Grubb CD, Zipp
J, Masuno MN, Molinski TF, Abel S (2005) Arabidopsis glucosyltransferase
UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis.
Plant J 40:893-908
Ebeler SE, Dingley
KH, Ubick E, Abel S, Mitchell AE, Burns SA, Steinberg FM, Clifford AJ (2005)
models and analytical approaches for understanding the relationships between
wine and cancer.
Drugs Exptl Clin Res 31:19-27
Dingley KH, Ubick
EA, Chiarappa-Zucca ML, Nowell S, Abel S, Ebeler SE, Mitchell AE, Burns
SA, Steinberg FM, AJ
Clifford (2003) Effect of dietary constituents with chemopreventive potential
on adduct formation of a low dose of the heterocyclic amines PhIP and IQ
and Phase II hepatic enzymes.
Nutr & Cancer 46:212-221
Li G, GS Goyal,
S Abel, Quiros CF (2001) Inheritance of three major genes involved in the
aliphatic glucosinolates in Brassica oleracea.
J Amer Soc Hort Sci 126:427-431
Our research on glucosinolate metabolism has been funded by the U.S. Department of Agriculture and the National Science Foundation.
Glucosinolates are a diverse class of plant-specific secondary metabolites synthesized mainly by cruciferous plants, including nutritionally important Brassica crops and the reference species Arabidopsis thaliana. Glucosinolate breakdown products exert a variety of activities and functions in plants, animals, and humans, which have been implicated in plant defense and chemical communication, in auxin homeostasis, as well as in the prevention of certain human cancers. Although the biosynthetic pathway is well understood in Arabidopsis, little is known about its regulation during plant development and in response to environmental cues (Grubb and Abel, 2006). We have taken a multifaceted approach to elucidating glucosinolate biosynthesis and its control in Arabidopsis. Functionally characterized genes related to glucosinolate metabolism will provide the tools to (i) understand the biological functions of glucosinolates in cruciferous plants, (ii) modify glucosinolate profiles for crop protection against pathogens and insects, and (iii) generate cruciferous crops with optimal content of cancer-preventive glucosinolates by molecular breeding or metabolic engineering.
1. Modifiers of Glucosinolate
To identify regulatory proteins of glucosinolate accumulation, we have taken a molecular genetics approach in Arabidopsis. Based on the anticarcinogenic properties of glucosinolate-derived isothiocyanates (Dingley et al., 2003), we previously developed a novel high-throughput bioassay in cultured mouse hepatoma cell cultures to screen for mutations that alter glucosinolate content and composition (gcc mutants) in A. thaliana (Gross et al., 2000; Grubb et al., 2002; Wang et al., 2002). Using a single leaf disk assay, we screened T-DNA activation tagged lines and isolated to date a number of lines with heritable gcc chemotypes. A major thrust of current research is the identification of GCC genes by positional cloning (EMS mutants) or plasmid rescue (T-DNA mutants) and the elucidation of the biochemical functions and biological roles of the encoded proteins in glucosinolate metabolism and plant defense.
Role of Calcium Signaling and IQD Proteins
We identified the first gene of the gcc mutant collection, IQD1, which stimulates production of glucosinolates and other defense-related compounds, as well as general-induced plant defense responses against herbivory and infection with necrotrophic fungi (Levy et al., 2005). The encoded protein is localized to the cell nucleus, binds to calmodulin in a calcium-dependent manner, and modulates glucosinolate pathway gene expression. Our data suggest that IQD1 is an RNA-binding protein that sensitizes jasmonate responses. IQD1 is the founding member of a new class of plant-specific calmodulin-binding proteins of unknown functions. We hypothesize that IQD1 and possibly related calmodulin-interacting proteins participate in the decoding of calcium-signitures elicited by abiotic and biotic challenge. IQD1 may integrate early wound- and pathogen/elicitor-induced changes in cytoplasmic calcium concentrations to stimulate and fine-tune an array of coordinated defense responses, including the upregulation of glucosinolate production. Elucidation of IQD1 function will provide an impetus for understanding the roles of calcium-calmodulin signaling and its intersection with plant defense responses.
The hallmark of IQD1 is a plant-specific domain of multiple calmodulin-binding motifs, called the IQ-Domain, which is the defining feature of 33 related proteins in Arabidopsis and of 29 IQD proteins in rice, the largest identifiable families of calmodulin-interacting proteins in plants (Abel et al., 2005). Our phylogenetic analyses suggest that IQD proteins are an ancient family of calmodulin targets and that exon shuffling and retention of paralogous genes, which are indicative of regulatory gene products, played a major role during their evolution. Insertional mutations in more than one-third of the Arabidopsis gene family members cause severe developmental defects. These observations emphasize the importance of IQD proteins, which are likely to link calcium signaling pathways to the regulation of nuclear gene expression in plant development and in response to various environmental cues.
3. The Glucosinolate-Auxin Connection
We previously identified Arabidopsis UGT74B1 as a major UDP-glucose-dependent glucosyltransferase catalyzing the penultimate reaction of the glucosinolate core pathway, i.e. the glucosylation of S-thiohydroximate intermediates (Grubb et al., 2004). Surprisingly, ugt74b1 knockout mutants show several morphological abnormalities consistent with IAA (indole-3-acetic acid or auxin) overproduction. Indeed, ugt74b1 plants have elevated levels of free and conjugated IAA and overexpress the DR5::GUS auxin reporter. Interestingly, several genetic screens for developmental aberrations in Arabidopsis identified mutations in genes of the glucosinolate core pathway that decrease production of tryptophan-derived (indolic) glucosinolates but concomitantly increase IAA accumulation, which points to a metabolic grid connecting biosynthesis of indolic glucosinolates and auxin. The simplest explanation for the high-auxin phenotypes of plants with blocked glucosinolate core pathway reactions is the diversion of excess indole-3-aldoxime (IAOx), a shared intermediate of both glucosinolate and auxin biosynthesis, into the IAA synthesis branch. Although some enzymatic activities that may function in the IAOx-to-IAA conversion have been described, none of the genes have been identified.
IAA is the principle auxin and indispensable for plant growth and development. Auxin biosynthesis is only incompletely understood because an intricate network of multiple biosynthetic pathways ensures biochemical redundancy for developmental plasticity and plant survival. Therefore, conventional forward genetic screens have met limited success in the dissection of auxin biosynthesis. However, screening for loss-of-function mutations in a genetic background that causes auxin overproduction is a promising and less exploited approach. We have established an ugt74b1 suppressor screen and tested its feasibility to identify genes of the IAOx-IAA pathway. Since the suppressor screen scores developmental aberrations caused by auxin overproduction, we expect isolation of various classes of suppressor mutations that reduce auxin accumulation or response by a variety of mechanisms. While genes related to auxin synthesis will be of primary interest, the prospect of identifying genes with novel functions in auxin metabolism and action is intriguing.