The indole-3-acetic acid (IAA) content of developing grains of Oryza sativa subsp. japonica was measured by combined liquid chromatography, tandem mass spectrometry in multiple-reaction-monitoring mode. The increase from 50 ng g(-1) fresh weight to 2.9 µg g(-1) fresh weight from 1 to 14 days after pollination was much larger than that previously reported by enzyme-linked immunoassay methods. The largest increase in IAA content coincided with the start of the major starch deposition phase of grain-fill. The increase in IAA content was strongly correlated with the expression of putative IAA biosynthesis genes, OsYUC9, OsYUC11 and OsTAR1, measured by quantitative reverse transcriptase polymerase chain reaction. These results confirm the importance of the tryptophan aminotransferase/YUCCA pathway in this system. All three genes were expressed in endosperm; expression of OsYUC11 appeared to be confined to endosperm tissue. Phylogenetic analysis indicated that OsYUC11 and AtYUC10 belong to a separate clade of YUCCAs, which do not have orthologues outside the Angiosperms. This clade may have evolved with a specific role in endosperm. Expression of tryptophan decarboxylase in developing rice grains did not correlate with IAA levels, indicating that tryptamine is unlikely to be important for IAA synthesis in this system. In light of these observations, we hypothesize that IAA production in developing rice grains is controlled via expression of OsTAR1, OsYUC9, OsYUC11 and that IAA may be important during starch deposition in addition to its previously suggested role early in grain development.

Indole-3-acetic acid (IAA) and its putative precursors, L- and D-tryptophan, indole-3-pyruvate, and tryptamine were isolated from tomato (Lycopersicon esculentum (L.) Mill.) shoots, identified by mass spectrometry, and measured using capillary gas chromatography with an electron capture detector and radioactive internal standards. Average amounts present were 7.9ng · (g FW)(--1) IAA, 5.7ng · (g FW)(--1) indole-3-pyruvate, 132 ng · (g FW)(--1) tryptamine, 103 ng · (g FW)(--1) D-tryptophan, and 2250 ng · (g FW)(--1) L-tryptophan. Indole-3-acetaldoxime was not found; detection limits were less than 1ng · (g FW)(--1). When tomato shoots were incubated for 6, 10 and 21 h in 30% (-2)H2O, up to four positions in IAA, L- and D-tryptophan, tryptamine and indole-3-pyruvate became labelled with (-2)H. Compounds became labelled rapidly with 10% of IAA molecules containing (-2)H after 6 h. The percentage of labelled molecules of IAA and L-tryptophan increased up to 10 h but then decreased again, correlating with an increase in the total shoot tryptophan and presumably a result of protein hydrolysis in the excised, slowly senescing tissue. The amount of (-2)H in D-tryptophan also showed an increase followed by a decrease, but the proportion of labelled molecules was much less than in L-tryptophan and IAA. Tryptamine became labelled initially at a similar rate to IAA but continued to accumulate (-2)H up to 21 h. We conclude that tryptamine is synthesized from a different pool of tryptophan from that used in IAA synthesis, and is not a major endogenous precursor of IAA in tomato shoots. Indole-3-pyruvate was the most heavily labelled compound after 6 and 10 h incubation (21-h data not available). Furthermore, the proportion of (-2)H-labelled indole-3-pyruvate molecules was quantitatively consistent with the amount of label in IAA. On the other hand, a quantitative comparison of the IAA turnover rate and the rate of (-2)H incorporation into both L- and D-tryptophan indicates that IAA is not made from the total shoot pool of either L- or D-tryptophan. Instead IAA appears to be synthesized from a restricted pool which is turning over rapidly and which has access to both newly synthesized tryptophan and that from protein hydrolysis.

The response of Sclerotinia sclerotiorum , the causal agent of stem rot of oilseed rape ( Brassica napus ), to toxic volatiles produced by the glucosinolate-myrosinase system was studied. Mycelium plugs were exposed to inoculated leaf discs of oilseed rape cultivars and two related species, black mustard ( Brassica nigra ) and white mustard ( Sinapis alba ). Growth of exposed colonies was inhibited by more than 87% compared with controls. Despite inhibition of exposed fungal colonies, the fungus continued to grow in infected tissue. Repeated exposure of the fungus to hydrated mustard powder (which contains both glucosinolates and myrosinase) or synthetic isothiocyanates (ITCs) resulted in growth inhibition decreasing from initial levels of up to 80% to insignificant levels after 2-3 days, suggesting that S. sclerotiorum has the ability to adapt to volatiles during the infection progress. This adaptation was studied by investigating induction of glutathione S-transferase-like genes identified from the S. sclerotiorum genome. Three genes, with locus numbers SS1G_07195.1, SS1G_01918.1 and SS1G_10295.1, appeared to be up-regulated following exposure of S. sclerotiorum to mustard powder or allyl ITC. A fourth gene, SS1G_07319.1, appeared to be down-regulated. In addition, glutathione S-transferase catalytic activity in crude mycelium extracts was doubled following 48 h of exposure to mustard powder volatiles. This adaptation could allow S. sclerotiorum to parasitize tissues of Brassica species despite the production of toxic metabolites.

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 xprG Δ null 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.

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