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.

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.

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.