The detection of ER stress and mediation of UPR signaling occurs via sensors located at the ER membrane. In mammals there are three transmembrane embedded sensors: IRE1, ATF6, and PERK (Walter and Ron, 2011). Each of these sensors initiate signaling pathways that can restore ER homeostasis or under conditions of chronic stress and increasing damage, activate alternative routes leading to cell death. The type 1 transmembrane protein, kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1 a and b), is a dual functioning protein. IRE1 has ribonuclease activity and is responsible for splicing X-box binding protein-1 (XBP) mRNA, generating the transcription factor XBP1 (Yoshida et al., 2001). XBP1 translocates to the nucleus where it activates the expression of cytoprotective genes, including members of the ER QC pathway. IRE1 is also capable of activating the apoptotic-signaling kinase 1 (ASK1) and Jun-N-terminal kinase (JNK) that promote apoptosis (Koizumi et al., 2001; Nagashima et al., 2011; Humbert et al., 2012).

In plants, IRE1a and IRE1b localize to the perinuclear ER and the signaling pathways that emanate from these sensors include a completely separate set of intermediary factors from those identified in mammals (Koizumi et al., 2001). In fact recent data suggest a distinct and specialized role for IRE1b in ER stressed but not nutrient stressed induced autophagy and further suggests that an alternative pathway is in play during autophagy responses during nutrient deprivation in plants (Liu et al., 2012).

IRE1 activates mRNA splicing of the transcription factor, bZIP60 which recognizes promoters with a recently identified ER stress responsive cis-element UPRE-III in the NAC103 promoter. The NAC103 transcription factor activates several UPR related foldases including CRT1, CNX, PDI-5 (Sun et al., 2013a). Both IRE1 proteins have been reported to be expressed throughout the plant (Noh et al., 2002). While autophagy markers including the formation of autophagosomes were observed following nutrient starvation, wild type, IRE1a and IRE1b mutants resulted in similar phenotypes; (Liu et al., 2012). Treatment of Arabidopsis IRE1a and IRE1b knock out mutants with the well-known ER stress inducers, tunicamycin (TM) and dithiothreitol (DTT), resulted in contrasting phenotypes. Application of DTT and TM induced the formation of autophagosomes in IRE1a mutants and wild type plants. In contrast, autophagy was not induced in IRE1b mutants under the same conditions (Liu et al., 2012). ER stress-induced autophagy in plants occurs only via the IRE1b-mediated pathway (Koizumi et al., 2001; Liu et al., 2012).

In mammals, ATF6 is a type II transmembrane basic leucine-zipper (bZIP) domain-containing activating transcription factor (Yoshida et al., 2001). Upon ER stress ATF6 moves through the Golgi compartment and is cleaved by cellular proteases for maturation. The plant equivalents to the mammalian ATF6 pathway, are two key ER-localized, membrane tethered transcription factors, bZIP17 and bZIP28 (Liu et al., 2007a,b). Similar to ATF6, bZIP17, and bZIP28 are activated following detection of accumulating unfolded proteins in the ER and then translocate to the Golgi apparatus where they are cleaved by Golgi-localized proteases for maturation. In the nucleus bZIP17 and bZIP28 activate expression of cytoprotective chaperones and foldases, facilitating the formation of correct macromolecular structures and protein folding, respectively (Liu et al., 2007a, 2008; Liu and Howell, 2010a; Srivastava et al., 2012).

The third ER resident sensor identified in mammals is the type I transmembrane protein kinase RNA-like ER kinase (PERK). Upon detection of unfolded proteins and ER stress, PERK phosporylates and inactivates the translation initiation factor eIF2a to shut down protein synthesis (Harding et al., 2000). PERK also activates the transcription factor CHOP which induces gene expression that leads to apoptosis. Translational regulation may not be entirely conserved since no obvious PERK homologs have as yet been identified in plants (Urade, 2009; Eichmann and Schafer, 2012).

Calcium stores in the ER are critical for the functioning of certain ER resident foldases. Calcium imbalance in the ER can disrupt the functioning of this protein folding pathway causing malformed proteins to accumulate. Release of Ca⁺² from the ER can interfere with protein folding and leads to increased Ca²⁺ levels in the mitochondria and can promote oxidative stress and ultimately cell death (Berridge, 2002). Given the critical role of calcium in protein folding, oxidative stress and programmed cell death, mammalian systems utilize several different regulators including Bcl-2 and family members (Bax, and Bak), which are cytoprotective calcium sensors that modulate the release of ER Ca⁺² stores and regulate cell death. Given the importantance of calcium in protein folding in the plant ER, it is in some ways surprising that genome sequence comparisons between plants and mammals indicate that these Bcl-2 family members are not present in plants, at least at the level of primary DNA sequence. Remarkably, transgenically expressed cytoprotective Bcl-2 and others (e.g., nematode Ced-9, chicken Bcl-xl; insect IAP; viral p35) function in plants in a similar manner to what occurs in animals including inhibiting PCD in response to pathogen invasion and abiotic/environmental stresses, in accordance with transkingdom pathway conservation (Dickman et al., 2001; Lincoln et al., 2002; Williams and Dickman, 2008; Dickman and Fluhr, 2013). Thus mammalian anti-apopotic machinery functions in plants and points to a conserved apoptotic-like PCD mechanism for death in plants. A key issue that is not entirely reconciled, is that there is little evidence for conservation at the DNA/gene level. We have suggested that structural homologies independent of sequence account for functional conservation, and indeed we have shown this to be the case when we uncovered the Arabidopsis BAG gene family (Doukhanina et al., 2006; Kabbage and Dickman, 2008; Williams et al., 2010). Initial blast analyses of Arabidopsis nucleotide and amino acid sequences failed to identify homologs of the mammalian BAG family. Therefore more sensitive methods were used based on higher level conservation including Hidden Markov Modelling (HMM) and profile-profile alignment algorithims to identify seven BAG members in Arabidopsis (Doukhanina et al., 2006). Unlike animals, plant BAGs display unique sub-cellular localisation; the three predicted calmodulin-binding BAGs are localized in the ER, mitochondria and vacuole, all of which are known Ca²⁺ reservoirs and mediators of cell death pathways (Kabbage and Dickman, 2008; Williams et al., 2010; Dickman and Fluhr, 2013).

Programmed cell death is essential for normal growth and development (Kerr et al., 1972), however, disease occurs when PCD is inappropriately regulated (too much, too little). In mammals this can lead to a range of pathologies including cancer to Parkinson’s disease. The UPR, autophagy and Ca²⁺ signaling between the ER and mitochondria are core components of the PCD machinery. These core components have recently been identified in plants thus linking the UPR to PCD and innate immunity (Ma and Berkowitz, 2007; Martinon and Glimcher, 2011; Stael et al., 2012).

In plants there are four key ER resident factors that link Ca⁺² stores to UPR and cell death regulation. These are calreticulin, calmodulin, ER-type IIA Ca²⁺ pump, and type IIB Ca⁺²-ATPase. Calreticulin is a Ca⁺² binding protein that chaperones maturation of glycoproteins in the ER (Jia et al., 2009). Calmodulin is a Ca⁺² sensor that recognizes changes in Ca⁺² levels in the ER and activates immune related PCD (Ranty et al., 2006; Ma et al., 2008; Du et al., 2009). Application of the ER-type IIA Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) leads to increased cytosolic Ca²⁺, cross-talk with mitochondria and PCD in soybean (Zuppini et al., 2004). The formation of the so-called mitochondrial permeability transition pore (PTP), an established marker of PCD, allows the selective leakage of several stress signals and cell death regulators sensitizing ER channels to further release of Ca²⁺ in a positive feedback loop (Berridge, 2002). In addition, the ER localized type IIB Ca⁺²-ATPase regulates N-mediated PCD in response to infection with Tobacco mosaic virus (TMV; Zhu et al., 2010). Perturbations in ER function, however, promote the release of Ca²⁺ from the ER into the mitochondria resulting in diminished protein folding capacity, ER stress, the formation of PTP and subsequent apoptosis (Berridge, 2002).

In addition to disruption of ER function and the accumulation of unfolded proteins, studies have shown that depletion/release of Ca²⁺ from the ER is promoted by pro-death members of the Bcl-2 family (Scorrano et al., 2003; Hammadi et al., 2013). Additionally, cytoprotective anti-PCD proteins such as Bcl-2 have been shown to prevent PCD by suppressing the release ER Ca²⁺ (Lam et al., 1994). One protein that may facilitate ER Ca²⁺ homeostasis by acting as a Ca²⁺ permeable channel is Bax inhibitor and is discussed below.

A plant-specific branch of the ER stress pathway was recently noted in soybean; N-rich protein (NRP)-mediated cell death. NRPs are asparagine rich proteins that represent the hallmarks of an integrative pathway linking ER and osmotic stress signaling (Reis and Fontes, 2012). The combination of ER-stress and osmotic stress activates the transcription factor GmERD15 which in turn binds to and triggers expression of N-rich protein genes including NRP-A and NRP-B (Reis and Fontes, 2012). Overexpression of these NRPs in soybean induces the cell death effector GmNAC81, activating a caspase-3-like activity promoting DNA fragmentation; features associated with PCD. The ER chaperone BiP limits spread of this death signal (Reis et al., 2011). In a yeast two-hybrid screen for GmNAC81 interactors, GmNAC30 was identified. Interestingly, a functionally relevant binding site for GmNAC81/ GmNAC30 was found in the promoter of vacuolar processing enzyme (VPE; Mendes et al., 2013). VPE is a plant protease the activity of which, is important for viral and bacterial-induced HR. VPEs have been reported on several occasions to induce PCD in plants following pathogen challenge. Although structurally unrelated to caspases, VPE has caspase-1 (an inflammatory caspase) activity (Hatsugai et al., 2004; Hara-Nishimura et al., 2005; Kuroyanagi et al., 2005). This system links ER stress, the UPR, and cell death regulation. Transgenic tobacco plants overexpressing BIP show enhanced water stress tolerance (Alvim et al., 2001; Valente et al., 2009), thus there is a potential biotechnological application by generating crop plants (e.g., soybean) overexpressing BiP that would mitigate ER stress and lead to enhanced water stress tolerance. Interestingly, NRP signaling is believed independent of the known ER resident stress sensor mechanisms; thus it will be of particular interest to identify key components and piece together these PCD pathway(s).

Members of the Bcl-2-associated athanogene (BAG) family are a conserved group of eukaryotic co-chaperones that assist in protein folding and diverse cellular processes including apoptosis and stress signaling. The Arabidopsis BAG family contains seven members and unlike its mammalian counterparts which are primarily cytosolic, different family members localize to different sub-cellular organelles (Doukhanina et al., 2006; Kabbage and Dickman, 2008; Williams et al., 2010). BAG proteins regulate Bcl-2, interact with Hsp70/Hsc70, and associate with the ubiquitin/proteasome systems in animals. BAG proteins function in processes that promote cell survival in both plants and animals (Kabbage and Dickman, 2008).

In the ER, AtBAG7 plays a key role in UPR pathways, particularly in response to heat and cold stress (Williams et al., 2010). AtBAG7 directly binds to the ER-resident sensor and chaperone AtBiP2 (Williams et al., 2010) and likely provides essential co-chaperoning activity needed to prevent the build-up of unfolded proteins and limit cell death as a result. Interestingly, AtBAG7 mutants display hypersensitivity to the ER-stressor and autophagy inducer TM, suggesting that AtBAG7 plays a role in the regulation of autophagy pathways (Williams et al., 2010). Moreover such mutants were also sensitive to the ER stress stimuli heat and cold.

Subsequent staining of TM treated wild type and AtBAG7 mutant plants with the autophagosome detecting dye monodansylcadaverine (MDC) however, showed autophagosomes are formed in the presence or absence of AtBag7. Thus, AtBag7 does not appear to be necessary for autophagy. Our preliminary data indicates that AtBAG7 translocates from the ER to the nucleus in response to heat stress. Yeast two hybrid and BIFC studies show that following nuclear translocation, AtBAG7 binds to the transcription factor BTF3, a member of the NAC family of stress responsive transcription factors. It is reasonable to hypothesize that ER-localized AtBAG7 activates stress response pathways via translocation to the nucleus and transcriptional activation of genes associated with abiotic stress protection. In this respect AtBAG7 may not only play a role as an important ER co-chaperone but may be an important mediator of ER – nucleus signaling during ER stress responses. Studies are in progress to determine the contributions of AtBAG7 to the UPR and PCD.

The ER luminal binding protein (BiP/Grp78) is a member of the heat shock protein 70 (HSP70) family (Haas, 1994). It is the most abundant ER-chaperone, and a key player of the ERQC machinery. BiP binds to and suppresses the activity of the mammalian ER stress sensors, PERK, IRE1, and ATF6 and the plant ER stress sensors IRE1, bZIP17, and bZIP28 (Srivastava et al., 2013). Upon detection of unfolded proteins, however, BiP is released and binds to unfolded proteins thus leaving IRE1 free to oligomerise and splice bZIP60 mRNA, the result of which induces transcription of UPR associated genes.

Three BiP isoforms have been identified in Arabidopsis (Lin et al., 2001). Expression of BiP is induced by environmental stressors such as heat, salinity and pathogen invasion (Sung et al., 2001). Expression profiling has shown that BiP 1 and 2 are constitutively expressed while activation of BiP3 is triggered by bZIP60 as part of the UPR (Iwata et al., 2008; Deng et al., 2011). BiP3 expression is also induced in the absence of bZIP60, suggesting additional factors mediate transcription of BiP3 in the IRE/bZIP60 pathway. As mentioned BiP mediated inhibition of UPR is a factor in drought in transgenic tobacco (Alvim et al., 2001). BiP is also a negative regulator of ER stress related cell death during pathogen invasion (Jelitto-Van Dooren et al., 1999). Surface exposed leucine rich receptors are responsible for recognition of the bacterial pathogen-associated molecular patterns (PAMPs) and are critical in plant defense. Complex formation between BiP, and other ER resident proteins such as stromal-derived factor-2 (SDF-2) and calreticulin is essential for the glycosylation, activity and accumulation of the receptor kinases EFR and Cf-9, respectively. Similarly, loss of BiP complex formation causes ER retention and reduced levels of EFR and Cf-9 (Nekrasov et al., 2009; Liebrand et al., 2012). Taken together, in addition to reducing unfolded protein levels, BiP stabilizes immune receptors to facilitate host defense.

Initially identified as a suppressor of BAX-induced (BI) cell death in yeast and mammalian cells, Bax-inhibitor proteins are highly conserved ER localized, multi-transmembrane proteins (Ishikawa et al., 2011). BI-1 genes are one of the few examples of a gene identified and characterized in both animals and plants that specifically regulate PCD. There is increasing evidence that BI-1 plays roles in both PCD and autophagy to regulate the switching of ER-mediated cell death pathways from autophagy to PCD (Castillo et al., 2011; Carvalho et al., 2013).

Over-expression of BI-1 reduces PCD as well as the cytosolic Ca²⁺ concentration, potentially by functioning as a pH sensitive regulator of ER calcium channel activity. Evidence for these functions is supported by topology studies which indicate that the C-terminus of BI-1 forms a Ca²⁺ pore that could act as the source for its Ca²⁺ -leaking properties (Bultynck et al., 2012). BI-1 also plays a role in autophagy responses via interaction with the ER stress sensor, IRE1; via interaction with BI-1, IRE-1 is activated leading to induction of UPR gene expression (Lisbona et al., 2009).

As in mammals Arabidopsis/plant Bax Inhibitor-1 (AtBI-1) also suppresses Bax when expressed in tobacco, as well as several abiotic and biotic stress-induced PCD (Watanabe and Lam, 2006, 2008). AtBI-1 mutants also display increased sensitivity to ER stress factors such as heat and TM. Conversely, Arabidopsis plants over expressing AtBI-1 are more tolerant to TM, heat and cold stress (Watanabe and Lam, 2008). Studies have indicated that BI-1 functions downstream of ROS generation; over-expression of BI-1 in Arabidopsis did not suppress BAX-induced ROS production but still prevented PCD (Kawai-Yamada et al., 2001, 2004). AtBI-1 prevents Bax-induced cell death at least in part, by maintaining ER Ca²⁺ homeostasis (Ihara-Ohori et al., 2007). AtBI-1 knockouts display accelerated methyl jasmonate-induced senescence pathways further linking the ER to autophagy (Yue et al., 2012).

A role for BI-1 in the regulation of plant autophagy responses is also suggested by starvation experiments. Carbon starvation is a known inducer of autophagy in plants and animals; down-regulation of the tobacco BI-1 homolog augments cell death upon starvation (Bolduc and Brisson, 2002). Importantly, Arabidopsis BI-1 knockouts did not display any developmental abnormalities suggesting a specific role for BI-1 during these stress conditions.