RNA‐seq analysis of laser microdissected Arabidopsis thaliana leaf epidermis, mesophyll and vasculature defines tissue‐specific transcriptional responses to multiple stress treatments

Abstract

SUMMARY Acclimation of plants to adverse conditions requires the coordination of gene expression and signalling pathways between tissues and cell types. As the energy and carbon capturing organs, leaves are significantly affected by abiotic and biotic stresses. However, tissue‐ or cell type‐specific analyses of stress responses have focussed on the Arabidopsis root. Here, we comparatively explore the transcriptomes of three leaf tissues (epidermis, mesophyll, vasculature) after induction of diverse stress pathways by chemical stimuli (antimycin A, 3‐amino‐1,2,4‐triazole, methyl viologen, salicylic acid) and ultraviolet light in Arabidopsis using laser capture microdissection followed by RNA sequencing. Stimulation of stress pathways caused an overall reduction in the number of genes expressed in a tissue‐specific manner, though a small subset gained or changed their tissue specificity. We find no evidence of a common stress response, with only a few genes consistently responsive to two or more treatments in the analysed tissues. However, differentially expressed genes overlap between tissues for individual treatments. A focussed analysis provided evidence for an interaction of auxin and ethylene that mediates retrograde signalling during mitochondrial dysfunction specifically in the epidermis, and a gene regulatory network defined the hierarchy of interactions. Taken together, we have generated an extensive reference dataset that will be valuable for future experiments analysing transcriptional responses on a tissue or single‐cell level. Our results will enable the tailoring of the tissue‐specific engineering of stress‐tolerant plants.

Single Cell App: An App for Single Cell RNA-sequencing Data Visualization, Comparison and Discovery

“…For example, stele-specific expression of PHO1;H3 was detected in plants grown in an zinc deficient environment while data analysed here was obtained from seedlings supplied with sufficient nutrients (Khan et al, 2014). Indeed, cell heterogeneity in response to environmental stimuli has been previously reported (Berkowitz et al, 2021;Jean-Baptiste et al, 2019;Shulse et al, 2019). We also observed here that a variety of genes encoding phosphate transporters were induced by protoplasting, which would normally be discarded in analysis.…”

“…Similarly, in a study examining tissue specificity of gene expression in Arabidopsis leaves the tissue specific expression decreased with applications of chemicals that mimicked adverse growth conditions (i.e. oxidative stress) or hormones (Berkowitz et al, 2021). Hence it is difficult for non-expert researchers to test the robustness of marker genes defined by earlier studies, and to evaluate how consistent they may be under different conditions.…”

Cross-species transcriptomic analyses reveals common and opposite responses in Arabidopsis, rice and barley following oxidative stress and hormone treatment

“…To ensure efficacy of application for the treatments (Table 1 ) in this study, the expression of marker genes was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in all three species. The starting point for the treatments was the concentration and time that had been previously extensively optimised in Arabidopsis [ 48 – 50 ], and 3 h was the time point at which transcript abundance for the mitochondrial stress marker Alternative oxidase (AOX) peaked. The induction of various marker genes was 16-fold or greater at 3 h in all species and treatments, except in barley, ABA treatment for the marker gene Beta-glucosidase 31 was > than 8-fold, and with the induction of AOX1a with ABA treatment in barley similar to that seen in rice and Arabidopsis this was considered sufficient.…”

Legume Alternative Oxidase Isoforms Show Differential Sensitivity to Pyruvate Activation

“…It is not known whether GmAOX1, GmAOX2A and GmAOX2D co-localize within the same cell types of soybean. However, in Arabidopsis thaliana shoots there is indirect evidence for AOX transcript co-localization in some cell types, based on laser microdissection transcriptomics that suggested both At Aox1a and AtAox1b were present in vasculature cells, while At Aox1a , AtAox1d and AtAox2 transcripts were all found in mesophyll ( Berkowitz et al, 2021 ). In addition, interactions between different AOX peptides have been suggested, based on Blue Native (BN)-PAGE experiments with isolated A. thaliana leaf mitochondrial proteins; specifically AtAOX1A, AtAOX1B, and AtAOX1D could be found in a complex of approximately 140 kDa ( Senkler et al, 2017 ).…”