HPLC Separation of Glucosinolates from Leaves and Seeds of Arabidopsis thaliana and Their Identification Using Thermospray Liquid Chramatography/Mass Spectrometry

Hogge, Lawrence; Reed, Darwin W.; Underhill, E. W.; Haughn, George W.

doi:10.1093/chromsci/26.11.551

Cited by 136 publications

(92 citation statements)

References 11 publications

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“…The analysis, in vivo, of individual members of the nitrilase family of A. tha/iana is problematic due to the similarity of the four isoforms, likely metabolic redundancy, multiple potential inputs into the pool of the IAA precursor IAN, and the presence of a large number of glucosinolates (Hogge et al, 1988) Thimann and Mahadevan, 1964). Additionally, levels of IAN in plant tissues are low (Normanly eta/., 1993, and vide infra).…”

Section: Discussionmentioning

confidence: 99%

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme

Schmidt

Müller

Hain³

et al. 1996

The Plant Journal

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Summary Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole‐3‐acetonitrile to the plant growth hormone, indole‐3‐acetic acid in vitro. To probe the capacity of this enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV‐35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg−1 protein with indole3‐acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1‐[13C]‐indole‐3‐acetonitrile to 1‐[13C]‐indole‐3‐acetic acid in vivo as determined by HPLC/ GC‐MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole‐3‐acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole‐3‐acetonitrile, a strong auxin‐overproducing phenotype developed resulting in increased lateral root formation (at 10 µM indole‐3‐acetonitrile) or stunted shoot growth, excessive lateral root initiation, inhibition of root out‐growth and callus formation at the root/shoot interface (at 100 µM indole‐3‐acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole‐3‐acetonitrile to indole‐3‐acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high‐auxin phenotype upon transgenic plants. Thus, the A. thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.

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Section: Discussionmentioning

confidence: 99%

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme

Schmidt

Müller

Hain³

et al. 1996

The Plant Journal

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“…Arabidopsis contains at least 23 different glucosinolates derived from tryptophan and from chain-elongated homologs of phenylalanine and methionine (Hogge et al, 1988). In Arabidopsis, at least six related members of the CYP79 family (http://ag.arizona.edu/p450) have been identified.…”

Section: Glucosinolate Biosynthesismentioning

confidence: 99%

“…The S-alkyl-thiohydroximate formed by CYP83B1 catalysis can be cleaved by a C-S lyase to generate thiohydroximates. It is well established that thiohydroximates are glucosylated by a soluble UDPG:thiohydroximate glucosyltransferase to form desulfoglucosinolates that are subsequently sulfated by a soluble 3Ј-phosphoadenosine 5Ј-phosphosulphate:desulphoglucosinolate sulphotransferase (PAPS transferase) (reviewed in Halkier and Du, 1997).Arabidopsis contains at least 23 different glucosinolates derived from tryptophan and from chain-elongated homologs of phenylalanine and methionine (Hogge et al, 1988). In Arabidopsis, at least six related members of the CYP79 family (http://ag.arizona.edu/p450) have been identified.…”

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confidence: 99%

CYP83B1, a Cytochrome P450 at the Metabolic Branch Point in Auxin and Indole Glucosinolate Biosynthesis in Arabidopsis

et al. 2001

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Auxins are growth regulators involved in virtually all aspects of plant development. However, little is known about how plants synthesize these essential compounds. We propose that the level of indole-3-acetic acid is regulated by the flux of indole-3-acetaldoxime through a cytochrome P450, CYP83B1, to the glucosinolate pathway. A T-DNA insertion in the CYP83B1 gene leads to plants with a phenotype that suggests severe auxin overproduction, whereas CYP83B1 overexpression leads to loss of apical dominance typical of auxin deficit. CYP83B1 N-hydroxylates indole-3-acetaldoxime to the corresponding aci -nitro compound, 1-aci -nitro-2-indolyl-ethane, with a K m of 3 M and a turnover number of 53 min Ϫ 1 . The aci -nitro compound formed reacts non-enzymatically with thiol compounds to produce an N -alkyl-thiohydroximate adduct, the committed precursor of glucosinolates. Thus, indole-3-acetaldoxime is the metabolic branch point between the primary auxin indole-3-acetic acid and indole glucosinolate biosynthesis in Arabidopsis. INTRODUCTIONApical dominance, cell expansion, vascular differentiation, lateral root and root hair formation, phototropism, and root gravitropism are among the many processes in plants controlled by auxins (Davies, 1995). The level of auxin is regulated by both de novo biosynthesis and reversible and irreversible conjugation to sugars, amino acids, and peptides as well as by degradation. Although the chemical structure of the primary auxin, indole-3-acetic acid (IAA), has been known since the 1930s (Wildman, 1997), not much is known about how plants actually synthesize this essential compound. Plants appear to be capable of synthesizing IAA by both tryptophan-dependent and tryptophan-independent pathways. Classical incorporation studies with radiolabeled compounds have not unambiguously identified the precursors or elucidated the biosynthetic pathway for IAA. (For a recent review of IAA metabolism, see Normanly and Bartel [1999].)Although a number of mutants in IAA metabolic pathways and perception have been described, the genes involved and their biochemical function and physiological relevance have not all been elucidated (reviewed in Bartel, 1997;Normanly and Bartel, 1999). For example, both the rty/sur1/hls3/alf1 (Boerjan et al., 1995;Celenza et al., 1995;King et al., 1995;Lehman et al., 1996) and sur2 mutants are known to accumulate increased levels of free auxin. Identification of the gene products affected and elucidation of the biochemical roles of these proteins should increase our limited knowledge of IAA biosynthesis and regulation.A link between indole glucosinolates and auxin has often been suggested in the literature. Glucosinolates are sulfurcontaining bioactive natural products derived from amino acids and sequestered in vacuoles of cruciferous plants (Halkier, 1999). It has recently been shown that the cytochromes CYP79B2 and CYP79B3 of Arabidopsis metabolize tryptophan to indole-3-acetaldoxime. This metabolite is often suggested to be the precursor of indole-3-acetonitrile (IAN...

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“…Aliphatic glucosinolate profiles vary between wildtype accessions of A. thaliana and in leaves comprise alkenyl, methylthioalkyl, methylsuphinylalkenyl and hydroxyalkyl homologues of propyl and butyl glucosinolates, with trace levels of other homologues with longer side chain lengths (Hogge et al, 1988;Haughn et a!., 1991). By analogy with studies in Brassica, a model can be developed that explains the variation in aliphatic glucosinolates on the basis of variation in…”

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confidence: 99%

Genetics of aliphatic glucosinolates. I. Side chain elongation in Brassica napus and Arabidopsis thaliana

et al. 1994

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Aliphatic glucosinolates are thioglucosides synthesized by genera within the Capparales including Arabidopsis and Brassica. They have been shown to mediate pest or pathogen interactions and to reduce the feeding quality of rapeseed meal. Their biological activity is largely dependent upon the structure of the side chain which determines the nature of hydrolysis products produced following tissue damage. A generalized model of the biosynthesis of aliphatic glucosinolates is proposed and tested with reference to the genetic regulation of side chain length by analysing recombinant populations of B. napus and A. thaliana developed from parental lines which varied in their glucosinolate content. The results of the B. napus studies were consistent with a model in which alleles at a single locus (Gsl-pro) regulate the presence or absence of propyl glucosinolates, and those at two other loci (Gsl-elong-C and Gsl-elong-A) (which map onto a pair of homoeologous linkage groups, one from the C genome and one from the A genome) regulate side chain elongation of the amino acid derivative which results in the production of butyl and pentyl glucosinolates. As null alleles do not occur at the Gsl-elong-A locus, the proportion of propyl glucosinolates is limited to below 30 per cent of total aliphatic glucosinolates. Alleles at a single locus in A. thaliana (GsIelong-Ar), which maps 1.3 cM from the RFLP locus m291 on chromosome 5, regulate side chain elongation of aliphatic glucosinolates in this species. It is suggested that the Gsl-elong-Ar gene is homologous to the Gsl-elong-A and Gsl-elong-C genes and, if cloned, could be used to downregulate the endogenous Gsl-eiong genes in B. napus.

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HPLC Separation of Glucosinolates from Leaves and Seeds of Arabidopsis thaliana and Their Identification Using Thermospray Liquid Chramatography/Mass Spectrometry

Cited by 136 publications

References 11 publications

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme

CYP83B1, a Cytochrome P450 at the Metabolic Branch Point in Auxin and Indole Glucosinolate Biosynthesis in Arabidopsis

Genetics of aliphatic glucosinolates. I. Side chain elongation in Brassica napus and Arabidopsis thaliana

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