Shrestha, Asis: Genetic and molecular analysis of drought stress adaptation in cultivated and wild barley. - Bonn, 2020. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Asis Shrestha}},
title = {Genetic and molecular analysis of drought stress adaptation in cultivated and wild barley},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2020,
month = oct,

note = {Drought tolerance breeding is an integral part of modern-day agriculture for sustained crop production. In this study, we employed forward and reverse genetics tools to understand and improve drought stress adaptation in barley (Hordeum vulgare L.). Using the forward genetics approach, we identified a novel allele of pyrroline-5-carboxylate synthase 1 (HvP5cs1) from wild barley ISR42-8 (H. vulgare spp. spontaneum). The putative functional mutations were detected in the HvP5cs1 promoter across ABA-responsive elements (ABREs) between ISR42-8 and cultivar Scarlett. Thereafter, allele mining of the HvP5cs1 promoter region in a barely diversity panel comprised of wild barley, landraces and cultivars identified novel variants of cis-acting elements. Proline measurements and expression analyses in the haplotypes based on promoter variation illustrated the significance of ABRE and MYB binding elements for HvP5cs1 transcriptional regulation.
Next, we quantified the HvP5cs1 promoter activity of ISR42-8 and Scarlett through transient expression of promoter::GUS construct in Arabidopsis protoplast (Col-0). GUS expression analysis revealed greater activation of ISR42-8 promoter than Scarlett upon ABA application. To test the role of ABRE binding factors (ABFs), we evaluated HvP5cs1 promoter activity of ISR42-8 in protoplast of loss-of-function abf1 abf2 abf3 abf4 quadruple mutant in Arabidopsis. Notably, ISR42-8 promoter activity diminished in the protoplast of the abf1 abf2 abf3 abf4 quadruple mutant indicating the promoter activity is regulated in an ABF-dependent manner.
Then, we developed a near-isogenic line (NIL-143) QPro.S42-1H in cultivated barley Scarlett through marker-assisted backcrossing (BC6). NIL-143 preserved the genetic competence of ISR42-8 to accumulate proline in higher concentrations under drought conditions. Under drought stress, NIL-143 maintained superior membrane integrity, reduced pigment damage, and sustained photosynthetic health compared to Scarlett. Further, NIL-143 presented a remarkable improvement in drought stress recovery than Scarlett. The introgression of QPro.S42-1H enhanced yield attributes in NIL-143 compared to Scarlett under drought stress in field conditions.
Hereafter, we generated barley mutant lines of putative ABFs using the CRISPR/Cas9 system in the background of cultivated barley Golden Promise. ABFs belongs to basic leucine zipper family, and four putative orthologs were identified in barley. We targeted the conserved C-terminus end, and site-directed mutation events were detected at the target site in HORVU3Hr1G084360 (HvAbi5) and HORVU6Hr1G080670 (HvAbf). Three allelic mutants were detected for HvABI5, resulting in a translational frameshift. One allelic mutant with 3 bp deletion in HvABF caused a loss of serine from the conserved domain. The morphological and physiological evaluation revealed that hvabf and hvabi5 were more sensitive to drought stress than wild type. Global transcriptome profiling demonstrated that stress-inducible genes were downregulated in hvabf and hvabi5 compared to wild type.
As a proof of concept, stress-inducible proline synthesis was evaluated in Col-0 and the abf1 abf2 abf3 abf4 quadruple mutant at different stress scenarios. Upon ABA application, P5CS1 mRNA expression and shoot proline content were significantly more upregulated in Col-0 as compared to the abf1 abf2 abf3 abf4 quadruple mutant.
In conclusion, the present data uncover the genetic regulation of drought-inducible proline accumulation and its role in drought stress adaptation in barley.},

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