Gene editing to improve stress resilience in wheat

Ashaduzzaman Sagar

doi:10.60867/00000169

Water deficit, salinity, and low-temperature stresses negatively affect wheat production worldwide, underscoring the need to develop stress-tolerant wheat varieties. This can be achieved by modifying the stress response mechanisms and regulations in plants. The recent DNA-editing approach, CRISPR/Cas editing, can accurately modify transcriptional and translational elements in organisms, thereby manipulating molecular processes. They have been used to generate mutants in plants and animals with loss-of-function, allowing rapid study of gene function. The most recent advance in editing involves modifying protein translation by editing non-coding regulatory elements, such as promoters, 5ʹ untranslated regions (UTRs), and upstream open reading frames (uORFs). This strategy is now being applied to change the turnover of specific proteins essential for the expression of traits in plants and animals. For plants, altering these proteins can change the molecular functions they are involved in, leading to changes in phenotypic expression that can translate into new agronomic traits or improvements of existing desirable ones. For wheat, one such trait is tolerance to abiotic stresses, which can be obtained by altering the translation of stress-responsive transcription factors (TFs). In this study, the general aim was to assess the potential to improve wheat stress tolerance by modulating the protein synthesis of TFs via CRISPR/Cas editing. This project identified and characterised two stress-responsive genes, ScLIP19 and ScbZIP15, in the Australian winter wheat cv. Scepter. Following identification of the two genes among 94 other stressresponsive genes, sequence analyses of these genes uncovered the presence of highly conserved bZIP motifs and complex uORF architectures. ScLIP19 and ScbZIP15 contained 33 and 15 uORFs, respectively, in their 5ʹ UTRs of the mature mRNA coding sequences. The complex uORF architecture of these two genes made them candidates for uORF editing to assess their potential to improve wheat stress tolerance via translational regulation. Expression of the genes in water-, salt-, and cold-stressed wheat plants at the vegetative, booting, anthesis, and grain-filling stages varied, with ScLIP19 showing consistent, steady upregulation across stresses, whereas ScbZIP15 showed stress- and stage-dependent responses. Both genes showed higher expression when plants were stressed at all growth stages. Heterologous promoter expression in tobacco via β-glucuronidase (GUS) reporter assay validated the stress-inducible nature of both the ScLIP19 and ScbZIP15 promoters. The GUS protein driven by the ScLIP19 promoter accumulated under all tested stresses with up to 3.59- fold increase. In contrast, ScbZIP15 promoter decreased GUS activity under water stress (0.44-fold) but increased under salinity (1.30-fold) and cold stress (2.46-fold). These diverse profiles confirm that the promoters were responsive to the three abiotic stresses. CRISPR/Cas9-mediated modifications of the uORF regions in transgenic tobacco carrying the promoter elements of the genes were conducted to investigate translational regulation. Editing the uORF region of ScLIP19 altered the uORF architecture (33 or 34 uORFs across transgenic events) in tobacco, resulting in modified GUS expression. The results indicated that even without precise mutation of specific uORFs, editing within the 5′ UTR can influence translational output by reshaping uORF configurations. Editing of the native promoter regions of ScLIP19 in wheat showed promise, with ambiguous sequences detected in the guide RNA target regions, although time did not permit a full analysis of the effects on protein synthesis. In contrast, targeted modifications in the 5′ UTR of ScbZIP15 in both tobacco and wheat did not result in any editing; possibly because of the efficiency of the guide RNAs rather than the nascent structure of the target region of the gene or its interaction with other editing elements. This project provides new information on ScLIP19 and ScbZIP15 in the commercial Australian winter wheat cv. Scepter, including their uORF architecture, patterns of response to salt, water, and cold stress, and the potential to increase tolerance to these stresses through editing of the promoters and 5ʹ UTRs to influence translation of the TFs they encode. It also establishes that modifying regulatory regions, rather than genes via transgenesis or gene knockout, can alter molecular processes, facilitating the development of new crop varieties, particularly climate-resilient wheat varieties.

Gene editing to improve stress resilience in wheat

Files and links (1)

Abstract

Details

UN Sustainable Development Goals (SDGs)

Metrics