Navegando por Autor "Carvalho, Humberto H."
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Item Functional and regulatory conservation of the soybean ER stress-induced DCD/NRP- mediated cell death signaling in plants(BioMed Plant Biology, 2016-07-12) Reis, Pedro A. B.; Carpinetti, Paola A.; Freitas, Paula P.J.; Santos, Eulálio G.D.; Camargos, Luiz F.; Oliveira, Igor H.T.; Silva, José Cleydson F.; Carvalho, Humberto H.; Dal-Bianco, Maximiller; Soares-Ramos, Juliana R.L.; Fontes, Elizabeth P. B.The developmental and cell death domain (DCD)-containing asparagine-rich proteins (NRPs) were first identified in soybean (Glycine max) as transducers of a cell death signal derived from prolonged endoplasmic reticulum (ER) stress, osmotic stress, drought or developmentally-programmed leaf senescence via the GmNAC81/GmNAC30/GmVPE signaling module. In spite of the relevance of the DCD/NRP-mediated signaling as a versatile adaptive response to multiple stresses, mechanistic knowledge of the pathway is lacking and the extent to which this pathway may operate in the plant kingdom has not been investigated. Here, we demonstrated that the DCD/NRP-mediated signaling also propagates a stress-induced cell death signal in other plant species with features of a programmed cell death (PCD) response. In silico analysis revealed that several plant genomes harbor conserved sequences of the pathway components, which share functional analogy with their soybean counterparts. We showed that GmNRPs, GmNAC81and VPE orthologs from Arabidopsis, designated as AtNRP-1, AtNRP-2, ANAC036 and gVPE, respectively, induced cell death when transiently expressed in N. benthamiana leaves. In addition, loss of AtNRP1 and AtNRP2 function attenuated ER stress-induced cell death in Arabidopsis, which was in marked contrast with the enhanced cell death phenotype displayed by overexpressing lines as compared to Col-0. Furthermore, atnrp-1 knockout mutants displayed enhanced sensitivity to PEG-induced osmotic stress, a phenotype that could be complemented with ectopic expression of either GmNRP-A or GmNRP-B. In addition, AtNRPs, ANAC036 and gVPE were induced by osmotic and ER stress to an extent that was modulated by the ER-resident molecular chaperone binding protein (BiP) similarly as in soybean. Finally, as putative downstream components of the NRP-mediated cell death signaling, the stress induction of AtNRP2, ANAC036 and gVPE was dependent on the AtNRP1 function. BiP overexpression also conferred tolerance to water stress in Arabidopsis, most likely due to modulation of the drought-induced NRP-mediated cell death response. Our results indicated that the NRP-mediated cell death signaling operates in the plant kingdom with conserved regulatory mechanisms and hence may be target for engineering stress tolerance and adaptation in crops.Item GmNAC30 and GmNAC81 integrate the endoplasmic reticulum stress- and osmotic stress-induced cell death responses through a vacuolar processing enzyme(PNAS, 2013-09-30) Mendes, Giselle C.; Reis, Pedro A. B.; Calil, Iara P.; Carvalho, Humberto H.; Aragão, Francisco J. L.; Fontes, Elizabeth P. B.Prolonged endoplasmic reticulum and osmotic stress synergistically activate the stress-induced N-rich protein-mediated signaling that transduces a cell death signal by inducing GmNAC81 (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 GmNAC81. We discovered another member of the NAC (NAM-ATAF1,2-CUC2) family, GmNAC30, which binds to GmNAC81 in the nucleus of plant cells to coordinately regulate common target promoters that harbor the core cis-regulatory element TGTG[TGC]. We found that GmNAC81 and GmNAC30 can function either as transcriptional repressors or activators and cooperate to enhance the transcriptional regulation of common target promoters, suggesting that heterodimerization may be required for the full regulation of gene expression. Accordingly, GmNAC81 and GmNAC30 display overlapping expression profiles in response to multiple environmental and developmental stimuli. Consistent with a role in PCD, GmNAC81 and GmNAC30 bind in vivo to and transactivate hydrolytic enzyme promoters in soybean protoplasts. A GmNAC81/GmNAC30 binding site is located in the promoter of the caspase-1–like vacuolar processing enzyme (VPE) gene, which is involved in PCD in plants. We demonstrated that the expression of GmNAC81 and GmNAC30 fully transactivates the VPE gene in soybean protoplasts and that this transactivation was associated with an increase in caspase-1–like activity. Collectively, our results indicate that the stress-induced GmNAC30 cooperates with GmNAC81 to activate PCD through the induction of the cell death executioner VPE.Item The molecular chaperone binding protein BiP prevents leaf dehydration-induced cellular homeostasis disruption(Plos One, 2014-01-29) Carvalho, Humberto H.; Brustolini, Otávio J. B.; Pimenta, Maiana R.; Mendes, Giselle C.; Gouveia, Bianca C.; Silva, Priscila A.; Silva, José Cleydson F.; Mota, Clenilso S.; Soares-Ramos, Juliana R. L.; Fontes, Elizabeth P. B.BiP overexpression improves leaf water relations during droughts and delays drought-induced leaf senescence. However, whether BiP controls cellular homeostasis under drought conditions or simply delays dehydration-induced leaf senescence as the primary cause for water stress tolerance remains to be determined. To address this issue, we examined the drought-induced transcriptomes of BiP-overexpressing lines and wild-type (WT) lines under similar leaf water potential (ψw) values. In the WT leaves, a ψw reduction of −1.0 resulted in 1339 up-regulated and 2710 down-regulated genes; in the BiP-overexpressing line 35S::BiP-4, only 334 and 420 genes were induced and repressed, respectively, at a similar leaf ψw = −1.0 MPa. This level of leaf dehydration was low enough to induce a repertory of typical drought-responsive genes in WT leaves but not in 35S::BiP-4 dehydrated leaves. The responders included hormone-related genes, functional and regulatory genes involved in drought protection and senescence-associated genes. The number of differentially expressed genes in the 35S::BiP-4 line approached the wild type number at a leaf ψw = −1.6 MPa. However, N-rich protein (NRP)- mediated cell death signaling genes and unfolded protein response (UPR) genes were induced to a much lower extent in the 35S::BiP-4 line than in the WT even at ψw = −1.6 MPa. The heatmaps for UPR, ERAD (ER-associated degradation protein system), drought-responsive and cell death-associated genes revealed that the leaf transcriptome of 35S::BiP-4 at ψw = −1.0 MPa clustered together with the transcriptome of well-watered leaves and they diverged considerably from the drought-induced transcriptome of the WT (ψw = −1.0, −1.7 and −2.0 MPa) and 35S::BiP-4 leaves at ψw = −1.6 MPa. Taken together, our data revealed that BiP-overexpressing lines requires a much higher level of stress (ψw = −1.6 MPa) to respond to drought than that of WT (ψw = −1.0). Therefore, BiP overexpression maintains cellular homeostasis under water stress conditions and thus ameliorates endogenous osmotic stress.