RESUMO
The growth-regulating factor (GRF) family of transcriptional factors are involved in the control of leaf size and senescence, inflorescence and root growth, grain size, and plant regeneration. However, there is limited information about the genes regulated by these transcriptional factors, which are in turn responsible for their functions. Using a meta-analysis approach, we identified genes encoding Arabidopsis (Arabidopsis thaliana) zinc-finger homeodomain (ZF-HD) transcriptional factors, as potential targets of the GRFs. We further showed that GRF3 binds to the promoter of one of the members of the ZF-HD family, HOMEOBOX PROTEIN 33 (HB33), and activates its transcription. Increased levels of HB33 led to different modifications in leaf cell number and size that were dependent on its expression levels. Furthermore, we found that expression of HB33 for an extended period during leaf development increased leaf longevity. To cope with the functional redundancy among ZF-HD family members, we generated a dominant repressor version of HB33, HB33-SRDX. Expression of HB33-SRDX from HB33 regulatory regions was seedling-lethal, revealing the importance of the ZF-HD family in plant development. Misexpression of HB33-SRDX in early leaf development caused a reduction in both cell size and number. Interestingly, the loss-of-function of HB33 in lines carrying a GRF3 allele insensitive to miR396 reverted the delay in leaf senescence characteristic of these plants. Our results revealed functions for ZF-HDs in leaf development and linked them to the GRF pathway.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Arabidopsis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , MicroRNAs/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Folhas de Planta/metabolismoRESUMO
KEY MESSAGE: Proper root growth depends on the clearance of TCP transcripts from the root apical meristem by microRNA miR319. The evolutionarily conserved microRNA miR319 regulates genes encoding TCP transcription factors in angiosperms. The miR319-TCP module controls cell proliferation and differentiation in leaves and other aerial organs. The current model sustains that miR319 quantitatively tunes TCP activity during leaf growth and development, ultimately affecting its size. In this work we studied how this module participates in Arabidopsis root development. We found that misregulation of TCP activity through impairment of miR319 binding decreased root meristem size and root length. Cellular and molecular analyses revealed that high TCP activity affects cell number and cyclin expression but not mature cell length, indicating that, in roots, unchecking the expression of miR319-regulated TCPs significantly affects cell proliferation. Conversely, tcp multiple mutants showed no obvious effect on root growth, but strong defects in leaf morphogenesis. Therefore, in contrast to the quantitative regulation of the TCPs by miR319 in leaves, our data suggest that miR319 clears TCP transcripts from root cells. Hence, we provide new insights into the functions of the miR319-TCP regulatory system in Arabidopsis development, highlighting a different modus operandi for its action mechanism in roots and shoots.
Assuntos
Proteínas de Arabidopsis/fisiologia , MicroRNAs/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , MicroRNAs/metabolismo , Microscopia Confocal , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
The characteristic leaf shapes we see in all plants are in good part the outcome of the combined action of several transcription factor networks that translate into cell division activity during the early development of the organ. We show here that wild-type leaves have distinct transcriptomic profiles in center and marginal regions. Certain transcripts are enriched in margins, including those of CINCINNATA-like TCPs (TEOSINTE BRANCHED, CYCLOIDEA and PCF1/2) and members of the NGATHA and STYLISH gene families. We study in detail the contribution of microRNA319 (miR319)-regulated TCP transcription factors to the development of the center and marginal regions of Arabidopsis (Arabidopsis thaliana) leaves. We compare in molecular analyses the wild type, the tcp2 tcp4 mutant that has enlarged flat leaves, and the tcp2 tcp3 tcp4 tcp10 mutant with strongly crinkled leaves. The different leaf domains of the tcp mutants show changed expression patterns for many photosynthesis-related genes, indicating delayed differentiation, especially in the marginal parts of the organ. At the same time, we found an up-regulation of cyclin genes and other genes that are known to participate in cell division, specifically in the marginal regions of tcp2 tcp3 tcp4 tcp10 Using GUS reporter constructs, we confirmed extended mitotic activity in the tcp2 tcp3 tcp4 tcp10 leaf, which persisted in small defined foci in the margins when the mitotic activity had already ceased in wild-type leaves. Our results describe the role of miR319-regulated TCP transcription factors in the coordination of activities in different leaf domains during organ development.
Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Plants have the ability to generate different and new organs throughout their life cycle. Organ growth is mostly determined by the combinatory effects of cell proliferation and cell expansion. Still, organ size and shape are adjusted constantly by environmental conditions and developmental timing. The plasticity of plant development is further illustrated by the diverse organ forms found in nature. MicroRNAs (miRNAs) are known to control key biological processes in plants. In this review, we will discuss recent findings showing the participation of miRNA networks in the regulation of cell proliferation and organ growth. It has become clear that miRNA networks play both integrative and specific roles in the control of organ development in Arabidopsis thaliana. Furthermore, recent work in different species demonstrated a broad role for miR396 in the control of organ size, and that specific tuning of the miR396 network can improve crop yield.
Assuntos
MicroRNAs/genética , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologiaRESUMO
The microRNA (miRNA) miR396 regulates GROWTH-REGULATING FACTORs (GRFs), a plant specific family of transcription factors. Overexpression of miR396 causes a decrease in the GRFs that has been shown to affect cell proliferation in the meristem and developing leaves. To bring further insights into the function of the miR396 regulatory network we performed a mutant enhancer screen of a stable Arabidopsis transgenic line expressing 35S:miR396b, which has a reduction in leaf size. From this screen we recovered several mutants enhancing this phenotype and displaying organs with lotus- or needle-like shape. Analysis of these plants revealed mutations in as2 and rdr6. While 35S:miR396b in an as2 context generated organs with lotus-like shape, the overexpression of the miRNA in an rdr6 mutant background caused more important developmental defects, including pin-like organs and lobed leaves. Combination of miR396 overexpressors, and rdr6 and as2 mutants show additional organ defects, suggesting that the three pathways act in concert. Genetic interactions during leaf development were observed in a similar way between miR396 overexpression and mutants in RDR6, SGS3 or AGO7, which are known to participate in trans-acting siRNA (ta-siRNA) biogenesis. Furthermore, we found that miR396 can cause lotus- and pin-like organs per se, once a certain expression threshold is overcome. In good agreement, mutants accumulating high levels of TCP4, which induces miR396, interacted with the AS1/AS2 pathway to generate lotus-like organs. The results indicate that the miR396 regulatory network and the ta-siRNA biogenesis pathway synergistically interact during leaf development and morphogenesis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Folhas de Planta , RNA Polimerase Dependente de RNA , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Meristema/genética , Meristema/metabolismo , MicroRNAs/genética , Morfogênese , Mutação , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
MicroRNAs (miRNAs) are major regulators of gene expression in multicellular organisms. They recognize their targets by sequence complementarity and guide them to cleavage or translational arrest. It is generally accepted that plant miRNAs have extensive complementarity to their targets and their prediction usually relies on the use of empirical parameters deduced from known miRNA-target interactions. Here, we developed a strategy to identify miRNA targets which is mainly based on the conservation of the potential regulation in different species. We applied the approach to expressed sequence tags datasets from angiosperms. Using this strategy, we predicted many new interactions and experimentally validated previously unknown miRNA targets in Arabidopsis thaliana. Newly identified targets that are broadly conserved include auxin regulators, transcription factors and transporters. Some of them might participate in the same pathways as the targets known before, suggesting that some miRNAs might control different aspects of a biological process. Furthermore, this approach can be used to identify targets present in a specific group of species, and, as a proof of principle, we analyzed Solanaceae-specific targets. The presented strategy can be used alone or in combination with other approaches to find miRNA targets in plants.
Assuntos
Regulação da Expressão Gênica de Plantas , MicroRNAs/química , RNA de Plantas/química , Arabidopsis/genética , Sequência de Bases , Sequência Conservada , Etiquetas de Sequências Expressas , MicroRNAs/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Alinhamento de Sequência , SolanaceaeRESUMO
Cell proliferation is an important determinant of plant form, but little is known about how developmental programs control cell division. Here, we describe the role of microRNA miR396 in the coordination of cell proliferation in Arabidopsis leaves. In leaf primordia, miR396 is expressed at low levels that steadily increase during organ development. We found that miR396 antagonizes the expression pattern of its targets, the GROWTH-REGULATING FACTOR (GRF) transcription factors. miR396 accumulates preferentially in the distal part of young developing leaves, restricting the expression of GRF2 to the proximal part of the organ. This, in turn, coincides with the activity of the cell proliferation marker CYCLINB1;1. We show that miR396 attenuates cell proliferation in developing leaves, through the repression of GRF activity and a decrease in the expression of cell cycle genes. We observed that the balance between miR396 and the GRFs controls the final number of cells in leaves. Furthermore, overexpression of miR396 in a mutant lacking GRF-INTERACTING FACTOR 1 severely compromises the shoot meristem. We found that miR396 is expressed at low levels throughout the meristem, overlapping with the expression of its target, GRF2. In addition, we show that miR396 can regulate cell proliferation and the size of the meristem. Arabidopsis plants with an increased activity of the transcription factor TCP4, which reduces cell proliferation in leaves, have higher miR396 and lower GRF levels. These results implicate miR396 as a significant module in the regulation of cell proliferation in plants.