RESUMO
Introduction: Plant growth is a plastic phenomenon controlled both by endogenous genetic programs and by environmental cues. The embryonic stem, the hypocotyl, is an ideal model system for the quantitative study of growth due to its relatively simple geometry and cellular organization, and to its essentially unidirectional growth pattern. The hypocotyl of Arabidopsis thaliana has been studied particularly well at the molecular-genetic level and at the cellular level, and it is the model of choice for analysis of the shade avoidance syndrome (SAS), a growth reaction that allows plants to compete with neighboring plants for light. During SAS, hypocotyl growth is controlled primarily by the growth hormone auxin, which stimulates cell expansion without the involvement of cell division. Methods: We assessed hypocotyl growth at cellular resolution in Arabidopsis mutants defective in auxin transport and biosynthesis and we designed a mathematical auxin transport model based on known polar and non-polar auxin transporters (ABCB1, ABCB19, and PINs) and on factors that control auxin homeostasis in the hypocotyl. In addition, we introduced into the model biophysical properties of the cell types based on precise cell wall measurements. Results and Discussion: Our model can generate the observed cellular growth patterns based on auxin distribution along the hypocotyl resulting from production in the cotyledons, transport along the hypocotyl, and general turnover of auxin. These principles, which resemble the features of mathematical models of animal morphogen gradients, allow to generate robust shallow auxin gradients as they are expected to exist in tissues that exhibit quantitative auxin-driven tissue growth, as opposed to the sharp auxin maxima generated by patterning mechanisms in plant development.
RESUMO
The intimate association of host and fungus in arbuscular mycorrhizal (AM) symbiosis can potentially trigger induction of host defence mechanisms against the fungus, implying that successful symbiosis requires suppression of defence. We addressed this phenomenon by using AM-defective vapyrin (vpy) mutants in Petunia hybrida, including a new allele (vpy-3) with a transposon insertion close to the ATG start codon. We explore whether abortion of fungal infection in vpy mutants is associated with the induction of defence markers, such as cell wall alterations, accumulation of reactive oxygen species (ROS), defence hormones and induction of pathogenesis-related (PR) genes. We show that vpy mutants exhibit a strong resistance against intracellular colonization, which is associated with the generation of cell wall appositions (papillae) with lignin impregnation at fungal entry sites, while no accumulation of defence hormones, ROS or callose was observed. Systematic analysis of PR gene expression revealed that several PR genes are induced in mycorrhizal roots of the wild-type, and even more in vpy plants. Some PR genes are induced exclusively in vpy mutants. Our results suggest that VPY is involved in avoiding or suppressing the induction of a cellular defence syndrome that involves localized lignin deposition and PR gene induction.
Assuntos
Micorrizas , Petunia , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Lignina , Micorrizas/genética , Petunia/genética , Raízes de Plantas , SimbioseRESUMO
The VAPYRIN (VPY) gene in Medicago truncatula and Petunia hybrida is required for arbuscular mycorrhizal (AM) symbiosis. The moss Physcomitrella patens has a close homolog (VPY-like, VPYL), although it does not form AM. Here, we explore the phylogeny of VPY and VPYL in land plants, and study the expression and developmental function of VPYL in Ppatens We show that VPYL is expressed primarily in the protonema, the early filamentous stage of moss development, and later in rhizoids arising from the leafy gametophores and in adult phyllids. Knockout mutants have specific phenotypes in branching of the protonema and in cell division of the leaves (phyllids) in gametophores. The mutants are responsive to auxin and strigolactone, which are involved in regulation of protonemal branching, indicating that hormonal signaling in the mutants is not affected in hormonal signaling. Taken together, these results suggest that VPYL exerts negative regulation of protonemal branching and cell division in phyllids. We discuss VPY and VPYL phylogeny and function in land plants in the context of AM symbiosis in angiosperms and development in the moss.
Assuntos
Bryopsida/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Bryopsida/metabolismo , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Compostos Heterocíclicos com 3 Anéis/metabolismo , Ácidos Indolacéticos/metabolismo , Lactonas/metabolismo , Mutagênese , Fenótipo , Filogenia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Bacterial lipo-chitooligosaccharides (LCOs) are key mediators of the nitrogen-fixing root nodule symbiosis (RNS) in legumes. The isolation of LCOs from arbuscular mycorrhizal fungi suggested that LCOs are also signaling molecules in arbuscular mycorrhiza (AM). However, the corresponding plant receptors have remained uncharacterized. Here we show that petunia and tomato mutants in the LysM receptor-like kinases LYK10 are impaired in AM formation. Petunia and tomato LYK10 proteins have a high affinity for LCOs (Kd in the nM range) comparable to that previously reported for a legume LCO receptor essential for the RNS. Interestingly, the tomato and petunia LYK10 promoters, when introduced into a legume, were active in nodules similarly to the promoter of the legume orthologous gene. Moreover, tomato and petunia LYK10 coding sequences restored nodulation in legumes mutated in their orthologs. This combination of genetic and biochemical data clearly pinpoints Solanaceous LYK10 as part of an ancestral LCO perception system involved in AM establishment, which has been directly recruited during evolution of the RNS in legumes.
Assuntos
Lipopolissacarídeos/metabolismo , Micorrizas/fisiologia , Rhizobium/metabolismo , Quitina/análogos & derivados , Quitina/metabolismo , Quitosana , Fabaceae/metabolismo , Fabaceae/microbiologia , Regulação da Expressão Gênica de Plantas/genética , Solanum lycopersicum/metabolismo , Micorrizas/metabolismo , Oligossacarídeos , Petunia/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/genética , Simbiose/genéticaRESUMO
How complex developmental-genetic networks are translated into organs with specific 3D shapes remains an open question. This question is particularly challenging because the elaboration of specific shapes is in essence a question of mechanics. In plants, this means how the genetic circuitry affects the cell wall. The mechanical properties of the wall and their spatial variation are the key factors controlling morphogenesis in plants. However, these properties are difficult to measure and investigating their relation to genetic regulation is particularly challenging. To measure spatial variation of mechanical properties, one must determine the deformation of a tissue in response to a known force with cellular resolution. Here, we present an automated confocal micro-extensometer (ACME), which greatly expands the scope of existing methods for measuring mechanical properties. Unlike classical extensometers, ACME is mounted on a confocal microscope and uses confocal images to compute the deformation of the tissue directly from biological markers, thus providing 3D cellular scale information and improved accuracy. Additionally, ACME is suitable for measuring the mechanical responses in live tissue. As a proof of concept, we demonstrate that the plant hormone gibberellic acid induces a spatial gradient in mechanical properties along the length of the Arabidopsis thaliana hypocotyl.
Assuntos
Arabidopsis/citologia , Microscopia Confocal/instrumentação , Células Vegetais/química , Automação , Fenômenos Biomecânicos , Parede Celular/efeitos dos fármacos , Parede Celular/fisiologia , Elasticidade , Giberelinas/farmacologia , Hipocótilo/citologia , Hipocótilo/efeitos dos fármacos , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/efeitos da radiação , Luz , Modelos Biológicos , Células Vegetais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
Arbuscular mycorrhiza (AM) is a mutual symbiosis that involves a complex symbiotic interface over which nutrients are exchanged between the plant host and the AM fungus. Dozens of genes in the host are required for the establishment and functioning of the interaction, among them nutrient transporters that mediate the uptake of mineral nutrients delivered by the fungal arbuscules. We have isolated in a genetic mutant screen a petunia (Petunia hybrida) Gibberellic Acid Insensitive, Repressor of Gibberellic Acid Insensitive, and Scarecrow (GRAS)-type transcription factor, Atypical Arbuscule (ATA), that acts as the central regulator of AM-related genes and is required for the morphogenesis of arbuscules. Forced mycorrhizal inoculations from neighboring wild-type plants revealed an additional role of ATA in restricting mycorrhizal colonization of the root meristem. The lack of ATA, which represents the ortholog of Required For Arbuscular Mycorrhiza1 in Medicago truncatula, renders the interaction completely ineffective, hence demonstrating the central role of AM-related genes for arbuscule development and function.
Assuntos
Regulação da Expressão Gênica de Plantas , Micorrizas/crescimento & desenvolvimento , Petunia/genética , Petunia/microbiologia , Proteínas de Plantas/metabolismo , Simbiose/genética , Fatores de Transcrição/metabolismo , Contagem de Colônia Microbiana , Genes de Plantas , Loci Gênicos , Medicago truncatula/genética , Medicago truncatula/microbiologia , Meristema/genética , Meristema/microbiologia , Dados de Sequência Molecular , Morfogênese , Mutação/genética , Fenótipo , Proteínas de Plantas/genética , Fatores de Transcrição/genéticaRESUMO
Plants engage in mutualistic interactions with microbes that improve their mineral nutrient supply. The most wide-spread symbiotic association is arbuscular mycorrhiza (AM), in which fungi of the order Glomeromycota invade roots and colonize the cellular lumen of cortical cells. The establishment of this interaction requires a dedicated molecular-genetic program and a cellular machinery of the plant host. This program is partially shared with the root nodule symbiosis (RNS), which involves prokaryotic partners collectively referred to as rhizobia. Both, AM and RNS are endosymbioses that involve intracellular accommodation of the microbial partner in the cells of the plant host. Since plant cells are surrounded by sturdy cell walls, root penetration and cell invasion requires mechanisms to overcome this barrier while maintaining the cytoplasm of the two partners separate during development of the symbiotic association. Here, we discuss the diverse functions of the cell wall compartment in establishment and functioning of plant symbioses with the emphasis on AM and RNS, and we describe the stages of the AM association between the model organisms Petunia hybrida and Rhizophagus irregularis.
RESUMO
Although genetic control of morphogenesis is well established, elaboration of complex shapes requires changes in the mechanical properties of cells. In plants, the first visible sign of leaf formation is a bulge on the flank of the shoot apical meristem. Bulging results from local relaxation of cell walls, which causes them to yield to internal hydrostatic pressure. By manipulation of tissue tension in combination with quantitative live imaging and finite-element modeling, we found that the slow-growing area at the shoot tip is substantially strain-stiffened compared with surrounding fast-growing tissue. We propose that strain stiffening limits growth, restricts organ bulging, and contributes to the meristem's functional zonation. Thus, mechanical signals are not just passive readouts of gene action but feed back on morphogenesis.
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Meristema/crescimento & desenvolvimento , Morfogênese , Brotos de Planta/crescimento & desenvolvimento , Solanum lycopersicum/crescimento & desenvolvimento , Parede Celular/fisiologia , Parede Celular/ultraestrutura , Elasticidade , Pressão Hidrostática , Solanum lycopersicum/citologia , Meristema/citologia , Modelos Biológicos , Concentração Osmolar , Pressão Osmótica , Brotos de Planta/citologiaRESUMO
Most terrestrial plants engage into arbuscular mycorrhizal (AM) symbiosis with fungi of the phylum Glomeromycota. The initial recognition of the fungal symbiont results in the activation of a symbiosis signalling pathway that is shared with the root nodule symbiosis (common SYM pathway). The subsequent intracellular accommodation of the fungus, and the elaboration of its characteristic feeding structures, the arbuscules, depends on a genetic programme in the plant that has recently been shown to involve the VAPYRIN gene in Medicaco truncatula. We have previously identified a mutant in Petunia hybrida, penetration and arbuscule morphogenesis 1 (pam1), that is defective in the intracellular stages of AM development. Here, we report on the cloning of PAM1, which encodes a VAPYRIN homologue. PAM1 protein localizes to the cytosol and the nucleus, with a prominent affinity to mobile spherical structures that are associated with the tonoplast, and are therefore referred to as tonospheres. In mycorrhizal roots, tonospheres were observed in the vicinity of intracellular hyphae, where they may play an essential role in the accommodation and morphogenesis of the fungal endosymbiont.
Assuntos
Micorrizas/fisiologia , Petunia/genética , Petunia/microbiologia , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Plantas/metabolismo , Clonagem Molecular , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Membrana , Dados de Sequência Molecular , Mutação , Proteínas de Transporte de Fosfato/genética , Proteínas de Plantas/genética , Raízes de Plantas/microbiologia , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae , SimbioseRESUMO
Several studies have suggested that debranching enzymes (DBEs) are involved in the biosynthesis of amylopectin, the major constituent of starch granules. Our systematic analysis of all DBE mutants of Arabidopsis thaliana demonstrates that when any DBE activity remains, starch granules are still synthesized, albeit with altered amylopectin structure. Quadruple mutants lacking all four DBE proteins (Isoamylase1 [ISA1], ISA2, and ISA3, and Limit-Dextrinase) are devoid of starch granules and instead accumulate highly branched glucans, distinct from amylopectin and from previously described phytoglycogen. A fraction of these glucans are present as discrete, insoluble, nanometer-scale particles, but the structure and properties of this material are radically altered compared with wild-type amylopectin. Superficially, these data support the hypothesis that debranching is required for amylopectin synthesis. However, our analyses show that soluble glucans in the quadruple DBE mutant are degraded by alpha- and beta-amylases during periods of net accumulation, giving rise to maltose and branched malto-oligosaccharides. The additional loss of the chloroplastic alpha-amylase AMY3 partially reverts the phenotype of the quadruple DBE mutant, restoring starch granule biosynthesis. We propose that DBEs function in normal amylopectin synthesis by promoting amylopectin crystallization but conclude that they are not mandatory for starch granule synthesis.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/metabolismo , Glicosídeo Hidrolases/fisiologia , Isoamilase/fisiologia , Amido/biossíntese , alfa-Amilases/fisiologia , Amilopectina/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestrutura , Microscopia Crioeletrônica , Glicosídeo Hidrolases/genética , Isoamilase/genética , Maltose/metabolismo , Oligossacarídeos/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/ultraestrutura , Espectroscopia de Infravermelho com Transformada de Fourier , Amido/genética , alfa-Amilases/genéticaRESUMO
The regulation of the arbuscular mycorrhizal (AM) symbiosis is largely under the control of a genetic programme of the plant host. This programme includes a common symbiosis signalling pathway that is shared with the root nodule symbiosis. Whereas this common pathway has been investigated in detail, little is known about the mycorrhiza-specific regulatory steps upstream and downstream of the common pathway. To get further insight in the regulation of the AM symbiosis, a transposon-mutagenized population of Petunia hybrida was screened for mutants with defects in AM development. Here, we describe a petunia mutant, penetration and arbuscule morphogenesis1 (pam1), which is characterized by a strong decrease in colonization by three different AM fungi. Penetrating hyphae are frequently aborted in epidermal cells. Occasionally the fungus can progress to the cortex, but fails to develop arbuscules. The resulting hyphal colonization of the cortex in mutant plants does not support symbiotic acquisition of phosphate and copper by the plant. Expression analysis of three petunia orthologues of the common SYM genes LjPOLLUX, LjSYMRK and MtDMI3 indicates that pam1 is not mutated in these genes. We conclude that the PAM1 gene may play a specific role in intracellular accommodation and morphogenesis of the fungal endosymbiont.
Assuntos
Micorrizas/crescimento & desenvolvimento , Petunia/microbiologia , Simbiose/genética , Expressão Gênica , Genes de Plantas , Mutação , Micorrizas/fisiologia , Petunia/genética , Petunia/fisiologia , Fenótipo , Fosfatos/metabolismo , Brotos de Planta/metabolismo , Simbiose/fisiologiaRESUMO
The shoot apical meristem contains cells that undergo continual growth and division to generate the building blocks for the aerial portion of the plant. As cells leave the meristem, they undergo differentiation to form specific cell types. Most notably, heterotrophic cells of the meristem rapidly gain autotrophic capability by synthesis and assembly of components of the chloroplast. At the same time, cells undergo enlargement via vacuolation. Despite significant advances in the characterization of transcriptional networks involved in meristem maintenance and leaf determination, our understanding of the actual mechanism of meristem cell differentiation remains very limited. Using a microinduction technique, we show that local, transient overexpression of a retinoblastoma-related (RBR) protein in the shoot apical meristem is sufficient to trigger cells in the meristem to undergo the initial stages of differentiation. Taken together with recent data showing that RBR protein plays a key role in restricting stem cell differentiation in the root apical meristem, our data contribute to an emerging picture of RBR proteins as a central part of the mechanism controlling meristem cell differentiation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Meristema/citologia , Brotos de Planta/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Ciclo Celular , Diferenciação Celular , Regulação da Expressão Gênica de Plantas , Marcadores Genéticos , Meristema/crescimento & desenvolvimento , Brotos de Planta/anatomia & histologia , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/metabolismo , Nicotiana/anatomia & histologia , Nicotiana/citologia , Nicotiana/genéticaRESUMO
The water pipes of elongating plant organs are the result of programmed cell death and are formed by the walls of dead and empty protoxylem elements. These protoxylem elements are passively elongated many times by the surrounding tissue before they are replaced and collapse. Well-known adaptations for this unique task include the characteristic secondary wall thickenings, forming rings and helices. A new, clearly distinct structural element containing glycine-rich proteins is now visualized for the first time, using confocal laser scanning microscopy in the mature protoxylem of elongating organs of seed plants. This structural element is arranged along the longitudinal axis of the protoxylem elements. It interconnects the secondary wall thickenings within and between protoxylem elements, as well as the protoxylem with other cell types such as xylem parenchyma cells and metaxylem elements. The structural element is stable against detergent extractions, proteinase, pectinase and cellulase hydrolysis, and is closely associated with rhamnogalacturonan-I, a pectic polysaccharide. The results clearly demonstrate that the cell wall of protoxylem cells is a highly dynamic and complex structure. The typical polysaccharide-rich primary wall of living and elongating plant cells is progressively modified and finally replaced by a protein-rich wall in the dead and passively stretched protoxylem elements. These glycine-rich walls originated early in the evolution of the seed plants as confirmed by the analysis of genomic information.