University of Tasmania
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Overgrowth mutants of barley and wheat with increased Gibberellin signalling

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posted on 2023-05-27, 14:47 authored by Mir Agha Zadeh, SA
DELLA proteins are a key component in the gibberellin (GA) signaling pathway that negatively regulates plant growth. In model plant systems such as Arabidopsis and rice, mutagenesis and screens for GA response mutants have led to the characterization of genes important in GA biosynthesis and GA signalling. In barley and wheat, screening for overgrowth mutants in a dwarf background (suppressor screen) resulted in the isolation of many mutants potentially involved in GA signalling. Characterization of these mutants showed that in barley and wheat nearly all of the mutations represent new Della alleles (Chandler and Harding 2013). This PhD project characterizes many of these overgrowth alleles, and addresses their potential application in cereal breeding. In barley, overgrowth mutants were isolated using dwarf mutants of the variety 'Himalaya' as starting material. They were characterized as plants with enhanced growth, which varied in extent among different mutants, but all retained the original dwarfing gene. These mutants carried new mutations elsewhere in the genome that resulted in enhanced GA signalling. Some overgrowth alleles led to the formation of larger grains, a trait that is important in barley breeding. To assess the effects of overgrowth alleles on grain size, they were back-crossed to a commercial variety (Sloop). In this thesis the agronomic characteristics of BC3F4 sister lines (homozygous for either the WT or mutant Della/Spy1 gene) were studied in detail in glass house and field experiments. The results indicated that overgrowth alleles were able to improve grain size of the commercial variety by about 22% across a range of environments compared to the wild type, but there was also a reduction in grain yield. The hormone physiology of overgrowth mutants was also investigated. Some alleles resulted in a high degree of resistance to growth inhibition caused by applied ABA. This result means that some selected overgrowth alleles are interesting material for studies on tolerance to factors such as drought. In wheat, overgrowth mutants were isolated as suppressors of the Rht-B1c dwarfing gene, which results from a 30 amino acid insertion in the DELLA protein encoded by the B-genome. The new mutants grew faster than the dwarf parent and at maturity they had a range of heights from slightly taller than Rht-B1c to as tall as Rht-B1a (tall isoline). More detailed characterization of these mutants showed that faster growth resulted from single nucleotide mutations in the dwarfing gene, or altered splicing of the transcript. This class of mutant includes about 15 different alleles that are semi-dwarf alleles of the Rht-1 gene, similar to Rht-B1b and Rht-D1b which formed the basis of the Green Revolution. Studies commenced with the characterization of overgrowth alleles that had single amino acid substitutions in the DELLA protein. Semi-dwarf overgrowth alleles with improved dormancy have been characterized. These mutants have significantly higher dormancy than Rht-B1b allele. This characteristic is important for wheat breeding in humid environment, where pre-harvest sprouting is a major issue. The screen for overgrowth mutants in wheat also resulted in 'tall' mutants that appeared to lack the Rht-B1 gene. Deletion of this gene was confirmed by Southern blot analysis. The extent of deletion in such lines is of interest since isolation of 4B deletion lines has not been very successful in other wheat genetic backgrounds. We used a newly developed technology, Single Nucleotide Polymorphism (SNP) chip analysis to assess the size of deletions. The results allowed us to classify the deletion lines into three classes: (i) interstitial deletions of 4BS (short arm of chromosome 4B), ranging from small to large deletions, (ii) lines that have lost all 4BS markers, but retain 4BL (long arm of chromosome 4B) markers, and (iii) lines that have lost all 4BS and all 4BL markers. Mutants that lacked the short arm and those that lacked the whole 4B chromosome were also studied with FISH (Fluorescent In Situ Hybridization) analysis (part of a collaborative cytogenetic study). Microscopy clearly showed the presence of acro-/telocentric 4B chromosomes for class (ii) mutants, while mutants in class (iii) had a chromosome count of 40, instead of 42, indicating the complete loss of 4B. The Rht-B1c allele contains one intron, and some overgrowth mutations have occurred in the nucleotides immediately flanking the exon-intron junction, making them potential splice site mutations. The expression of the Rht-B1c gene in these mutants was studied with semi-quantitative and real-time PCR techniques. The results of semi-quantitative PCR showed that splicing site alleles had less normal Rht-B1c mRNA relative to Rht-B1c (dwarf parent), and they also had mRNAs that differed in length. It was also shown that different-sized mRNAs in some cases resulted in a change of the open reading frame. qPCR showed that splicing site mutants had reduced expression levels of the gene, similar to Rht-B1c. However, some mutants had longer stem length (about 84% of the tall parent) which is difficult to reconcile with the similar gene expression levels of the dwarf parent (dwarf stem length is 42% of tall isoline). Our results suggested that altered Rht-B1ctranscripts produce DELLA proteins with less activity compared to the wild type, which in turn had less effect on growth repression. Overgrowth alleles enhance growth due to changes in DELLA proteins, one of the key components of the GA signalling pathway. These mutations might result in DELLA proteins with reduced activity in terms of their physical interactions with other proteins important in growth responses. They are also potentially useful in barley and wheat breeding, offering a range of semi-dwarf plants with characteristics important in breeding such as grain size (in barley) and improved dormancy (in wheat).


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