Transcriptome profiling was conducted to detect genes whose expression is significantly changed in an Arabidopsis mutant deficient in S-adenosylhomocysteine hydrolase1 (SAHH1) during early seedling development when mutant phenotypes could be clearly observed. A total of 2,040 differentially expressed genes were identified, representing approximately 6.7% of the 30,385 DNA oligonucleotide targets on the microarray. Among these differential expressed genes, many were mapped to pathways essential to plant growth and development including those of primary, secondary and hormone metabolisms. A significant proportion of up-regulated genes encoded transposable elements which were mapped to the centromeric and pericentromeric regions of the Arabidopsis chromosomes that were analyzed. A number of down-regulated genes were found to be involved in root hair formation, which might have contributed to the root hair defective phenotype of the mutant. Analysis of genes encoding transposable elements and those associating with root hair development indicated that these genes were highly co-expressed during seedling development. Despite SAHH1 deficiency, the expression of genes encoding methyltransferase remained largely unchanged in the sahh1 mutant. Bisulfite sequencing analysis of the transposable elements and the FWA gene revealed that their sequences in the mutant were deficient of 5-methylcytosines. Analysis of mutant genomic DNA using restriction endonucleases that were unable to cut methylated DNA suggested a genome-wide hypomethylation had occurred in the mutant. These results indicated that SAHH1 plays a critical role in methyl homeostasis, and its deficiency is a major contributing factor to the change of global gene expression, metabolic pathways and activation of transposable elements in the sahh1 mutant.

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Preparing homogeneous UDP-g1ucose:thiohydroximate S-glucosyltransferase (S-CT), the penultimate biosynthetic enzyme of glucosinolates, by standard chromatographic methods has yielded too little protein for adequate purity evaluation, identity verification, and structural analysis. l h e low yields were apparently due to low abundance in source tissues, aggravated by enzyme instability. Here we describe an immunological method for purification of-workable quantities from florets of Brassica oleracea ssp. botrytis (cauliflower). Florets that had undergone browning dueto exposure to sunlight contained higher S-CT activities than are normally found in Brassica tissues. S-CT was adsorbed from crude tissue extracts onto an agarose-monoclonal antibody complex. Elution from the complex required harsh alkaline conditions (pH 11.51, giving extremely variable activity recoveries (maximum 20%). l h e eluate contained two proteins that could be separated readily by preparative polyacrylamide gel electrophoresis or anion-exchange chromatography. The overall S-CT protein recovery was estimated at less than 200 pg/kg of cauliflower tissue. Molecular weight determinations with homogeneous cauliflower S-CT gave relative molecular weight (M,) values of 55,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 57,600 by gel chromatography; isoenzymes with isoelectric point values of 4.80 and 4.95 were identified. A polyclonal antibody raised against denatured enzyme showed broad cross-reactivity in immunoblots with S-CT from a number of Brassica species and other crucifers. l h e monoclonal antibody that was used in the immunopurification was much more specific; it exclusively precipitated S-CT isoenzymes that had their genomic origin in the primary diploids B. oleracea and Brassica campestris. Thus, all of the S-CT was precipitated from the amphidiploid Brassica napus, which is a hybrid of B. oleracea and B. campestris. About half of the S-CT was precipitated from the amphidiploids Brassica carinata and Brassica juncea, which have B. oleracea and B. campestris as one of their parents, respectively. It was shown that the S-CT isoenzymes of B. juncea with M, 55,500 and about 57,000 originate from the parents B. campestris and B. nigra, respectively.GS (sulfated S-glucosyl thiohydroximates) constitute a large group of secondary metabolites occurring in severa1 plant families, including a11 species of the Cruciferae. Their presence in oilseed Brassica crops (Brassica napus and Brassica campestris) has been of great nutritional and therefore economic concern, since the meal fraction is directed to animal feed markets as a protein source. GS have antinutritional properties and cause acute and chronic diseases, particularly * Corresponding author; fax 1-306-975-4839. 425

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