Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.     

Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase in transgenic alfalfa affects lignification, development and forage quality

The recently discovered enzyme hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) catalyzes the reactions both immediately preceding and following the insertion of the 3-hydroxyl group into monolignol precursors. A number of independent transgenic lines of alfalfa (Medicago sativa L.) were generated in which the levels of HCT were reduced through antisense HCT expression under control of the bean PAL2 promoter which is preferentially expressed in vascular tissue. Reduction of enzyme activity in these lines was from at least 15-50%. The most severely down-regulated lines exhibited significant stunting, reduction of biomass and delayed flowering. HCT down-regulation resulted in strongly reduced lignin content and striking changes in lignin monomer composition, with predominant deposition of 4-hydroxyphenyl units in the lignin. Vascular structure was impaired in the most strongly down-regulated lines. Analysis of forage quality parameters showed strong reductions of neutral- and acid-detergent fiber in the down-regulated lines, in parallel with large increases (up to 20%) in dry matter forage digestibility. Although manipulation of lignin biosynthesis can greatly improve forage digestibility, accompanying effects on plant development need to be better understood.     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

Improving Saccharification Efficiency of Alfalfa Stems Through Modification of the Terminal Stages of Monolignol Biosynthesis

A series of transgenic lines of alfalfa (Medicago sativa) were generated in which either one of the two potentially terminal enzymes of the monolignol pathway, cinnamoyl CoA reductase (CCR) or cinnamyl alcohol dehydrogenase (CAD) was down-regulated by expression of antisense transgenes. Levels of CCR enzymatic activity were reduced to between 10% to 65% of the control level, and levels of CAD activity were similarly reduced to between 5% to 40% of the control. Biomass yields were reduced in the most strongly down-regulated lines for both transgenes, but many of the lines exhibited reduced lignin levels but normal biomass and flowering time. In vitro dry matter digestibility was increased for most transgenic lines compared to controls. Saccharification efficiency was determined by measuring the release of sugars from cell walls directly, or after sulfuric acid pre-treatment and subsequent digestion with a mixture of cellulase and cellobiase. Several CCR down-regulated lines had significantly enhanced saccharification efficiency with both pre-treated and untreated tissues, whereas CAD down-regulation had less impact on sugar release when compared to that from CCR lines with similar lignin contents. One CCR line with a 50–60% improvement in saccharification efficiency exhibited normal biomass production, indicating the potential for producing high yielding, improved feedstocks for bioethanol production through genetic modification of the monolignol pathway.     

Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.     

Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase

The composition of lignin in tobacco stems has been altered by genetic engineering. Antisense expression of sequences encoding cinnamyl alcohol dehydrogenase (CAD), the enzyme catalysing the final step in lignin precursor synthesis, leads to the production of a modified lignin in otherwise normal plants. Although Klason and acetyl bromide lignin determinations show little quantitative change in lignin deposition in CAD antisense plants, a number of qualitative changes have been identified. The lignin is altered in both composition and structure and is more susceptible to chemical extraction. Consistent with a block in CAD activity, antisense plants incorporate less cinnamyl alcohol monomers and more cinnamyl aidehyde monomers into lignin than corresponding control plants. Antisense plants with very low levels of CAD activity also show a novel phenotype with the appearance of a red-brown colour in xylem tissues. A similar phenotype is correlated with altered lignification and improved digestibility in brownmidrib mutants of maize and sorghum. The improved chemical extractability of lignin in CAD antisense plants supports a role for this technology in improving the pulp and paper-making value of forest trees while the similarity with brown-midrib mutants suggests a route to more digestible forage crops.     

Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants

Different transgenic tobacco lines down-regulated for either one or two enzymes of the monolignol pathway were compared for their lignin content and composition, and developmental patterns. The comparison concerned CCR and CAD down-regulated lines (homozygous or heterozygous for the transgene) and the hybrids resulting from the crossing of transgenic lines individually altered for CCR or CAD activities. Surprisingly, the crosses containing only one allele of each antisense transgene, exhibit a dramatic reduction of lignin content similar to the CCR down-regulated parent but, in contrast to this transgenic line, display a normal phenotype and only slight alterations of the shape of the vessels. Qualitatively the lignin of the double transformant displays characteristics more like the wild type control than either of the other transgenics. In the transgenics with a low lignin content, the transformations induced other biochemical changes involving polysaccharides, phenolic components of the cell wall and also soluble phenolics. These results show that the ectopic expression of a specific transgene may have a different impact depending on the genetic background and suggest that the two transgenes present in the crosses may operate synergistically to reduce the lignin content. In addition, these data confirm that plants with a severe reduction in lignin content may undergo normal development at least in controlled conditions.     

In situ analysis of lignins in transgenic tobacco reveals a differential impact of individual transformations on the spatial patterns of lignin deposition at the cellular and subcellular levels

Using tobacco transgenic lines altered in the monolignol biosynthetic pathway and which differ in their lignin profiles we have evaluated lignin deposition at the cellular and subcellular levels using several microanalytical techniques. Surprisingly, whereas a Cinnamoyl CoA reductase (CCR) down-regulated line with a strong decrease in lignin content exhibited an overall reduction in lignin deposition in the walls of the different xylem cell types, this reduction was selectively targeted to the fibers in a double transformant (down-regulated for both CCR and Cinnamyl alcohol dehydrogenase (CAD)) displaying a similar degree of global lignin content decrease. Fiber and vessel secondary walls of the transgenic tobacco line homozygous for the ccr antisense gene (CCR.H) down-regulated plants were dramatically destructured, particularly in the S2 sublayer, whereas the deposition of lignins in the S1 sublayer was not significantly modified. In contrast, cell wall organization was slightly altered in xylem cells of the double transformant. The relative distribution of non-condensed and condensed units in lignin, evaluated microscopically with specific antibodies, was differentially affected in the transgenics studied and, in a general way, a drop in non-condensed lignin units (beta- 0-4 interunit linkages) was associated with a loss of cohesion and extensive disorganization of the secondary wall. These results demonstrate that lignification is tightly and independently regulated in individual cell types and cell wall sublayers. They also show that down-regulation of specific genes may induce targeted changes in lignin structure and in spatial deposition patterns of the polymer.     

Lignin genetic engineering

Although lignins play important roles in plants, they often represent an obstacle to the utilization of plant biomass in different areas: pulp industry, forage digestibility. The recent characterization of different lignification genes has stimulated research programmes aimed at modifying the lignin profiles of plants through genetic engineering (antisense and sense suppression of gene expression). The first transgenic plants with a modification of monomeric composition of lignins and lignin content have been recently obtained. Down regulation of the OMT gene induces dramatic reduction of syringyl units. CAD down regulated plants exhibit a unusual red phenotype associated with the developing xylem and several chemical modifications of their lignins including an increase in cinnamaldehydes in the polymer structure. Interestingly this novel lignin is removed more easily during the pulping process. In both OMT and CAD down regulated plants no changes in phenotypic characteristics such as growth architecture and morphology were observed. More recent experiments have shown that a reduction of CCR activity determines specific changes in the coloration of the xylem area suggesting significant chemical modifications which are currently being studied.These different results show that it is possible to manipulate lignins through targeted genetic transformation of plants and that lignins exhibit a relative flexibility of their chemical structure. Future developments should probe the impact of down regulating the expression of other recently characterized lignification genes such as F5H and CCoAOMT and also of a combination of genes in order to tailor lignins more adapted to specific purposes. In addition to biotechnological applications which should provide important economical benefits for utilization of wood in the pulp industry, genetic engineering of lignins offer very promising perspectives for the understanding of lignin synthesis, structure and properties.     

Improvement of cell wall digestibility in tall fescue by <Emphasis Type="Italic">Oryza sativa</Emphasis> SECONDARY WALL NAC DOMAIN PROTEIN2 chimeric repressor

Forage digestibility is one of the most important factors in livestock performance. As grasses grow and mature, dry matter increases but they become fibrous with secondary cell wall deposition and lignification of sclerenchyma cells, and forage quality drops. In rice (Oryza sativa), the SECONDARY WALL NAC DOMAIN PROTEIN2 fused with the modified EAR-like motif repression domain (OsSWN2-SRDX) reduces secondary cell wall thickening in sclerenchyma cells. We introduced OsSWN2-SRDX under the control of the OsSWN1 promoter into tall fescue (Festuca arundinacea Schreb.) to increase cell wall digestibility. Of 23 transgenic plants expressing OsSWN2-SRDX, nine had brittle internodes that were easily broken by bending. Their secondary cell walls were significantly thinner than those of the wild type in interfascicular fibers of internodes and in cortical fiber cells between leaf epidermal cells and vascular bundles. The dry matter digestibility increased by 11.8% in stems and by 6.8% in leaves compared with the wild type, and therefore forage quality was improved. In stem interfascicular fibers, acid detergent fiber and acid insoluble lignin were greatly reduced. Thus, the reduction of indigestible fiber composed of cellulose and lignin increased the degradability of sclerenchyma cell walls. OsSWN2-SRDX plants offer great potential in the genetic improvement of forage digestibility.     

Viability and longevity of pollen from transgenic and nontransgenic tall fescue (Festuca arundinacea) (Poaceae) plants

Pollen is an important vector of gene flow in plants, particularly for outcrossing species like tall fescue. Several aspects of pollination biology were investigated using pollen from transgenic and nontransgenic plants of tall fescue (Festuca arundinacea Schreb.), the most important forage species worldwide of the Festuca genus. To effectively assess in vitro pollen viability in tall fescue, an optimized germination medium (0.8 mol/L sucrose, 1.28 mmol/L boric acid and 1.27 mmol/L calcium nitrate) was developed. Treatment with relatively high temperatures (36° and 40°C) and high doses of UV-B irradiation (900-1500 μW/cm(2)) reduced pollen viability, while relative humidity did not significantly influence pollen viability. Viability of pollen from transgenic progenies (T1 and T2) was similar to that from seed-derived control plants. Pollen from primary transgenics (T0) and primary regenerants (R0) had various levels of viability. Hand pollination using the primary regenerants and transgenics revealed that no seed set could be obtained when pollen viability was lower than 5%. Pollen from transgenic progenies and nontransgenic control plants could survive up to 22 h under controlled conditions in growth chamber. However, under sunny atmospheric conditions, viability of transgenic and nontransgenic pollen reduced to 5% in 30 min, with a complete loss of viability in 90 min. Under cloudy atmospheric conditions, pollen remained viable up to 240 min, with about 5% viability after 150 min. This report is the first on pollen viability and longevity in transgenic forage grasses and could be useful for risk assessment of transgenic plants.     

Simultaneous regulation of F5H in COMT‐RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis

Ferulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.     

Downregulation of Cinnamyl Alcohol Dehydrogenase (CAD) Leads to Improved Saccharification Efficiency in Switchgrass

The bioconversion of carbohydrates in the herbaceous bioenergy crop, switchgrass (Panicum virgatum L.), is limited by the associated lignins in the biomass. The cinnamyl alcohol dehydrogenase (CAD) gene encodes a key enzyme which catalyzes the last step of lignin monomer biosynthesis. Transgenic switchgrass plants were produced with a CAD RNAi gene construct under the control of the maize ubiquitin promoter. The transgenic lines showed reduced CAD expression levels, reduced enzyme activities, reduced lignin content, and altered lignin composition. The modification of lignin biosynthesis resulted in improved sugar release and forage digestibility. Significant increases of saccharification efficiency were obtained in most of the transgenic lines with or without acid pretreatment. A negative correlation between lignin content and sugar release was found among these transgenic switchgrass lines. The transgenic materials have the potential to allow for improved efficiency of cellulosic ethanol production.     

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.     

Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway

Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.     

Impact of different levels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants

The effects of cinnamyl alcohol dehydrogenase (CAD, EC.1.1.1.195) down-regulation on lignin profiles of plants were analysed in four selected transgenic lines of tobacco (Nicotiana tabacum L. cv. Samsun) exhibiting different levels of CAD activity (8–56% of the control). A significant decrease in thioacidolysis yields (i.e. yield of β-O-4 linked monomers) and in the ratio of syringyl to guaiacyl monomers (S/G) was observed for three transgenic lines and the most drastic reduction (up to 50%) was correlated with the lowest level of CAD activity. Higher lignin extractability by mild alkali treatment was confirmed, and, in addition to a tenfold increase in C₆-C₁ aldehydes, coniferyl aldehyde was detected by high-performance liquid chromatography in the alkali extracts from the xylem of transgenic plants. In-situ polymerisation of cinnamyl aldehydes in stem sections of untransformed tobacco gave a xylem cell wall coloration strikingly similar to the reddish-brown coloration of the xylem of antisense CAD-down-regulated plants. Overall, these data provide new arguments for the involvement of polymerised cinnamyl aldehydes in the formation of the red-coloured xylem of CAD-down-regulated plants. Received: 24 January 1997 / Accepted: 14 May 1997