The unfolded protein response (UPR) is a highly conserved response that protects plants from adverse environmental conditions. The UPR is elicited by endoplasmic reticulum (ER) stress, in which unfolded and misfolded proteins accumulate within the ER. Here, we induced the UPR in maize (Zea mays) seedlings to characterize the molecular events that occur over time during persistent ER stress. We found that a multiphasic program of gene expression was interwoven among other cellular events, including the induction of autophagy. One of the earliest phases involved the degradation by regulated IRE1-dependent RNA degradation (RIDD) of RNA transcripts derived from a family of peroxidase genes. RIDD resulted from the activation of ZmIRE1 for promiscuous ribonuclease activity that attacks the mRNAs of secreted proteins. This was followed by an upsurge in expression of the canonical UPR genes indirectly driven by ZmIRE1 due to its splicing of Zmbzip60 to make an active transcription factor that directly upregulates many of the UPR genes. At the peak of UPR gene expression, a global wave of alternative RNA processing led to the production of many aberrant UPR gene transcripts, likely tempering the ER stress response. During later stages of ER stress, ZmIRE1’s activity declined as did the expression of survival modulating genes, Bax inhibitor1 and Bcl-2-associated athanogene7, amidst a rising tide of cell death. Thus, in response to persistent ER stress, maize seedlings embark on a course of gene expression and cellular events progressing from adaptive responses to cell death.
Suppl dataset 1 RNAseq
The ER stress transcriptome was profiled in maize B73 seedlings treated with 5 g ml-1 TM for 0, 3, 6, 12, 24, 36, and 48 h. (A 48-h mock-treated sample was included.) RNA was extracted from root samples and cDNA libraries were generated and subjected to DNA sequencing. Read sequences from three biological replicates (each with two technical replicates) were aligned to the maize genome v4. Read counts were normalized and differential gene expression was assessed using generalized linear models. Tab 1: Cluster analysis. Tab 2: RPKM values during time course following TM treatment. Tab 3: Mean and standard error of RPKM values.
Suppl dataset 2 CHIPseq bZIP60 & 17
Chromatin immunoprecipitation analysis was carried out in triplicate to identify the direct transcriptional targets for ZmbZIP60 and ZmbZIP17. GFP tagged forms of the bZIP transcription factors (minus their transmembrane and lumen-facing domains) were transfected into maize seedling mesophyll protoplasts and after incubation nuclei were isolated, chromatin extracted and fragmented by sonication. GFP tagged ZmbZIP60 and ZmbZIP91 were immunoprecipitated with anti-GFP antibody and the coimmunoprecipitated DNA fragments were eluted, used to construct DNA libraries and subject to DNA sequencing. Read sequences were aligned to maize genome version 4. Peaks were identified and their intersection with 500 bp upstream in the promoter regions of the various genes was computed to give a peak score.
Suppl dataset 3 ATACseq
ATACseq analysis was conducted in triplicate to identify changes in chromatin “openness” or accessibility in response to ER stress. Maize B73 seedlings were treated with 5 g ml-1 TM for 0, 6 and 12 h. Nuclei were isolated from root samples and incubated with Tn5 transposase to tag DNA fragments in regions of open chromatin. The tagged DNA was PCR amplified with primers directed against the Tn5 inserts and used to construct a cDNA library, which was subject to DNA sequencing. Short read sequences were aligned to the maize genome version 4, and peaks were called for each replicate at every time point. Differential peak scores representing changes in promoter openness in the 500 or 5000 bp upstream region for each gene for 6 h vs 0 h and 12 h vs 0 h were then calculated. Tab 1: from the start of transcription to 500 bp upstream. Tab 2: from the start of transcription to 5000 bp upstream.
Suppl dataset 4 RNA isoforms (3 h to 0 h)
RNA isoforms were analyzed using the RNAseq data to gauge the level of alternate RNA splicing. Differential exon usage was computed using a flattened maize gene model file with collapsed exon counting bins. For alternative splicing analysis, the number of reads were counted that overlaped with each of the exon counting bins. To identify exons skipped, each sample was compared to the sample from next immediate time point i.e. 3 h and 6 h, 6 h and 12 h, 12 h and 24 h, etc. Attached data compares time points 3 h to 0 h. Other time point comparisons are available on request.
Suppl dataset 5 Metabolic pathways
Metabolic pathway enrichment for the various gene clusters was analyzed according to Corncyc https://corncyc-b73-v4.maizegdb.org). The analysis was based on RNAseq data for differentially expressed genes in the different clusters.
