Phillip Morris

The colors of autumn leaves as symptoms of cellular recycling and defenses against environmental stresses.

The color changes that occur during foliar senescence are directly related to the regulation of nutrient mobilization and resorption from leaf cells, often under conditions of biotic and abiotic stress. Chlorophyll is degraded through a metabolic pathway that becomes specifically activated in senescence. Chlorophyll catabolic enzymes and genes have been identified and characterized and aspects of their regulation analyzed. Particular genetic interventions in the pathway lead to disruptions in protein mobilization and increased sensitivity to light-dependent cell damage and death. The chemistry and metabolism of carotenoid and anthocyanin pigments in senescing leaves are considered. Bright autumn colors observed in the foliage of some woody species have been hypothesized to act as a defense signal to potential insect herbivores. Critical consideration of the biochemical and physiological features of normal leaf senescence leads to the conclusion that accumulating or unmasking compounds with new colors are unlikely to represent a costly investment on the part of the tree. The influences of human evolutionary and social history on our own perception of autumn coloration are discussed. The possibility that insect herbivores may respond to volatiles emitted during leaf senescence, rather than to bright colors, is also presented. Finally, some new approaches to the analysis of protein recycling in senescence are briefly considered.

Transgenic plants of Lolium multiflorum, Lolium perenne, Festuca arundinacea and Agrostis stolonifera by silicon carbide fibre-mediated transformation of cell suspension cultures

Seven Lolium multiflorum, one Lolium perenne, 12 Festuca arundinacea and six Agrostis stolonifera plants were regenerated following transformation with a hygromycin resistance gene and hygromycin selection, from cell suspension colonies treated with silicon–carbide whiskers. Transformation was confirmed by PCR and Southern blotting; the latter also showed that six of the L. multiflorum plants were independent transformants (insufficient molecular evidence was obtained for the seventh), nine of the 12 F. arundinacea plants were independent transformants, but that all the A. stolonifera plants were derived from a single transformation event. Most plants tested contained fewer than five integrated transgene copies. Transgene expression was confirmed by reverse transcriptase-PCR (RT-PCR). Of the one A. stolonifera and three L. multiflorum transformants regenerated after co-transformation with both the hygromycin resistance gene and the β-glucuronidase (gusA) gene, none were found to express GUS activity. L. multiflorum regenerants from older (14–16 week old) cell suspensions showed loss of female fertility, but analysis of the progeny from three plants showed that the transgenes were being inherited as a single dominant allele with a high frequency of transmission of hygromycin resistance.

Agrobacterium tumefaciens-mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.)

Abstract.Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and β-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24–68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.

Co-transformed, diploid Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass) and Lolium temulentum (darnel) plants produced by microprojectile bombardment

Abstract A total of 37 plants (30 Lolium multiflorum Lam., 6 L. perenne L., 1 L. temulentum L.) were regenerated from cell suspension colonies bombarded with plasmid DNAs encoding a hygromycin resistance gene (HYG) expressed under a CaMV35S promoter and a β-glucuronidase (GUS) gene expressed under a truncated rice actin1 promoter and first intron, or a maize ubiquitin promoter and first intron. Resistant plants were regenerated under hygromycin selection and transferred to soil. PCR analysis showed that the co-transformation frequency of the GUS gene varied from 33% to 78% of transformants, while histochemical staining of leaf tissue from soil-grown plants showed that the co-expression frequency varied from 37% to 50%. The transgenic nature of the plants was demonstrated by Southern hybridisation analysis, which also showed that the non-selected (GUS) gene was generally present at a higher copy number than the selected (HYG) gene.

Latent and Active Polyphenol Oxidase (PPO) in Red Clover (Trifolium pratense) and Use of a Low PPO Mutant To Study the Role of PPO in Proteolysis Reduction

Polyphenol oxidase (PPO) activity in leaf extracts of wild type (WT) red clover and a mutant line expressing greatly reduced levels of PPO (LP red clover) has been characterized. Both latent and active forms of PPO were present, with the latent being the predominant form. PPO enzyme and substrate (phaselic acid) levels fluctuated over a growing season and were not correlated. Protease activation of latent PPO was demonstrated; however, the rate was too low to have an immediate effect following extraction. A novel, more rapid PPO activation mechanism by the enzymes own substrate was identified. Rates of protein breakdown and amino acid release were significantly higher in LP red clover extracts compared with WT extracts, with 20 versus 6% breakdown of total protein and 1.9 versus 0.4 mg/g FW of free amino acids released over 24 h, respectively. Inclusion of ascorbic acid increased the extent of protein breakdown. Free phenol content decreased during a 24 h incubation of WT red clover extracts, whereas protein-bound phenol increased and high molecular weight protein species were formed. Inhibition of proteolysis occurred during wilting and ensilage of WT compared with LP forage (1.9 vs 5 and 17 vs 21 g/kg of DM free amino acids for 24 h wilted forage and 90 day silage, respectively). This study shows that whereas constitutive red clover PPO occurs predominantly in the latent form, this fraction can contribute to reducing protein breakdown in crude extracts and during ensilage.

Manipulating the phenolic acid content and digestibility of Italian ryegrass (Lolium multiflorum) by vacuolar targeted expression of a fungal ferulic acid esterase

In grass cell walls, ferulic acid esters linked to arabinosyl residues in arabinoxylans play a key role in crosslinking hemicellulose. Although such crosslinks have a number of important roles in the cell wall, they also hinder the rate and extent of cell wall degradation by ruminant microbes and by fungal glycohydrolyase enzymes. Ferulic acid esterase (FAE) can release both monomeric and dimeric ferulic acids from arabinoxylans making the cell wall more susceptible to further enzymatic attack. Transgenic plants of Lolium multiflorum expressing a ferulic acid esterase gene from Aspergillus niger, targeted to the vacuole under a constitutive rice actin promoter, have been produced following microprojectile bombardment of embryogenic cell cultures. The level of FAE activity was found to vary with leaf age and was highest in young leaves. FAE expression resulted in the release of monomeric and dimeric ferulic acids from cell walls on cell death and this was enhanced severalfold by the addition of exogenous β-1,4-endoxylanase. We also show that a number of plants expressing FAE had reduced levels of cell wall esterified monomeric and dimeric ferulates and increased in vitro dry-matter digestibility compared with nontransformed plants.

Expression of an Antirrhinum dihydroflavonol reductase gene results in changes in condensed tannin structure and accumulation in root cultures of Lotus corniculatus (bird's foot trefoil)

Condensed tannins (proanthocyanidins) are an important factor in the nutritive and dietary quality of many forage crops. We report here experiments aimed at altering the levels and monomer composition of condensed tannins (CTs) in ‘hairy root’ cultures of Lotus corniculatus (birds foot trefoil) using genetic manipulation. An Antirrhinum majus dihydroflavonol reductase (DFR) cDNA was expressed in sense in L. corniculatus and CT levels in transgenic root cultures were analysed. Two co-transformed lines were noted with decreased CT content relative to controls and these levels were comparable with antisense-DFR phenotypes. In ADFR10, a co-transformed line with the highest CT levels, CT structure was altered in a manner consistent with the substrate specificity of the introduced gene; that is an increase in pro-pelargonidin monomers noted after hydrolysis of CTs. RT-PCR confirmed the expression of endogenous DFR gene(s) in both putatively co-suppressed lines and also in ADFR10. Analysis of selected root culture lines indicated that the monomer composition of CTs did not change during growth and development but that levels of CTs varied in a regulated manner.

The effect of selection pressure on transformation frequency and copy number in transgenic plants of tall fescue (Festuca arundinacea Schreb.)

Abstract Protoplasts of Festuca arundinacea (Schreb.) were transformed with plasmids containing a hygromycin resistance gene, and plants were regenerated under a variety of selection regimes. Plant regeneration was highest under discontinuous selection, but resulted in the production of significant numbers of escapes. Continuous selection at low concentrations of hygromycin produced the highest numbers of transgenic plants without escapes. Transgene copy numbers for 83 plants were estimated and related to the selection regime under which the plants were regenerated. Discontinuous application of hygromycin produced a high proportion of plants containing two or fewer inserted copies of the transgene (87%), as did low levels (50 mg·l−1) of continuous selection on hygromycin (88%). Continuous selection at high concentrations of hygromycin biased selection in favour of multiple-copy containing plants. Using a plasmid encoding a hygromycin phosphotransferase enzyme of higher specific activity (pROB5) resulted in the production of significantly more transgenic plants.

Targeting expression of a fungal ferulic acid esterase to the apoplast, endoplasmic reticulum or golgi can disrupt feruloylation of the growing cell wall and increase the biodegradability of tall fescue (Festuca arundinacea).

In the cell walls of grasses, ferulic acid is esterified to arabinoxylans and undergoes oxidative reactions to form ferulates dimers, trimers and oligomers. Feruloylation of arabinoxylan is considered important not only because it leads to cross-linked xylans but also because ferulates may act as a nucleating site for the formation of lignin and hence link arabinoxylans to lignin by forming a lignin-ferulate-arabinoxylan complex. Such cross-linking is among the main factors inhibiting the release of fermentable carbohydrates from grasses either for ruminant nutrition or for biofuel production. We have found that significant reductions in the levels of monomeric and dimeric phenolics can be achieved in the growing cell walls during plant development in leaves of Festuca arundinacea by constitutive intracellular targeted expression of Aspergillus niger ferulic acid esterase (FAEA). We propose that this occurred by directly disrupting ester bonds linking phenolics to cell wall polysaccharides by apoplast targeting or by preventing excessive feruloylation of cell wall carbohydrates prior to their incorporation into the cell wall, by targeting to the Golgi membrane system. Plants with lower cell wall ferulate levels, which showed increased digestibility and increased rates of cellulase-mediated release of fermentable sugars, were identified. Targeting FAE to the Golgi was found to be more effective than targeting to the ER, which supports the current theories of the Golgi as the site of feruloylation of arabinoxylans. It is concluded that targeting FAEA expression to the Golgi or apoplast is likely to be an effective strategy for improving wall digestibility in grass species used for fodder or cellulosic ethanol production.

Phenolic Compounds: extraction and analysis

Phenolic compounds make up one of the major families of secondary metabolites in plants, and they represent a diverse group of compounds. Phenolics can be broadly divided into non-soluble compounds such as condensed tannins, lignins, and cell-wall bound hydroxycinammic acids, and soluble phenolics such as phenolic acids, phenylpropanoids, flavonoids and quinones. In this article, a method for extracting and analysing the soluble fraction of these phenolics, using HPLC coupled to photodiode array (PDA) detector, is described along with its uses to profile the flavonoid content of Lotus japonicus.