Investigating the role of F-group bZIP transcription factors in Zn homeostasis in barley using CRISPR-Cas9 gene editing

Abstract

Zinc (Zn) is one of the essential micronutrients required for successful plant growth and development. Zn deficiency in plants due to lack of availability in soil is a significant problem in crop plants worldwide, leading to reduced growth and Zn content of the edible grain. To better understand the regulation of Zn homeostasis in plants, this thesis studied the seven F-group bZIP transcription factors(HvbZIP1, 10, 55,56, 57, 58 and 62) in the crop plant barley (Hordeumvulgare). Mutants in these bZIPs have been isolated using CRISPR-Cas9 gene editing and their role in the Zn-deficiency response was investigated. Novel single mutants for bzip55,bzip57 and bzip58 along with double mutants for bzip58 bzip1and bzip55 bzip57 have been successfully isolated using vectors assembled by Golden Gate assembly. Additional bzip56 bzip62 and bzip56 bzip62 bzip10 mutants were isolated and compared alongside the previously isolated bzip56-3bzip62-3mutant.  The bzip56 bzip62 andbzip56bzip62 bzip10 mutants grew similarly to the Wt under normal soil conditions but there was an impact on seed production, with a greater percentage of empty spikes produced in the mutants. Under Zn deficiency in hydroponics, bzip56and bzip56 bzip62 mutants showed increased stunted growth and chlorosis compared to Wt, while bzip62,bzip58, bzip58 bzip1 and bzip55 bzip57mutants showed no marked differences. Additional mutation of bzip10alongside bzip56 and bzip62 did not increase sensitivity to Zn deficiency. Defects were also present in bzip56bzip62 mutants under control Zn but were rescued with increased Zn supplementation. Decreased levels of Fe, Mn and P were present in bzip56 and bzip56bzip62 mutants under Zn deficiency compared to Wt and bzip62 mutants, while no difference in Zn content was observed in shoot tissue. Bioinformatic analysis has identified further genes in barley that contain Zn-deficiency response elements(ZDREs) in their promoters. ZDRE-containing genes bZIP1 and ZIP8 were upregulated under Zn deficiency in Wt but not in the bzip56-3 bzip62-3 mutant, indicating that these transcription factors regulate these genes and that this reduction in expression could contribute to the growth phenotype seen in the bzip56-3 bzip62-3 double mutant. In contrast, Asparagine Synthetase (HvASN1) contains a ZDRE and was up-regulated under Zn deficiency in Wt plants, however its expression was enhanced in the bzip56-3bzip62-3 mutant. Further analysis is required to understand the regulation of this gene and others identified in this study. This work has provided further support for a role of F-group bZIP transcription factors in responding to Zn deficiency in crops.  

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