RESUMO
Sucrose non-fermenting 1 (SNF1)-related protein kinase 1.1 (SnRK1.1; also known as KIN10 or SnRK1α) has been identified as the catalytic subunit of the complex SnRK1, the Arabidopsis thaliana homologue of a central integrator of energy and stress signalling in eukaryotes dubbed AMPK/Snf1/SnRK1. A nuclear localization of SnRK1.1 has been previously described and is in line with its function as an integrator of energy and stress signals. Here, using two biological models (Nicotiana benthamiana and Arabidopsis thaliana), native regulatory sequences, different microscopy techniques, and manipulations of cellular energy status, it was found that SnRK1.1 is localized dynamically between the nucleus and endoplasmic reticulum (ER). This distribution was confirmed at a spatial and temporal level by co-localization studies with two different fluorescent ER markers, one of them being the SnRK1.1 phosphorylation target HMGR. The ER and nuclear localization displayed a dynamic behaviour in response to perturbations of the plastidic electron transport chain. These results suggest that an ER-associated SnRK1.1 fraction might be sensing the cellular energy status, being a point of crosstalk with other ER stress regulatory pathways.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/citologia , Cloroplastos/metabolismo , Transporte de Elétrons , Metabolismo Energético , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais/fisiologia , Estresse Fisiológico , Nicotiana/citologia , Nicotiana/metabolismo , Fatores de Transcrição/metabolismoRESUMO
To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Proteínas de Membrana/metabolismo , Nicotiana/citologia , Folhas de Planta/citologia , Vacúolos/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Fluorescência , Genes Reporter , Microscopia Confocal , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/metabolismo , Transporte Proteico , Nicotiana/metabolismoRESUMO
Root system architecture depends on lateral root (LR) initiation that takes place in a relatively narrow developmental window (DW). Here, we analyzed the role of auxin gradients established along the parent root in defining this DW for LR initiation. Correlations between auxin distribution and response, and spatiotemporal control of LR initiation were analyzed in Arabidopsis thaliana and tomato (Solanum lycopersicum). In both Arabidopsis and tomato roots, a well defined zone, where auxin content and response are minimal, demarcates the position of a DW for founder cell specification and LR initiation. We show that in the zone of auxin minimum pericycle cells have highest probability to become founder cells and that auxin perception via the TIR1/AFB pathway, and polar auxin transport, are essential for the establishment of this zone. Altogether, this study reveals that the same morphogen-like molecule, auxin, can act simultaneously as a morphogenetic trigger of LR founder cell identity and as a gradient-dependent signal defining positioning of the founder cell specification. This auxin minimum zone might represent an important control mechanism ensuring the LR initiation steadiness and the acropetal LR initiation pattern.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Solanum lycopersicum/crescimento & desenvolvimento , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular , Ácidos Indolacéticos/farmacologia , Solanum lycopersicum/citologia , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/metabolismo , Microscopia Confocal , Morfogênese , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Transporte Proteico , Transdução de SinaisRESUMO
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.
Assuntos
Arabidopsis/citologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/fisiologia , MicroRNAs/metabolismo , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Hibridização In Situ , Meristema/citologia , Meristema/genética , Meristema/metabolismo , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Brotos de Planta/citologia , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Transativadores , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Inositol plays a role in membrane trafficking and signaling in addition to regulating cellular metabolism and controlling growth. In plants, the myo-inositol-1-phosphate is synthesized from glucose 6-phosphate in a reaction catalyzed by the enzyme myo-inositol-1-phosphate synthase (EC 5.5.1.4). Inositol can be converted into phytic acid (phytate), the most abundant form of phosphate in seeds. The path to phytate has been suggested to proceed via the sequential phosphorylation of inositol phosphates, and/or in part via phosphatidylinositol phosphate. Soybean [Glycine max (L.) Merrill] lines were produced using interfering RNA (RNAi) construct in order to silence the myo-inositol-1-phosphate (GmMIPS1) gene. We have observed an absence of seed development in lines in which the presence of GmMIPS1 transcripts was not detected. In addition, a drastic reduction of phytate (InsP6) content was achieved in transgenic lines (up to 94.5%). Our results demonstrated an important correlation between GmMIPS1 gene expression and seed development.