Grain size (GS) and grain weight (GW) are two key components of cereal yield that have been the subject of extensive research. Several candidateGS and GW genes are associated with quantitative trait loci (QTL) for grain weight. However, it is important to validate the precise roles of these genes. THOUSAND-GRAIN WEIGHT 6 (TGW6) is one such gene found in both rice (Oryza sativa) and wheat (Triticum aestivum). Inactive TGW6 alleles were reported to result in lower levels of the plant hormone, indole-3-acetic acid (IAA) and higher grain weight. The active allele was proposed to encode an IAA-glucose (IAA-Glc) hydrolase. Conversely, IAA biosynthesis mutants of rice (tsg1) and maize (de18) with reduced IAA levels have small or defective grains. Furthermore, most IAA in cereals is produced from tryptophan via tryptophan aminotransferase (TAR) and indole-3-pyruvate monooxygenase (YUCCA). The TGW6 work overlooked this source of IAA although TAR and YUCCA genes are also expressed in wheat grains. My study aimed to investigate whether TGW6 is the main source of IAA in developing wheat grains and whether inhibiting IAA production can increase grain size. I examined the expression of all genes contributing to IAA production during grain fill and compared this with grain IAA content. I also investigated the availability of IAA-Glc in grains to act as a substrate for TaTGW6. Lastly, a pilot experiment investigated whether inhibition of IAA biosynthesis has a positive or negative effect on grain yield. Expression of TaTAR2-B3, TaYUC9-1 and TaYUC10 increased 7–52 fold from 5 to 15 days after anthesis (DAA) correlating with a 30-fold increase in grain IAA content over the same period. On the other hand, TaTGW6 expression was not detected in grains. This was confirmed using published RNA-sequencing data, which showed TaTGW6 and OsTGW6 are both expressed in the inflorescence. In addition, TGW6 in cereals are part of a large protein family; TaTGW6 has eight homologues with over 80% amino acid identity. Finally, inhibition of IAA biosynthesis and IAA action had a negative effect on spike yield, but this was primarily due to increased grain abortion rather than an effect on grain size. My results show that TaTGW6 is unlikely to have any effect on the IAA content of grains or on grain size. On the other hand, I demonstrate that IAA is likely to have a positive effect on grain yield and primarily is produced from TAR/YUCCA pathway in developing wheat grains.

Plant disease management is key to sustainable production of staple food crops. Calcium (Ca²⁺) signal and phytohormones play critical roles in regulating plant defense responses against pathogens. The Ca²⁺ signals are sensed, decoded and transduced by calmodulin and other Ca²⁺ -binding proteins, followed by interaction with and modulation of activities of target proteins such as calmodulin-binding proteins (CBPs). Members of the Arabidopsis CBP60 gene family, AtCBP60g and AtSARD1, have emerged as major regulators of immune responses. In this study, we identified a 15 member CBP60 gene family in rice (Oryza sativa) of which OsCBP60g-3, OsCBP60g-4, OsCBP60a and OsSARD-like1 genes were consistently upregulated in rice seedlings in response to infection with both fungal (Magnaporthe oryzae) and bacterial (Xanthomonas oryzae) pathogens as well as by salicylic acid (SA). OsCBP60g-4 and OsCBP60g-3 were induced maximally by SA and brassinosteroid (BR), respectively, and OsCBP60g-4 was expressed at 3-fold higher levels in the M. oryzae resistant rice genotype (IC-346004) as compared to the susceptible rice genotype (Rajendra Kasturi). The considerable expansion of the immunity clade and the up-regulation of several OsCBP60 genes in response to pathogens and defense hormones supports the importance of further investigating OsCBP60 genes as targets for increasing disease resistance in rice.

Brassinosteroids (BRs) are plant steroid hormones that not only play vital roles in plant growth and development, but also in mediating stress responses. A group of calmodulin-binding proteins, known as CBP60s are also involved in mediating the response of plants to stress. The aims of the present study were: (1) to investigate the effect of exogenous 24-epibrassinolide (EBR) on the phenotype of cotton (Gossypium hirsutum) seedlings under mild to moderate biotic and abiotic stresses, (2) to find and characterise cotton CBP60-encoding genes, orthologous to Arabidopsis CBP60s with known involvement in stress responses, and to investigate whether EBR may act by modulating the expression of GhCBP60 genes in cotton leaf tissue under salt stress. Experiments were designed to demonstrate the effects of EBR application from 0.1 to 2 µM on the phenotypic responses of cotton seedlings to mild/moderate salt, drought and pathogen (Verticillium dahliae) stresses. Results show that the exogenous application of EBR at low concentrations of 0.1 and 0.2 µM had no positive effect on seedling growth under all stresses. In addition, EBR at a higher concentration (0.5 µM) or with the surfactant Tween 20 caused toxic effects. Bioinformatics approaches revealed the presence of GhCBP60 orthologues of AtCBP60. Phylogenetic analysis indicated that CBP60a, CBP60g, andSARD1 from Arabidopsis each have four co-orthologues in cotton. AtCBP60f has two coorthologues, whereas CBP60b/c/d have nine co-orthologues. Multiple amino acid sequence alignments indicate that the DNA-binding and CaM-binding domains of AtCBP60 are highly conserved in GhCBP60, suggesting similar protein structures to AtCBP60. Prediction of subcellular localisation suggested that all GhCBP60 proteins contain a nuclear localisation signal. This, together with the highly conserved putative DNA binding region, suggests that all GhCBP60 are transcription factors. The results of qRT-PCR demonstrated that EBR treatment of cotton up-regulated the expression of GhCBP60a/f/g. On the other hand, salt down-regulated the expression of GhCBP60a but up-regulated the expression of GhCBP60f/g. Interestingly, treatment with EBR in the absence of salt restored the expression of GhCBP60a to levels similar to the control tissue. Analysis of promoters of GhCBP60 genes for putative BR-related transcription factor binding motifs indicated the presence of CANNTG and GGTCC elements. However, these were not significantly enriched in stress-regulated genes. Furthermore, higher stringency BR-signalling-related elements: BRRE (CGTGTG/CGTGCG), G-box (CACGTG) and transcription factors TGA 1/TGA4 (TGACG) sense strands were absent in stress responsive genes GhCBP60a/f/g and GhSARD1 as compared to other groups. In the light of these results, I concluded that brassinosteroids (BRs) positively regulates the expression of novel GhCBP60 genes suggesting a possible connection between BR signalling and GhCBP60 transcription factors in mediating abiotic stress responses in cotton. However, the results from the cis-element search suggest that this connection is likely to be indirect rather than via a direct interaction with the BR signal transduction pathway.

Teff (Eragrostis tef) is a small grain, highly nutritious, low-risk, and warm-season annual cereal crop. Small grain size is the most important problem for teff production next to lodging. Because of this, teff experiences high post-harvest loss. Therefore, the objective of this project is to investigate the use of plant growth regulators (PGRs) to increase the grain size of teff directly and indirectly. The ethylene action inhibitor, 1-Methylcyclopropene (1-MCP) for direct effects on grain size, synthetic strigolactone (GR24) and synthetic auxin 1- Naphthaleneacetic acid (NAA) for indirect effects on grain size via the reduction in tillering. Teff is a semi-domesticated crop that produces an excessive number of tillers, with flowering and grain maturation occurring over an extended period. I demonstrated that application of PGRs at the appropriate time relative to panicle or plant growth stage, concentration, frequency, and interval increased grain size and thousand-grain weight (TGW). 1-MCP treatment of panicles increased grain size and TGW by 22% and 29%, and 10% and 16% with and without water stress, respectively. The effect was higher under moderate water stress. Unfortunately, to be effective, I had to treat individual panicles and treatment of whole plants was not effective. This is likely to be because of the phenology of teff flowering over an extended period (13-23 days). The difficulty of using 1-MCP at the prolonged flowering led me to look at a different strategy. Reducing tillering by GR24 and NAA treatment increase grain size and TGW by 12% and 8% and TGW by 20% and 14%, respectively. This effect is comparable with 1-MCP under normal growth conditions. Reducing tillering has also a small but positive effect on whole plant grain yield and harvest index. This is because the later tillers tend to have very small grains, with fewer tillers the plant is putting its resources into the earliest and most productive tillers/panicles. The reduction in tiller number has additional benefits like reducing lodging and seed shattering by making uniform panicle maturation. This shows targeting tillering could bring multiple trait improvements in teff. To obtain a major effect on grain size, I had to treat plants from an early stage (2-4 tillers) and for an extended period (8 times at 4-day intervals). This could make the treatment of plants in the field impractical. The positive effect of tiller reduction on grain size and TGW indicates that refocusing breeding strategies to reduce tillering or direct genetic manipulation to increase SL production would have a major positive effect on teff production. Understanding the molecular mechanism of tiller inhibition is vital to reduce tiller number via GR24 treatment, but not a lot is known in cereals, particularly in teff. To address this, I did RNA-seq upon GR24 treatment. I also investigate SL-responsive genes and other genes that are differentially expressed in actively growing tiller buds from other plants. I did not find any differential expressed genes (DEGs) 24 hours upon GR24 treatment, but I found several potential teff orthologues of plant hormone-related genes, cell cycle, sugar, transcription factor, and other genes involved in diverse functions. Cytokinin signalling and perception, auxin signalling, cell cycle, sugar, and other genes involved in diverse functions were highly expressed in growing tiller buds of teff. Particularly, HK4.2, AHP1.1, AHP1.2, IAA11, IAA30, IAA31, ARF3, ARF7.1, ARF7.2, ARF17.3, ARF21, His4, PCNA, PFPA1, Et_s981-0.21-1.path1, CTP synthase, DRM1, TPSS11, and SVP/AGL22 were expressed at high levels. The high expression of ARF7, DRM1, SVP/AGL22, and TPSS11 in growing tiller buds was reported for the first time in cereal crops. In contrast, SL transcription factor (GT1), most Arabidopsis SL responsive genes (except VLG), CK catabolism (CKX1/CKX2/CKX4/CKX9.1), transcription factor, auxin-responsive, sucrose starvation inducible (ASN1/DIN6) genes were expressed at low levels. The expression of VLG was identified for the first time in tiller buds. Exceptionally, RR4.2/RR9.2, GT1, PAP1/IAA26/IAA18, and CKX1/CKX2/CKX4/CKX9.1 genes were expressed in growing tiller buds similarly in more than three species including teff, suggesting these genes would be the primary target genes for tiller inhibition in teff. Therefore, GT1, IPA1, RR4.2/RR9.2, HK4.2, AHP1.1, AHP1.2, CKX1/CKX2/CKX4/CKX9.1, IAA11, IAA30, IAA31, ARF3, ARF7.1, ARF7.2, ARF17.3, ARF21, and PAP1/IAA26/IAA18 would be the main target genes for tiller inhibition in teff as these genes were reported functionally involved in tillering in other species. Thus, downregulating the expression of the highly expressed genes in growing tiller buds might inhibit tiller bud outgrowth. On the other hand, upregulated genes expressed at low levels in teff tiller buds might also inhibit tillering. Bud transcriptome analysis is the first study in teff and this study will be a base for teff breeders as there is a knowledge gap in a molecular study in teff particularly in tiller inhibition. Although treatment of teff in the field with 1-MCP, GR24, and NAA is unlikely to be effective to improve grain size, I have shown that research focusing on altering endogenous ethylene, SL, and auxin production or response is likely to have a positive effect on grain size and TGW with positive side benefits of reduced lodging, fewer late-flowering tillers of low productivity and a shorter season. Such plants would have major benefits both to poor farmers in Ethiopia as well as making teff more attractive to farmers outside Ethiopia seeking to diversify into specialist high-value crops.

The effect of auxin on wheat (Triticum aestivum L.) grain size is contentious. Additionally, the contributions to the IAA pool from de novo synthesis versus hydrolysis of IAA-glucose are unclear. Here, we describe the first comprehensive study of tryptophan aminotransferase and indole-3-pyruvate mono-oxygenase expression from 5 to 20 days after anthesis. A comparison of expression data with measurements of endogenous IAA via combined liquid chromatography–tandem mass spectrometry using heavy isotope labelled internal standards indicates that TaTAR2-B3, TaYUC9-A1, TaYUC9-B, TaYUC9-D1, TaYUC10-A and TaYUC10-D are primarily responsible for IAA production in developing grains. Furthermore, these genes are expressed specifically in developing grains, like those found in rice (Oryza sativa L.) and maize (Zea mays L.). Our results cast doubt on the proposed role of THOUSAND-GRAIN WEIGHT gene, TaTGW6, in promoting larger grain size via negative effects on grain IAA content. Work on this gene overlooked the contribution of IAA biosynthesis from tryptophan. Although IAA synthesis occurs primarily in the endosperm, we show the TaYUC9-1 group is also strongly expressed in the embryo. Within the endosperm, TaYUC9-1 expression is highest in aleurone and transfer cells, suggesting that IAA has a key role in differentiation of these tissues as has been proposed for other cereals.