Plant Biology

Taxadiene synthase gene from Taxus brevifolia was constitutively expressed in the moss Physcomitrella patens using a ubiquitin promoter to produce taxa-4(5),11(12)-diene, the precursor of the anticancer drug paclitaxel. In stable moss transformants, taxa-4(5),11(12)-diene was produced up to 0.05% fresh weight of tissue, without significantly affecting the amounts of the endogenous diterpenoids (ent-kaurene and 16-hydroxykaurane). Unlike higher plants that had been genetically modified to produce taxa-4(5),11(12)-diene, transgenic P. patens did not exhibit growth inhibition due to alteration of diterpenoid metabolic pools. Thus we propose that P. patens is a promising alternative host for the biotechnological production of paclitaxel and its precursors.

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k cat /K m values of 1050, 7650, and 1560 M ؊1 s ؊1 for arogenate versus 38, 240, and 16 M ؊1 s ؊1 for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k cat /K m values of 1140, 490, and 620 M ؊1 s ؊1 , with prephenate not serving as a substrate unless excess recombinant protein (>150 g/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.

Floral scent is likely important to the pollination of parasitic plants, despite that it has not been wellstudied. We studied the pollination ecology of the North American stem holoparasite Pilostyles thurberi (Apodanthaceae) at two field sites in Texas. To identify effective pollinators, we collected floral visitors to P. thurberi flowers, observed their foraging behavior, and looked for P. thurberi pollen on their bodies. Augochloropsis metallica bees (Halictidae) and eumenine potter wasps (Vespidae) were pollinators. P. thurberi flowers are visually inconspicuous but produce a strong fruity fragrance. GC/MS analysis of whole floral extracts and dynamic headspace samples revealed the fragrance to be an unusually simple bouquet of raspberry ketone and several eugenols. Comparison of scent profiles to those from uninfected host plants (Dalea formosa) allowed putative separation of parasite and host volatiles. This is the first report of the constituents of floral fragrance in Apodanthaceae.

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k cat /K m values of 1050, 7650, and 1560 M ؊1 s ؊1 for arogenate versus 38, 240, and 16 M ؊1 s ؊1 for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k cat /K m values of 1140, 490, and 620 M ؊1 s ؊1 , with prephenate not serving as a substrate unless excess recombinant protein (>150 g/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.

Taxol is a cancer fighting drug that was initially isolated from the Pacific Yew. However, the isolation process is not very efficient and the tree is being excessively harvested and faces extinction. To synthetically make Taxol is an inefficient and costly process. If the precursor taxadiene-5 alpha-acetoxy-10 beta-ol can be produced with ease, then the synthetic modification of that precursor would be an efficient way to produce the potent cancer fighting drug. Several genes from the Pacific Yew were isolated and amplified so that they could be inserted into the moss Physcomitrella patens. Using competent E. coli cells as entry vectors, the genes were transferred so that the metabolic pathway responsible for taxadiene-5 alpha-acetoxy-10 beta-ol synthesis could be replicated in Physcomitrella patens. When the final transfer was made to the moss, a transient expression of the genes resulted in small amounts of product being obtained. After gas chromatography mass spectrometry analysis, the chromatogram plots showed a few more promising peaks representing other Taxol precursors. With a permanent transfer to the moss, a much larger sample could be analyzed and more Taxol precursor could be produced. Figure 5. Electrophoresis Gel After PCR Amplification

Loblolly pine (Pinus taeda L.) cell suspension cultures secrete monolignols when placed in 8% sucrose/20 mM KI solution, and these were used to identify phenylpropanoid pathway flux-modulating steps. When cells were provided with increasing amounts of either phenylalanine (Phe) or cinnamic acid, cellular concentrations of immediate downstream products (cinnamic and p-coumaric acids, respectively) increased, whereas caffeic and ferulic acid pool sizes were essentially unaffected. Increasing Phe concentrations resulted in increased amounts of p-coumaryl alcohol relative to coniferyl alcohol. However, exogenously supplied cinnamic, p-coumaric, caffeic, and ferulic acids resulted only in increases in their intercellular concentrations, but not that of downstream cinnamyl aldehydes and monolignols. Supplying p-coumaryl and coniferyl aldehydes up to 40,000-320,000-fold above the detection limits resulted in rapid, quantitative conversion into the monolignols. Only at nonphysiological concentrations was transient accumulation of intracellular aldehydes observed. These results indicate that cinnamic and p-coumaric acid hydroxylations assume important regulatory positions in phenylpropanoid metabolism, whereas cinnamyl aldehyde reduction does not serve as a control point.

Taxol is a cancer fighting drug that was initially isolated from the Pacific Yew. However, the isolation process is not very efficient and the tree is being excessively harvested and faces extinction. To synthetically make Taxol is an inefficient and costly process. If the precursor taxadiene-5 alpha-acetoxy-10 beta-ol can be produced with ease, then the synthetic modification of that precursor would be an efficient way to produce the potent cancer fighting drug. Several genes from the Pacific Yew were isolated and amplified so that they could be inserted into the moss Physcomitrella patens. Using competent E. coli cells as entry vectors, the genes were transferred so that the metabolic pathway responsible for taxadiene-5 alpha-acetoxy-10 beta-ol synthesis could be replicated in Physcomitrella patens. When the final transfer was made to the moss, a transient expression of the genes resulted in small amounts of product being obtained. After gas chromatography mass spectrometry analysis, the chromatogram plots showed a few more promising peaks representing other Taxol precursors. With a permanent transfer to the moss, a much larger sample could be analyzed and more Taxol precursor could be produced. Figure 5. Electrophoresis Gel After PCR Amplification

The Arabidopsis genome sequencing in 2000 gave to science the first blueprint of a vascular plant. Its successful completion also prompted the US National Science Foundation to launch the Arabidopsis 2010 initiative, the goal of which is to identify the function of each gene by 2010. In this study, an exhaustive analysis of The Institute for Genomic Research (TIGR) and The Arabidopsis Information Resource (TAIR) databases, together with all currently compiled EST sequence data, was carried out in order to determine to what extent the various metabolic networks from phenylalanine ammonia lyase (PAL) to the monolignols were organized and/or could be predicted. In these databases, there are some 65 genes which have been annotated as encoding putative enzymatic steps in monolignol biosynthesis, although many of them have only very low homology to monolignol pathway genes of known function in other plant systems. Our detailed analysis revealed that presently only 13 genes (two PALs, a cinnamate-4hydroxylase, a p-coumarate-3-hydroxylase, a ferulate-5-hydroxylase, three 4-coumarate-CoA ligases, a cinnamic acid O-methyl transferase, two cinnamoyl-CoA reductases) and two cinnamyl alcohol dehydrogenases can be classified as having a bona fide (definitive) function; the remaining 52 genes currently have undetermined physiological roles. The EST database entries for this particular set of genes also provided little new insight into how the monolignol pathway was organized in the different tissues and organs, this being perhaps a consequence of both limitations in how tissue samples were collected and in the incomplete nature of the EST collections. This analysis thus underscores the fact that even with genomic sequencing, presumed to provide the entire suite of putative genes in the monolignol-forming pathway, a very large effort needs to be conducted to establish actual catalytic roles (including enzyme versatility), as well as the physiological function(s) for each member of the (multi)gene families present and the metabolic networks that are operative. Additionally, one key to identifying physiological functions for many of these (and other) unknown genes, and their corresponding metabolic networks, awaits the development of technologies to comprehensively study molecular processes at the single cell level in particular tissues and organs, in order to establish the actual metabolic context.

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 a-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, a-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred a-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from a-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.