Auxin biosynthesis in maize.

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.

Evolution of benzoxazinone biosynthesis and indole production in maize.

The synthesis of a diverse spectrum of secondary metabolites has allowed plants to develop sophisticated chemical defense mechanisms. Maize (Zea mays L.), for example, releases a cocktail of volatile compounds when attacked by a caterpillar. These compounds attract a parasitic wasp, which deposits its eggs in the larvae, thereby controlling the population size of the herbivore. Indole, which is part of the cocktail, is produced by an enzyme recruited from primary metabolism. Indole can either function as a volatile signal or be converted by specific cytochrome P450 enzymes into benzoxazinoids, which function as important defense chemicals.

Studies on the biosynthesis of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) from 3-hydroxyindolin-2-one in Zea mays.

The ring expansion of 3-hydroxyindolin-2-one to 2-hydroxy-1,4-benzoxazin-3-one (HBOA) was investigated by labelling experiments. Action of the cytochrome P450 enzyme BX4 from maize on 3-hydroxyindolin-2-one under an 18O2 atmosphere induced production of 2-hydroxy-1,4-benzoxazin-3-one in which the ring oxygen--but not the 2-hydroxy group of HBOA--is labelled. A mechanism for this transformation is proposed.

Retrobiosynthetic nuclear magnetic resonance analysis of amino acid biosynthesis and intermediary metabolism. Metabolic flux in developing maize kernels.

Information on metabolic networks could provide the basis for the design of targets for metabolic engineering. To study metabolic flux in cereals, developing maize (Zea mays) kernels were grown in sterile culture on medium containing [U-(13)C(6)]glucose or [1,2-(13)C(2)]acetate. After growth, amino acids, lipids, and sitosterol were isolated from kernels as well as from the cobs, and their (13)C isotopomer compositions were determined by quantitative nuclear magnetic resonance spectroscopy. The highly specific labeling patterns were used to analyze the metabolic pathways leading to amino acids and the triterpene on a quantitative basis. The data show that serine is generated from phosphoglycerate, as well as from glycine. Lysine is formed entirely via the diaminopimelate pathway and sitosterol is synthesized entirely via the mevalonate route. The labeling data of amino acids and sitosterol were used to reconstruct the labeling patterns of key metabolic intermediates (e.g. acetyl-coenzyme A, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, and Rib 5-phosphate) that revealed quantitative information about carbon flux in the intermediary metabolism of developing maize kernels. Exogenous acetate served as an efficient precursor of sitosterol, as well as of amino acids of the aspartate and glutamate family; in comparison, metabolites formed in the plastidic compartments showed low acetate incorporation.

Two glucosyltransferases are involved in detoxification of benzoxazinoids in maize.

Benzoxazinoids are major compounds involved in chemical defence in grasses. These toxins are stored in the vacuole as glucosides. Two glucosyltransferases, BX8 and BX9, that catalyse this last step of benzoxazinoid biosynthesis have been isolated via functional cloning. No close relative of these maize genes was found among the known glucosyltransferases. The enzymes display a very high degree of substrate specificity. DIMBOA, the major benzoxazinoid in young maize, is the preferred substrate. Both genes are highly expressed in young maize seedlings, the developmental stage with the highest activity of benzoxazinoid biosynthesis. Bx8 is included in the cluster of DIMBOA biosynthesis genes located on the short arm of chromosome 4. Hence, the gene cluster comprises three different enzymatic functions and a complete set of genes for the biosynthesis of DIBOA glucoside. Bx9 mapped to chromosome 1. Expression of Bx8 and Bx9 in Arabidopsis corroborated the potency of the enzymes in detoxification of their substrates. This capacity might have implications for allelopathic interactions.

An herbivore elicitor activates the gene for indole emission in maize.

Maize and a variety of other plant species release volatile compounds in response to herbivore attack that serve as chemical cues to signal natural enemies of the feeding herbivore. N-(17-hydroxylinolenoyl)-l-glutamine is an elicitor component that has been isolated and chemically characterized from the regurgitant of the herbivore-pest beet armyworm. This fatty acid derivative, referred to as volicitin, triggers the synthesis and release of volatile components, including terpenoids and indole in maize. Here we report on a previously unidentified enzyme, indole-3-glycerol phosphate lyase (IGL), that catalyzes the formation of free indole and is selectively activated by volicitin. IGL's enzymatic properties are similar to BX1, a maize enzyme that serves as the entry point to the secondary defense metabolites DIBOA and DIMBOA. Gene-sequence analysis indicates that Igl and Bx1 are evolutionarily related to the tryptophan synthase alpha subunit.

Auxin biosynthesis in maize kernels.

Auxin biosynthesis was analyzed in a maize (Zea mays) kernel culture system in which the seeds develop under physiological conditions similar to the in vivo situation. This system was modified for precursor feeding experiments. Tryptophan (Trp) is efficiently incorporated into indole-3-acetic acid (IAA) with retention of the 3, 3' bond. Conversion of Trp to IAA is not competed by indole. Labeling with the general precursors [U-(13)C(6)]glucose and [1, 2-(13)C(2)]acetate followed by retrobiosynthetic analysis strongly suggest that Trp-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. The synthesis of IAA from indole glycerol phosphate and IAA formation via condensation of indole with an acetyl-coenzyme A or phosphoenolpyruvate derived metabolite can be excluded.

Role of natural benzoxazinones in the survival strategy of plants.

Benzoxazinoid acetal glucosides are a unique class of natural products abundant in Gramineae, including the major agricultural crops maize, wheat, and rye. These secondary metabolites are also found in several dicotyledonous species. Benzoxazinoids serve as important factors of host plant resistance against microbial diseases and insects and as allelochemicals and endogenous ligands. Interdisciplinary investigations by biologists, biochemists, and chemists are stimulated by the intention to make agricultural use of the benzoxazinones as natural pesticides. These natural products are not only constituents of a plant defense system but also part of an active allelochemical system used in the competition with other plants. This review covers biological and chemical aspects of benzoxazinone research over the last decade with special emphasis on recent advances in the elucidation of the biosynthetic pathway.

The hydroxamic acid pathway.

An important component of general defence mechanisms of plants are toxic secondary metabolites that function as natural pesticides. The cyclic hydroxamic acids DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one) and DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) play an important role in the chemical defence of cereals against pests such as insects and pathogenic fungi and bacteria. Five genes that are clustered on chromosome four are sufficient to encode the enzymes to synthesize DIBOA. The first gene in the pathway, Bx1, encodes an enzyme resembling a tryptophan synthase alpha subunit that catalyses the formation of indole and thereby establishes the branchpoint that leads to the secondary metabolites. Four cytochrome P450-dependent monooxygenases encoded by Bx2-Bx5 catalyse consecutive hydroxylations to form DIBOA. This pathway can be generalized for grasses, since identical enzyme activities have been found in rye. The pathway is relatively short and begins with a metabolite ubiquitous to plants. Therefore DIBOA biosynthesis could be introduced into other plant species to confer improved disease resistance.

Cytochrome P450 monooxygenases of DIBOA biosynthesis: specificity and conservation among grasses.

DIBOA and DIMBOA are secondary metabolites of grasses which function as natural pesticides. The four maize genes BX2 through BX5 encode cytochrome P450-dependent monooxygenases that catalyse four consecutive reactions in the biosynthesis of these secondary products. Although BX2-BX5 share significant sequence homology, the four enzymes have evolved into specific enzymes each catalysing predominantly only one reaction in the pathway. In addition to these natural reactions, BX3 hydroxylates 1,4-benzoxazin-3-one and BX2 shows pCMA demethylase activity. With respect to DIBOA biosynthesis, identical enzymatic reactions have been found in rye as compared to maize, indicating early evolution of the P450 enzymes in the grasses.

Three new dominant C1 suppressor alleles in Zea mays.

Three new dominant suppressor mutations of the C1 transcription regulator gene in maize--C1-I delta 1, C1-I delta 2 and C1-I delta 3--are described that suppress anthocyanin colouration in kernels similar to the function of the C1-I standard inhibitor. The C1-I delta mutations were induced by imprecise excision of an En/Spm transposon in the third exon of the C1 gene. These transposon footprints cause a frameshift in the C1 open reading frame that leads to truncated proteins due to an early stop codon 30 amino acids upstream of the wild-type C1 protein. Therefore, the C1-I delta gene products lack the carboxy-terminal transcriptional activation domain of C1. The C1-I standard allele also lacks this domain and in addition differs in 17 amino acids from the wild-type C1 allele. The new C1-I delta alleles provide evidence that deletion of the carboxy-terminal activation domain alone is sufficient to generate a dominant suppressive effect on the function of wild-type C1.

Analysis of a chemical plant defense mechanism in grasses.

In the Gramineae, the cyclic hydroxamic acids 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one (DIMBOA) form part of the defense against insects and microbial pathogens. Five genes, Bx1 through Bx5, are required for DIBOA biosynthesis in maize. The functions of these five genes, clustered on chromosome 4, were demonstrated in vitro. Bx1 encodes a tryptophan synthase alpha homolog that catalyzes the formation of indole for the production of secondary metabolites rather than tryptophan, thereby defining the branch point from primary to secondary metabolism. Bx2 through Bx5 encode cytochrome P450-dependent monooxygenases that catalyze four consecutive hydroxylations and one ring expansion to form the highly oxidized DIBOA.

Expression of a cytochrome P450 gene family in maize.

Maize seedlings, like seedlings of many other plants, are rich in cytochrome P450 (P450) enzyme activity. Four P450 genes (CYPzm1-4), isolated from a seedling-specific cDNA library, are characterized by a transient and seedling-specific expression pattern. The maximum steady state mRNA levels are reached at 3 days in root and at 7 days in shoot tissue, respectively. All four genes belong to one gene family and are closely related to the CYP71 family of plant P450 genes, which includes the enzymes of the ripening avocado fruit (CYP71A1) and eggplant hypocotyls (CYP71A2, A3, A4). The expression of these related P450 genes in monocot and dicot plants indicates that these enzymes play a significant role in plants; however, the in vivo enzyme functions are unknown. The divergence of the four members of the maize gene family is sufficiently high to account for different substrate and/or reaction specificity. Although the general expression pattern of the four genes is identical, the maximum steady-state mRNA levels vary in different maize lines. In situ hybridisation reveals the highest mRNA levels in the coleoptile, the first developed leaflets, the ground tissue of the nodular complex, and in the cortex and pith of the region of cell division in the root. The mapping of the maize CYPzm genes shows that, as in animals, P450 genes of the same family can be clustered. The presence of the CYPzm gene cluster in maize argues for generation of distinct plant P450 gene families by gene duplication.

Insulin therapy increases low plasma growth hormone binding protein in children with new-onset type 1 diabetes.

This study was undertaken (1) to evaluate growth hormone binding protein (GHBP) levels in newly diagnosed patients with Type 1 diabetes before and after insulin therapy and (2) to determine the relationship of GHBP to glycaemic control, C-peptide level and blood pH. GHBP, expressed as a percentage of (125I)GH bound, was determined in 33 patients with Type 1 diabetes (M/F = 19/14, 12.3 +/- 0.4 years) before (day 0), after 5 days (day 5) and after 3 months (month 3) of insulin therapy. At day 0, GHBP was lower in Type 1 diabetes compared with 38 matched healthy control subjects (3.9 +/- 0.4 vs 8.2 +/- 0.4%, p < 0.001). There was no significant improvement in GHBP at day 5 (4.4 +/- 0.3%). At month 3, GHBP increased to (6.0 +/- 0.4%, p < 0.001 vs day 0), but was still lower than controls, p < 0.001. At day 0 GHBP correlated with BMI (r = 0.50, p = 0.001), blood glucose (r = -0.43 p = 0.006) and pH (r = 0.48, p = 0.004), but not HbA1. GHBP at month 3 correlated with day 0 C-peptide (r = 0.41, p = 0.02). Thus, (1) circulating GHBP is low in newly diagnosed patients with Type 1 diabetes, and increases after 3 months of insulin therapy but does not normalize and (2) the severity of biochemical derangement and residual beta-cell function at diagnosis may determine GHBP status and its recovery. We conclude that insulin is an important modulator of GH binding protein in newly diagnosed children with Type 1 diabetes.

Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco.

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5'-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.

Mobility of the maize transposable element En/Spm in Arabidopsis thaliana.

The autonomous element En-1 of the maize En/Spm transposable element system is capable of frequent somatic and germinal excision in the heterologous host Arabidopsis thaliana. The pattern of En-homologous transcripts generated in transgenic Arabidopsis resembles En transcription in maize. An excision reporter construct based on NPT-II gene (pKEn2) can be used reliably for the isolation of En-1 germinal revertants by seed germination on kanamycin-containing medium. Re-insertion after germinal excision is apparently frequent. A dSpm receptor element can be efficiently trans-activated in Arabidopsis either by En-1 or by expressing cDNAs of tnpA and tnpD. Excision and re-insertion of En/Spm take place with similar characteristics as in maize. This is the first description of En/Spm transposition in Arabidopsis and the parameters analysed here suggest that transposon tagging with En should be feasible in this species.

The transposable element En/Spm-encoded TNPA protein contains a DNA binding and a dimerization domain.

The En/Spm-encoded TNPA protein binds to 12-bp DNA sequence motifs that are present in the subtermini of the transposable element. DNA binding of TNPA to monomeric and dimeric forms of the binding motif was analyzed by gel retardation and cross-linking studies. A DNA binding domain at the N-terminal and a dimerization domain at the C-terminal portion of TNPA were localized using deletion derivatives of TNPA. These domains are novel since no apparent homology has been found in the data bases. The stoichiometry of the TNPA-DNA complexes was analyzed. A special complex is formed with a tail-to-tail dimeric DNA binding motif, most probably involving two DNA-bound TNPA molecules that interact via their dimerization domains. In redox reactions the requirement for one or two disulfide bonds for DNA binding of TNPA was shown. The implications of these findings for the excision mechanism of En/Spm are discussed.

Genetic and molecular analysis of a three-component transposable-element system in maize.

Two different factors control the mutability of an unstable allele (c2-m881058Y) of the C2 gene of maize. Both an autonomous En/Spm element and an unrelated independent factor, named Mediator, are coordinately required for the excision of the insert in c2-m881058Y. According to genetic analysis, Mediator does not have the suppressor (S) function or mutator (M) function of En/Spm. Mediator has no effect on the timing or frequency of excision of En1, En-low, or various I/dSpm elements. Hence, Mediator only mediates a specific interaction between En and the insert at c2-m881058Y. Molecular analysis of c2-m881058Y has revealed a 3.3 kb, complex, En-related receptor element inserted into the second exon of the C2 gene. The ends of this element are homologous to the ends of En/Spm, but an internal 1.7 kb region shows no En/Spm homology. A great degree (11-14%) of nucleotide changes, relative to En1, occur within and between the 12 bp TNPA binding motifs. Alterations of these critical cis-determinants may account for the need for a "helper" factor for excision. This element is named Irma, for Inhibitor that requires Mediator also, and represents a unique, low copy number class of receptor element.