Impaired Expression of Chloroplast HSP90C Chaperone Activates Plant Defense Responses with a Possible Link to a Disease-Symptom-Like Phenotype

Abstract

RNA-seq analysis of a transgenic tobacco plant, i-hpHSP90C, in which chloroplast HSP90C genes can be silenced in an artificially inducible manner resulting in the development of chlorosis, revealed the up- and downregulation of 2746 and 3490 genes, respectively. Gene ontology analysis of these differentially expressed genes indicated the upregulation of ROS-responsive genes; the activation of the innate immunity and cell death pathways; and the downregulation of genes involved in photosynthesis, plastid organization, and cell cycle. Cell death was confirmed by trypan blue staining and electrolyte leakage assay, and the H2O2 production was confirmed by diaminobenzidine staining. The results collectively suggest that the reduced levels of HSP90C chaperone lead the plant to develop chlorosis primarily through the global downregulation of chloroplast- and photosynthesis-related genes and additionally through the light-dependent production of ROS, followed by the activation of immune responses, including cell death.

Transcriptome Analysis Shows Activation of Stress and Defense Responses by Silencing of Chlorophyll Biosynthetic Enzyme CHLI in Transgenic Tobacco

“…Previously, we described the transcriptome changes preceding the chlorosis developments and relevant biological events in another chlorosis model, i-hpHSP90C transgenic plant system [ 20 ], which is similar to the present i-amiCHLI plants but differs in the silencing target. To find out the common features in the chlorosis development in these two transgenic systems, we compared the results from GO enrichment analyses with up- and down-regulated genes.…”

“…We have previously generated two inducible silencing systems for CHLI and HSP90C in tobacco and found that the silencing of these genes could stimulate the plants to develop chlorosis [ 18 , 19 , 20 ]. As discussed previously [ 20 ], these systems have advantages over the experimental systems with the virus- or viroid-infected plants because, in these transgenic lines, it is possible to analyze those plant cells that have committed to developing but not exhibited chlorosis. Utilizing the advantages of these systems, in our previous and current studies, an RNA-seq method was implemented to explore the early molecular changes, which lead to chlorosis development.…”

“…Despite that the altered transcriptome of CC group plants increased the difficulty of RNA-seq data analysis, we identified several characteristic transcriptome changes in Dex-treated C-1 plants (CD group) through two types of analysis. One was pairwise analysis using DESeq2 as we used in the previous study [ 20 ], and the other is expression profile analysis using the STEM program. Because the latter could statistically be less stringent than the former, we carefully compared the GO terms enriched by the DEGs identified in those analyses.…”

“…In this study, we first describe the results in the CHLI system of an RNA-seq analysis, H 2 O 2 , and cell death assays, which were similar to what we found in HSP90C-silenced chlorosis model plants [ 20 ]. The observation led us to a comparative transcriptome study of these two systems.…”

“…These findings suggested that the chlorosis in these model systems develops through the compromised biogenesis of chloroplast and the activation of plant reactions to the affected chloroplast functioning. Moreover, we recently reported that, in the HSP90C model, the genes related to the plant innate immune response, oxidative stress response, and cell death were upregulated, hydrogen peroxide (H 2 O 2 ) was produced in chloroplasts, resulting in sporadic cell death in plants with chlorosis [ 20 ].…”