Weiting Ni

Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase.

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.

Reduced lignin in transgenic plants containing a caffeic acidO-methyltransferase antisense gene

Lignin is a major structural polymer of secondarily thickended plant vascular tissue and fibres, imparting mechanical strength to stems and trunks and hydrophobicity to conducting vessels. Constitutive expression of a lucerne caffeic acid 3-O-methyltransferase antisense RNA in transgenic tobacco leads to a significant reduction in lignin content, particularly in the younger parts of the stems, without apparent alterations in lignin monomer composition. These observations open up the possibility of genetically manipulating plants with reduced lignin for improved processing and biomass digestibility.

Stress responses in alfalfa (Medicago sativa L.) XIX. Transcriptional activation of oxidative pentose phosphate pathway genes at the onset of the isoflavonoid phytoalexin response

We have isolated cDNA clones encoding the pentose phosphate pathway enzymes 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from alfalfa (Medicago sativa L.). These exhibit extensive nucleotide and amino acid sequence similarity to the corresponding genes from bacteria, Drosophila and mammals. Transcripts encoding both enzymes are expressed at high levels in roots and nodules. Exposure of alfalfa suspension cells to an elicitor from yeast cell walls results in co-ordinated increases in transcription rates for both genes, followed by increased steady state transcript levels but only slightly increased extractable enzyme activities, at the onset of accumulation of isoflavonoid phytoalexins. Levels of NADPH and NADP remain relatively constant in alfalfa cells following elicitation. The rapid transcriptional activation of 6PGDH and G6PDH does not therefore appear to be a response to altered pyridine nucleotide redox state. These genes appear to respond to early events in elicitor-mediated signalling rather than to subsequent elicitor-induced changes in secondary metabolism. Hydrogen peroxide, a potential signal for elicitation of anti-oxidative genes in biologically stressed plant cells, did not induce 6PGDH or G6PDH transcripts or enzymatic activity.

STRESS RESPONSES IN ALFALFA (MEDICAGO SATIVA L.). XX. TRANSCRIPTIONAL ACTIVATION OF PHENYLPROPANOID PATHWAY GENES IN ELICITOR-INDUCED CELL SUSPENSION CULTURES

Nuclear transcript run-on analysis was used to investigate the relative transcription rates of genes encoding enzymes of isoflavonoid phytoalexin biosynthesis and related pathways in elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures. Genes encoding L-phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and chalcone reductase (CHR) were most rapidly activated, with increases in transcription measurable within 10–20 min after elicitation. Cinnamic acid 4-hydroxylase (C4H), chalcone isomerase (CHI), isoflavone reductase (IFR) and caffeic acid 3-O-methyltransferase (COMT) genes were also rapidly activated, but at a slower initial rate. Transcription of chalcone 2′-O-methyltransferase (CHOMT), and 1,3-β-D-glucanase genes was less rapid, with lag periods of 60 and 30 min post-elicitation, respectively. Treatment of cells with the PAL inhibitor L-α-aminooxy-β-phenylpropionic acid (AOPP) resulted in increased transcription of PAL, CHS and CHR, but reduced transcription of CHOMT, indicating a role for phenylpropanoid products as both negative and positive regulators of gene expression within the phenylpropanoid pathway.

Stress Responses in Alfalfa (XXI. Activation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl Coenzyme A 3-O-Methyltransferase Genes Does Not Contribute to Changes in Metabolite Accumulation in Elicitor-Treated Cell-Suspension Cultures)

Transcription of genes encoding L-phenylalanine ammonia-lyase (PAL), the first enzyme of the phenylpropanoid pathway, and caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT), enzymes involved in the synthesis of lignin and wall-esterified phenolic compounds, was strongly activated in elicitor-treated cell-suspension cultures of alfalfa (Medicago sativa L.). However, consequent changes in the extractable activities of COMT and CCOMT were small to nonexistent compared with a 15- to 16-fold increase in PAL activity. Only low levels of COMT and CCOMT transcripts were reflected in the total and polysomal RNA fractions compared with PAL transcripts. Elicited cell cultures did not accumulate lignin or the products of COMT and CCOMT in the soluble and wall-esterified phenolic fractions. In one alfalfa cell line in which elicitation resulted in very high PAL activity and increased deposition of methoxyl groups in the insoluble wall fraction, there was still no change in COMT and CCOMT activities. Overall, these results indicate that the initial gene transcription events in elicited cells may be less selective than the subsequent metabolic changes, highlighting the importance of posttranscriptional events in the control of phenylpropanoid biosynthesis.

Genetic Manipulation of Lignin and Phenylpropanoid Compounds Involved in Interactions with Microorganisms

Increasing knowledge of the biochemistry of plant secondary product synthesis, and the cloning of biosynthetic pathway genes, has opened up the possibility of engineering novel pathways or reducing unwanted metabolites by genetic engineering strategies. Such approaches should lead to significant improvements in agronomic performance and post-harvest processing. At present, the necessary knowledge base is most advanced in the area of phenylpropanoid derivatives and certain alkaloids. Further advances in our understanding of terpenoid biochemistry, and our ability to genetically transform cereals and large seeded legumes, are required to underpin fuller exploitation of genetic manipulation of secondary metabolism for plant improvement.

Transcriptional Regulation of Phytoalexin Biosynthetic Genes

In legumes, isoflavonoid derivatives function as antimicrobial phytoalexins, whereas phytoalexins of solanaceous species are of terpenoid origin. The phenylpropanoid and isoprenoid pathways leading to these phytoalexins are involved in the synthesis of a wide range of secondary metabolites with important functions in plant growth, development and responses to the environment. Elicitation of phytoalexin biosynthesis involves transcriptional activation of the genes encoding enzymes of general phenylpropanoid/terpenoid biosynthesis, and of the genes for the specific branch pathways leading to antimicrobial compounds. In order to understand the molecular controls determining the developmental and environmental regulation of the general and specific enzymes of phytoalexin synthesis, we are studying the promoter regions of three elicitor inducible genes, chalcone synthase (chs, isoflavonoid pathway, general), isoflavone reductase (ifr,isoflavonoid pathway, specific) and 3-hydroxy-3-methylglutaryl CoA reductase (hmgr,terpenoid pathway, general). We describe cis-elements and trans-factors involved in the expression of these genes in relation to their tissue specific expression and response to biotic stress. Two elements, the G-box and H-box, located within 50 bp of the TATA box, are important for regulation of expression of chs and probably hmgr, but are not present in the alfalfa ifr promoter.