Abstract
The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a. Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves. Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive. AtST5a and its close homologs AtST5b and AtST5c were purified as His6-tagged proteins from Escherichia coli. The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs). They accept a broad range of dsGSs as substrates. However, in a competitive situation, AtST5a clearly prefers tryptophan- and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c. Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin. Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo.
Highlights
Compared with the animal cell, very little is known regarding the structural and regulatory roles of the sulfate group in plants
These fall into three subgroups: the flavonol sulfotransferases described for Flaveria species [1], the steroid sulfotransferases identified in Brassica napus [2, 3], and a hydroxyjasmonic acid-specific sulfotransferase from Arabidopsis thaliana [4]
Much interest has been directed to the Brassicaceae family: the genus Brassica alone contains a large number of agriculturally important crops, including many vegetables and one of the most important oilseed crops, oilseed rape (B. napus), the defatted seed meal of which is fed to animals
Summary
Wasternack (Institute of Plant Biochemistry, Halle an der Saale, Germany). 11-Hydroxyjasmonic acid was purified from suspension cultures of Eschscholtzia californica as described by Xia and Zenk [24]. Application of compounds dissolved in 40% (v/v) acetone and 0.1% (v/v) Tween 20 was performed by spraying plant rosettes until leaves were wet. Plant RNA was prepared according to Barkan [28]. CDNA synthesis, PCR amplification, and DNA restriction and ligation followed the manufacturers’ protocols. Contaminating DNA in the RNA preparations was digested with the RQ1 RNase-free DNase (Promega, Mannheim, Germany). This step was critical because the AtST5 genes contain no introns, and amplification could result from genomic DNA.
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