Arabidopsis thaliana GAMT1 and GAMT2 encode enzymes that catalyze formation of the methyl esters of gibberellins (GAs). Ectopic expression of GAMT1 or GAMT2 in Arabidopsis, tobacco (Nicotiana tabacum), and petunia (Petunia hybrida) resulted in plants with GA deficiency and typical GA deficiency phenotypes, such as dwarfism and reduced fertility. GAMT1 and GAMT2 are both expressed mainly in whole siliques (including seeds), with peak transcript levels from the middle until the end of silique development. Within whole siliques, GAMT2 was previously shown to be expressed mostly in developing seeds, and we show here that GAMT1 expression is also localized mostly to seed, suggesting a role in seed development. Siliques of null single GAMT1 and GAMT2 mutants accumulated high levels of various GAs, with particularly high levels of GA 1 in the double mutant. Methylated GAs were not detected in wild-type siliques, suggesting that methylation of GAs by GAMT1 and GAMT2 serves to deactivate GAs and initiate their degradation as the seeds mature. Seeds of homozygous GAMT1 and GAMT2 null mutants showed reduced inhibition of germination, compared with the wild type, when placed on plates containing the GA biosynthesis inhibitor ancymidol, with the double mutant showing the least inhibition. These results suggest that the mature mutant seeds contained higher levels of active GAs than wild-type seeds.

In plants, benzoic acid (BA) is believed to be synthesized from Phe through shortening of the propyl side chain by two carbons. It is hypothesized that this chain shortening occurs via either a β-oxidative or non-β-oxidative pathway. Previous in vivo isotope labeling and metabolic flux analysis of the benzenoid network in petunia (Petunia hybrida) flowers revealed that both pathways yield benzenoid compounds and that benzylbenzoate is an intermediate between l-Phe and BA. To test this hypothesis, we generated transgenic petunia plants in which the expression of BPBT, the gene encoding the enzyme that uses benzoyl-CoA and benzyl alcohol to make benzylbenzoate, was reduced or eliminated. Elimination of benzylbenzoate formation decreased the endogenous pool of BA and methylbenzoate emission but increased emission of benzyl alcohol and benzylaldehyde, confirming the contribution of benzylbenzoate to BA formation. Labeling experiments with 2H5-Phe revealed a dilution of isotopic abundance in most measured compounds in the dark, suggesting an alternative pathway from a precursor other than Phe, possibly phenylpyruvate. Suppression of BPBT activity also affected the overall morphology of petunia plants, resulting in larger flowers and leaves, thicker stems, and longer internodes, which was consistent with the increased auxin transport in transgenic plants. This suggests that BPBT is involved in metabolic processes in vegetative tissues as well.

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta.

http://www.daylab.plp.msu.edu/pseudoperonospora-cubensis/ (Day Laboratory website with research advances in downy mildew); http://veggies.msu.edu/ (Hausbeck Laboratory website with downy mildew news for growers); http://cdm.ipmpipe.org/ (Cucurbit downy mildew forecasting homepage); http://ipm.msu.edu/downymildew.htm (Downy mildew information for Michigan's vegetable growers).

To elucidate the genetic and biochemical regulation of elicitor-induced p-coumaraldehyde accumulation in plants, we undertook a multifaceted approach to characterize the metabolic flux through the phenylpropanoid pathway via the characterization and chemical analysis of the metabolites in the p-coumaryl, coniferyl, and sinapyl alcohol branches of this pathway. Here, we report the identification and characterization of four cinnamyl alcohol dehydrogenases (CADs) from cucumber (Cucumis sativus) with low activity toward p-coumaraldehyde yet exhibiting significant activity toward other phenylpropanoid hydroxycinnamaldehydes. As part of this analysis, we identified and characterized the activity of a hydroxycinnamoyl-coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) capable of utilizing shikimate and p-coumaroyl-coenzyme A to generate p-coumaroyl shikimate. Following pectinase treatment of cucumber, we observed the rapid accumulation of p-coumaraldehyde, likely the result of low aldehyde reductase activity (i.e. alcohol dehydrogenase in the reverse reaction) of CsCAD enzymes on p-coumaraldehyde. In parallel, we noted a concomitant reduction in the activity of CsHCT. Taken together, our findings support the hypothesis that the up-regulation of the phenylpropanoid pathway upon abiotic stress greatly enhances the overall p-coumaryl alcohol branch of the pathway. The data presented here point to a role for CsHCT (as well as, presumably, p-coumarate 3-hydroxylase) as a control point in the regulation of the coniferyl and sinapyl alcohol branches of this pathway. This mechanism represents a potentially evolutionarily conserved process to efficiently and quickly respond to biotic and abiotic stresses in cucurbit plants, resulting in the rapid lignification of affected tissues.

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