DOI: 10.5061/dryad.kn95474
title: Data from: Response to persistent er stress in plants: a multiphasic process that transitions cells from prosurvival activities to cell death

description: 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.
description: Suppl dataset 1
 RNAseqThe 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 & 17Chromatin
 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
 ATACseqATACseq 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 pathwaysMetabolic 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.