SummaryUbiquitin E3 ligases are a diverse family of protein complexes that mediate the ubiquitination and subsequent proteolytic turnover of proteins in a highly speci®c manner. Among the several classes of ubiquitin E3 ligases, the Skp1-Cullin-F-box (SCF) class is generally comprised of three`core' subunits: Skp1 and Cullin, plus at least one F-box protein (FBP) subunit that imparts speci®city for the ubiquitination of selected target proteins. Recent genetic and biochemical evidence in Arabidopsis thaliana suggests that post-translational turnover of proteins mediated by SCF complexes is important for the regulation of diverse developmental and environmental response pathways. In this report, we extend upon a previous annotation of the Arabidopsis Skp1-like (ASK) and FBP gene families to include the Cullin family of proteins. Analysis of the protein interaction pro®les involving the products of all three gene families suggests a functional distinction between ASK proteins in that selected members of the protein family interact generally while others interact more speci®cally with members of the F-box protein family. Analysis of the interaction of Cullins with FBPs indicates that CUL1 and CUL2, but not CUL3A, persist as components of selected SCF complexes, suggesting some degree of functional specialization for these proteins. Yeast two-hybrid analyses also revealed binary protein interactions between selected members of the FBP family in Arabidopsis. These and related results are discussed in terms of their implications for subunit composition, stoichiometry and functional diversity of SCF complexes in Arabidopsis.

Elicitor-induced sanguinarine accumulation in opium poppy (Papaver somniferum) cell cultures provides a responsive model system to profile modulations in gene transcripts and metabolites related to alkaloid biosynthesis. An annotated expressed sequence tag (EST) database was assembled from 10,224 random clones isolated from an elicitor-treated opium poppy cell culture cDNA library. The most abundant ESTs encoded defense proteins, and enzymes involved in alkaloid metabolism and S-adenosylmethionine-dependent methyl transfer. ESTs corresponding to 40 enzymes involved in the conversion of sucrose to sanguinarine were identified. A corresponding DNA microarray was probed with RNA from cell cultures collected at various time-points after elicitor treatment, and compared with RNA from control cells. Several diverse transcript populations were coordinately induced, with alkaloid biosynthetic enzyme and defense protein transcripts displaying the most rapid and substantial increases. In addition to all known sanguinarine biosynthetic gene transcripts, mRNAs encoding several upstream primary metabolic enzymes were coordinately induced. Fourier transform-ion cyclotron resonance-mass spectrometry was used to characterize the metabolite profiles of control and elicitor-treated cell cultures. Principle component analysis revealed a significant and dynamic separation in the metabolome, represented by 992 independent detected analytes, in response to elicitor treatment. Identified metabolites included sanguinarine, dihydrosanguinarine, and the methoxylated derivatives dihydrochelirubine and chelirubine, and the alkaloid pathway intermediates N-methylcoclaurine, N-methylstylopine, and protopine. Some of the detected analytes showed temporal changes in abundance consistent with modulations in the profiles of alkaloid biosynthetic gene transcripts.

Unacceptably dark bran color has prevented the white-kernelled variety Argent from meeting grain color marketing standards for hard white wheats (Triticum aestivum L.). The objective of this research was to identify phenolic compounds that negatively affect bran color in white wheat using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and vanillin-HCl and NaOH staining methods. In mature bran, FT-ICR-MS detected derivatives of the flavonol quercetin in varieties Argent and RL4137 (red-kernelled wheat) but not in W98616, a white wheat variety with acceptable grain color. Derivatives of the isoflavone formononetin were more abundant in W98616 relative to RL4137 and Argent. Vanillin-HCl staining indicated that RL4137 sequestered high levels of proanthocyanidin (PA) throughout its entire seed coat, whereas white wheats sequestered PAs as discrete speckles. Argent possessed abundant speckles over its entire seed coat, whereas speckles were almost undetectable in W98616. In mature kernels, flavonoids throughout the seed coat of RL4137 reacted with NaOH, but only the speckles appeared to react in white wheats. W98616 consistently had lighter grain than Argent before and after NaOH treatment. Free and bound phenolic differences in bran samples confirmed that the darker seed coat color of Argent, relative to W98616, was likely due to higher total phenolic acid content. Although isoflavones accumulated in Argent and RL4137, it appears that the majority of the flux through the flavonoid pathway ultimately accumulates quercetin derivatives and PAs. In W98616, PAs accumulate, but it appears that flavonoid biosynthesis ultimately accumulates isoflavones. Argent, compared to W98616, generally accumulated higher levels of total phenolics (flavonols, stilbenes, and PAs) within its darker pigmented bran.

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