Eukaryotes have developed multiple UPR pathways that communicate with the nucleus and vary by evolutionary lineage [6]. In vascular plants, two eukaryote-wide pathways have been identified with some plant-specific features. Each UPR pathway consists of unique components, including stress sensors, activators, and transcription factors. A dedicated subclade of basic leucine zipper (bZIP)-type transcription factors is involved in the UPR, of which bZIP17, bZIP28, and bZIP60 have been identified in Arabidopsis. These bZIPs are localized to the ER membrane, where two distinct machineries activate them for translocation into the nucleus (Figure 1).

Arabidopsis bZIP17 and bZIP28 are counterparts of mammalian activating transcription factor 6 (ATF6) and mediate a UPR pathway via post-translational proteolysis [5]. In each of these transcription factors, the N-terminus containing the bZIP domain faces the cytosol and is connected by a transmembrane domain to the C-terminal tail, which is exposed to the ER lumen [6,7]. In response to increasing ER stress levels, these two bZIPs are secreted into the Golgi apparatus, where proteases SITE-1 PROTEASE (S1P) and S2P cleave the proteins between the bZIP and transmembrane domains. The released N-terminus possessing the bZIP domain is freely translocated into the nucleus and activates downstream genes involved in the UPR [6,7] (Figure 1b).

In a paralogous manner to that observed in human ATF6, the lumen-facing region of Arabidopsis bZIP28 functions as a stress sensor [8]. During ER homeostasis, ER-resident chaperone BINDING PROTEIN 3 (BiP3) binds to the lumen-facing region of bZIP28, preventing its activation. When ER stress increases, BiP3 preferentially binds to unfolded proteins, allowing bZIP28 to be secreted into the Golgi apparatus, where it is activated (Figure 1b). Further details have not been fully elucidated, but recent studies identified a key ER-resident chaperone and a novel chemical affecting ATF6 trafficking in mammals [9,10]. This could provide insight into the underlying mechanism in plants.

The other UPR pathway is mediated by Arabidopsis bZIP60, which is activated by alternative splicing [11,12,13]. bZIP60 counterparts exist in most eukaryotes, from budding yeast to mammals, and are known as X-box binding protein 1 (XBP1) [6]. In the absence of activation by ER stress, the major isoform of bZIP60 mRNA encodes a protein with a transmembrane domain that anchors it to the ER membrane, thus compromising its transcription factor activity. When ER stress is elevated, stress sensor and activator INOSITOL-REQUIRING ENZYME 1 (IRE1) in the ER lumen specifically splices the bZIP60 mRNA to remove its transmembrane domain, allowing for bZIP60 to migrate into the nucleus to activate the UPR (Figure 1b). Two major IRE1 homologs (IRE1a and IRE1b) were extensively studied in Arabidopsis [13,14,15,16]. Both IRE1s are transmembrane proteins that localize to the ER membrane, where their ER lumen-facing domain functions as a stress sensor, and their cytosol-facing domain splices or degrades target mRNAs. These two homologs are functionally redundant during bZIP60 activation in response to ER stress. A third IRE1 homolog in Arabidopsis was recently characterized. Unlike IRE1a and IRE1b, IRE1c lacks the lumen-facing domain that functions as the ER stress sensor; however, biochemical and genetic experiments revealed that IRE1c couples with IRE1b to play critical roles in gametogenesis [17,18]. Arabidopsis lyrata, a close relative of Arabidopsis thaliana, contains a fourth IRE1c-like homolog whose role in the UPR is unclear [17]. By contrast, only a single IRE1 copy has been identified in rice (Oryza sativa subsp. japonica), and the knockout of this gene is lethal [19].

In addition to bZIPs, three NAC (NAM, ATAC, and CUC)-type transcription factors (NAC062, NAC089, and NAC103) play regulatory roles in the plant UPR [20,21,22]. The expression of these genes is induced by ER stress, and their expression is diminished in bzip60 single mutants [20,22,23], suggesting that these NACs may function as subordinates of the IRE1-bZIP60 pathway. However, the translocation mechanism of these NACs from the ER to the nucleus remains uncertain; novel plant-specific pathways are expected to be discovered upon further investigation.

bZIP transcription factors form dimers that bind to their target DNA sequences [24]. These transcription factors can form homo- and heterodimers even across subclade boundaries. Different dimers are thought to have different target preferences that could result in diverse downstream gene regulatory pathways. The three UPR bZIPs (bZIP17, bZIP28, and bZIP60) also form both homo- and heterodimers in every possible combination [23,25]; their different modes of action have been analyzed using combinations of their knockout mutants.

In Arabidopsis, bZIP28 and bZIP60 are commonly accepted as the foremost combination governing canonical UPR downstream regulation [7,26]. The expression of most known ERQC component genes was impaired by the double mutation of bZIP28 and bZIP60 (bzip2860). A significant number of genes, including BiP3, CNX1 (CALNEXIN 1), and ERO1 (ER OXIDOREDUCTIN 1), also fully lost their stress-inducible expression in the double mutant [23]. Moreover, these two bZIPs have their own specific targets. A single bzip28 mutation was sufficient to inhibit the stress-responsive expression of CRT2 (CALRETICULIN 2), SHD (SHEPHERD), and SDF2 (STOMATAL-DERIVED FACTOR2), whereas the single bzip60 mutation abolished the stress-responsive induction of PDI9 (PROTEIN DISULFIDE ISOMERASE 9), SAR1A (SECRETION-ASSOCIATED RAS 1A), and SEC31A (SECRETORY 31A) [23,25,27]. Consistently, bzip2860 plants showed reduced tolerance to chemical-induced ER stress, and the bzip28 and bzip60 single mutants had a similar, but weaker, phenotype [28].

bZIP17, a homolog of bZIP28, is also a transcriptional activator that responds to ER stress [29], although its role in the canonical UPR appears to be auxiliary to that of other UPR bZIPs. Under ER stress conditions, transcriptomic changes in vegetative tissues caused by the double mutation of bZIP17 and bZIP60 (bzip1760) are only slightly different from those caused by the single bzip60 mutation, and the single bzip17 mutation also resulted in only subtle changes [23]. Recent studies revealed that bZIP17 plays important roles in inflorescence tissues [30,31,32], with specific target genes including HOMEOBOX 7 (HB-7) and DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) [32,33].

3. UPR Action in Stress Responses