Molecular insights into abiotic stress responses in barley and Arabidopsis

Abstract

This cumulative thesis, titled "Molecular insights into abiotic stress responses in barley and Arabidopsis", investigates two critical aspects of plant responses to abiotic stress. The first part explores hydrogen peroxide (H₂O₂) and its interaction with calcium (Ca²⁺) signalling in barley (<em>Hordeum vulgare</em> L.), while the second part examines drought stress regulation in <em>Arabidopsis thaliana</em>, revealing a novel mechanism involving the genes <em>GASA3</em> and <em><em>AFP1</em></em>. <br/> H₂O₂ plays a pivotal role in signalling pathways that enable plants to adapt to environmental challenges. Despite its importance, its transcriptomic impact remains underexplored. To address this, RNA-Seq analysis was used to examine changes in gene expression in barley roots and leaves after H₂O₂ treatment. This revealed 1883 differentially expressed genes (DEGs) in roots and 1001 in leaves, with most responses being tissue-specific. Only 209 DEGs were commonly regulated, and 37 showed opposing regulation across tissues. Gene ontology (GO) analysis highlighted the organ-specific nature of the response: leaf DEGs were enriched in hormone signalling, H₂O₂ response, and abiotic stress adaptation, while root DEGs were associated with H₂O₂ detoxification, glutathione metabolism, and cell wall modifications. A follow-up study examined the cross-talk between H₂O₂ and Ca²⁺ signalling using RNA-Seq under conditions that blocked Ca²⁺-transients. By comparing expression profiles from H₂O₂-only and LaCl₃+H₂O₂ treatments, 331 Ca²⁺-dependent H₂O₂-responsive genes in leaves and 1320 in roots were identified and grouped into five and four clusters, respectively. A SKM network analysis further revealed transcription factors potentially governing this H₂O₂–Ca²⁺ cross-talk. <br/> Drought is one of the most severe abiotic stresses impacting plant growth, development, and reproduction. Like H₂O₂, drought triggers extensive transcriptomic reprogramming. In this study, two strongly drought-induced genes, <em>GASA3</em> and <em>AFP1</em>, were identified. Loss-of-function mutants showed enhanced drought resistance, while constitutive overexpression of either gene led to reduced tolerance. The <em>gasa3afpl</em> double mutant exhibited even greater resistance than single mutants. Both genes are also ABA-inducible, though <em>GASA3</em> expression remained low in the absence of <em>AFP1</em>. The improved drought tolerance in mutants was linked to higher leaf water content due to smaller stomatal apertures and reduced transpiration. Additionally, ABA levels were elevated in mutants under drought stress- not due to increased biosynthesis, but via the release of conjugated vacuolar ABA-GE through β-glucosidase BG2. Consistent with this, ABA-responsive genes such as <em>RD29A/B</em> and <em>ABF2/3</em> were more strongly upregulated in mutants than in wild type (WT). Conversely, <em>PP2CA</em>, which encodes a phosphatase involved in ABA negative feedback, was repressed in the absence of <em>GASA3</em> and <em>AFP1</em>. These results suggest that both genes act as negative regulators of drought tolerance, with <em>AFP1</em> influencing <em>GASA3</em> expression. <br/> Altogether, this thesis offers new insights into plant abiotic stress responses and provides a foundation for future functional studies in barley and other crops, potentially guiding breeding strategies for improved stress resilience under changing climates.