The SNAC-A (ATAF) subfamily in plants: possible downstream components of the NRP/DCDs-mediated cell death signaling

A via de sinalização de morte celular mediada por proteínas NRP/DCDs foi primeiramente descrita em soja como uma via integrativa de resposta a múltiplos estresses. A falta de ferramentas moleculares adequadas à caracterização dessa via em soja levou ao estudo de sua conservação em espécies modelo. Em Arabidopsis, os membros da via foram descritos, no entanto não foi definido o ortólogo do gene GmNAC30. Neste trabalho, foi conduzida uma análise in silico da subfamília SNAC-A (ATAF), à qual pertence GmNAC30, demonstrando ser ela amplamente distribuída no reino vegetal. Em comparação com Arabidopsis, foi verificada uma expansão dessa subfamília em soja, o que pode estar relacionado à pressão seletiva que espécies cultiváveis sofrem a favor da manutenção de genes capazes de conferir tolerância a estresses. Consistente com essa ideia, a análise da expressão de alguns membros desse subgrupo em soja demonstrou que todos eles respondem a múltiplos estresses. Os genes duplicados da subfamília SNAC-A em soja exibiram padrão e cinética de indução por polietilenoglicol (PEG) e Tunicamicina (TUN) similares; porém, os pares apresentaram diferenças sutis de indução por AS, indicando que devem possuir funções parcialmente sobrepostas, mas não absolutamente idênticas. A fim de verificar a hipótese difundida de ser ATAF1 o ortólogo de GmNAC30 foram realizados ensaios de interação de ATAF1 com ANAC036 (ortólogo de GmNAC081) pelo sistema de duplo-híbrido de leveduras e análises de expressão gênica de ATAF1 em resposta a estresses osmótico e no retículo endoplasmático (RE). Os resultados obtidos, no entanto, indicaram não ser ATAF1 o ortólogo de GmNAC30, uma vez que não interagiu com ANAC036 pelo sistema de duplo- híbrido, e ao contrário de GmNAC030, não foi induzido por tunicamicina (TUN), embora, assim como GmNAC030, ATAF1 tenha exibido atividade transcricional pelo sistema de mono-híbrido de leveduras. Uma possível interação do gene ATAF1 com a via de morte celular induzida por estresses no RE e osmótico foi avaliada por genética reversa no mutante ataf1-2 que exibiu 75% de redução na expressão de ATAF1. Tanto a expressão basal quanto a expressão induzida por PEG e TUN dos genes NRP-1 e ANAC036, componentes da via de morte celular, foi superior no mutante ataf1-2 do que em Col-0, implicando ATAF1 como possível regulador negativo da via de morte celular mediada por NRPs. Sendo ATAF2 um outro membro do subgrupo SNAC-A que está filogeneticamente muito próximo a GmNAC30, foi avaliada a sua resposta a tratamentos com PEG e tunicamicina em plântulas das linhagens Col-0 e ataf1-2. Foi observada a indução de ATAF2 em resposta a ambos os estresses, sendo que a indução é aumentada no mutante ataf1-2, consistente com o perfil de expressão gênica de integrantes da via de morte celular mediada por NRPs. Coletivamente, estes resultados substanciam o argumento de que ATAF2 possa ser ortólogo de GmNAC030. No entanto, experimentos complementares são necessários para a confirmação dessa hipótese.The NRP/DCDs- mediated cell death signaling was first described in soybean as an integrative pathway for multiple stresses. The lack of adequate molecular tools for characterization of this pathway in soybean led to the demonstration of its conservation in plant model systems. In Arabidopsis, several members of this family have been described, but the GmNAC30 ortholog remains to be identified. In this investigation, an in silico analysis of the SNAC-A (ATAF) subfamily of GmNAC30 was conducted and demonstrated that SNAC-A is largely distributed in the plant kingdom. As compared to Arabidopsis, the SNAC-A subfamily was expanded in soybean, more likely due to the selective pressure undergone by economically relevant crops towards the maintenance and duplication of stress related genes. Accordingly, an expression analysis of some soybean genes of this subfamily demonstrated that they respond to multiple stresses. The duplicated genes from the soybean SNAC-A subfamily displayed similar profile and induction kinetics by PEG and tunicamycin (TUN). Nevertheless, the pairs exhibited slightly differences in salicylic acid (SA) induction, indicating that they may display partially overlapping, but not absolutely identical functions. To confirm that ATAF1 is a GmNAC030 ortholog, yeast two-hybrid assays for ATAF1-ANAC036 (GmNAC081 otrholog) interaction and analysis of ATAF1 gene expression in response to osmotic and endoplasmic reticulum (ER) stresses were conducted. However, the results indicated that ATAF1 is not likely a GmNAC30 ortholog, as ATAF1 did not interact with ANAC036 in yeast and, opposing to the GmNAC030 expression profile, it was not induced by TUN, although like GmNAC030, ATAF1 was transcriptionally active by the yeast mono-hybrid assay. A possible interaction of ATAF1 with the ER stress- and osmotic stress-induced cell death signaling was monitored by reverse genetic in ataf1-2 mutant, which displayed a 75% reduction in the accumulation of ATAF1 transcripts. Both basal and stress-induced expressions of NRP-1 and ANAC036, components of the cell death pathway, were higher in ataf1-2 than in Col-0, implicating ATAF1 as a possible negative regulator of the NRPs- mediated cell death signaling. Because ATAF2 is another member of the SNAC-A subfamily, which is phylogenetically close related to GmNAC30, its response to PEG and TUN was monitored in Col-0 and ataf1-2 seedlings. ATAF2 was induced by both treatments and the transcript accumulation was further increased in ataf1-2, which was consistent with the expression profile of components of the NRPs-mediated cell death signaling. Collectively, these results substantiate the argument that ATAF2 may be a GmNAC030 ortholog. However, complementary experiments are necessary to confirm this hypothesis.Conselho Nacional de Desenvolvimento Científico e Tecnológic

The stress-induced soybean NAC transcription factor GmNAC81 plays a positive role in developmentally programmed leaf senescence

The onset of leaf senescence is a highly regulated developmental change that is controlled by both genetics and the environment. Senescence is triggered by massive transcriptional reprogramming, but functional information about its underlying regulatory mechanisms is limited. In the current investigation, we performed a functional analysis of the soybean (Glycine max) osmotic stress- and endoplasmic reticulum (ER) stress-induced NAC transcription factor GmNAC81 during natural leaf senescence using overexpression studies and reverse genetics. GmNAC81-overexpressing lines displayed accelerated flowering and leaf senescence but otherwise developed normally. The precocious leaf senescence of GmNAC81-overexpressing lines was associated with greater Chl loss, faster photosynthetic decay and higher expression of hydrolytic enzyme-encoding GmNAC81 target genes, including the vacuolar processing enzyme (VPE), an executioner of vacuole-triggered programmed cell death (PCD). Conversely, virus-induced gene silencing-mediated silencing of GmNAC81 delayed leaf senescence and was associated with reductions in Chl loss, lipid peroxidation and the expression of GmNAC81 direct targets. Promoter–reporter studies revealed that the expression pattern of GmNAC81 was associated with senescence in soybean leaves. Our data indicate that GmNAC81 is a positive regulator of age-dependent senescence and may integrate osmotic stress- and ER stress-induced PCD responses with natural leaf senescence through the GmNAC81/VPE regulatory circuit