Remobilization of stem WSC is well known to contribute to grain yield in wheat. There is, however, extensive genetic variation in the contribution of stem WSC to grain yield under post-anthesis water-deficit. Fructan 1-exohydrolase (1-FEH) is one of the major enzymes contributing to WSC remobilisation and the maintenance of grain yield under water-deficit. 1-FEH has three isoforms (1-FEH w1, w2 and w3) that degrade β - (2-1) fructan linkages thus contributing to fructan remobilization to grain. This thesis investigated the functional role of the three isoforms of the 1-FEH gene in WSC remobilisation under post anthesis water-deficit. Individual performance of the three isoforms was investigated using the corresponding isoform mutation lines derived from the Australian wheat variety Chara. Results from glasshouse experiments showed that the mutation of isoform 1-FEH w3 slowed down WSC remobilisation under post anthesis water-deficit and reduced grain filling and yield. In contrast, mutations of 1-FEH w1 and w2 did not affect WSC remobilisation under water-deficit. This means that 1-FEH w3 plays the leading functional role in WSC remobilisation during grain filling under water-deficit.
This differences in remobilisation of WSC components between the mutation lines correlated with the expressional differences of the three isoforms of the 1-FEH gene across the lines. In the 1-FEH w3 mutation line, the expression of the other two isoforms (1-FEH w2 and w1) had the same level as the non-mutated parental cultivar Chara. However, in the 1-FEH w2 and w1 mutation lines, 1-FEH w3 showed significantly higher expression compared to Chara. The results indicated that the functional loss of the isoforms 1-FEH w2 and w1 was made up by the higher expression of the isoform 1-FEH w3 but the functional loss of the 1-FEH w3 isoform was not compensated by the other isoforms. This explains the ability of 1-FEH w2 and w1 mutation lines to maintain the same pattern of WSC remobilisation as the non-mutated parental cultivar. It was also, revealed that the expressional differences of the isforms of the 1-FEH gene across different mutation lines significantly influenced the degradation of WSC and its components under post anthesis water-deficit.
Fructan, a fructose-based polymer synthesized from sucrose by fructosyltransferases (FTs), is the main component of wheat stem WSC and is a major source of sugar supply under post anthesis water-deficit when photosynthesis is reduced. Quick degradation of fructan is essential to remobilise sugar to developing grain under water-deficit and this is facilitated by FEHs. The 1-FEH w3 mutation line showed slower degradation and remobilization of fructan compared to the 1-FEH w2 and w1 mutation lines and Chara. This slow degradation made the 1-FEH w3 mutation line partially susceptible to post anthesis water-deficit. Noticeably, differences in WSC component degradation and gene expression of 1-FEH isoforms only became evident under post anthesis water-deficit and not in well-watered plants.
This thesis also characterised the 1-FEH gene mutation, by mapping and annotating the mutated region. The F1 seeds, developed by back crossing the 1-FEH w1, w2 and w3 mutation lines with Chara, were genotyped using the Infinium 90K SNP iSelect platform. Putative deletions were identified in the FEH mutation lines encompassing the FEH genomic regions. A total of 15, 20 and 15SNPs were identified within the mutation regions of 1-FEH w1 w2, and w3, respectively. Mapping analysis demonstrated that the mutation affected significantly longer regions than the target gene regions of 1-FEH w1, w3 and w2. From the annotation of the mutation regions, 8 and 6 non-target genes were discovered on chromosomes 6A and 6B, respectively. The annotation of the 1-FEH w2 mutated region was complicated by the presence of an extra three copies of the gene on chromosome 6D. Functional roles of the non-target genes was carried out following computational biology approaches and confirmed that none of the affected non-target genes were expected to have a direct influence on 1-FEH gene function.
This study also ratified the association of the distinct role of the 1-FEH w3 gene in sugar remobilisation to the developing wheat grain. Accumulation of oligosaccharides at two seed developmental stages were examined in the 1-FEH w3 mutation line in comparison to Chara under well-watered and water-deficit conditions. This study successfully overcome the challenge of preparing 25 μm seed sections by adopting cryosectioning using egg white which provided compatibility with the mass spectrometric equipment and enabled the production of ions from the oligosaccharides by the laser. Hexose and its polymers were detected separately by the mass spectrometry imaging (MSI) without any enzymatic digestion thus providing information regarding the localisation of sugar accumulation within the tissues of developing seeds. The abundance and localisation pattern of the identified oligosaccharides was influenced by the post anthesis water-deficit treatment. Under water-deficit, the mutation of the 1-FEH w3 reduced the abundance of oligosaccharide accumulation in two stages of seed development (17 DAA and 22 DAA) indicating it pivotal role under post anthesis water-deficit. This is the first study to use MSI to explore sugar accumulation directly within the tissue of developing seeds of wheat.
This thesis established the individual role of three isoforms of 1-FEH in remobilising WSC under post anthesis water-deficit and provides unequivocal evidence that 1-FEH w3 is taking the most vital role. This new insight into the distinct role of the 1-FEH gene isoforms under post anthesis water-deficit should assist in providing new gene targets for water-deficit tolerant wheat breeding in the future
