due to the onset of senescence, which results in the degradation of host plant cells and symbiosomes. A number of transcription factors, proteases, and functional genes have been associated with nodule senescence; however, whether other proteases or transcription factors are involved in nodule senescence remains poorly understood. In this study, we identified an early nodule senescence mutant in Medicago truncatula, denoted basic helix-loop-helix transcription factor2 (bhlh2), that exhibits decreased nitrogenase activity, acceleration of plant programmed cell death (PCD), and accumulation of reactive oxygen species (ROS). The results suggest that MtbHLH2 plays a negative role in nodule senescence. Nodules of wild-type and bhlh2-TALEN mutant plants at 28 d postinoculation were used for transcriptome sequencing. The transcriptome data analysis identified a papain-like Cys protease gene, denoted MtCP77, that could serve as a potential target of MtbHLH2. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis demonstrated that MtbHLH2 directly binds to the promoter of MtCP77 to inhibit its expression. MtCP77 positively regulates nodule senescence by accelerating plant PCD and ROS accumulation. In addition, the expression of MtbHLH2 in the nodules gradually decreased from the meristematic zone to the nitrogen fixation zone, whereas the expression of MtCP77 showed enhancement. These results indicate that MtbHLH2 and MtCP77 have opposite functions in the regulation of nodule senescence. These results reveal significant roles for MtbHLH2 and MtCP77 in plant PCD, ROS accumulation, and nodule senescence, and improve our understanding of the regulation of the nodule senescence process.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cisteína Proteases/metabolismo , Medicago truncatula/crescimento & desenvolvimento , Medicago truncatula/genética , Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Sequência de Aminoácidos , Apoptose/genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Meristema/genética , Mutação/genética , Fixação de Nitrogênio/genética , Fenótipo , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Nicotiana , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Transcrição GênicaRESUMO
Gibberellins (GAs), a class of phytohormones, act as an essential natural regulator of plant growth and development. Many studies have shown that GA is related to rhizobial infection and nodule organogenesis in legume species. However, thus far, GA metabolism and signaling components are largely unknown in the model legume Medicago truncatula. In this study, a genome-wide analysis of GA metabolism and signaling genes was carried out. In total 29 components, including 8 MtGA20ox genes, 2 MtGA3ox genes, 13 MtGA2ox genes, 3 MtGID1 genes, and 3 MtDELLA genes were identified in M. truncatula genome. Expression profiles revealed that most members of MtGAox, MtGID1, and MtDELLA showed tissue-specific expression patterns. In addition, the GA biosynthesis and deactivation genes displayed a feedback regulation on GA treatment, respectively. Yeast two-hybrid assays showed that all the three MtGID1s interacted with MtDELLA1 and MtDELLA2, suggesting that the MtGID1s are functional GA receptors. More importantly, M. truncatula exhibited increased plant height and biomass by ectopic expression of the MtGA20ox1, suggesting that enhanced GA response has the potential for forage improvement.