Functional Characterization of Mitochondrial Small Heat Shock Protein Genes in Eggplant for Abiotic Stress Tolerance

Abstract

Plants are sessile organisms and have evolved with numerous mechanisms for overcoming various environmental stresses. In response to various abiotic stresses, plants are used to transcribe a class of proteins known as heat shock proteins (HSPs). The mysterious and relatively understudied mitochondrial small heat shock proteins (msHSPs) evolved in the plant lineage, which are the extensive variety of HSPs found in all three domains of life, are intimately linked to protein homeostasis and survival under stress conditions. The present investigation systematically characterized a novel mitochondrial sHSP (SmsHSP24.1) from Eggplant (Solanum melongena L.) by transgenic overexpression and CRISPR/Cas9 mediated genome editing method. Differential expression of SmsHSP24.1 suggested that it plays a constructive role under stressful circumstances. The SmsHSP24.1 overexpressed Escherichia coli-BL21 cell line demonstrated exceptional thermo-tolerance, withstanding temperatures of up to 52ºC. The protein had a multimeric structure that was unveiled by spectrometry and electron microscopy assay, acting as a molecular chaperone at high temperatures. In constitutively overexpressed Eggplant lines, overexpression of SmsHSP24.1 dramatically improved resilience to heat, drought, and salt stresses and demonstrated quick germination and seedling vigour in Eggplant. According to the RNAseq data, the glutathione (GHS) pathway's reactive oxygen species (ROS) scavenging enzymes appear to be upregulated. Genes essential to the mitochondrial electron transport chain (ETC) were also subject to transcriptional modifications. Auxin biosynthesis, cell wall transport, and repairs-related genes were also found to be at elevated levels. Quantitative PCR (qPCR), biochemical, and physiological parameters of transgenic Eggplants were found to play a crucial role in a range of stress responses and plant growth and development. Apart from that, approximately 2.0 kb upstream region predicted as SmsHSP24.1 promoter was cloned, followed by the presence of numerous stress-related cisregulatory elements has been confirmed by GUS expression assay, indicating that the protein is

likely to be expressed as inducible

way in transgenic Eggplant lines. Furthermore, CRISPR/Cas9 mediated knocked out of SmsHSP24.1 protein also indicated the indispensable role of this protein in stress response. Our results indicated an entirely novel source of the mitochondrial small heat shock protein (SmsHSP24.1) and its positive physiological role in transgenic Eggplant. Consequently, this gene has enormous potential in engineering stressresilient crop plants to boost agricultural production and ensure our food security.