Compositional analysis of Chinese water chestnut (Eleocharis dulcis) cell-wall material from parenchyma, epidermis, and subepidermal tissues.

Chinese water chestnut (Eleocharis dulcis (Burman f.) Trin ex Henschel) is a corm consumed globally in Oriental-style cuisine. The corm consists of three main tissues, the epidermis, subepidermis, and parenchyma; the cell walls of which were analyzed for sugar, phenolic, and lignin content. Sugar content, measured by gas chromatography, was higher in the parenchyma cell walls (931 µg/mg) than in the subepidermis (775 µg/mg) or epidermis (685 µg/mg). The alkali-extractable phenolic content, measured by high-performance liquid chromatography, was greater in the epidermal (32.4 µg/mg) and subepidermal cell walls (21.7 µg/mg) than in the cell walls of the parenchyma (12.3 µg/mg). The proportion of diferulic acids was higher in the parenchyma. The Klason lignin content of epidermal and subepidermal cell walls was ~15%. Methylation analysis of Chinese water chestnut cell-wall polysaccharides identified xyloglucan as the predominant hemicellulose in the parenchyma for the first time, and also a significant pectin component, similar to other nongraminaceous monocots.

HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester, and linkage of pHBA into cell walls.

As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase, was introduced into B. vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analysed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14% on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.

A novel polyamine acyltransferase responsible for the accumulation of spermidine conjugates in Arabidopsis seed.

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4'-O-glycosyl)-sinapoyl),N'-sinapoylspermidine and N,N'-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.

AtMYB12 regulates caffeoyl quinic acid and flavonol synthesis in tomato: expression in fruit results in very high levels of both types of polyphenol.

Plant polyphenolics exhibit a broad spectrum of health-promoting effects when consumed as part of the diet, and there is considerable interest in enhancing the levels of these bioactive molecules in plants used as foods. AtMYB12 was originally identified as a flavonol-specific transcriptional activator in Arabidopsis thaliana, and this has been confirmed by ectopic expression in tobacco. AtMYB12 is able to induce the expression of additional target genes in tobacco, leading to the accumulation of very high levels of flavonols. When expressed in a tissue-specific manner in tomato, AtMYB12 activates the caffeoyl quinic acid biosynthetic pathway, in addition to the flavonol biosynthetic pathway, an activity which probably mirrors that of the orthologous MYB12-like protein in tomato. As a result of its broad specificity for transcriptional activation in tomato, AtMYB12 can be used to produce fruit with extremely high levels of multiple polyphenolic anti-oxidants. Our data indicate that transcription factors may have different specificities for target genes in different plants, which is of significance when designing strategies to improve metabolite accumulation and the anti-oxidant capacity of foods.

Convergent evolution in the BAHD family of acyl transferases: identification and characterization of anthocyanin acyl transferases from Arabidopsis thaliana.

Members of the BAHD family of plant acyl transferases are very versatile catalytically, and are thought to be able to evolve new substrate specificities rapidly. Acylation of anthocyanins occurs in many plant species and affects anthocyanin stability and light absorption in solution. The versatility of BAHD acyl transferases makes it difficult to identify genes encoding enzymes with defined substrate specificities on the basis of structural homology to genes of known catalytic function alone. Consequently, we have used a modification to standard functional genomics strategies, incorporating co-expression profiling with anthocyanin accumulation, to identify genes encoding three anthocyanin acyl transferases from Arabidopsis thaliana. We show that the activities of these enzymes influence the stability of anthocyanins at neutral pH, and some acylations also affect the anthocyanin absorption maxima. These properties make the BAHD acyl transferases suitable tools for engineering anthocyanins for an improved range of biotechnological applications.

Dihydrocaffeoyl polyamines (kukoamine and allies) in potato (Solanum tuberosum) tubers detected during metabolite profiling.

Four related phenolic amides previously undescribed from the species were revealed during metabolic profiling of potato (Solanum tuberosum) tubers. N(1),N(12)-Bis(dihydrocaffeoyl)spermine (kukoamine A) and N(1),N(8)-bis(dihydrocaffeoyl)spermidine were positively identified by comparison with authentic standards, while the structures N(1),N(4),N(12)-tris(dihydrocaffeoyl)spermine and N(1),N(4),N(8)-tris(dihydrocaffeoyl)spermidine are proposed for the other two metabolites. Each amide was present at several tens of micrograms per gram of dry matter. Several of these compounds were subsequently detected in other solanaceous species, such as tomato (Lycopersicon esculentum) and Nicotiana sylvestris. They appeared not to be present in Arabidopsis thaliana or Beta vulgaris. Bis(dihydrocaffeoyl)spermine isomers have previously been identified in only a single plant, the Chinese medicinal species Lycium chinense (Solanaceae), where they may account for some of the described biological activity. The other compounds have not until now been reported in vivo, though some of the equivalent hydroxycinnamoyl derivatives are known. The surprising discovery of kukoamine and allies in a range of solanaceous species including potato, a common food crop that has a long history of scientific investigation, provides exemplary evidence for the potential of the nontargeted techniques of metabolomics in studying plant metabolites.

NMR and HPLC-UV profiling of potatoes with genetic modifications to metabolic pathways.

Metabolite profiling has been carried out to assess the compositional changes occurring in potato tubers after genetic modifications have been made to different metabolic pathways. Most major features in the (1)H NMR and HPLC-UV profiles of tuber extracts have been assigned. About 40 GM lines and controls belonging to 4 groups of samples (derived from cv. Record or cv. Desirée and modified in primary carbon metabolism, starch synthesis, glycoprotein processing, or polyamine/ethylene metabolism) were analyzed. Differences were assessed at the level of whole profiles (by PCA) or individual compounds (by ANOVA). The most obvious differences seen in both NMR and HPLC-UV profiles were between the two varieties. There were also significant differences between two of the four Desirée GM lines with modified polyamine metabolism and their controls. Compounds notably affected were proline, trigonelline, and numerous phenolics. However, that modification gave rise to a very abnormal phenotype. Certain lines from the other groups had several compounds present in significantly higher or lower amounts compared to the control, but the differences in mean values amounted to no more than a 2-3-fold change: in the context of variability in the whole data set, such changes did not appear to be important.

4-Hydroxycinnamoyl-CoA hydratase/lyase, an enzyme of phenylpropanoid cleavage from Pseudomonas, causes formation of C(6)-C(1) acid and alcohol glucose conjugates when expressed in hairy roots of Datura stramonium L.

4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL), a crotonase homologue of phenylpropanoid catabolism from Pseudomonas fluorescens strain AN103, led to the formation of 4-hydroxybenzaldehyde metabolites when expressed in hairy root cultures of Datura stramonium L. established by transformation with Agrobacterium rhizogenes. The principal new compounds observed were the glucoside and glucose ester of 4-hydroxybenzoic acid, together with 4-hydroxybenzyl alcohol- O-beta- D-glucoside. In lines actively expressing HCHL, these together amounted to around 0.5% of tissue fresh mass. No protocatechuic derivatives were found, although a trace of vanillic acid-beta- D-glucoside was detected. There was no accumulation of 4-hydroxybenzaldehydes, whether free or in the form of their glucose conjugates. There was some evidence suggesting a diminished availability of feruloyl-CoA for the production of feruloyl putrescine and coniferyl alcohol. The findings are discussed in the context of a diversion of phenylpropanoid metabolism, and the ability of plants and plant cultures to conjugate phenolic compounds.

Prior exposure to lipopolysaccharide potentiates expression of plant defenses in response to bacteria.

Lipopolysaccharide (LPS) is a ubiquitous component of Gram-negative bacteria which has a number of diverse biological effects on eukaryotic cells. In contrast to the large body of work in mammalian and insect cells, the effects of LPS on plant cells have received little attention. LPS can induce defense-related responses in plants, but in many cases these direct effects are weak. Here we have examined the effects of prior inoculation of LPS on the induction of plant defense-related responses by phytopathogenic xanthomonads in leaves of pepper (Capsicum annuum). The resistance of pepper to incompatible strains of Xanthomonas axonopodis pv. vesicatoria or to X. campestris pv. campestris is associated with increased synthesis of the hydroxycinnamoyl-tyramine conjugates, feruloyl-tyramine (FT) and coumaroyl-tyramine (CT). FT and CT are produced only in trace amounts in response to compatible strains of X. axonopodis pv. vesicatoria. Treatment of leaves with LPS from a number of bacteria did not induce the synthesis of FT and CT but altered the kinetics of induction upon subsequent bacterial inoculation. In incompatible interactions FT and CT synthesis was accelerated, whereas in compatible interactions synthesis was also considerably enhanced. The ability of the tissue to respond more rapidly was induced within 4 h of LPS treatment and the potentiated state was maintained for at least 38 h. Earlier treatment with LPS also potentiated the expression of other defense responses such as transcription of genes encoding acidic beta-1,3-glucanase. Our findings indicate a wider role for LPS in plant-bacterial interactions beyond its limited activity as a direct inducer of plant defenses.