The involvement of RS2Z proteins in tomato heat stress response and thermotolerance

Abstract

Due to their sessile nature, plants are constantly exposed to an everchanging environment. When these changes exceed certain limits, they can significantly impact plant growth and development, which, in case of crop plants, has consequences on food security. Exposure to high temperatures causes heat stress (HS), one of the most devastating stresses that plants can face. The survival and recovery from HS are dependent on the activation of the HS response (HSR), a collection of molecular mechanisms conferring HS tolerance by maintaining the cellular homeostasis. Stress responses follow a strictly orchestrated network of signal perception and -transduction, ultimately resulting in an adaptive cellular output. Thereby, the massive reshaping of the transcriptome plays a major part, in which heat stress transcription factors (HSFs) play the key role by inducing the expression of HS-responsive genes, including heat shock proteins and other transcription factors. Additionally, alternative splicing (AS), the selective usage of splice sites, contributes to the rapid adjustment of the transcriptome landscape by producing different mRNA variants from a single gene. Consequently, this results in the reduction of translatable transcripts by nonsense-mediated mRNA-decay or nuclear retention, but also enhances the proteome diversity by allowing the synthesis of protein isoforms with distinct functions. AS thereby modulates the activity of important regulatory factors like HSFA2 in Solanum lycopersicum (tomato). HSFA2 is the key factor of acquired thermotolerance (ATT), which enables the ability to survive a potentially lethal HS through pre-exposure to a preceding mild HS. Temperature-dependent AS leads to the synthesis of two HSFA2 protein variants, whereby inhibition of splicing ensures the synthesis of the stable isoform HSFA2-I that is required for ATT. Transcriptome analysis of several plant species exposed to HS has highlighted the strong impact of high temperatures on the regulation of pre-mRNA splicing. Despite its importance, little is known about the molecular basis of the AS regulation in plants. Particularly for an economically important crop like tomato, understanding the regulation of HS-sensitive AS will contribute to the description of such an important regulatory mechanism but also might offer new insights for increasing HS resilience. Serine/arginine-rich proteins (SR proteins) are central regulators of constitutive and AS by modulating the splice site selection by the spliceosome. This study describes two members of the RS2Z subfamily of SR proteins in tomato, namely RS2Z35 and RS2Z36, which act as core regulators of AS under HS and consequently as central factors for thermotolerance. This study investigates the interaction of the two RS2Z proteins with the HSFA2 pre-mRNA and provides evidence for their function as splicing repressors in this particular AS event. Thereby, RS2Z proteins play an important role in the HSR by modulating the AS of the key factor of the ATT. Furthermore, based on global transcriptome analysis of knockout mutants of single or both RS2Z genes, it is demonstrated that RS2Z proteins are involved in the splicing of pre-mRNAs of almost 2000 genes. Moreover, RS2Z proteins act as splicing regulators and take part in a large portion of HS-induced AS events, thus playing a broader role in AS regulation. Furthermore, the HS-induced RS2Z36 is involved in basal thermotolerance (BTT), highlighting its importance for the basic HS resilience capacity of tomato. In addition, RNA sequencing demonstrates that RS2Z proteins–especially RS2Z36–regulate the expression of proteins involved in plant immunity. The study thereby provides experimental evidence for the important and essential role of SR proteins for plant thermotolerance and suggests the existence of RS2Z-mediated crossroads of different stress responses.