**Acknowledgements**

*Plant Science - Structure, Anatomy and Physiology in Plants Cultured in Vivo and in Vitro*

between age- and stress-induced cell death pathways.

DCD-NRP/NAC/VPE signaling module may integrate stress-induced with natural leaf senescence and (ii) other NAC genes may be involved in integrated circuits

Regarding the first hypothesis, several lines of evidence indicate that the regulatory circuit NRPs/GmNAC81:GmNAC30/VPE integrates osmotic stress- and ER stress-induced PCD response with natural leaf senescence. First, not only *GmNAC30* and *GmNAC81* but also the other cell death pathway components, *NRP-A*, *NRP-B*, and *VPE*, are induced by leaf senescence [43, 59, 70]. Second, the activity of VPE is also induced during the onset of leaf senescence [59]. Third, transient expression of the soybean components of ER stress- and osmotic stressinduced cell death response, NRP-A, NRP-B, GmNAC81, and GmNAC30, as well as the *Arabidopsis* orthologs AtNRP1, AtNRP2, ANAC36, and γVPE, in protoplasts and *in planta* induce a cell death response bearing the hallmarks of leaf senescence and PCD. These symptoms include the induction of caspase 1-like activity and DNA fragmentation, chlorophyll loss, protein degradation, enhanced lipid peroxidation, and the induction of senescence-associated marker genes [36–38, 40, 55]. Fourth, enhanced accumulation of BiP, which negatively regulates the NRPs/GmNAC81:GmNAC30/VPE signaling module, also promotes a delay in leaf senescence in transgenic plants [59]. Finally, GmNAC81 is a positive regulator of naturally programmed leaf senescence [70]. Although leaf senescence is genetically programmed in an age-dependent manner, it can be triggered by environmental cues and is also positively and negatively regulated by various plant hormones. *GmNAC81* and *GmNAC30* are induced by the phytohormones ABA, jasmonic acid (JA) and salicylic acid (SA), which are positive regulators of senescence, and GmNAC81-overexpressing lines display high levels of ABA, mimicking the enhanced endogenous levels of this hormone during leaf senescence [70, 71]. Consistent with a role in leaf senescence, the overexpression of *GmNAC81* in soybean plants accelerates leaf senescence, a phenotype associated with extensive leaf yellowing, increased chlorophyll loss, faster photosynthetic decay, and enhanced expression and activity of the GmNAC81 direct target VPE, than untransformed, wild-type plants. Conversely, suppressing *GmNAC81* expression delays leaf senescence and decreases the expression of GmNAC81 direct target genes, including *VPE* [70]. Therefore, GmNAC81 is involved in developmentally programmed leaf senescence. Furthermore, ER stress- and osmotic stress-induced PCD is integrated with natural leaf senescence through the NRPs/NACs/VPE regulatory circuit.

Since the discovery of the ER stress- and osmotic stress-induced DCD/NRPmediated cell death response, considerable progress has been achieved toward deciphering the components and regulation of the pathway (**Figure 3**). We now know that the combination of multiple stresses synergistically activates a plant-specific PCD response that is initiated by induction of the stress-responsive transcription factor GmERD15, which, in turn, binds and activates the DCD/NRP promoter. Induction of the DCD/NRP genes *NRP-A* and *NRP-B* leads to the activation of a signal cascade that culminates with the upregulation of the transcription factors GmNAC81 and GmNAC30. The NAC transcription factors form a heterodimer to activate the expression of hydrolytic enzymes, including VPE, an executioner of vacuole-triggered programmed cell death. The stress-induced DCD/NRP-mediated cell death response is conserved in plants with similar regulatory mechanisms and represents a shared response to multiple stress signals. As a negative regulator of the stress-induced DCD/NRP-mediated cell death response, overexpression of the

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**7. Conclusion**

We thank the Brazilian government agencies, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), for financial support.
