What if a protein involved in programmed cell death also acts like a tuning dial for the unfolded protein response? In Arabidopsis, BAP2 helps cells survive stress—but when the main stress pathway fails, it can instead push them toward death. ER stress occurs when the balance between protein synthesis and the folding capacity of the endoplasmic reticulum is challenged, and it can be potentially lethal.

The unfolded protein response, or UPR, can restore endoplasmic-reticulum homeostasis or actuate programmed cell death when ER stress is unresolved. The unresolved question is how the UPR determines cell fate, especially in plants.

To address that question, the study combines genetics, ER stress profiling, natural variation, and quantitative trait locus analysis across 350 natural Arabidopsis thaliana accessions. Arabidopsis encodes two functional IRE1 paralogs, IRE1A and IRE1B, which resemble IRE1 in yeast and mammals.

Under chronic ER stress, losing both IRE1A and IRE1B, or both bZIP28 and bZIP60, accelerates progression to cell death, while losing either bZIP60 or bZIP28 is viable. Together, these observations indicate that IRE1 and bZIP28 or bZIP60 protect cells by antagonizing endogenous cell-death processes during chronic ER stress.

During transient stress, IRE1 is activated temporarily and eventually inactivated, whereas during chronic stress its activity remains sustained and can trigger cell death. IRE1 therefore has a biphasic role: it directs pro-life activities while stress remains resolvable, but ignites pro-death processes when the UPR is insufficient.

Coordinating adaptive and pro-death responses is crucial for UPR sufficiency in cell-fate determination, yet the underlying mechanisms remain poorly established, especially in plants. Because IRE1 is essential for cell-fate determination, the amplitude and duration of IRE1 signaling must be tightly regulated.

Several mammalian cofactors regulate IRE1 signaling outputs, but similar cofactors in plants remain largely unknown. The mammalian ER-membrane protein BAX inhibitor 1 negatively modulates IRE1 endonuclease activity and XBP1 splicing, while Arabidopsis BI1 does not reduce IRE1 splicing activity.

The study combines genetics and quantitative genomics by leveraging natural variation in Arabidopsis. It establishes BAP2, a general programmed-cell-death regulator in plants, as a cellular rheostat that monitors the sufficiency of the IRE1-bZIP60 UPR arm during ER stress.

Under conditions of UPR insufficiency, BAP2 acts as a pro-death effector. The study hypothesized that the plant UPR would show natural variation that could help identify modulators of ER stress responses. To test this, sensitivity to chronic ER stress was analyzed in 350 natural Arabidopsis accessions.

Each accession was grown for 10 days on solid media containing tunicamycin, an ER stress inducer, or DMSO as the solvent control. The accessions showed a highly significant accession-by-treatment interaction, and the 350 accessions displayed a 10-fold range of natural variation in ER stress sensitivity.

Figure one establishes substantial natural variation in Arabidopsis responses to ER stress. Across three hundred fifty accessions, relative biomass after tunicamycin treatment spans from about twenty percent to roughly one hundred seventy percent of untreated growth, with Na-1 and Est-0 marking contrasting responses.

The photographs and scatter plots confirm these differences at twenty-five and fifty nanograms per milliliter, providing the basis for QTL mapping and later candidate-gene analysis. Na-1 had the highest relative growth ratio compared with Col-0, at one point seven three plus or minus zero point one three, while Est-0 had one of the lowest, at zero point two one plus or minus zero point zero three.

The two accessions differed four point six-fold in tunicamycin-induced ER stress sensitivity at twenty-five or fifty nanograms per milliliter tunicamycin. To compare the accessions, the study examined three distinct genes involved in the UPR: spliced bZIP60, BiP3, and ERdj3B.

They used quantitative reverse-transcription PCR on seedlings after three or six hours of tunicamycin treatment to quantify the transcript levels. Compared with Col-0, Est-0 had higher BiP3 and spliced bZIP60 expression at both treatment times, while Na-1 had significantly higher levels at six hours; the results indicate natural variation in UPR activation, especially in genes controlled by the IRE1-bZIP60 arm.

To identify the polymorphism associated with the different responses, the study used QTL-seq, bulked-segregant analysis, and high-throughput resequencing in an F3 population from a Na-1 by Est-0 cross. Relative growth ratios were estimated for 400 F3 progenies, and the 10 percent tail, consisting of 42 individuals, was classified as hyper-sensitive or hyper-resistant.

DNA from the extreme groups was pooled and sequenced to approximately seventy-four-fold coverage, then compared using SNP-index and delta SNP-index values. Panel a shows the distribution of relative biomass ratios across four hundred F3 lines, with orange and green cutoffs marking the hyper-sensitive and hyper-resistant ten-percent tails.

Panel b then compares allele frequencies between these pooled extremes across the genome; the red delta SNP index departs from the confidence boundaries most clearly on chromosome two, identifying a candidate region for ER-stress sensitivity. Panels c and d expand this region and show stress-responsive expression of BAP2, linking the genetic signal to a testable candidate gene.

The analysis used a ten-kilobase step as one of its reported settings, with the result shown in Figure two and Supplementary Data three. This analysis identified one genomic region contributing to ER stress sensitivity at the end of chromosome 2, within the ninety-five-percent confidence interval.

Among the candidate genes was BON-ASSOCIATED PROTEIN2, or BAP2, which is involved in programmed cell death. The study measured transcript levels of six candidate genes in Col-0 seedlings treated with tunicamycin, using an extended time course of six, twenty-four, or forty-eight hours.

Except for BAP2, whose expression increased over the ER stress time course, the other selected genes were unchanged. This result led the study to focus on BAP2 for downstream analyses. BAP2 encodes a twenty-three-kilodalton protein containing a calcium-dependent phospholipid-binding C2 domain, and it is believed to regulate programmed cell death in plants.

The study tested an established bap2 knockout mutant during chronic tunicamycin-induced ER stress, measuring shoot fresh weight and chlorophyll content after ten days. The ire1a ire1b control showed a strong reduction in shoot fresh weight and chlorophyll content, while bap2 also showed a significant reduction compared with Col-0.

The same pattern appeared with dithiothreitol, another ER stress inducer: ire1a ire1b showed a drastic reduction, while bap2 showed a moderate but significant reduction compared with Col-0. Figure three tests whether BAP2 helps Arabidopsis tolerate chronic ER stress induced by tunicamycin.

The images and shoot-weight measurements show the bap2 knockout response alongside Col-0 and ire1a ire1b, while complementation experiments compare genomic BAP2 fragments from Na-1 and Est-0. The Na-1 transgene restores the bap2 stress response, whereas the Est-0 BAP2 N67S version does not, linking natural BAP2 variation to ER-stress sensitivity.

To test temporary rather than unresolved ER stress, the study used a six-hour tunicamycin pulse, drug washout, and root-growth measurement after four days on drug-free media. The bzip28 knockout control showed significant inhibition of root growth compared with Col-0.

In contrast, bap2 root growth was similar to Col-0, indicating that BAP2 is not necessary for recovery from ER stress and has a predominant role in unresolved ER stress conditions. In BAP2, Est-0 carries a nonsynonymous change that substitutes serine for aspartate at position sixty-seven compared with Col-0 and Na-1.

The substitution occurs in the C2 functional domain of BAP2, which is highly conserved with its homolog BAP1. The study transformed bap2 with either the Na-1 or Est-0 BAP2 genomic clone, including a one-kilobase promoter region. The Na-1 BAP2 clone complemented bap2 ER stress sensitivity, whereas the Est-0 BAP2 N67S lines showed sensitivity similar to bap2, demonstrating reduced function of the Est-0 allele.

Figure four tests whether BAP2 alleles explain Est-0’s sensitivity to tunicamycin-induced ER stress. After ten days, images and relative shoot fresh weight show that introducing the Na-1 BAP2 allele into Est-0 partially improves stress performance, whereas the Est-0 BAP2 N67S allele produces responses similar to Est-0.

This supports a contribution from the C2-domain substitution, while the incomplete rescue indicates that additional genetic factors also affect ER-stress sensitivity. In the Est-0 background, lines expressing Est-0 BAP2 N67S responded like Est-0, while lines expressing Na-1 BAP2 showed significantly enhanced ER stress resistance.

The transgenic lines had similar BAP2 expression levels, indicating that the differences in ER stress response were due to BAP2 sequence variation rather than transgene expression differences. Na-1 and Est-0 also showed no substantial differences in BAP2 expression under ER stress compared with Col-0.

Because BAP2 regulates programmed cell death in other abiotic and biotic conditions, the study tested whether it also regulates cell death caused by ER stress. Cell death was measured through electrolyte leakage in seven-day-old Col-0, bap2, and ire1a ire1b seedlings treated with one microgram per milliliter tunicamycin or DMSO for forty-eight hours.

Compared with wild type, both ire1a ire1b and bap2 showed a significant increase in electrolyte leakage. Figure five tests whether BAP2 limits ER stress-induced cell death in seven-day-old seedlings. After forty-eight hours with tunicamycin, the chart reports electrolyte leakage and hydrogen peroxide accumulation for Col-0, bap2, and the ire1a ire1b control, while the images show corresponding seedling and trypan-blue staining patterns.

The increased damage-associated readouts in the mutant backgrounds support the authors’ conclusion that BAP2 antagonizes programmed cell death during ER stress. Tunicamycin-treated shoots of bap2 and ire1a ire1b seedlings showed more extensive trypan-blue-positive areas than Col-0.

These results indicate that BAP2 is required to antagonize programmed cell death when the UPR is sufficient during ER stress. Because BAP2 was induced during prolonged ER stress in Col-0, the study asked which UPR signaling arm controls that induction. BAP2 messenger RNA levels were compared in ire1a ire1b and bzip28 mutants with Col-0 after twenty-four or forty-eight hours of tunicamycin treatment using quantitative reverse-transcription PCR.

Panels a and b measure BAP2 transcripts after tunicamycin-induced ER stress, using Col-0, bzip28, and ire1a ire1b seedlings. The figure shows that BAP2 induction occurs in the bzip28 background, while the ire1a ire1b mutant also displays a marked response during prolonged treatment; short pulse treatments further examine this regulation.

This matters because it supports the authors’ conclusion that IRE1 controls BAP2 induction and that BAP2 may help modulate UPR activation during ER stress. The results suggest that BAP2 negatively modulates IRE1 splicing activity, but BAP2 does not modulate IRE1 activity at the transcriptional level because IRE1A and IRE1B transcript levels did not significantly differ between bap2 and Col-0.

An in-vitro interaction assay found an association between BAP2 and the cytosolic region of IRE1B, which contains the kinase and endonuclease domains. The assay did not detect an interaction between IRE1B and BAP2N67S, suggesting that BAP2 may interact with IRE1B to modulate its activity and that the structure of the BAP2 C2 domain may be involved.

Under chronic ER stress with a sufficient UPR, BAP2 negatively modulates expression of UPR genes primarily controlled by the IRE1-bZIP60 arm, while IRE1 suppresses BAP2 induction. To test BAP2 during UPR insufficiency, the study generated an ire1a ire1b bap2 triple mutant.

The ire1a ire1b mutant was lethal under chronic ER stress, but removing BAP2 produced a slight yet significant increase in tolerance at twenty-five nanograms per milliliter tunicamycin, not at fifty nanograms per milliliter. Under prolonged but mild stress at five, ten, or twenty nanograms per milliliter tunicamycin, the triple mutant partially reverted the lethal phenotype, increased shoot fresh weight and chlorophyll content, and showed reduced electrolyte leakage compared with ire1a ire1b.

Figure seven tests how BAP2 contributes to cell death during chronic ER stress caused by tunicamycin, or TM. Seedling images, shoot fresh weight, electrolyte leakage, and hydrogen peroxide measurements compare Col-0, bap2, IRE1-deficient, and combined mutants, while the combined mutant provides a direct test of BAP2 function when IRE1 is dysfunctional.

The figure also measures NAC089 expression after stress, connecting these physiological outcomes to a transcriptional regulator of programmed cell death and supporting the authors’ conclusion that BAP2 acts as a pro-death effector when IRE1 signaling is insufficient. Natural genetic variation in Arabidopsis identified BAP2 as a critical plant UPR modulator and linked a BAP2 SNP polymorphism to contrasting ER stress sensitivity in Est-0 and Na-1.

BAP2 is induced by ER stress in an IRE1-regulated manner and is necessary to control UPR activity during ER stress responses. The study identifies BAP2 as a rheostat between programmed cell death and the plant UPR: it modulates IRE1 activity during ER stress and executes pro-death processes when the UPR is insufficient.

Figure eight presents a model of how BAP2 and IRE1 jointly determine cell fate as ER stress intensifies. During UPR sufficiency, IRE1 supports pro-survival signaling while BAP2 acts like a rheostat, with feedback between the two helping optimize the response and potentially induce NAC089.

When the UPR becomes insufficient, BAP2 promotes hydrogen peroxide accumulation and programmed cell death, showing how pro-life and pro-death processes form a stress-dependent tug-of-war. BAP2 is an IRE1-linked rheostat: it supports tolerance during chronic ER stress when the UPR is sufficient, but becomes a pro-death effector when IRE1 signaling is insufficient.

This connects natural genetic variation to plant cell-fate decisions.