The IRE1 endonuclease domain catalyzes the non-conventional cytoplasmic splicing of the mRNA encoding bZIP60/XBP1/HAC1 (in Arabidopsis, metazoans and budding yeast respectively), leading to the translation of a transcription factor (bZIP60s/XBP1s/HAC1s) that mainly upregulates the expression of ERQC and ERAD-related genes (Yoshida et al., 2001; Iwata et al., 2008; Deng et al., 2011). Among the species, the amino acid sequences of these transcription factors are not highly conserved; however, a two stem–loop structure accompanied by a consensus sequence in each loop of the IRE1-splicing mRNA substrates is remarkably conserved and associated with IRE1-mediated cleavage (Oikawa et al., 2010; Deng et al., 2011; Nagashima et al., 2011). In yeast and metazoans, the spliced substrate becomes a potent transcriptional activator, since it gains a transcriptional activation domain in the new C-terminal tail (Mori et al., 2000; Yoshida et al., 2001). It is noteworthy that in the absence of induced ER stress in budding yeasts, the unspliced HAC1 (HAC1u) mRNA is not translated, since an intron in the HAC1u mRNA blocks its translation (Chapman and Walter, 1997), while in mammalian cells the unspliced XBP1 (XBP1u) mRNA is translated. XBP1u protein along with the XBP1u mRNA is associated peripherally with the ER membrane through an amphipathic region, where it facilitates the targeting of XBP1u mRNA to IRE1 presumably to increase the cytoplasmic splicing efficiency providing a rapid response to ER stress (Yanagitani et al., 2009). Upon prolonged ER stress, XBP1u forms a complex with XBP1s, leading it to be exported from the nucleus to the cytoplasm and rapidly degraded by the proteasome, presumably shutting down the transcription of the XBP1-target genes (Yoshida et al., 2006). Intriguingly, the IRE1-spliced XBP1s mRNA loses the ER membrane-anchor domain and it is released in the cytosol, indicating a different translational place for XBP1u and XBP1s mRNAs that presumably prevents the excess of degradation of the XBP1s by XBP1u protein during ER stress (Yanagitani et al., 2009). In plants, experiments based on the expression of in-frames fluorescent protein fusion with unspliced bZIP60u, have shown an association of bZIP60u with the ER through its putative C-terminal transmembrane domain (Deng et al., 2011). However, the biological roles of unspliced bZIP60 (bZIP60u) on the ER membrane are currently unknown. Unlike yeast and metazoans, spliced bZIP60 (bZIP60s) does not gain a transcriptional activation activity, since the transcriptional activation domain is located in the N-terminal tail along with a nuclear localization signal. The IRE1-splicing produces instead a new protein deprived of the transmembrane domain (Deng et al., 2011; Nagashima et al., 2011) and characterized by an improved fine regulatory modulation of its transcription activity as recently reported in rice (Lu et al., 2012). However, how this tuning is achieved is still not clear.

Under ER stress, the transcription factor domain of bZIP28/ATF6 increases the expression of genes involved in protein folding and of other ER-stress related transcription factors such as bZIP60/XBP-1, providing a positive feedback for augmenting the UPR (Yoshida et al., 2001; Liu and Howell, 2010). Moreover, in metazoans, ATF6 enhances the expression of genes involved in ERAD, lipid biosynthesis and ER expansion, which are required to improve the capacity of the secretory pathway (Bommiasamy et al., 2009).

The UPR genes are induced through the recognition of cis-acting elements on their promoter regions by the UPR transcription factors. In budding yeast, HAC1s binds and activates the UPR element-I (UPRE-I, consensus region CAGNGTG; Mori et al., 1996) and UPRE-II (TACGTG; Fordyce et al., 2012). In mammalians, XBP1 efficiently binds the UPRE-I (GA-TGACGT-G[G/A]; Wang et al., 2000; Yamamoto et al., 2004) and the ER stress responsive element-II (ERSE-II, ATTGG–N–CCACG; Kokame et al., 2001; Yamamoto et al., 2004), while ATF6 binds the ERSE-I (ERSE-I, CCAAT–N9–CCACG; Roy and Lee, 1999) and the ERSE-II (ATTGG–N–CCACG; Kokame et al., 2001) only in the presence of the nuclear transcription factor NF-Y (Yamamoto et al., 2004). Interestingly, the mammalian ERSE-I and UPRE-I elements are conserved in plants (Oh et al., 2003; Iwata et al., 2008; Liu and Howell, 2010), where other cis-elements have also been found, such as the pUPRE-II (GATGACGCGTAC; Hayashi et al., 2013) and the pUPRE-III (TCATCG; Sun et al., 2013). Similar to ATF6 in mammals, bZIP28 binds the ERSE-I element with assistance from the transcription factors NF-Y (Liu and Howell, 2010), while bZIP60s directly binds the pUPRE-III (Sun et al., 2013) and it regulates also promoters containing the cis-element ERSE-I and UPRE-I (Iwata and Koizumi, 2005). The multiple cis-elements involved in the ER stress response and their differential binding affinities for the UPR transcription factors presumably fine temporal modulate the UPR signaling and the ER stress response. Moreover, plants have evolved an additional layer of UPR regulation. The plant-specific nuclear transcription factor NAC103 is indeed induced by ER stress presumably through bZIP60s, and the encoded protein NAC103 in turn regulates the UPR downstream genes (Sun et al., 2013). However, the precise mechanisms of the gene regulation networks are largely unknown.

In metazoans, although PERK induces general protein translation attenuation, it also favors selective protein translation. Specifically, the PERK-phosphorylated eIF2α activates the translation of mRNAs with uORF (upstream open reading frame) within their 5’ untranslated region (UTR), like the activating transcription factor 4 (ATF4; Harding et al., 2000). ATF4 protects cells against oxidative stress and ensures the supply of reducing substances (i.e., glutathione) by enhancing the metabolism of their precursors (i.e., sulfur-containing amino acids; Harding et al., 2003). Also PERK phosphorylates the transcription factor Nrf2 (nuclear factor erythroid2-releated factor 2), which translocates to the nucleus, heterodimerizes with the small Maf proteins and activates the transcription of genes involved in the redox homeostasis by binding to the antioxidant response elements on the target gene promoters (Cullinan et al., 2003).

Autophagy is an evolutionarily conserved process of bulk degradation, whereby large portions of cytoplasmic and organellar components are engulfed by double membrane vesicles (termed as “autophagosome”) and delivered to the lysosome in metazoan, or to the vacuole in yeast and plants, for degradation and recycling of macromolecules (Liu and Bassham, 2012). During ER stress, autophagy is activated in yeast, mammals and plants, and it is involved in clearing unfolded protein from the ER by supplementing the ERAD pathway and, in turn, alleviating stress (Ding et al., 2007; Liu et al., 2012). In yeast, IRE1 regulates autophagy through the splicing of HAC1, which induces the production of Atg8p, an ubiquitin-like protein required for autophagosome formation (Yorimitsu et al., 2006). Unlike yeasts, in metazoans, autophagy is triggered by the kinase activity of IRE1 and PERK. IRE1, indeed, recruits the adaptor protein TNFR-associated factor2 (TRAF2) on the ER membranes, thus triggering the activation of the apoptosis signal regulating kinase1 (ASK1), which in turn regulates the activation of the c-Jun-N-terminal kinase (JNK), whose pathway induces the autophagosome formation (Pattingre et al., 2009). Moreover, PERK promotes autophagy through the phosphorylation of eIF2α, which induces the expression of ATF4, recently considered a key signal for autophagy activation (Matsumoto et al., 2013). ATF4 in turn activates the expression of autophagy-related (ATG) genes and of C/EBP-homologous protein (CHOP) transcription factor. CHOP and ATF4 together promote and modulate the induction of genes implicated in the formation, elongation and function of the autophagosome (B’chir et al., 2013). Similar to metazoans, in plants, it has been recently found that IRE1, specifically the IRE1b isoform, activates autophagy upon ER stress response independently from the IRE1-mediated bZIP60 mRNA splicing (Liu et al., 2012). However, the mechanistic features of plant autophagy under ER stress are mainly unknown, in terms of the upstream regulator/s of IRE1b as well as its downstream targets.

Upon excessive and prolonged ER stress, in metazoans, all the three UPR signaling pathways lead to cell death through apoptosis via the intrinsic mitochondrial pathway. Indeed, UPR regulates the activity of the pro-apoptotic members of the Bcl-2 family via transcriptional and post-transcriptional mechanisms, leading the BAX/BAK-mediated pore formation in the mitochondrial outer membrane, release of cytochrome c from the mitochondria, and subsequent activation of caspases, which are critical regulators of apoptosis via their role in propagating apoptotic signaling cascades (Rodriguez et al., 2011). However, it is not yet clear whether other types of cell death occur to eliminate terminally compromised cells under irreversible ER stress. Intriguingly in metazoans, under prolonged ER stress autophagy may switch from a pro-survival process to apoptosis. Several regulators of autophagy machinery are indeed involved in the apoptosis, such as JNK and CHOP (Rodriguez et al., 2011).

In mammals RIDD activity mediated by IRE1α enhances (1) ER stress intensity through the decay of mRNA encoding UPR target genes during the transition phase between the adaptive and apoptotic response (Han et al., 2009), and (2) expression of pro-apoptotic proteases, like the Caspase 2 (Upton et al., 2008), through the decay of selected antiapoptotic pre-miRNAs during the apoptotic response (Upton et al., 2012). In plants, the biological significance of RIDD activity in cell fate determination is still unknown.

In yeast, ER stress can induce PCD with apoptotic phenotypes (Hauptmann et al., 2006), as well as in a non-apoptotic process, where vacuole fragmentation and leaking of vacuolar materials are cell death features (Kim et al., 2012). Plant cell death executioners in the UPR are instead largely unknown. Unlike in metazoans, the plant IRE1 does not seem to have a pro-apoptotic role, given that the Arabidopsis ire1 double mutants display compromised ER stress tolerance, instead of a greater survival rate (Nagashima et al., 2011; Chen and Brandizzi, 2012). Although neither homologs of Bcl-2 family proteins nor components of the PERK-CHOP pathways have been identified in plants yet, some regulators of ER-PCD seem to be conserved across kingdoms. These include the Bax inhibitor1 (BI-1)-like protein, an ER transmembrane protein that protects cells against ER-stress induced-cell death (Chae et al., 2003; Watanabe and Lam, 2008), and the chaperone BiP, that has a protective function against ER stress induced-cell death in both mammalian and plant cells (Kishi et al., 2010; Reis et al., 2011).