CRISPR-Cas9 gene editing provides evidence for a role of F-group bZIP transcription factors in Zn homeostasis in barley

Abstract

Zinc (Zn) is an essential micronutrient for all living organisms. Human Zn deficiency is a serious health concern in many populations around the world, due partly to heavy reliance on nutrient-poor cereal crops as a main calorie source, which are often grown on Zn-poor soils. Developing cereal crops that maintain high yields and suitable grain Zn content without the intensive application of fertiliser is important for sustainable agriculture. This thesis investigates the role of F-group bZIP transcription factors (bZIP1,10, 55, 56, 57, 58 and 62) in the barley Zn-deficiency response. This research progresses previous work on the generation and isolation of bzip knockout mutants using CRISPR-Cas9 gene editing, and functional characterisation on Zn-deficient conditions. The bzip56 single mutant and the bzip56bzip62 double mutant are detrimentally affected on Zn deficiency, in the form of pronounced stunted growth, chlorosis and necrosis. The bzip62 single mutant grows similarly to WT. Mutants of bZIP10, which resides in the same clade as bZIP56 and 62, were not detrimentally affected by Zn deficiency but bZIP10 overexpression improves barley low-Zn tolerance. Single and double mutants of remaining bZIPs, bzip55, bzip57, bzip58 and bzip1 were also not markedly affected by Zn deficiency, suggesting hierarchal regulatory roles within the F-group. Growing bzip56 and bzip56bzip62 mutants on Zn deficiency showed increased levels of Zn in the roots and shoots compared to WT, and differences in Fe, Mn, Cu and P levels which could explain the detrimental growth on low Zn. Increasing Zn concentration rescues this severe phenotype.

In Arabidopsis, AtbZIP19 and AtbZIP23 regulate Zn homeostasis under low Zn conditions by binding to Zn-Deficiency Response Elements (ZDREs) in the promoters of ZIP transporter genes, thereby increasing Zn uptake and distribution within the plant. We demonstrate through gene expression studies in bzip56 and bzip56bzip62 mutants that primarily bZIP56 regulates ZDRE-containing genes under Zn deficiency including HvZIP7 and HvZIP8, ZDRE-genes of varying physiological function like HvGR-RBP1, HvNAS1, and the ZDRE-containing bZIPs, HvbZIP10 and 1. Specifically, HvGR-RBP1 showed marked upregulation under Zn deficiency and downregulation upon Zn supplementation, suggesting activity of this gene is sensitive to Zn status. This research has generated novel hvgr-rbp1 single mutants to investigate the role of this gene in Zn-deficiency responses, however no distinctive phenotype was observed in these mutants on low Zn. Further work is required to fully elucidate the involvement of this gene in Zn deficiency. Overall, this research has provided further evidence that F-group bZIPs are involved in the barley Zn-deficiency response.

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Identifiers

ORCID for Kathryn Elisa De Abreu: orcid.org/0000-0002-4748-4060

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