Caracterização fisiológica de plantas superexpressando GmNAC6 em soja e seus efeitos na morte celular programada

• Autor(a) principal: Mendes, Giselle Camargo
• Data de Publicação: 2013
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/997
• topic: GmNAC6; Soja; Morte celular programada; GmNAC6; Soybean; Programmed cell death; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

Prolonged ER stress and osmotic stress synergistically activate the stress-induced N-rich protein (NRP)-mediated signaling that transduces a cell death signal by inducing GmNAC6 in soybean. To identify novel regulators of the stress-induced programmed cell death (PCD) response, we screened a two-hybrid library for partners of GmNAC6. Here we discovered another member of the NAC family, GmNAC30, which binds to GmNAC6 in the nucleus of plant cells to coordinately regulate common target promoters, which harbor the core cisregulatory element TGTG[TGC]. We found that GmNAC6 and GmNAC30 can function either as transcriptional repressors or activators and cooperate to each other to enhance transcriptional regulation of common target promoters, suggesting that heterodimerization may be required for full regulation of gene expression. Accordingly, GmNAC6 and GmNAC30 display an overlapped expression profile in response to multiple environmental and developmental stimuli. Consistent with a role in programmed cell death, GmNAC6 and GmNAC30 bind in vivo to and transactivate hydrolytic enzyme promoters in soybean protoplasts. A GmNAC6/GmNAC30 binding site is found in the promoter of the caspase-1 like vacuolar processing enzyme (VPE) gene, an executioner of programmed cell death in plants. We showed that expression of GmNAC6 along with GmNAC30 fully transactivate VPE gene in soybean protoplasts. Collectively our results indicate that GmNAC30 cooperates with GmNAC6 to activate PCD through induction of the cell death executioner VPE. The interpretation that GmNAC6 functions as a regulator of stress-induced PCD, likely via induction of VPE, a key enzyme involved in vacuole-mediated PCD, raised the possibility that GmNAC6 would function as a regulator of leaf senescence as well. To address this issue, soybean plants, variety BR16, were transformed with GmNAC6 under the control of the 35S promoter and analysis of transgene incorporation was carried out by PCR for three generations. Three homozygous transgenic lines (T3 generation) were selected as they display similar levels of GmNAC6 expression. All three transgenic lines (GmNAC6.1, GmNAC6.2 and GmNAC6.3) were phenotypically identical to the control, untransformed line during the vegetative phase of development and flowered with similar rate and period. However, at the R3 stage of development, the leaf senescence in the transgenic lines was accelerated as compared to control leaves. In all three transgenic lines, ectopic expression of GmNAC6 accelerated leaf yellowing that was associated with a greater loss of pigments (Chlorophyll a and b, and carotenoids) and a higher accumulation of ROS, as compared to the control plants. Furthermore the early senescence phenotype displayed by the transgenic lines was associated with a higher induction of senescence marker genes in their leaves at the R3 developmental stage in comparison with the levels in the control leaves. Consistent with a role as a transcriptional regulator of VPE gene expression, the overexpression of GmNAC6 induced VPE transcript accumulation in the transgenic lines to a much higher extent than those in the control lines at the R3 developmental stage. Given that VPE is an executioner of vacuolemediated PCD, GmNAC6 induces VPE expression and GmNAC6-mediated leaf senescence was directly correlated with temporal and spatial expression of VPE, it is reasonable to propose that GmNAC6 regulates leaf senescence via induction of VPE, a kind of PCD that is mediated by the vacuolar collapse. As further evidence that GmNAC6 may regulate developmentally programmed leaf senescence, the GmNAC6-overexpressing transgenic lines displayed an enhanced sensitivity to the abiotic stresses, osmotic stress, RE stress and drought. Therefore, the results of the present investigation further support the notion that plant tolerance to abiotic stresses is genetically linked to leaf longevity.