**6. The stress-induced DCD/NRP-mediated cell death signaling positively regulates leaf senescence**

Leaf senescence is a natural process in plant development, which begins with a physiological transition between active photosynthetic leaves to degenerative and nutrient-recycling leaves. The classical age senescence-related symptom is the leaf yellowing caused by generalized chlorophyll loss. The age-induced senescence or naturally programmed leaf senescence, hereafter referred to as leaf senescence, occurs by plant aging and is precisely regulated by senescence-associated genes (SAGs) [66, 67].

Many SAGs are environmental- and stress-responsive genes, integrating a convergent regulatory cascade between natural plant development and stress-induced PCD [68]. At the molecular level, the onset of senescence is accompanied by a massive reprogramming of gene expression, probably controlled by senescenceassociated transcription factors. Among these, several NAC transcription factors have been associated with senescence regulation based on high-resolution temporal expression profiles [69].

In soybean, a transcriptomic analysis of senescing leaves reveals that 44% of the *GmNAC* genes were differentially expressed at the onset of leaf senescence. The most representative subfamilies of soybean senescence-associated *NAC* genes were the abiotic stress-induced SNAC-A (ATAF) subfamily, in which 90% of the members were differentially expressed during senescence, followed by the biotic stress-induced TERN subfamily, displaying 80% of the members differentially expressed during leaf senescence [43]. *GmNAC30* and *GmNAC81*, which belong to the SNAC-A and TERN subfamilies, respectively, are among the upregulated genes by leaf senescence [43, 59]. These results raise the hypotheses that the (i)

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

related to AtNRP-2. GmNAC81 and its paralog share sequence conservation with the *Arabidopsis* ortholog ANAC36 (At2G17040), whereas the predicted *Arabidopsis* ortholog of soybean VPE was identified as At4G32940/γVPE. Transient expression of the selected *Arabidopsis* orthologs of pathway components (*AtNRP-1*, *AtNRP-2*, *ANAC36*, and *γVPE)* induces cell death in *Nicotiana benthamiana* leaves with the appearance of hallmarks of PCD and leaf senescence, including DNA fragmentation, leaf yellowing, chlorophyll loss, and lipid peroxidation [38]. In addition, knockout lines for each one of pathway genes in *Arabidopsis* display enhanced tolerance to ER stress-mediated cell death induction. Very importantly, the stress induction of *AtNRP2*, *ANAC36*, and *γVPE* was dependent on the AtNRP1 function, confirming the upstream position of AtNRP1 in the cell death pathway. Therefore, in *Arabidopsis*, the execution of the cell death program has been proposed to occur through AtNRP1-mediated induction of the AtNRP2-ANAC36-γVPE signaling module. Nevertheless, functional information about the GmERD15 and GmNAC30 orthologs in *Arabidopsis* is lacking, and these pathway components have not been identified yet in *Arabidopsis*. Both in soybean and *Arabidopsis*, the DCD/NRPmediated cell death pathway is modulated by the ER-resident molecular chaperone BiP, which negatively regulates the gene expression and activity of these cell death-

**5. A negative regulator of the NRP/NAC081/VPE signaling module** 

Plants can negatively modulate the NRP/DCD-mediated cell death response to suit the cellular balance during the stress conditions. Moreover, this modulation improves the cellular stableness and consequently increases the plant tolerance to stress conditions in an essential process that is required for plant acclimatization and development. The molecular chaperone BiP plays a crucial role as a negative regulator of NRP/DCD-mediated cell death response. BiP belongs to the HSP70 family, which is essential to protect the cells against environmental stresses and to

The molecular chaperone BiP has a catalytic site at the amino-terminal region and a substrate-binding site at the carboxy-terminal region [60]. BiP is involved in the regulation of several processes in the endoplasmic reticulum, a critical organelle that is related to responses to abiotic and biotic stress in plants. In the ER, BiP acts as a sensor that responds to quantitative and qualitative changes in the ER by regulating the activity of ER stress transducers [61]. Furthermore, BiP coordinately regulates the cell death signaling, which connects the signals from osmotic and ER

BiP attenuates the NRP/DCD-mediated cell death signal propagation by the modulation of expression and activity of the pathway signaling components (**Figure 3**). BiP overexpression in soybean attenuates ER stress- and osmotic stress-mediated cell death, a phenotype that is linked to a delay in the induction of *GmNRP-A*, *GmNRP-B*, and *GmNAC81* under ER stress and osmotic stress [38]. Furthermore, enhanced accumulation of BiP in tobacco (*Nicotiana tabacum*) prevents the GmNRP- and GmNAC81-mediated induction of cell death-associated physiological and molecular markers, whereas silencing of endogenous BiP

In addition to alleviating ER and osmotic stress-mediated cell death, the *BiP* overexpression in plants has also been shown to increase their tolerance to water deficits [62–64]. Enhanced accumulation of BiP in soybean, tobacco, and *Arabidopsis* promotes a delay in drought-induced senescence and wilting of leaves

**70**

inducing genes [13, 40].

**confers tolerance to drought**

restore the cell homeostasis [59].

enhances the cell death response.

stress in a DCD/NRP-dependent manner [35, 36, 38].

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 between age- and stress-induced cell death pathways.

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.
