The endosperm of cereal grain forms the staple diet for most of the world's population, and feeds much of their stock. Grain size and quality are determined largely by events taking place during coenocytic nuclear division, endosperm cellularisation and cell differentiation, and the production of storage molecules. Thus, understanding the complex signalling processes occurring at each of these steps is essential for maintaining and improving our food supply. Here, we critically review evidence for the effects of phytohormones on grain size, as well as hormone homeostasis, signalling and crosstalk. We focus on rice endosperm due to the importance of rice as a food crop and a model grass, as well as its relative neglect in recent reviews; however, data from other cereals are also discussed due to strong evidence for conserved signalling networks operating during grain development. Discussion is restricted to auxin, cytokinin, ethylene, abscisic acid and gibberellin. Our review highlights the need for accurate hormone determinations combined with information on gene expression. We present evidence for separate, localised signalling roles for auxin at different stages of grain development and highlight key research questions for other hormones where much less data are available.

The glucosinolate-myrosinase defence system, specific to Brassicales plants, produces toxic volatile compounds during mechanical injury or pathogen attack. The reaction of this system to oxalic acid, known as a pathogenicity factor of 'Sclerotinia sclerotiorum', is not fully understood. The hydrolysis of glucosinolates was studied at varying conditions in the presence of oxalic acid in the substrate. In a bioassay, colonies of the pathogen were exposed to volatiles from hydrated mustard powder used as a myrosinase and glucosinolate source. The glucosinolate-myrosinase (GSL-M) system was activated in the presence of oxalic acid at a concentration and pH similar to that expected in vivo. Volatile production was inhibited only when the pH fell to 3 or below. It is unlikely that oxalic acid plays a significant role in disarming the GSL-M system during infection of 'Brassica' hosts.

Oilseed rape stem rot disease caused by 'Sclerotinia sclerotiorum' causes serious yield losses worldwide. Glucosinolates as specific secondary metabolites of 'Brassicaceae' are produced in various parts of the host plants. Their enzymatic hydrolysis releases chemical components, particularly isothiocyanates, with fungitoxic activity and volatile characteristics. To investigate the effect of volatiles derived from 'Brassica' tissues, the pathogen was exposed to hydrolysis products of 'Brassica' shoot parts as sources of glucosinolates including oilseed rape varieties and two species, black and white mustard. The results showed significant differences in inhibition of 'S. sclerotiorum' growth between varieties and species. All tissues of black mustard inhibited completely the exposed colonies of the pathogen and oilseed rape varieties Dunkeld, Oscar and Rainbow had significant inhibitory effect on the fungus. The genotypes demonstrated significant differences for the production of toxic volatiles, indicating that GSL contents in Brassica species and even cultivars have different potentials for toxic products.

Current understanding of the role of plant hormones during cereal grain filling is confounded by contradictory reports on hormone production that is based on poor methodology. We report here on the accurate measurement of indole-3-acetic acid (IAA) and abscisic acid (ABA) by combined liquid chromatography-tandem mass spectrometry in multiple reaction-monitoring mode with heavy isotope labelled internal standards. ABA and IAA contents of superior versus inferior rice grains (ABA maxima 159 ng g^–1 FW and 109 ng g^–1 FW, IAA maxima 2 µg g^–1 FW and 1.7 µg g^–1 FW respectively) correlated with the expression of biosynthetic genes and with grain fill. Results confirm that grain ABA is produced primarily by OsNCED2(5), but suggest that ABA import and metabolism also play important roles in ABA regulation. The IAA content of grains is primarily influenced by OsYUC9 and OsYUC11. However, the distinct expression profile of OsYUC12 suggests a specific role for IAA produced by this enzyme. Co-expression of OsYUC12 with OsIAA29 indicates their involvement in a common signalling pathway. Co-expression and cis-element analysis identified several aleurone-specific transcriptional regulators as well as glutelin as strong candidates for detailed investigation for direct regulation by the auxin-signalling pathway.

The glucosinolate-myrosinase (GSL-M) system in oilseed rape and other members of the family Brassicaceae produces toxic products which can limit fungal pathogen attacks on the host. The role of this system in resistance of oilseed rape to 'Sclerotinia sclerotiorum', causal agent of stem rot, was investigated. Mustard powder was used as a GSL and myrosinase source in bioassays. The effect of toxic volatiles derived from hydrolysis of glucosinolates was observed as inhibition of fungal growth. Oxalic acid, a pathogenicity factor of the pathogen, did not affect production of toxic volatiles and inhibition occurred only at very acidic pH levels, regardless of the presence of oxalic acid. This indicated that oxalic acid at physiological concentrations and pH did not affect the GSL-M defense system. Exposure of 'S. sclerotiorum' colonies to inoculated leaves or leaf discs of host species or cultivars revealed that volatiles derived from infected leaf tissues have a toxic effect. This suggested that the GSL-M system is activated during infection of leaves and disease development. Freeze-dried powders of shoot parts of brassica species and cultivars including leaf, petiole, and stem demonstrated significant differences in producing toxic volatiles through their inhibitory effects on 'S. sclerotiorum' mycelial growth 'in vitro', indicating that GSL contents in brassica species and even cultivars have different potentials for toxic products. Tolerance of 'S. sclerotiorum' to toxic volatiles derived from mustard powder and also synthetic isothiocyanates developed during repeated exposure of mycelium to these biocidal chemicals.

The 'Aspergillus nidulans' xprG gene encodes a putative transcriptional activator that is a member of the Ndt80 family in the p53-like superfamily of proteins. Previous studies have shown that XprG controls the production of extracellular proteases in response to starvation. We undertook transcriptional profiling to investigate whether XprG has a wider role as a global regulator of the carbon nutrient stress response. Our microarray data showed that the expression of a large number of genes, including genes involved in secondary metabolism, development, high-affinity glucose uptake and autolysis, were altered in an xprGΔnull mutant. Many of these genes are known to be regulated in response to carbon starvation. We confirmed that sterigmatocystin and penicillin production is reduced in xprG⁻ mutants. The loss of fungal mass and secretion of pigments that accompanies fungal autolysis in response to nutrient depletion was accelerated in an xprG1 gain-of-function mutant and decreased or absent in an xprG⁻ mutant. The results support the hypothesis that XprG plays a major role in the response to carbon limitation and that nutrient sensing may represent one of the ancestral roles for the p53-like superfamily. Disruption of the AN6015 gene, which encodes a second Ndt80-like protein, showed that it is required for sexual reproduction in 'A. nidulans'.

Oilseed rape is economically affected by stem rot caused by 'Sclerotinia sclerotiorum' worldwide. Glucosinolates are the specific secondary metabolites of Brassica plants that appear in different profiles of each species. Their hydrolysis products have biocidal activity and may play a role in resistance against plant pathogenic fungi. The resistance of oilseed rape ('Brassica napus') cultivars and two other Brassica species ('B. nigra' and 'Sinapis alba') was evaluated employing leaf disc inoculation, and oxalic acid and fungal inoculums on leaves of intact plants under controlled conditions. By using leaf disc inoculation, three plant ages were used to compare their reactions against the pathogen. No significant differences between genotypes were observed in this method. However, results demonstrated significant differences in main effects of wounding and plant age. The two intact plant inoculation techniques (oxalic acid and fungal mycelium) resulted in significant differences between genotypes in reaction to the disease. Furthermore, the oxalic acid assay followed the same pattern as fungal inoculations. Among the oilseed rape cultivars, AV-Sapphire and AG-Castle were the most resistant and susceptible genotypes, respectively. Brassica species differed significantly in their reaction to disease, in both wounded and non-wounded leaves with fungal mycelium inoculation and oxalic acid. Overall, non-significant differences between Brassica genotypes showed the unreliability of the leaf disc assay, whereas leaf inoculation of intact plants by means of either oxalic acid or fungal mycelium demonstrated significant differences in lesion size among Brassica cultivars and species.

The importance of flavin monooxygenases (OsYUCCA), tryptophan decarboxylases (OsTDC), nitrilases (OsNIT), and aldehyde oxidases (OsAO) for auxin production in developing rice grains was investigated. Indole-3-acetic acid (IAA) levels in grains increased from approximately 20ng/g FW to 2 μg/g FW during 14 days after flowering (DAF), with the largest increase in IAA (4 and 7 days DAF) correlating with the major gain in grain fresh weight. The rice genome was found to contain 14 OsYUCCA, 7 OsTDC, 4 OsAO, and 2 OsNIT genes. Phylogenetic analysis showed that OsTDC1 has orthologues across the plant kingdom. OsTDC1 was expressed in developing grains at 1, 7, and 21 DAF, however quantitative RT-PCR analysis did not show a clear correlation between OsTDC1 expression and IAA synthesis. Phylogenetic analysis of OsYUCCAs classified OsYUCCAs 9-14, in the same clade as AtYUCCA10 and AtYUCCA11, which are reported to be involved in seed development. A strong correlation between expression of OsYUCCAs 9 & 11 and IAA content suggested these genes are crucial for IAA synthesis in rice grains. Phylogenetic analysis of AOs suggested that major plant groups inherited one AO sequence with isoforms being products of recent gene duplication. This was surprising as AOs have proposed involvement in both abscisic acid and IAA synthesis. Expression analysis and sequencing showed that of four rice AOs, two (OsAO1 and OsAO2) were expressed in grains at 1, 7, and 21 DAF. OsNIT1 and OsNIT2 are part of a conserved clade with members from a diverse group of plants. RT-PCR results as well as on-line microarray data showed expression of OsNITs in grains at 1, 7, and 21 DAF. Despite evidence of expression there was no clear correlation observed between AO or NIT transcripts and IAA content. Data thus provided strong evidence for the involvement of YUCCA in auxin synthesis in developing grains, but only weak evidence for involvement of TDC, AO and NIT. The observed correlation between expression of tryptophan amino transferase (OsTAA1), and OsYUCCA 9 & 11 however suggested that an alternative pathway which involves both YUCCA and TAA should be considered. This would require a different catalytic activity for YUCCA than that previously proposed.

Rice endosperm feeds more than half of the world's population. Its development is a complex process with multiple layers of regulation. The molecular events occurring during its early stages of development determine largely the final grain size and weight, which are key components of grain yield. A large and rapid increase in the content of indole-3-acetic acid (IAA), the main in planta auxin, occurs during early endosperm development. However, auxin action in early endosperm is poorly understood. In this study, I aimed to explore spatio-temporal expression of IAA biosynthesis and signalling genes during early stages of endosperm development in rice. My other aim was to assess the effects of IAA on grain fill and to determine the auxin-response genes in early grains. I found that OsYUC12, one of the three grain-specific OsYUCCAs, was expressed in the aleurone, sub-aleurone and embryo at 3-8 days after pollination (DAP), suggesting early aleurone, sub-aleurone and embryo as sites of IAA biosynthesis. The non-canonical OsIAA29 was strongly co-expressed with OsYUC12. Its spatial expression was restricted to dorsal aleurone, where it could be part of an auxin signalling pathway. OsIAA29 has orthologues only in cereal and non-cereal grass species; expression of the orthologues is early grain- or endosperm-specific. This gene may have evolved independently in the grass family (Poaceae) and acquired conserved functions related to aleurone development. Furthermore, a cluster of MYB transcription factors orthologous to maize Myb-Related Protein-1 (ZmMRP-1) may act downstream of the auxin signalling in dorsal aleurone and regulate its differentiation in relation to apoplastic nutrient transfer. Taking my cue from IAA biosynthesis and signalling, I used a chemical approach to determine its function in early rice grains. Application of two IAA biosynthesis inhibitors, Lkynurenine and 4-phenoxyphenylboronic acid (PPBo), to rice spikelets from 3 to 10 DAP reduced significantly grain IAA levels. Genes encoding small cysteine-rich peptides, seed storage proteins and amino acid transporters were down-regulated in auxin-deficient grains at 5 DAP. These genes are expressed specifically in the aleurone, sub-aleurone and embryo. Auxin may also regulate homeostasis of ethylene, cytokinins, gibberellins and abscisic acid in early grains. Inhibitor-treated panicles showed extensive post-fertilisation seed abortion, which consequently led to significant reduction in their total weight at maturity. This suggested a crucial role of auxin in grain retention, probably through regulation of coenocyte cellularisation. Furthermore, I showed short-lived expression of three OsARFs (OsARF13, OsARF14 and OsARF16) and two OsAUX/IAAs (OsIAA8 and OsIAA14) in early grains at 2-6 DAP. OsARF16 is most likely to interact with OsIAA8 and OsIAA14 in early endosperm. Thus, the results from this study suggested auxin action during key events of early endosperm development such as cellularisation of coenocyte nuclei, early aleurone differentiation and initiation of starch and storage protein biosynthesis, which take place at 3-7 DAP. These findings will point towards new areas of research that will further our understanding of the role of auxin in cereal endosperm development.