Connective auxin transport contributes to strigolactone-mediated shoot branching control independent of the transcription factor BRC1.
ABSTRACT: The shoot systems of plants are built by the action of the primary shoot apical meristem, established during embryogenesis. In the axil of each leaf produced by the primary meristem, secondary axillary shoot apical meristems are established. The dynamic regulation of the activity of these axillary meristems gives shoot systems their extraordinary plasticity of form. The ability of plants to activate or repress these axillary meristems appropriately requires communication between meristems that is environmentally sensitive. The transport network of the plant hormone auxin has long been implicated as a central player in this tuneable communication system, with other systemically mobile hormones, such as strigolactone and cytokinin, acting in part by modulating auxin transport. Until recently, the polar auxin transport stream, which provides a high conductance auxin transport route down stems dominated by the auxin export protein PIN-FORMED1 (PIN1), has been the focus for understanding long range auxin transport in the shoot. However, recently additional auxin exporters with important roles in the shoot have been identified, including PIN3, PIN4 and PIN7. These proteins contribute to a wider less polar stem auxin transport regime, which we have termed connective auxin transport (CAT), because of its role in communication across the shoot system. Here we present a genetic analysis of the role of CAT in shoot branching. We demonstrate that in Arabidopsis, CAT plays an important role in strigolactone-mediated shoot branching control, with the triple pin3pin4pin7 mutant able to suppress partially the highly branched phenotype of strigolactone deficient mutants. In contrast, the branchy phenotype of mutants lacking the axillary meristem-expressed transcription factor, BRANCHED1 (BRC1) is unaffected by pin3pin4pin7. We further demonstrate that mutation in the ABCB19 auxin export protein, which like PIN3 PIN4 and PIN7 is widely expressed in stems, has very different effects, implicating ABCB19 in auxin loading at axillary bud apices.
Project description:Plants continuously extend their root and shoot systems through the action of meristems at their growing tips. By regulating which meristems are active, plants adjust their body plans to suit local environmental conditions. The transport network of the phytohormone auxin has been proposed to mediate this systemic growth coordination, due to its self-organising, environmentally sensitive properties. In particular, a positive feedback mechanism termed auxin transport canalization, which establishes auxin flow from active shoot meristems (auxin sources) to the roots (auxin sinks), has been proposed to mediate competition between shoot meristems and to balance shoot and root growth. Here we provide strong support for this hypothesis by demonstrating that a second hormone, strigolactone, regulates growth redistribution in the shoot by rapidly modulating auxin transport. A computational model in which strigolactone action is represented as an increase in the rate of removal of the auxin export protein, PIN1, from the plasma membrane can reproduce both the auxin transport and shoot branching phenotypes observed in various mutant combinations and strigolactone treatments, including the counterintuitive ability of strigolactones either to promote or inhibit shoot branching, depending on the auxin transport status of the plant. Consistent with this predicted mode of action, strigolactone signalling was found to trigger PIN1 depletion from the plasma membrane of xylem parenchyma cells in the stem. This effect could be detected within 10 minutes of strigolactone treatment and was independent of protein synthesis but dependent on clathrin-mediated membrane trafficking. Together these results support the hypothesis that growth across the plant shoot system is balanced by competition between shoot apices for a common auxin transport path to the root and that strigolactones regulate shoot branching by modulating this competition.
Project description:In seed plants, shoot branching is initiated by the formation of new meristems in the axils of leaves, which subsequently develop into new axes of growth. This study describes the genetic control of axillary meristem formation by the LATERAL SUPPRESSOR (LAS) gene in Arabidopsis thaliana. las mutants show a novel phenotype that is characterized by the inability to form lateral shoots during vegetative development. The analysis shows that axillary meristem formation is differently regulated during different phases of development. During reproductive development, axillary meristems initiate in close proximity to the shoot apical meristem and do not require LAS function. In contrast, during the vegetative phase, axillary meristems initiate at a distance to the SAM and require LAS function. This control mechanism is conserved between the distantly related species tomato and Arabidopsis. Monitoring the patterns of LAS and SHOOT MERISTEMLESS transcript accumulation allowed us to identify early steps in the development of leaf axil identity, which seem to be a prerequisite for axillary meristem initiation. Other regulators of shoot branching, like REVOLUTA and AUXIN RESISTANT 1, act downstream of LAS. The results are discussed in the context of the "detached meristem" and the "de novo formation" concepts of axillary meristem formation.
Project description:Shoot branching requires the establishment of new meristems harboring stem cells; this phenomenon raises questions about the precise regulation of meristematic fate. In seed plants, these new meristems initiate in leaf axils to enable lateral shoot branching. Using live-cell imaging of leaf axil cells, we show that the initiation of axillary meristems requires a meristematic cell population continuously expressing the meristem marker SHOOT MERISTEMLESS (STM). The maintenance of STM expression depends on the leaf axil auxin minimum. Ectopic expression of STM is insufficient to activate axillary buds formation from plants that have lost leaf axil STM expressing cells. This suggests that some cells undergo irreversible commitment to a developmental fate. In more mature leaves, REVOLUTA (REV) directly up-regulates STM expression in leaf axil meristematic cells, but not in differentiated cells, to establish axillary meristems. Cell type-specific binding of REV to the STM region correlates with epigenetic modifications. Our data favor a threshold model for axillary meristem initiation, in which low levels of STM maintain meristematic competence and high levels of STM lead to meristem initiation.
Project description:Axillary meristems (AMs) are secondary shoot meristems whose outgrowth determines plant architecture. In rice, AMs form tillers, and tillering mutants reveal an interplay between transcription factors and the phytohormones auxin and strigolactone as some factors that underpin this developmental process. Previous studies showed that knockdown of the transcription factor gene RFL reduced tillering and caused a very large decrease in panicle branching. Here, the relationship between RFL, AM initiation, and outgrowth was examined. We show that RFL promotes AM specification through its effects on LAX1 and CUC genes, as their expression was modulated on RFL knockdown, on induction of RFL:GR fusion protein, and by a repressive RFL-EAR fusion protein. Further, we report reduced expression of auxin transporter genes OsPIN1 and OsPIN3 in the culm of RFL knockdown transgenic plants. Additionally, subtle change in the spatial pattern of IR4 DR5:GFP auxin reporter was observed, which hints at compromised auxin transport on RFL knockdown. The relationship between RFL, strigolactone signalling, and bud outgrowth was studied by transcript analyses and by the tillering phenotype of transgenic plants knocked down for both RFL and D3. These data suggest indirect RFL-strigolactone links that may affect tillering. Further, we show expression modulation of the auxin transporter gene OsPIN3 upon RFL:GR protein induction and by the repressive RFL-EAR protein. These modified forms of RFL had only indirect effects on OsPIN1. Together, we have found that RFL regulates the LAX1 and CUC genes during AM specification, and positively influences the outgrowth of AMs though its effects on auxin transport.
Project description:The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS.
Project description:In plants, small groups of pluripotent stem cells called axillary meristems are required for the formation of the branches and flowers that eventually establish shoot architecture and drive reproductive success. To ensure the proper formation of new axillary meristems, the specification of boundary regions is required for coordinating their development. We have identified two maize genes, BARREN INFLORESCENCE1 and BARREN INFLORESCENCE4 (BIF1 and BIF4), that regulate the early steps required for inflorescence formation. BIF1 and BIF4 encode AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, which are key components of the auxin hormone signaling pathway that is essential for organogenesis. Here we show that BIF1 and BIF4 are integral to auxin signaling modules that dynamically regulate the expression of BARREN STALK1 (BA1), a basic helix-loop-helix (bHLH) transcriptional regulator necessary for axillary meristem formation that shows a striking boundary expression pattern. These findings suggest that auxin signaling directly controls boundary domains during axillary meristem formation and define a fundamental mechanism that regulates inflorescence architecture in one of the most widely grown crop species.
Project description:Dwarfism traits in Zea mays are regulated by multiple factors including the hormone auxin. Dwarf brachytic2 (br2) mutants harbour lesions in the gene encoding an orthologue of Arabidopsis thaliana ABCB1 which functions in auxin efflux out of meristematic regions in the shoot and root. br2 mesocotyls and coleoptiles exhibit reduced auxin transport. However, the dwarf stature of br2 derives from shortened lower internodes whilst the upper portion of the plant is completely normal. As such, it is counter-intuitive to attribute br2 dwarfism exclusively to reduced auxin export out of the shoot apex. Arabidopsis abcb1 mutants exhibit only minor reductions in auxin transport and plant height unless combined with mutations in the ABCB19 auxin transporter. Phylogenetic modelling analysis excludes the possibility that BR2 is more closely related to ABCB19 which has three more closely related orthologues in maize. BR2 is expressed in nodal meristems, and analyses of auxin transport and content indicate that BR2 function in these grass-specific tissues is analogous to ABCB1 function in the shoot and root apex of Arabidopsis. These results indicate that ABCB1/BR2 function is conserved between dicots and monocots, but also suggests that this function must be understood in the context of the segmental organization of grass plants.
Project description:Plant cytosolic ribosomal proteins are encoded by small gene families. Mutants affecting these genes are often viable, but show growth and developmental defects, suggesting incomplete functional redundancy within the families. Dormancy to growth transitions, such as the activation of axillary buds in the shoot, are characterised by co-ordinated upregulation of ribosomal protein genes.A recessive mutation in RPS10B, one of three Arabidopsis genes encoding the eukaryote-specific cytoplasmic ribosomal protein S10e, was found to suppress the excessive shoot branching mutant max2-1. rps10b-1 mildly affects the formation and separation of shoot lateral organs, including the shoot axillary meristems. Axillary meristem defects are enhanced when rps10b-1 is combined with mutations in REVOLUTA, AUXIN-RESISTANT1, PINOID or another suppressor of max2-1, FAR-RED ELONGATED HYPOCOTYL3. In some of these double mutants, the maintenance of the primary shoot meristem is also affected. In contrast, mutation of ALTERED MERISTEM PROGRAMME1 suppresses the rps10b-1axillary shoot defect. Defects in both axillary shoot formation and organ separation were enhanced by combining rps10b-1 with cuc3, a mutation affecting one of three Arabidopsis NAC transcription factor genes with partially redundant roles in these processes. To assess the effect of rps10b-1 on bud activation independently from bud formation, axillary bud outgrowth on excised cauline nodes was analysed. The outgrowth rate of untreated buds was reduced only slightly by rps10b-1 in both wild-type and max2-1 backgrounds. However, rps10b-1 strongly suppressed the auxin resistant outgrowth of max2-1 buds. A developmental phenotype of rps10b-1, reduced stamen number, was complemented by the cDNA of another family member, RPS10C, under the RPS10B promoter.RPS10B promotes shoot branching mainly by promoting axillary shoot development. It contributes to organ boundary formation and leaf polarity, and sustains max2-1 bud outgrowth in the presence of auxin. These processes require the auxin response machinery and precise spatial distribution of auxin. The correct dosage of protein(s) involved in auxin-mediated patterning may be RPS10B-dependent. Inability of other RPS10 gene family members to maintain fully S10e levels might cause the rps10b-1 phenotype, as we found no evidence for unique functional specialisation of either RPS10B promoter or RPS10B protein.
Project description:Elongation of the Arabidopsis hypocotyl pushes the shoot-producing meristem out of the soil by rapid expansion of cells already present in the embryo. This elongation process is shown here to be impaired by as much as 35% in mutants lacking ABCB19, an ATP-binding cassette membrane protein required for polar auxin transport, during a limited time of fast growth in dim white light beginning 2.5 days after germination. The discovery of high ectopic expression of a cyclin B1;1-based reporter of mitosis throughout abcb19 hypocotyls without an equivalent effect on mitosis prompted investigations of the endoreplication variant of the cell cycle. Flow cytometry performed on nuclei isolated from upper (growing) regions of 3-day-old hypocotyls showed ploidy levels to be lower in abcb19 mutants compared with wild type. CCS52A2 messenger RNA encoding a nuclear protein that promotes a shift from mitosis to endoreplication was lower in abcb19 hypocotyls, and fluorescence microscopy showed the CCS52A2 protein to be lower in the nuclei of abcb19 hypocotyls compared with wild type. Providing abcb19 seedlings with nanomolar auxin rescued their low CCS52A2 levels, endocycle defects, aberrant cyclin B1;1 expression, and growth rate defect. The abcb19-like growth rate of ccs52a2 mutants was not rescued by auxin, placing CCS52A2 after ABCB19-dependent polar auxin transport in a pathway responsible for a component of ploidy-related hypocotyl growth. A ccs52A2 mutation did not affect the level or pattern of cyclin B1;1 expression, indicating that CCS52A2 does not mediate the effect of auxin on cyclin B1;1.
Project description:In the production and breeding of Chrysanthemum sp., shoot branching is an important quality aspect as the outgrowth of axillary buds determines the final plant shape. Bud outgrowth is mainly controlled by apical dominance and the crosstalk between the plant hormones auxin, cytokinin and strigolactone. In this work the hormonal and genetic regulation of axillary bud outgrowth was studied in two differently branching cut flower Chrysanthemum morifolium (Ramat) genotypes. C17 is a split-type which forms an inflorescence meristem after a certain vegetative period, while C18 remains vegetative under long day conditions. Plant growth of both genotypes was monitored during 5 subsequent weeks starting one week before flower initiation occurred in C17. Axillary bud outgrowth was measured weekly and samples of shoot apex, stem and axillary buds were taken during the first two weeks. We combined auxin and cytokinin measurements by UPLC-MS/MS with RT-qPCR expression analysis of genes involved in shoot branching regulation pathways in chrysanthemum. These included bud development genes (CmBRC1, CmDRM1, CmSTM, CmLsL), auxin pathway genes (CmPIN1, CmTIR3, CmTIR1, CmAXR1, CmAXR6, CmAXR2, CmIAA16, CmIAA12), cytokinin pathway genes (CmIPT3, CmHK3, CmRR1) and strigolactone genes (CmMAX1 and CmMAX2). Genotype C17 showed a release from apical dominance after floral transition coinciding with decreased auxin and increased cytokinin levels in the subapical axillary buds. As opposed to C17, C18 maintained strong apical dominance with vegetative growth throughout the experiment. Here high auxin levels and decreasing cytokinin levels in axillary buds and stem were measured. A differential expression of several branching genes accompanied the different hormonal change and bud outgrowth in C17 and C18. This was clear for the strigolactone biosynthesis gene CmMAX1, the transcription factor CmBRC1 and the dormancy associated gene CmDRM1, that all showed a decreased expression in C17 at floral transition and an increased expression in C18 with continuous vegetative growth. These results offer a case study for Chrysanthemum, showing an altered cytokinin to auxin balance and differential gene expression between vegetative growth with apical dominance and transition to generative growth with loss of apical dominance and axillary bud outgrowth. This suggests a conservation of several aspects of the hormonal and genetical regulation of bud outgrowth in Chrysanthemum. Furthermore, 15 previously uncharacterised genes in chrysanthemum, were described in this study. Of those genes involved in axillary bud outgrowth we identified CmDRM1, CmBRC1 and CmMAX1 as having an altered expression preceding axillary bud outgrowth, which could be useful as markers for bud activity.