Auxins are hormones that regulate many aspects of plant growth and development. The main plant auxin is indole-3-acetic acid (IAA), whose biosynthetic pathway is not fully understood. Indole-3-acetaldoxime (IAOx) has been proposed to be a key intermediate in the synthesis of IAA and several other indolic compounds. Genetic studies of IAA biosynthesis in Arabidopsis have suggested that 2 distinct pathways involving the CYP79B or YUCCA (YUC) genes may contribute to IAOx synthesis and that several pathways are also involved in the conversion of IAOx to IAA. Here we report the biochemical dissection of IAOx biosynthesis and metabolism in plants by analyzing IAA biosynthesis intermediates. We demonstrated that the majority of IAOx is produced by CYP79B genes in Arabidopsis because IAOx production was abolished in CYP79B-deficient mutants. IAOx was not detected from rice, maize, and tobacco, which do not have apparent CYP79B orthologues. IAOx levels were not significantly altered in the yuc1 yuc2 yuc4 yuc6 quadruple mutants, suggesting that the YUC gene family probably does not contribute to IAOx synthesis. We determined the pathway for conversion of IAOx to IAA by identifying 2 likely intermediates, indole-3-acetamide (IAM) and indole-3-acetonitrile (IAN), in Arabidopsis. When 13 C6-labeled IAOx was fed to CYP79B-deficient mutants, 13 C 6 atoms were efficiently incorporated to IAM, IAN, and IAA. This biochemical evidence indicates that IAOx-dependent IAA biosynthesis, which involves IAM and IAN as intermediates, is not a common but a species-specific pathway in plants; thus IAA biosynthesis may differ among plant species.indole-3-acetic acid ͉ plant hormone

The phytohormone auxin plays a central role in many aspects of plant growth and development. IAA is the most studied natural auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin for >40 years, but its role in plant growth and development remains unclear. In this study, we show that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. PAA is widely distributed in vascular and non-vascular plants. Although the biological activities of PAA are lower than those of IAA, the endogenous levels of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin-responsive genes through the TIR1/AFB pathway in Arabidopsis. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA (YUC) genes increases PAA metabolite levels in Arabidopsis, indicating that YUC flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by different types of auxins.

Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with 13 C in Arabidopsis seedlings. Efficient 13 C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a 13 C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.Cytokinins (CKs), 1 a group of phytohormones, have profound physiological roles in plants, e.g. promotion of cell division, release of lateral buds from apical dominance, and delay of senescence. The biological activity, signal transduction, and metabolism of CKs have long been studied (1). However, it remains unclear how different classes of CKs are produced in plants and whether such classes of CKs play different roles in plant development. Most natural CKs are derivatives of N 6

Degradation of arylglycerol-␤-aryl ether is the most important process in bacterial lignin catabolism. Sphingobium sp. strain SYK-6 degrades guaiacylglycerol-␤-guaiacyl ether (GGE) to ␣-(2-methoxyphenoxy)-␤-hydroxypropiovanillone (MPHPV), and then the ether linkage of MPHPV is cleaved to generate ␣-glutathionyl-␤-hydroxypropiovanillone (GS-HPV) and guaiacol. We have characterized three enantioselective glutathione S-transferase genes, including two genes that are involved in the ether cleavage of two enantiomers of MPHPV and one gene that is involved in the elimination of glutathione from a GS-HPV enantiomer. However, the first step in the degradation of four different GGE stereoisomers has not been characterized. In this study, three alcohol dehydrogenase genes, ligL, ligN, and ligO, which conferred GGE transformation activity in Escherichia coli, were isolated from SYK-6 and characterized, in addition to the previously cloned ligD gene. The levels of amino acid sequence identity of the four GGE dehydrogenases, which belong to the short-chain dehydrogenase/ reductase family, ranged from 32% to 39%. Each gene was expressed in E. coli, and the stereospecificities of the gene products with the four GGE stereoisomers were determined by using chiral high-performance liquid chromatography with recently synthesized authentic enantiopure GGE stereoisomers. LigD and LigO converted (␣R,␤S)-GGE and (␣R,␤R)-GGE into (␤S)-MPHPV and (␤R)-MPHPV, respectively, while LigL and LigN transformed (␣S,␤R)-GGE and (␣S,␤S)-GGE to (␤R)-MPHPV and (␤S)-MPHPV, respectively. Disruption of the genes indicated that ligD is essential for the degradation of (␣R,␤S)-GGE and (␣R,␤R)-GGE and that both ligL and ligN contribute to the degradation of the two other GGE stereoisomers.

Agrobacterium tumefaciens infects plants and induces the formation of tumors called ''crown galls'' by integrating the transferred-DNA (T-DNA) region of the Ti-plasmid into the plant nuclear genome. Tumors are formed because the T-DNA encodes enzymes that modify the synthesis of two plant growth hormones, auxin and cytokinin (CK). Here, we show that a CK biosynthesis enzyme, Tmr, which is encoded by the Agrobacterium T-DNA region, is targeted to and functions in plastids of infected plant cells, despite having no typical plastid-targeting sequence. Evidence is provided that Tmr is an adenosine phosphate-isopentenyltransferase (IPT) that creates a new CK biosynthesis bypass by using 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP) as a substrate. Unlike in the conventional CK biosynthesis pathway in plants, transzeatin-type CKs are produced directly without the requirement for P450 monooxygenase-mediated hydroxylation. Consistent with the plastid localization of Tmr, HMBDP is an intermediate in the methylerythritol phosphate pathway, a plastid-localized biosynthesis route for universal isoprenoid precursors. These results demonstrate that A. tumefaciens modifies CK biosynthesis by sending a key enzyme into plastids of the host plant to promote tumorigenesis.crown gall ͉ isopentenyltransferase ͉ methylerythritol phosphate pathway C ytokinins (CKs) are a group of plant hormones essential for cell division and differentiation in plants (1). Most natural CKs, including isopentenyladenine (iP) and trans-zeatin (tZ) (Fig. 1), are derivatives of N 6 -prenylated adenine (1). The prenyl side chain of CKs can be derived from the methylerythritol phosphate (MEP) pathway or the mevalonate (MVA) pathway, both of which supply common C 5 units for isoprenoid biosynthesis (2, 3). The MEP pathway widely occurs in the bacterial kingdom and the plastids of plants, whereas the MVA pathway is commonly found in the cytosol of eukaryotes (2, 3). Thus, plants have the two different isoprenoid pathways in separate subcellular compartments. Recent work has shown that the majority of the prenyl side chain of iP and tZ is derived from the MEP pathway in Arabidopsis seedlings (4).To initiate CK biosynthesis, an isoprenoid precursor is transferred to AMP, ADP or ATP by adenosine phosphateisopentenyltransferase (IPT, EC 2.5.1.27) ( Fig. 1) (5-7). At least two routes have been proposed for the formation of tZ, an active CK, in plants. In the conventional iP riboside 5Ј-monophosphate (iPRMP)-dependent pathway, dimethylallyl diphosphate (DMAPP) is used as the side chain precursor for iPRMP, which is then converted to tZ riboside 5Ј-monophosphate (tZRMP) by P450 monooxygenases (8) (CYP735A1 and CYP735A2 in Arabidopsis; Fig. 1). The other proposed pathway is an iPRMPindependent bypass, in which an unidentified hydroxylated derivative of DMAPP is directly transferred to the adenine moiety (9). A candidate for this putative substrate is 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP) (10), which has recently been shown to occur as an int...

Lignin accumulates in the cell walls of specialized cell types to enable plants to stand upright and conduct water and minerals, withstand abiotic stresses, and defend themselves against pathogens. These functions depend on specific lignin concentrations and subunit composition in different cell types and cell wall layers. However, the mechanisms controlling the accumulation of specific lignin subunits, such as coniferaldehyde, during the development of these different cell types are still poorly understood. We herein validated the Wiesner test (phloroglucinol/HCl) for the restrictive quantitative in situ analysis of coniferaldehyde incorporation in lignin. Using this optimized tool, we investigated the genetic control of coniferaldehyde incorporation in the different cell types of genetically-engineered herbaceous and woody plants with modified lignin content and/or composition. Our results demonstrate that the incorporation of coniferaldehyde in lignified cells is controlled by (a) autonomous biosynthetic routes for each cell type, combined with (b) distinct cell-to-cell cooperation between specific cell types, and (c) cell wall layer-specific accumulation capacity. This process tightly regulates coniferaldehyde residue accumulation in specific cell types to adapt their property and/or function to developmental and/or environmental changes.

An enantio-specific polyphenol oxidase (PPO) was purified Ϸ1,700-fold to apparent homogeneity from the creosote bush (Larrea tridentata), and its encoding gene was cloned. The posttranslationally processed PPO (Ϸ43 kDa) has a central role in the biosynthesis of the creosote bush 8 -8 linked lignans, whose representatives, such as nordihydroguaiaretic acid and its congeners, have potent antiviral, anticancer, and antioxidant properties. The PPO primarily engenders the enantio-specific conversion of (؉)-larreatricin into (؉)-3-hydroxylarreatricin, with the regiochemistry of catalysis being unambiguously established by different NMR spectroscopic analyses; the corresponding (؊)-enantiomer did not serve as a substrate. This enantio-specificity for a PPO, a representative of a widespread class of enzymes, provides additional insight into their actual physiological roles that hitherto have been difficult to determine.

Transgenic tobacco (Nicotiana tabacum 1.) plants in which the activity of 4-coumarate:coenzyme A ligase is very low contain a novel lignin in their xylem. Details of changes in hydroxycinnamic acids bound to cell walls and in the structure of the novel lignin were identified by base hydrolysis, alkaline nitrobenzene oxidation, pyrolysis-gas chromatography, and '3C-nuclear magnetic resonance analysis. In the brownish tissue of the transgenic plants, the levels of three hydroxycinnamic acids, p-coumaric, ferulic, and sinapic, which were bound to cell walls, were apparently increased as a result of down-regulation of the expression of the gene for 4-coumarate:coenzyme A ligase. Some of these hydroxycinnamic acids were linked to cell walls via ester and ether linkages. The accumulation of hydroxycinnamic acids also induced an increase in the leve1 of condensed units in the novel lignin of the brownish tissue. Our data indicate that the behavior of some of the incorporated hydroxycinnamic acids resembles lignin monomers in the brownish tissue, and their accumulation results in dramatic changes in the biosynthesis of lignin in transgenic plants.The deposition of lignin in vascular cell walls is a key step in the differentiation of these cells into the functional elements that participate in the transport of water and in the structural support of plants. The biosynthesis of lignin has been studied extensively and the major enzymes involved in the synthesis of precursors to lignin have been identified (Higuchi, 1985; Lewis and Yamamoto, 1990). In the past decade many genes for enzymes related to the biosynthesis of lignin have been cloned from a variety of plant species, and the expression of these genes at various stages of lignification has been characterized (Sederoff et al., 1994; Whetten and Sederoff, 1995). In several recent studies transgenic plants carrying chimeric genes for the enzymes involved in lignin biosynthesis were generated and the altered lignins in these transformants were analyzed (Elkind et al