Characterization of four bifunctional plant IAM/PAM-amidohydrolases capable of contributing to auxin biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed.

YUCCA8 and YUCCA9 overexpression reveals a link between auxin signaling and lignification through the induction of ethylene biosynthesis.

Auxin is associated with the regulation of virtually every aspect of plant growth and development. Many previous genetic and biochemical studies revealed that, among the proposed routes for the production of auxin, the so-called indole-3-pyruvic acid (IPA) pathway is the main source for indole-3-acetic acid (IAA) in plants. The IPA pathway involves the action of 2 classes of enzymes, tryptophan-pyruvate aminotransferases (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1(TAA1)/TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR)) and flavin monooxygenases (YUCCA). Both enzyme classes appear to be encoded by small gene families in Arabidopsis consisting of 5 and 11 members, respectively. We recently showed that it is possible to induce transcript accumulation of 2 YUCCA genes, YUC8 and YUC9, by methyl jasmonate treatment. Both gene products were demonstrated to contribute to auxin biosynthesis in planta.1 Here we report that the overexpression of YUC8 as well as YUC9 led to strong lignification of plant aerial tissues. Furthermore, new evidence indicates that this abnormally strong secondary growth is linked to increased levels of ethylene production.

Azotobacter vinelandii nitrogenase: “Kinetics of nif gene expression and insights into the roles of FdxN and NifQ in FeMo-co biosynthesis”

The Molybdenum-nitrogenase is responsible for most biological nitrogen fixation activity (BNF) in the biosphere. Due to its great agronomical importance, it has been the subject of profound genetic and biochemical studies. The Mo nitrogenase carries at its active site a unique iron-molybdenum cofactor (FeMoco) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase through a complex and highly regulated pathway involving proteins acting as molecular scaffolds, metallocluster carriers or enzymes that provide substrates in appropriate chemical forms. Specific expression regulatory factors tightly control the accumulation levels of all these other components. Insertion of FeMo-co into a P-cluster containing apo-NifDK polypeptide results in nitrogenase reconstitution. Investigation of FeMo-co biosynthesis has uncovered new radical chemistry reactions and new roles for Fe-S clusters in biology.

The role of the secondary cell wall in plant resistance to pathogens

Plant resistance to pathogens relies on a complex network of constitutive and inducible defensive barriers. The plant cell wall is one of the barriers that pathogens need to overcome to successfully colonize plant tissues. The traditional view of the plant cell wall as a passive barrier has evolved to a concept that considers the wall as a dynamic structure that regulates both constitutive and inducible defense mechanisms, and as a source of signaling molecules that trigger immune responses. The secondary cell walls of plants also represent a carbon-neutral feedstock (lignocellulosic biomass) for the production of biofuels and biomaterials. Therefore, engineering plants with improved secondary cell wall characteristics is an interesting strategy to ease the processing of lignocellulosic biomass in the biorefinery. However, modification of the integrity of the cell wall by impairment of proteins required for its biosynthesis or remodeling may impact the plants resistance to pathogens. This review summarizes our understanding of the role of the plant cell wall in pathogen resistance with a focus on the contribution of lignin to this biological process.

A mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids accumulates meromycolates

Mycolic acids are a major constituent of the mycobacterial cell wall, and they form an effective permeability barrier to protect mycobacteria from antimicrobial agents. Although the chemical structures of mycolic acids are well established, little is known on their biosynthesis. We have isolated a mycolate-deficient mutant strain of Mycobacterium smegmatis mc2-155 by chemical mutagenesis followed by screening for increased sensitivity to novobiocin. This mutant also was hypersensitive to other hydrophobic compounds such as crystal violet, rifampicin, and erythromycin. Entry of hydrophobic probes into mutant cells occurred much more rapidly than that into the wild-type cells. HPLC and TLC analysis of fatty acid composition after saponification showed that the mutant failed to synthesize full-length mycolic acids. Instead, it accumulated a series of long-chain fatty acids, which were not detected in the wild-type strain. Analysis by 1H NMR, electrospray and electron impact mass spectroscopy, and permanganate cleavage of double bonds showed that these compounds corresponded to the incomplete meromycolate chain of mycolic acids, except for the presence of a β-hydroxyl group. This direct identification of meromycolates as precursors of mycolic acids provides a strong support for the previously proposed pathway for mycolic acid biosynthesis involving the separate synthesis of meromycolate chain and the α-branch of mycolic acids, followed by the joining of these two branches.

Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis

Vitamin C (l-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and l-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains ≈25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells

Phosphatidylserine (PtdSer) synthesis in Chinese hamster ovary (CHO) cells occurs through the exchange of l-serine with the base moiety of phosphatidylcholine or phosphatidylethanolamine. The synthesis is depressed on the addition of PtdSer to the culture medium. A CHO cell mutant named mutant 29, whose PtdSer biosynthesis is highly resistant to this depression by exogenous PtdSer, has been isolated from CHO-K1 cells. In the present study, the PtdSer-resistant PtdSer biosynthesis in the mutant was traced to a point mutation in the PtdSer synthase I gene, pssA, resulting in the replacement of Arg-95 of the synthase by lysine. Introduction of the mutant pssA cDNA, but not the wild-type pssA cDNA, into CHO-K1 cells induced the PtdSer-resistant PtdSer biosynthesis. In a cell-free system, the serine base-exchange activity of the wild-type pssA-transfected cells was inhibited by PtdSer, but that of the mutant pssA-transfected cells was resistant to the inhibition. Like the mutant 29 cells, the mutant pssA-transfected cells grown without exogenous PtdSer exhibited an ≈2-fold increase in the cellular PtdSer level compared with that in CHO-K1 cells, although the wild-type pssA-transfected cells did not exhibit such a significant increase. These results indicated that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of a normal PtdSer level in CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for the inhibition.

The specific features of methionine biosynthesis and metabolism in plants

Plants, unlike other higher eukaryotes, possess all the necessary enzymatic equipment for de novo synthesis of methionine, an amino acid that supports additional roles than simply serving as a building block for protein synthesis. This is because methionine is the immediate precursor of S-adenosylmethionine (AdoMet), which plays numerous roles of being the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of polyamines and of the phytohormone ethylene. In addition, AdoMet has regulatory function in plants behaving as an allosteric activator of threonine synthase. Among the AdoMet-dependent reactions occurring in plants, methylation of cytosine residues in DNA has raised recent interest because impediment of this function alters plant morphology and induces homeotic alterations in flower organs. Also, AdoMet metabolism seems somehow implicated in plant growth via an as yet fully understood link with plant-growth hormones such as cytokinins and auxin and in plant pathogen interactions. Because of this central role in cellular metabolism, a precise knowledge of the biosynthetic pathways that are responsible for homeostatic regulation of methionine and AdoMet in plants has practical implications, particularly in herbicide design.

A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: Architecture of metabolic diversity

In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.

Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes

A collection of 8,000 Arabidopsis thaliana plants carrying 48,000 insertions of the maize transposable element En-1 has been generated. This population was used for reverse genetic analyses to identify insertions in individual gene loci. By using a PCR-based screening protocol, insertions were found in 55 genes. En-1 showed no preference for transcribed or untranscribed regions nor for a particular orientation relative to the gene of interest. In several cases, En-1 was inserted within a few kilobases upstream or downstream of the gene. En-1 was mobilized from such positions into the respective gene to cause gene disruption. Knock-out alleles of genes involved in flavonoid biosynthesis were generated. One mutant line contained an En-1 insertion in the flavonol synthase gene (FLS) and showed drastically reduced levels of kaempferol. Allelism tests with other lines containing En-1 insertions in the flavanone 3-hydroxylase gene (F3H) demonstrated that TRANSPARENT TESTA 6 (TT6) encodes flavanone 3-hydroxylase. The f3h and fls null mutants complete the set of A. thaliana lines defective in early steps of the flavonoid pathway. These experiments demonstrate the efficiency of the screening method and gene disruption strategy used for assigning functions to genes defined only by sequence.

Parallel-up structure evidences the molecular directionality during biosynthesis of bacterial cellulose

The “parallel-up” packing in cellulose Iα and Iβ unit cells was experimentally demonstrated by a combination of direct-staining the reducing ends of cellulose chains and microdiffraction-tilting electron crystallographic analysis. Microdiffraction investigation of nascent bacterial cellulose microfibrils showed that the reducing end of the growing cellulose chains points away from the bacterium, and this provides direct evidence that polymerization by the cellulose synthase takes place at the nonreducing end of the growing cellulose chains. This mechanism is likely to be valid also for a number of processive glycosyltransferases such as chitin synthases, hyaluronan synthases, and proteins involved in the synthesis of nodulation factor backbones.

Terpenoid biosynthesis from 1-deoxy-d-xylulose in higher plants by intramolecular skeletal rearrangement

The incorporation of [1-13C]- and [2,3,4,5-13C4]1-deoxy-d-xylulose into β-carotene, lutein, phytol, and sitosterol in a cell culture of Catharanthus roseus was analyzed by NMR spectroscopy. The labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach. 13C Enrichment and 13C13C coupling patterns showed conclusively that 1-deoxy-d-xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, β-carotene, and lutein. Label from 1-deoxyxylulose was also diverted to phytosterols to a minor extent (6% relative to carotene and phytol formation). The data demonstrate that the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an intramolecular rearrangement process.

Glutamyl-tRNAGln amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis

Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis. They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS). The alternative route involves transamidation of incorrectly charged tRNA. Examination of the preliminary genomic sequence of the radiation-resistant bacterium Deinococcus radiodurans suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation. Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS). Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present. Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to Bacillus subtilis gatCAB. However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating D. radiodurans GluRS to produce the required mischarged Glu-tRNAGln substrate. The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the D. radiodurans genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in D. radiodurans extracts, suggests that the gatCAB genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.

Control of membrane phosphatidylcholine biosynthesis by diacylglycerol levels in neuronal cells undergoing neurite outgrowth

Phospholipids are the major components of cell membranes and are required for cellular growth. We studied membrane phosphatidylcholine (PtdCho) biosynthesis in neuronal cells undergoing neurite outgrowth, by using PC12 cells as a model system. When neurite outgrowth was induced by exposing PC12 cells to nerve growth factor for 2 and 4 days, the amounts of [14C]choline incorporated into [14C]phosphatidylcholine per cell (i.e., per DNA) increased approximately 5- and 10-fold, respectively, as compared with control cells, reflecting increases in the rate of PtdCho biosynthesis. [14C]choline uptake was not affected. Analysis of the three major PtdCho biosynthetic enzymes showed that the activity of CDPcholine:1,2-diacylglycerol cholinephosphotransferase was increased by approximately 50% after nerve growth factor treatment, but the activities of choline kinase or choline-phosphate cytidylyltransferase were unaltered; the cholinephosphotransferase displayed a high Km value (≈1,200 μM) for diacylglycerol. Moreover, free cellular diacylglycerol levels increased by approximately 1.5- and 4-fold on the second and fourth days, respectively. These data indicate that PtdCho biosynthesis is enhanced when PC12 cells sprout neurites, and the enhancement is mediated primarily by changes in cholinephosphotransferase activity and its saturation with diacylglycerol. This suggests a novel regulatory role for diacylglycerol in membrane phospholipid biosynthesis.

Genetic control of abscisic acid biosynthesis in maize

Abscisic acid (ABA), an apocarotenoid synthesized from cleavage of carotenoids, regulates seed maturation and stress responses in plants. The viviparous seed mutants of maize identify genes involved in synthesis and perception of ABA. Two alleles of a new mutant, viviparous14 (vp14), were identified by transposon mutagenesis. Mutant embryos had normal sensitivity to ABA, and detached leaves of mutant seedlings showed markedly higher rates of water loss than those of wild type. The ABA content of developing mutant embryos was 70% lower than that of wild type, indicating a defect in ABA biosynthesis. vp14 embryos were not deficient in epoxy-carotenoids, and extracts of vp14 embryos efficiently converted the carotenoid cleavage product, xanthoxin, to ABA, suggesting a lesion in the cleavage reaction. vp14 was cloned by transposon tagging. The VP14 protein sequence is similar to bacterial lignostilbene dioxygenases (LSD). LSD catalyzes a double-bond cleavage reaction that is closely analogous to the carotenoid cleavage reaction of ABA biosynthesis. Southern blots indicated a family of four to six related genes in maize. The Vp14 mRNA is expressed in embryos and roots and is strongly induced in leaves by water stress. A family of Vp14-related genes evidently controls the first committed step of ABA biosynthesis. These genes are likely to play a key role in the developmental and environmental control of ABA synthesis in plants.