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The effects of viral infection on plant cell phenolics in wheat and tobacco Gibney, Blair 1995

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THE EFFECTS OF VIRAL INFECTION ON PLANT C E L L PHENOLICS IN WHEAT AND TOBACCO by BLAIR GIBNEY B.Sc, The University of British Columbia, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Botany) We accept this thesis as conforming to the Med standard T H E UNIVERSITY OF BRITISH COLUMBIA November 1995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of BOTflM^ The University of British Columbia Vancouver, Canada Date DE-6 (2/88) Abstract The study compared the effects on free and cell wall bound phenolics of Triticum aestivum cv. Katepwa inoculated with Tobacco Necrosis Virus ( T N V ) , Barley Stripe Mosaic Virus ( B S M V ) or distilled water. In addition, the phenolic compounds in Nicotiana tabacum cv. Xanthi n.c. inoculated with T N V , Tobacco Mosaic Virus or distilled water were examined. Analysis of the free phenolics consisted o f methanol extracts of freeze-dried plant leaves. Samples of the methanol extracts were subjected to hydrolysis by N a O H and B-glucosidase. Cell wall bound phenolics were determined from hydrolysis of the cell walls with 2 M N a O H . Identification and separation of the various phenolic compounds was attained by use of H P L C . In unhydrolysed wheat methanol extracts, no phenolics were identified. In the tobacco samples, protocatechuic acid was found in mock samples. In J3-glucosidase hydrolysed wheat samples ferulic acid was found in both virally challenged treatments but not in mock inoculated plants. In the tobacco, T M V infected samples contained vanillic, caffeic, salicylic, ferulic and possibly truxinic or truxillic acids. In T N V inoculated samples 4-hydroxybenzoic acid was found. Mock samples contained no identifiable peaks. A l k a l i hydrolysis methanol extracts contained vanillic, caffeic, benzoic, salicylic and truxinic/truxillic acids. In the T N V infected plants benzoic acid was identified. N o compounds were identified in mock inoculated samples. Wheat cell walls released vanillic, p-coumaric, ferulic, and diferulic acids as well as vanillin and p-hydroxybenzaldehyde. Levels released decreased late in the infection process. The mock infected tobacco cell walls released p-coumaric acid. The T N V inoculated samples released p-hydroxybehzaldehyde and vanillin as well as p-coumaric acid. The T M V exposed plants produced caffeic acid in addition to the compounds produced by TNV. i v Contents Abstract p. ii Contents p. iv List of Figures p. vii Acknowledgements p. ix 1.0 Introduction p.l 1.1 The plant cell wall p. 2 1.2 Cell Wall Polysaccharides p. 2 1.3 Other Non-Phenolic Cell Wall Components p. 5 1.4 Cell Wall Phenolics p. 7 1.4.1 Lignins p. 7 1.4.2 Wood Lignins p. 10 1.4.3 Monocot Lignins p. 13 1.5 Biosynthesis p. 19 1.6 Monolignol Transport p. 29 1.7 Polymerization p. 29 1.8 Soluble Phenolics p. 34 1.9 Polyamines p. 35 2.0 General Viral Symptoms p. 35 2.1 Chlorosis p. 37 2.2 Hypersensitive Response p. 38 V 2.2.1 Early Events p. 40 2.2.2 Oxidative Burst p. 41 2.2.3 Structural Proteins p. 43 2.2.4 Polyamines p. 43 2.2.5 Ethylene p. 45 2.2.6 Phenolic Accumulation p. 45 2.2.7 Lignins p. 48 2.3 Phytoalexins p. 49 2.4 Systemic Acquired Resistance p. 50 2.5 Pathogenesis Related Proteins p. 51 2.6 Salicylic Acid p. 52 3.0 The Viruses p. 54 3.1 Tobacco Necrosis Virus p. 54 3.2 Barley Stripe Mosaic Virus p. 55 3.3 Tobacco Mosaic Virus p. 56 3.4 Viral Movement p. 56 4.0 Materials and Methods p. 60 5.0 Results p. 66 5.1 General Pathology p. 66 5.1.1 Wheat p. 66 5.1.2 Tobacco p. 66 5.2 Systemic Acquired Resistance in Wheat p. 67 v i 5.3 Analysis of Phenolic Compounds Induced by Infection p. 68 5.3.1 Free Phenolics from Methanol Extracts p. 68 5.3.1.1 Wheat p. 68 5.3.1.2 Tobacco p. 68 5.3.2 f3-Glucosidase Treated Methanol Extracts p. 68 5.3.2.1 Wheat p. 68 5.3.2.2 Tobacco p. 68 5.3.3 Alkaline Hydrolysis of Methanol Extracts p. 72 5.3.3.1 Wheat p. 72 5.3.3.2 Tobacco p. 72 5.3.4 Butanol Extracts of Methanol Extracts p. 77 5.3.4.1 Wheat p. 77 5.3.4.2 Tobacco p. 77 5.4 Cell Wall Associated Phenolics p. 77 5.4.1 Alkaline Hydrolysis of Cell Walls p. 77 5.4.1.1 Wheat p. 77 5.4.1.2 Tobacco p. 86 5.4.2 Lignin Analysis p. 86 5.4.2.1 Wheat p. 86 6.0 Discussion p. 90 References p. 98 Appendix 1 p. 120 V l l List of Figures. Figure 1. Lignin Bonding Patterns. pg. 11 Figure 2. Cinnamate Pathway pg. 21 Figure 3. HPLC Profile of Free Phenolics in Mock Inoculated Tobacco. pg. 69 Figure 4. HPLC Profile of Phenolics from B-Glucosidase Hydrolysed Methanol Extracts of BSMV and TNV Inoculated Wheat, pg. 70 Figure 5 HPLC Profile of Phenolics from B-Glucosidase Hydrolysed Methanol Extracts of T M V Inoculated Tobacco. pg. 71 Figure 6 HPLC Profile of Phenolics from B-Glucosidase Hydrolysed Methanol Extracts of TNV Inoculated Tobacco. pg. 73 Figure 7 HPLC Profile of Phenolics from Alkaline Hydrolysed Methanol Extracts of BSMV Inoculated Wheat. pg. 74 Figure 8 HPLC Profile of Phenolics from Alkaline Hydrolysed Methanol Extracts of Mock Inoculated Wheat. pg. 75 Figure 9 HPLC Profile of Phenolics from Alkaline Hydrolysed Methanol Extracts of T M V Inoculated Tobacco. pg. 76 Figure 10 HPLC Profile of Phenolics from Alkaline Hydrolysed Methanol Extracts of TNV Inoculated Tobacco. pg. 77 Figure 11 HPLC Profile of Phenolics from Alkaline Hydrolysed Cell Walls of Inoculated Wheat. pg. 79 Figure 12 Levels of Vanillic Acid in Inoculated Wheat Cell Walls pg. 80 VI11 Figure 13 Levels of p-Hydroxybenzoic Acid in Inoculated Wheat Cell Walls. pg. 81 Figure 14 Levels of Vanillin in Inoculated Wheat Cell Walls pg. 82 Figure 15 Levels of P-Coumaric Acid in Inoculated Wheat Cell Walls pg. 83 Figure 16 Levels of Ferulic Acid in Inoculated Wheat Cell Walls pg. 84 Figure 17 Levels of Diferulic Acid in Inoculated Wheat Cell Walls pg. 85 Figure 18 HPLC profile of Phenolics from Alkaline Hydrolyzed Cell Walls of T M V inoculated Tobacco. pg. 87 Figure 19 Structures of Benzoic Acid Derivatives. pg. 88 Figure 20 Structures of Other Compounds Found. pg. 89 i x Acknowledgements Dr. Chris French, Dr. Neil Towers, Dr. Bruce Bohm, Jon Page, Dr. Phil Gunning, Rob Hill, Dr. Santokh Singh, Shona Ellis, Dr. Ramon Razal, Dr. Vic Rathore and my friends and family for emotional and, of late, financial support. This work is dedicated to my grandfather Everett Hudson Vollans. 1 1.0 Introduction Plants synthesize a great diversity of compounds through the cinnamate pathway. The major products are lignins followed by the flavonoids. In addition, cinnamic acid derivatives are incorporated into a tremendous variety of terpenoids, carbohydrates, alkaloids and polyketides either as acyl substituents or as major components. Considerable amounts of these phenylpropanoids, generated from L-phenylalanine, are incorporated into the structure of the plant cell wall as lignins as well as conjugates with cell wall polysaccharides such as hemicelluloses and pectins. The present study is concerned with the effects of viral infections on plant cell wall and soluble pools of phenolic acids. Plants commonly respond to bacterial and fungal infections by accumulating cinnamic derivatives e.g.. isoflavonoids (Zaprometov, 1993). Much less is known about the consequences of viral infection. This lack of information suggested the present research. In the work reported here, the effects of Tobacco Necrosis Virus (TNV) and Barley Stripe Mosaic Virus (BSMV) on the levels of phenolic compounds in Triticum aestivum cv. Katepwa were examined. In addition the effects of TNV, Tobacco Mosaic Virus (TMV), and Potato Virus X (PVX) on three varieties of Nicotiana tabacum (White Burley, Samsun, and Xanthi n.c.) were examined. The levels of free and cell wall bound cinnamic and benzoic acid derivatives were determined. 2 1.1 The plant cell wall In studying viral effects on plant cell wall phenolics, it is important to understand the basic structure of cell walls. The wall is composed of a complex group of compounds including lignins, cellulose, pectins, proteins and many other components. Bolwell (1988) points to a shifting trend in the understanding of cell walls, moving from the concept of a passive matrix to a dynamic structure. Roland et al. (1989) proposes that the cell wall can be considered a composite with cellulose as the reinforcing fibre embedded in a complex matrix including lignin. Thus the cell can use the synergy of the cellulose and the surrounding matrix to improve tensile strength. A comparison could be drawn to fibreglass strands in a plastic resin or rebar in a concrete matrix. However, clearly the structure must be more dynamic to allow for growth, a point recognized by Roland et al. (1989), who compares the cell wall to cholesteric liquid crystals which allow for growth. Essentially, the cell wall has to be viscoplastic, in that it must be resistant for support but still fluid enough for elongation or oscillation in plasticity over time. Once growth is completed, the network can become more stabilized. 1.2 Cell Wall Polysaccharides The cell wall polysaccharides consist of polymers of one or more of nine common sugars; D-glucose, D-xylose, D-galactose, L-arabinose, L-rhamnose, D-galacturonic acid, D-mannose, L-fucose, and D-glucuronic acid. Traces of other sugars can also be found. Through various glycosidic linkages, these sugars form the major polysaccharide cell wall polymers including cellulose, pectins, 3 arabinans, arabinogalactans, galactans, xyloglucans, glucomannans, galactoglucomannans, mannan galactomannans, heteroxylans, B-D-glucans and rhamnogalacturonans (Tucker and Mitchell, 1993). These polymers vary in concentration between different species and between tissues within the plant. The initial thin primary wall laid down during cell growth includes the middle lamella. A secondary wall is added after the completion of cell growth in cells that undergo secondary thickening. Unfortunately, not a single synthase of plant polysaccharides has been unequivocally identified (Gibeaut and Carpita, 1994). Thus, the precise mechanism and regulation of the formation of the carbohydrate based wall components remains difficult to study. The primary cell walls of most dicots are similar in polymer type but the concentration of polymers vary (Selvendran,1985 ). Cellulose (13-1-4 glucosyl links) is present, comprising 9-40% of the primary wall. The other major polymers are pectic substances such as rhamnogalacturonans, arabinans, galactans and type I arabinogalactans (Bl-4 linked galactosyl backbone with 5-linked and terminal arabinosyl residues) (Aspinall, 1980). Pectins combine with Ca2+ions to form a gel and may control wall porosity, matrix charge, pH modulation and ion balance (Gibeaut and Carpita, 1994). Xyloglucans are the major hemicelluloses in dicotyledons (Hori and Elbein, 1985) representing around 20% of the dry weight while xylans make up approximately 2% (McNeil et al., 1984). In the lignified secondary walls of dicots and gymnosperms, cellulose represents 40-60% of the cell walls. Other non-cellulosic components are glucomannans, galactomannans 4 and a smaller component of 4-O-methylglucuronoarabinoxylans in gymnosperms and 4-O-methylglucuronoxylans with a minor component of glucomannans in dicots (Tucker and Mitchell, 1993). Monocots can be divided into two groups, the Gramineae and the rest of the monocots (Tucker and Mitchell, 1993), depending on wall structure. In addition to 35-40% cellulose, the Gramineae have low levels of pectic substances, the major non-cellulosic components being hemicelluloses, such as arabinoxylans and glucuronoarabinoxylans (GAX) (Varner and Lin, 1989). In dividing and elongating cells, GAXs are highly branched. Later in development, unbranched GAXs accumulate (Gibeaut and Carpita, 1994). Xylans make up 15-20% of the dry weight of monocots, while xyloglucans makes up only 2%. The secondary walls of the Gramineae are also lignified. McCann, Wells and Roberts, (1989) suggest that hemicellulose plays an important role in spacing cellulose fibrils. Additional non-cellulosic polysaccharides are glucuronarabinoxylans, heteroglucans and B-glucans which consist of 30% B-3 and 70% B-4 D-glucans (Tucker and Mitchell, 1993). Monocotyledons other than grasses have walls ranging from grass-like to the types found in the dicotyledons. Plant cell wall formation responds to environmental stimuli. For example, Iraki et al. (1989) examined the changes caused by saline and water stress adaptation and showed that the overall wall mass was decreased by 50%, cellulose synthesis was inhibited, and certain pectic substances were more readily extracted. In addition, there was an increase in rhamnose units as well as an increase in side 5 chain substitution of the rhamnogalacturonan polymers, although the side chains were shorter than in normal cells. Using labelled sugars, Gibeaut et al. (1991) have shown a turnover of polysaccharides in the cell wall, particularly arabinose residues and glucose in B-D-glucans. This finding suggests that the cell wall polysaccharides bound into the wall are not just a passive matrix. Northcote (1985) notes that since the wall is formed by components in the cytoplasm and is able to be continually modified during development, there must be information exchanged between the wall and cytoplasm. This communication may be important when addressing pathogenic effects as well. The composition of the cell wall varies dramatically between primary and secondary walls, tissue type and species. Even where similarities exist there can be confounding variables. An example is that xyloglucans of the Gramineae have a lower xylose contribution than those of dicots (Hayashi, 1989). The significance of these differences is not known. 1.3 Other Non-Phenolic Cell Wall Components The plant cell wall also contains structural proteins, some waxes, and various cations such as Ca 2 + which can bind to pectins forming bridges between carboxyl groups (Varner and Lin, 1989). Norway spruce (Picea abies) cuttings grown in low Ca 2 + environments exhibited a decrease in lignin and non-cellulosic polysaccharides, while in higher Ca 2 + situations these compounds increased with a corresponding decrease in cellulose production (Eklund and Eliasson, 1990). This situation may signify that the plant often has many approaches available to attain a 6 similar result and the path utilized depends on the availability of certain compounds in the environment,. In the cell wall, the best known of the structural proteins are the extensins. These proteins are basic because of their high lysine content and are rich in hydroxyproline residues that are glycosylated with 1 -4 arabinosyl groups (Cooper et al., 1987; Tierney and Varner, 1987; Cassab and Varner, 1988 ). Extensins are unusual proteins consisting of 50% protein and 50% carbohydrate. The carbohydrate portion of a typical extensin is made up of 97% arabinose chains (mainly 3-4 linked residues) attached to hydroxyproline and 3% galactose joined as single units to serine. The protein component of extensins often consists of amino acids organized into repeat units with a high level (33-42%) of hydroxyproline (Cassab and Varner, 1988). Again there are distinct differences between species. For example, hydroxyproline groups in monocots are much less frequently glycosylated than they are in dicots (Tucker and Mitchell, 1993). Cooper (1994) analysed cell wall regeneration in tobacco protoplasts exposed to a hydroxyproline rich glycoprotein (HRGP) inhibitor. The larger fibrils were coated with globular knobs and smaller fibrils were absent or not visible, suggesting that HRGP may be important in the structural organization of other wall components. When the protein is synthesized, it is released as a soluble polypeptide. The polypeptide rapidly becomes insoluble in the wall, likely via covalent cross-linking with other wall components. 7 Another HRGP type is an arabinogalactan protein (Marcus, Greenberg and Averyhart-Fullard, 1991) composed of 10% protein and 90% carbohydrate. Arabinogalactan proteins are more acidic in nature than the extensins. The carbohydrate structure is much more complex with more variety in glycosyl residues. The protein is more associated with the extracellular space than the cell wall. There are various other proteins that have been described including a r proline rich protein in soybean (Averyhart-Fullard et al., 1988) and a glycine rich protein in Petunia (Condit and Meagher, 1987 ) and rice (Lei and Wu, 1991). 1.4 Cell Wall Phenolics 1.4.1 Lignins Lignin is the term given to the complex phenylpropanoid polymers laid down in the web of cellulosic fibres, proteins and other carbohydrate polymers in the maturing plant cell wall. Considering the quantity of the phenylpropanoid monomers that are utilized in lignification and the carbon commitment to the process, lignin synthesis is still surprisingly poorly understood. In large part the lack of understanding stems from a technical inability to isolate native lignins from the cell. The name lignins is probably the best term for these polymers as it reflects their structural heterogeneity (Monties, 1991). Basically lignification consists of the polymerization of three lignol precursors, p-coumaryl alcohol (PCA1), coniferyl alcohol (CA1) and sinapyl alcohol (SA1). Once polymerized , the lignin moieties are referred to as p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) 8 lignins. The polymers are characteristic for various species and tissues. Gymnosperms have primarily a G-type, most Angiosperms generally have GS, and grasses have primarily GSH. This classification is an oversimplification as exceptions occur (Lewis and Yamamoto, 1990). Lignification is important to the plant because it strengthens the cell wall, thus enabling the plant to grow tall and also make it more resistant to predation by herbivores and pathogens (Akin et al., 1990). The lignin also decreases the permeability of the cell wall in water conducting cells. Two current areas in lignin research focus on wood products and animal forage. Consequently, the technology of lignin removal has been the primary concern. The general recalcitrance of lignified tissues to extraction has required that harsh chemical treatments be utilized to examine partially degraded lignin structures. The problem with this type of approach is that the formation of chemical artefacts is virtually unavoidable. One method of avoiding chemical artefacts has been to study lignins released into solution by cultured cells (Brunow et al., 1990). Importantly, Brunow et al. (1993) showed that these lignins had a larger content of uncondensed B-O-4 type lignin bonds suggesting similarities with proposed models of early development lignin which are believed to be less condensed than more mature lignins. Boudet, Lapierre and Grima-Pettenati (1995) suggest that the alterations in released lignins compared to normal lignins from within cell walls will help display the contribution of the cell wall matrix. Studies by Agarwal and Atalla 9 (1986) using Raman spectroscopy showed that the aromatic rings of lignins were oriented parallel to the cell wall surface which suggests the cell wall matrix may be very important in determining the form of lignification. In addition, Taylor et al. (1993) inhibited cellulose and xylan production in differentiating Zinnia tracheids which led to the formation of dispersed lignins compared to controls where lignins were localized to the secondary wall. Donaldson (1994) examined lignification in the middle lamella and secondary wall of Pinus radiata. In the middle lamella, the carbohydrates have little order, Donaldson found that lignols added to this region were polymerized to lignin spheres. In the secondary wall, where carbohydrates are strictly oriented, lignols were added to lignin lamellae between cellulose fibrils. Lignin deposition was increased along rather than across cellulose fibrils. This study provided important evidence that the cellulose matrix may control lignin deposition. Lignins appear to be bound directly to polysaccharides, Minor (1991) found that arabinose was bound to lignins in secondary walls of loblolly pine. Another promising technique, 1 3 C-NMR, has been utilized to assist in examining lignins in a more natural state. However this technique is still very complicated and not without flaws. Most lignin evaluations overemphasize ether and ester bound molecules at the expense of condensed carbon-carbon (C-C) bonded structures which are more resistant to degradation. H (p-coumaryl) lignol units are likely to be especially overlooked as these units appear to be more 10 condensed with both 3 and 5 positions available for the formation of C-C bonds (Boudet, Lapierre, and Grima-Pettenati, 1995). As mentioned earlier, there is great heterogeneity between lignins in different species. There is also great variation between tissue structures e.g. between leaves and stems or compression and normal wood. Because there is such variation, woody and grass lignins will be discussed separately. 1.4.2 Wood Lignins In trees, the lignin content varies between 15-36% (Higuchi,1985). As there is a large economic return from the conversion of heavily lignified wood into non-lignified paper, it is not surprising that the lignins of woody angiosperms and gymnosperms are the best studied of the lignins. Gymnosperms have G (guaiacyl) type lignins. However some species have high S (syringyl) contents (Obst and Landucci, 1986). In general, there is a diverse binding pattern for softwood lignins (Figure 1). The major substructure present is the uncondensed guaiacyl glycerol (B-O-4') which comprises 40-60% of the lignin bonding. The remainder of the lignin substructures are made up of the more resilient phenylcoumaran (B-5') at 10%, diarylpropane (B-T) at 5-10%, pinoresinol (fl-B') at 5%, biphenyl (5-5") at 10% and diphenyl ether (4-0-5') at 5% (Higuchi,1985). Freudenberg et al. (1958) showed that the rate of monomer addition in model lignins led to variations in the type of bonds formed. When monomers were added dropwise (zutropfverfahren) the resulting polymer was 11 OH O C H 3 *OCH 3 0-0-4' (40-60%) O C H 3 OH )CH 3 O C H 3 O C H 3 P-P' (5%) C H 3 O ' OH O C H 3 OH O C H 3 4-0-5 (5%) 5-5' (10%) Figure 1. Substructure of lignin (after Higuchi, 1985) 12 comprised of B-O-4 links whereas when the monomers were added in bulk (zulaufVerfahren) the links consisted of the condensed style B-B' Heterogeneity of lignins within the plant is well documented. In the case of compression wood in Pinus radiata and Tsuga heterophylla there is an increase in lignin and a decrease in methoxyl content (Bland 1958; 1961; Fukushima and Terashima,1991) suggesting an increase in H lignins (Lapierre, Monties and Rolando, 1988). Takabe et al. (1992) showed that G/S distribution varies between early and latewood in beech. At a subcellular level there is even greater heterogeneity. The lignins in the cell corners and middle lamella show an increased H content over secondary wall lignins (Whiting and Goring, 1982). The increase in H content is best described by developmental studies. Terashima and Fukushima (1989) using microradiography in Pinus thunbergii showed that H units were added initially in cell wall lignification. These units were added in the cell corners and in the middle lamellae. Then two waves of G monomers were added to the secondary wall. The early H and first wave of G were likely to be in condensed form whereas the later G wave was less condensed. In the angiosperms, the lignin type is referred to as GS although again this trend is over simplified. The angiosperm lignins have twice as many labile units as those of grasses or gymnosperms (Boudet, Lapierre, and Grima-Pettenati, 1995). The general trend is for the monomers to be added in the order H,G,S (Terashima and Fukushima, 1989 ). The H units were found to add only in early lignification, forming a highly condensed deposit within pectic substances and the 13 hemicellulose gel (Fukushima and Terashima, 1990). In poplar, Terashima (1989) showed that the addition of labelled ferulic acid and sinapic acid led to increased incorporation of ferulic acid derived residues in vessels and more sinapic acid derived residues in fibres. In the same study, the addition of coniferyl alcohol to Magnolia showed an increase in incorporation into cell walls of vessels and the middle lamellae early in lignification and another increase in the secondary wall of fibres late in lignification. The addition of labelled sinapyl alcohol showed initial incorporation into the secondary wall of fibres followed by vessels. The final levels showed S concentrations slightly higher than G levels in fibre and vessel secondary walls and virtually equal in the middle lamella. Lai and Guo (1991) showed that free phenolic hydroxyl groups were at similar levels in four gymnosperm species (12-13%). However in six hardwood species, the level ranged from 7% in white birch to 11 % in red oak, thus displaying a potential for heterogeneity between species. 1.4.3 Monocot Lignins Monocots provide some of the most interesting lignins. Within the group there are large variations in lignin concentration. For example, reed canary grass contains only 1.2 % lignin (Burritt et al., 1984,) whereas bamboo contains 26% Higuchi et al., 1967 ). Monocot lignins are derived from all three monolignols. Grasses also contain ester and/or ether linked hydroxycinnamic acids (HCAs) (Scalbert et al., 1985). The presence and function of these acids has become one of the major areas of cell wall research. 14 In wheat, Whitmore (1971) reported that growth decreased as lignin levels increased. In a later paper (1974), he showed that an increase in hydroxycinnamic acids (HCAs) rather than lignins correlated with a decrease in growth. HCAs, which bind to arabinoxylans, are the acid analogues of the lignol monomers. p-Coumaric acid (PCA) is analogous to PCA1, while ferulic acid (FA) corresponds to CA1 and sinapic acid (SinA) is analogous to SA1. Unfortunately, the presence of the HCAs renders it difficult to identify H and G lignins by 1 3 C- NMR due to an overlap with PCA and FA assignments respectively (Boudet, Lapierre, and Grima-Pettenati, 1995). In herbaceous dicots, FA is also found bound to pectins (Hatfield et al., 1991). In wheat, it was shown (Lam et al., 1992a) that PCA and FA formed esters with arabinoxylans while thirty to seventy five percent of FA was bound in ether links. Lam et al. (1992a) showed that FA formed esters in the primary wall, then as lignification proceeded, ether bonds were increasingly formed. The HCA molecules can cross-link in light to form bridges (cyclobutane dimers) that strengthen the cell wall (Hartley et al., 1990). A study by Lam et al. (1994) indicated that almost all FA bridges in wheat are ester-ether bound. An earlier study (Lam et al., 1992b) revealed that 9-10 FA bridges occurred per 100 C 6 C 3 lignin monomers in wheat. Both cis and trans isomers have been found but the trans form is much more abundant. Dimers can bind head to head or head to tail forming truxinic or truxillic acids respectively (Hartley et al., 1990). This heterogeneity leads to a possibility of 36 theoretical isomers, many of which occur naturally (Morrison, Hartley and 15 Himmelsbach, 1992). Light also increases the amount of FA-FA and PCA-FA cyclodimers (Hartley and Ford, 1989). McCallum et al. (1992) noted that these dimers are generally associated with tropical grass species such as Setaria anceps, Digitara decumbens, Heteropogon contortus (Ford and Hartley, 1990), Cynodon dactylon (Hartley et al., 1990) Festuca arundinacea (Hartley and Morrison, 1991) and Phyllostachys edulis (Tachibana, Ohkubo and Towers, 1992) rather than temperate species. Ford and Hartley (1990) found that dimers are much more abundant in stems than leaves. In sunlight dimerization PAXX-PAXX formed the fastest with PAXX-FAXX next and FAXX-FAXX the slowest (Hartley and Ford, 1989). FA bridges are only present in non-woody species (Iiyama, Lam and Stone, 1990). Stafford and Brown (1976) found evidence for a ferulic dimerase producing a B-B dimer. The enzyme did not appear to be a laccase or a peroxidase. A later paper (1977), determined the final oxidant for the enzyme to be H 2 0 2 produced by light reactions in the chloroplast. The enzyme involved appeared to be a peroxidase acting differently from horseradish peroxidase. Bound hydroxycinnamic acids are a potential starting point for lignification (Harris and Hartley, 1976; Scalbert et al., 1985). In wheat, Iiyama, Lam and Stone (1990) showed that FA initially formed ester bonds with carbohydrate and later formed ether bonds to lignin. Kondo et al. (1990) showed that arabinose is the key sugar in feruloylation. Hartley et al. (1990) isolated PAXX and F A X X which are fragments of esterified arabinoxylan polymers. 16 PAXX is p-coumaroyl arabinosyl xylosyl xylose and F A X X is feruloyl arabinosyl xylosyl xylose. Ishii, Hiroi and Thomas (1990) and Ishii and Hiroi (1990) found FA bound to a xyloglucan as well as arabinoxylans in bamboo. In the dicots spinach and sugar beet, Ishii (1994) found that FA bound to combinations of either 2 or 3 arabinoses, 2 galactoses or arabinoxylans. Izydorczyk and Biliaderis (1991) found that arabinoxylans with a low arabinose to xylose ratio were more likely feruloylated in wheat. Myton and Fry (1994) examined feruloylation of arabinoxylans in Festuca arundinaceae and found that labelled arabinose appeared to be feruloylated at 1 -3 minutes after application and appeared in feruloylated form in culture media at 20-35 minutes. Over time there was no appreciable increase in feruloylation of labelled arabinose suggesting that feruloylation occurs in the protoplast and not outside the cell. This finding disagrees with Yamamoto and Towers (1985) who found an increase in feruloylation with time . Myton and Fry (1994) explain the discrepancy by suggesting that either the rate of synthesis of feruloylated polysaccharides may increase as a proportion of polysaccharide production as cells age while overall polysaccharide production slows or the FA group protects the polysaccharide against enzyme mediated degradation leading to an overall increase in degree of feruloylation. Harris and Hartley (1976) examined diferulic acid (DFA), a C-C dimer of FA formed in various plants. In Avena a constant DFA:FA ratio was found throughout development (Kamisaka et al., 1990).This trend was also seen in rice (Tan et al., 1991; 1992). Growth of rice in air or aerated water (rather than still 17 water) or in light led to an increase in DFA and FA and a decrease in extensibility. Similarly, when airgrown rice was submerged, cellulose, hemicellulose, FA and DFA decreased (Kutschera et al., 1993). Studies of FA:DFA ratios in light and dark (Miyamoto et al., 1994), suggested that in the dark the rate limiting step in DFA formation was feruloylation of hemicellulose, whereas in light the rate limiting step appeared to be peroxidase catalysed coupling. Cleavage of ether bonds in cell walls at 170°C with 4M NaOH releases 20 fold more DFA compared to the amounts released at room temperature (Ford and Hartley, 1990). Most studies of DFA only consider the 5,5 bonded form. However, Ralph et al. (1994) have found 8,5, 8-8,8-0-4, and 4-0-5 bonding patterns as well. These forms of DFA are in some cases greater than or equal to concentrations of the 5,5 form. Thus DFA levels are likely to be underestimated. Stewart, Robertson and Morrison, (1994) found six peaks in GC-MS of barley, and suggested that they are possible isomers of 5,5 DFA. Ralph et al. (1994) suggest that they may represent the other bonding patterns. He and Terashima (1990) in a study of lignification in sugar cane found differences in monomer composition between cell types. In the secondary wall of fibres and in metaxylem the monomer concentration was S>G>H, whereas in protoxylem G=H>S. For the HCAs , it was found that FA was added early while PCA and SinA appeared later. In a following study (1991), the same group found that the PCA addition rate was quite constant but the FA incorporation rate surged early in lignification and then decreased. The different cell types also lignified at 18 different rates. The process first occurs in the walls of protoxylem cells then proceeds in the middle lamellae of fibres and in the cell walls of metaxylem vessels. Finally lignification occurs in the secondary walls of fibres. Boudet, Lapierre and Grima-Pettenati (1995) suggested that analysis of thioacidolysis dimers should show increased levels of H. Their results showed a limited amount of H-H and H-G dimers but no H-S dimers. The lack of H-S dimers supports the findings of Terashima et al. (1993) that H and S addition are temporally separated. Based on the limited number of H dimers, Boudet, Lappierre and Grima-Pettenati (1995) hint that the unproven assumption that H molecules are simply condensed and thus not apparent in monomer analysis may be incorrect. However they may underplay the fact that thioacidolysis affects only uncondensed B-O-4 bonds, thus highly condensed material would be unlikely to be affected substantially. The level of HCAs was found to be higher in parenchyma cells than in the more heavily lignified vascular tissue (He and Terashima, 1991). Thus it would appear that cross-links might replace some "woody" function. Many groups have examined digestibility in forage grasses (Buxton and Russell, 1988; Akin et al., 1990; 1992) and found that an increase in lignin caused a decrease in digestibility. The non-woody plant may need increased stabilization of the cell wall, perhaps as a defense against herbivores. The plant may not be able to afford the extensive metabolic costs of lignin and/or the time required for lignification. Free HCAs have been implicated in allelopathy (Glass, 1977, Blum, Dalton and Shann, 1985). In cucumber exposed to FA, Booker, Blum and Fiscus 19 (1992) showed that nitrate uptake was inhibited and potassium efflux increased. Similar results were obtained with corn (Bergmark et al., 1992). 1.5 Biosynthesis Monolignols are a major product of the shikimate/chorismate pathway which is involved in the synthesis of the amino acids phenylalanine (Phe) and tyrosine (Tyr). The heterogeneity between lignins of various species is likely caused by differences in substrate specificities and activities in enzymes of biosynthesis and polymerization (Lewis and Yamamoto, 1990). The shikimate pathway is known to occur in the chloroplast although some evidence exists for a parallel pathway in the cytosol (Jensen et al., 1989). An important recent development in Phe/Tyr biosynthesis was the determination that arogenate is an intermediate between prephenate and the amino acids (Siehl, Connelly and Conn, 1986). It was formerly believed that in plants a transamination of phenylpyruvic acid or p-hydroxyphenylpyruvic acid was involved in the generation of Phe and Tyr (Byng et al., 1981; Gaines et al., 1982 ). The enzymes of this process between prephenate and Phe and Tyr are prephenate aminotransferase (Siehl, Connelly and Conn, 1986) arogenate dehydratase (Jung, Zamir and Jensen, 1986) and arogenate dehydrogenase (Byng et al., 1981; Connelly and Conn, 1986). The last two enzymes are subject to feedback inhibition by their products, Phe and Tyr respectively. From Phe, cinnamate is produced, leading to the production of the other HCAs. Hydroxycinnamic-CoA molecules are used to form flavonoids, stilbenes, 20 lignans, phenolamides and esters. The pathway (see Figure 2) may be viewed as constitutive (Boudet, Lapierre, and Grima-Pettenati, 1995) with levels of activity depending on environmental stimuli and cell type and age. The first step of the cinnamate pathway (Fig. 2) is achieved with phenylalanine ammonia lyase (PAL) (E.C.4.3.1.5) which catalyses the deamination of L-Phe to trans-cinnamate and NEL*. PAL consists of 3-4 isozymes in bean (Cramer et al., 1989), parsley (Lois et al.,1989; Appert, 1994), rice (Minami, 1989), Arabidopsis (Ohl et al., 1990) and pea (Yamada et al., 1992). Transcripts of individual genes show very different patterns of accumulation (Liang et al., 1989; Lois et al., 1989). Appert (1994) found that all four enaymes in parsley had similar properties. In potato (Joos and Hahlbroch, 1992) ten isozymes were found. In woody species, fewer PAL isozymes are found, e.g. two in poplar (Subramainian et al., 1993) and only one in Pinus banksiana (Campbell and Ellis, 1992a) and Pinus taeda (Whetten and Sedoroff, 1992). Bevan et al. (1989) examined GUS fusions using the PAL 2 promoter from Phaseolus vulgaris in potato and tobacco and found high activity in developing xylem and xylem parenchyma but not in older or differentiated xylem. This result supports the suggestion that xylem parenchyma may provide lignin precursors to developing xylem (Pickett-Heaps, 1968). The PAL 2 promoter was also active in other tissues and cell types that accumulate phenylpropanoids. It was also activated by wounding in potato with no corresponding increase in lignification. Shufflebottom et al. (1993) compared PAL 2 and 3 in Arabidopsis, potato and tobacco and found COOH NH3' COOH COOH P A L OH Phenylalanine trans-Cinnamic acid p-Coumaric acid COOH COOH C 3 H COOH F 5 H O M T HO y 0CH3 OH 5-Hydroxyferulic acid O C H 3 OH Ferulic acid OH OH Caffeic acid O M T COOH H 3 C 0 ' y "0CH3 OH Sinapic acid Figure 2. Biosynthesis of hydroxycinnamic acids 22 differential and overlapping expression. PAL 3 was not present in differentiating xylem and vascular tissues. Cinnamate 4-hydroxylase (E.C. 1.14.13.11) is a cytochrome P-450 enzyme which hydroxylates cinnamate to form PCA. Much less is known about this enzyme. InHelianthus tuberosus (Werck-Reichhardt et al., 1993), it was shown, via polyclonal antibodies, that the enzyme was induced by various environmental factors. The enzyme has been cloned from Jerusalem artichoke (Teusch et al., 1993), alfalfa (Fahrendorf and Dixon, 1993) and mung bean (Mizutani, 1993). Pierrel et al. (1994) showed that the enzyme was highly specific for its natural substrate but not active with FA, PCA or benzoic acid (BA) Hydroxylation of coumarate and ferulate to produce caffeate and sinapate occurs via coumarate 3-hydroxylase and ferulate 5-hydroxylase. The mechanisms and enzyme system(s) are unclear. It is unknown whether free acids or some conjugate are the enzyme substrates (Boudet, Lapierre, and Grima-Pettenati, 1995). Heller and Kiihnl (1985) and Kiihnl et al. (1987) demonstrated the hydroxylation of 5-0-(4-coumaroyl) D- shikimate in parsley and 5-0-(4-coumaroyl) D-quinate in carrot to their respective 5-O-caffeoyl esters. In Cichorium endivia, Lotfy, Fleuriet and Macheix (1994) found both quinate and shikimate specific hydroxycinnamoyl - CoA transferases. Bolwel and Butt, (1983) examined phenolases (E.C. 1.10.3.1,1.14.18.1) that may hydroxylate free p-coumarate to caffeate. The specificity of this involvement was not determined. In Silene dioica, Kamsteeg et al. (1981) showed that p-coumaroyl-CoA was a 23 substrate for an FAD dependent monooxygenase that produced caffeoyl-CoA. An elicitor- induced coumaroyl-CoA hydroxylase was characterized from parsley (Kneusel, Matern and Nicolay, 1989). Grand (1984) characterized ferulate 5-hydroxylase (F5H) in a microsomal fraction from poplar but the enzyme has not been further studied. The methylation step is performed by O-methyl transferases (OMTs), e.g. 5-adenosyl-L-methionine:0-diphenol-0-methyl transferase (E.C. 2.1.1.6). In gymnosperms OMTs are monofunctional methylating only caffeate (Higuchi, 1981). In angiosperms, the OMTs are bifunctional, methylating both caffeate and 5-hydroxyferulate e.g. in soybean (Poulton, Hahlbrock and Grisebach, 1976) and tobacco (Hermann et al., 1987). Hermann et al. isolated three isoforms, OMT I, II, III. OMT I is associated with lignification. The other OMTs have differing substrate specificities and differing responses to pathogenesis. Bugos, Chiang, and Campbell (1991) cloned OMT from aspen and found it had an increased specificity toward 5-hydroxyferulate as compared with caffeate. While no gymnosperm OMTs have been cloned, cloning has been achieved with poplar (Dumas et al., 1992), tobacco (Jaeck et al., 1992) and corn (Collazo et al., 1992). Dwivedi et al. (1994) used an antisense OMT in transgenic tobacco plants which resulted in lignins with a decreased S content. Also using tobacco, Pellegrini et al. (1993) showed that OMT I was the major lignin associated form. OMTII and III were encoded by the same gene (allozymes) and were induced by hypersensitive response to TMV (Jaeke et al., 1992 and Pellegrini et al., 1993) but not apparent in 24 lignifying tissues. The separation of the OMT forms suggests that separate pathways for lignins and other phenolics are induced by developmental and stress regulation. Feruloyl CoA is produced from p-coumaroyl CoA via caffeoyl-CoA through the action of caffeoyl-CoA -3-O-methyl transferase (CCoAOMT) in parsley (Kneusel, Matern and Nicolay, 1989) and carrot (Kiihnl et al., 1989). Characterization of the enzyme by Schmitt, Pakusch, and Matern (1991) revealed no extensive homology with known OMTs. CCoAOMT and its mRNA increase in response to elicitation leading to the suggestion that the enzyme modulates formation of ferulate esters (Pakusch, Matern, and Schiltz, 1991; Schmitt, Pakusch and Matern, 1991). The enzyme was also present in tissues undergoing normal development. Ye et al. (1994) cloned CCoAOMT from Zinnia and proposed that the enzyme is involved in an alternate methylation pathway. They suggested that CCoAOMT was important in tracheary differentiation whereas COMT, which acts directly on the acid instead of the CoA derivative, was postulated to be a stress response. They also found that distribution of CCoAOMT was similar to that of cinnamate 4 hydroxylase. In a subsequent paper (Ye and Varner, 1995), CCoAOMT was shown to be more prevalent in xylem in young internodes whereas COMT was more prevalent in phloem. In older tissues, both forms were present. Kamsteeg et al. (1981) found p-coumaroyl-CoA 3- hydroxylase activity in Silene dioica. This activity was also found in parsley (Kneusel et al., 1989) and was elicitor induced. This activity, combined with CCoAOMT induction, was 25 proposed to play a defence role. Ye et al. (1994) found the 3-hydroxylase activity in Zinnia but state that it cannot be separated from caffeoyl-CoA formation by a CoA ligase of caffeic acid or trans esterification of caffeoyl shikimate to caffeoyl CoA by hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (Kiihnl et al., 1989). Ni, Paiva and Dixon (1994) suggest the presence of CCoAOMT accounts for the maintenance of lignin formation in transgenic tobacco with an antisense OMT. The known methylation pathway (Ye et al., 1994) continues with 4-coumarate-CoA ligase (4CL)(EC6.2.1.12) which was considered the branch point enzyme between general phenylpropanoid biosynthesis and lignin biosynthesis (although the demonstration of the alternate methylation pathway may make the branch point less precise). The enzyme is induced co-ordinately with PAL in several plants by different stimuli (Hahlbrock et al., 1983). 4CL has several isoforms in soybean (Knoblach and Hahlbrock, 1975), Petunia (Ranjeva, Boudet and Faggion, 1976), pea (Wallis and Rhodes, 1977) and poplar (Grand, Boudet and Boudet, 1983). The isoforms have different specificities and catalytic properties which may distinguish lignin monomer composition (Grand, Boudet, and Boudet, 1983). The enzymes were shown to have regulation features similar to those of PAL (Hauffe et al., 1991 and Hauffe et al., 1993). Elicitation of soybean showed that isoforms of 4CL were differentially induced (Uhlmann and Ebel, 1993) 26 The next enzyme, cinnamoyl CoA reductase (CCR) (E.C. 1.2.1.44) marks the beginning of the lignin branch pathway. The enzyme catalyses the conversion of CoA esters to aldehydes. The enzyme has been purified in a variety of species, including spruce (Luderitz and Grisebach, 1981), poplar (Sarni, Grand, and Boudet, 1984) and soybean (Wegenayer, Ebel and Grisebach, 1976), but little is known regarding its regulation. Goffner et al. (1994), using CCR from Eucalyptus, determined that the affinities for PCA-CoA, FA-CoA and SinA-CoA were approximately equal. The conversion of FA was about three times more effective than the conversion of other cinnamic acids. The next enzyme is much better understood. Cinnamyl alcohol dehydrogenase (CAD) (EC 1.1.1.195) occurs in one form in gymnosperms (O'Malley, Porter, and Sederoff, 1992; Galiano et al , 1993) which utilizes coniferyl but not syringyl substrates. In angiosperms, isoforms which vary in substrate specificity, are evident in Eucalyptus (Goffner et al., 1992; Hawkins and Boudet, 1994), bean (Grima-Pettenati et al., 1994) and wheat (Pillonel, Hunziter and Binder, 1992). In tobacco, Halpin et al. (1992) found a dimeric form which may or may not equate to the isoforms. Umezawa, Davin and Lewis (1988) suggest that one isoform is constitutive for lignification whereas the other is applied to defence lignins and lignans. Similarly, Walter (1992) suggests there are two pathways of signal transduction, one for vascular tissue and the other for defence. In spruce culture, Messner and Bol (1993) tried to show elicitation and found only slight induction. They speculate that the constitutive form is unaffected 27 while the induced form is masked. Mitchell, Hall and Barber (1994) found a substrate specific induction of CAD in wheat that increased the S content of defence lignins. Studies with CAD-GUS constructs in tobacco (Walter, Schaff and Hess, 1994) and two eucalypts (Hibino et al., 1994; Feuillet et al., 1995) showed more activity in roots than in stems and leaves. The enzyme appears early in parenchyma cells between xylem fibres. Later in development, the enzyme appeared in xylem ray cells and parenchyma around phloem. Again this pattern is suggestive of the export of lignin precursors although this occurrence has never been shown. Pillonel et al. (1991) determined that a brown midrib mutant of sorghum was a CAD mutant and that the plant contained cinnamaldehydes in its core lignins. The mutant plant also had a lower PCA:FA ratio and reduced amount of pentosans with increased hexosan residues. The formation of benzoic acid derivatives is less well understood. Cvikrova et al. (1990) found that plants placed in the dark accumulated benzoic derivatives at the expense of HCAs. Initial theories (Zenk, 1965) proposed a J3-oxidation of HCA-CoA to their respective benzoic-CoA equivalent. Evidence for this theory came from feeding studies (Zenk , 1965) in which vanillin was produced from FA by Vanilla planifolia without free vanillic acid as an intermediate. Additional support came from similar studies by El-Basyouni et al. (1964) and from the findings of Vollmer, Reisener and Grisebach (1965) that Ci and C 2 of the phenylpropanoid precursors were removed as acetate or acetyl-CoA. 28 Subsequent studies in potato (French et al., 1976), carrot (Schnitzler et al., 1992) and Lithospermum (Yazaki, Heide and Tabata, 1991) found evidence for p-hydroxy benzaldehyde as an intermediate in the formation of p-hydroxy benzoic acid. The reaction was found to be independent of ATP or CoA addition thus contradicting the B-oxidation mechanism. The alternative mechanism proposed in its place involves a retro-aldol reaction, cleaving acetate, followed by reduction of the resulting aldehyde. Another mechanistic model, suggested by Funk and Brodelius (1990a; b; 1992), involves methylation of the p-hydyroxy group of the HCAs, activation by glucosides followed by side chain degradation and demethylation to form p-hydroxy benzoic acid. Recently, Loscher and Heidi (1994) found in Lithospermum cultures that p-hydroxybenzoate (P-HBA) was formed from PCA via PCA-CoA and P-HBA-CoA apparently similar to the B-oxidation method of Zenk (1965). Loscher and Heidi (1994) proposed a 3 step mechanism consisting of the addition of water to the double bond, oxidation to a keto function, followed by thioclastic cleavage. One other interesting factor is the formation of cis isomers of cinnamyl derivatives. These monomers accumulate in the bark of Fagus (Morelli et al., 1986, Lewis et al., 1987). It was determined that the isomerization occurred at the monolignol level. While Lewis et al. (1989) showed that the conversion could occur photochemically, cis isomer formation could also occur in vivo in the dark. 29 Braun and Tevini (1993) showed that the conversion of FA and PCA to cis forms by UV light led to an increase in flavonoid production which they ascribe to a stimulation of PAL, which is feedback inhibited by trans cinnamic acids, 1.6 Monolignol Transport The inter and intracellular transport of lignols is not well understood. It is postulated that monomers from neighbouring cells move as glycosides. Primary transport within the lignifying cell is via endomembrane vesicles (Yamamoto, Bokelman and Lewis, 1989) which fuse with the cell membrane and release the monomers into the wall. Glycosides of monolignols are much more prevalent in softwoods. 13-Glucosidases act to release monolignols from the glucosides. Dharmawardhana, Ellis and Carlson (1995) have reported the isolation of a J3-glucosidase specific for coniferin in Pinus contorta. Marcinowski and Grisebach (1978) have shown however that little of the coniferyl alcohol used in lignification proceeds through the glycoside pool. 1.7 Polymerization The mechanism of lignin polymerization remains unclear. Classically, the process has been viewed as involving wall oxidases that convert monolignols to mesomeric free radicals that spontaneously polymerize. Chain propagation is likely to occur and peroxidases have been viewed as the critical enzymes. Lewis and Yamamoto (1990) set out a list of criteria to allow for identification of lignin peroxidases, including substrate specificity, primary structure, sub-cellular location and temporal correlation. None of the many peroxidases identified has 30 met these criteria (Boudet, Lapierre, and Grima-Pettenati. 1995). Plants have many forms of peroxidases (Lewis and Yamamoto, 1990) . Specific peroxidases are capable of generating peroxide and monolignol radicals . Hwang et al. (1991) proposed a model for polymerization by peroxidase in vitro. The first step, initiation, involved radical production by abstraction of phenolic hydrogen. Propagation followed via the radical combining with another monomer or X-mer. Termination completes the process when the radical receives a hydroxyl radical released by peroxidase. The process of polymerization converts the slightly soluble coniferyl alcohol to a completely insoluble polymer. The requirement of H 2 0 2 for the polymerization of lignins by peroxidases is well-known. As peroxide is too reactive to be transported in membrane vesicles it is likely to be produced in the cell wall. The natural presence of H 2 0 2 in the cell wall was shown histochemically by Olsen and Varner (1993). It has also been demonstrated in electron micrographs of lignifying poplar (Czaninski, Sachot, and Catesson, 1993). Studies in bean (Schoppfer, 1994) found that peroxide could be detected in phloem and schlerenchyma but not in xylem. The addition of peroxide scavengers to cell cultures of Pinus taeda inhibited lignification (Nose et al., 1995). An increase in lignification was seen in transgenic tobacco overexpressing a peroxidase (Lagrimini, 1991). Plants underexpressing peroxidase displayed normal lignification (Chabbert et al., 1992). Tsutsumi (1993) showed that lignins and peroxidases increased but PAL levels remained constant under conditions of water stress,. 31 The energy supply for polymerization was thought to be oxidation of malate to oxaloacetate by malate dehydrogenase forming NADPH. Peroxidases could then oxidize NADPH to form peroxide (Elstner and Heupal, 1976; Gross and Janse, 1977; Gross et al., 1977). More recent evidence indicates some problems with the model. Goldberg, Le and Catesson (1985) found only low levels of malate dehydrogenase and no evidence for a malate/oxaloacetate shuttle in the apoplast. Other studies also found little indication of NADPH in the wall in vivo (McNeil et al., 1984; Crane and Barr, 1989). Alternatively, the oxidation of putrescine or polyamines by diamine oxidase (DAO) (E.C. 1.4.3.6) or polyamine oxidase (PAO) (E.C. 1.5.3.3) to produce peroxide could occur (Angelini and Federico, 1989). Slocum and Furey (1990) found a strong association between PAO/DAO and lignification localized to the middle lamella, while blockage of polyamine biosynthesis inhibited xylogenesis (Phillips et al., 1988). Flores and Filmer (1985) provide valuable evidence showing that the necessary transport mechanisms are present. Polyamine concentration can vary between seasons (Konigshofer, 1990) or as a result of stress (Dohman, Koppers and Langebartek, 1990) and may reach millimolar levels in some plants (Friedman, Levin and Altman, 1986). Plants also have several peroxide scavenging mechanisms (Castillo and Greppin, 1986; Rennenberg and Polle, 1989) which tend to inhibit lignification. Thus, during lignification, these mechanisms must be decreased in the apoplast or at least reduced in a localised manner. Ascorbate has been proposed as a major 32 free radical scavenger in the apoplast (Castillo, Miller and Greppin, 1987; Polle et al., 1990). Ascorbate may regulate the types of links formed in the wall by limiting the action of long - lifetime phenolic radicals and/or interacting with H 2 0 2 (Chinoy, 1984). Ascorbate levels are regulated by ascorbate oxidase which is found in the cell wall of many plants (Butt, 1980; Kroneck et al., 1982 and Lamport, 1965) and increases in elongating cells (Mertz, 1961). Ascorbate oxidase is related to laccase (Ohkawa et al., 1989). Dehydroascorbate, the oxidized form of ascorbate, can be regenerated to ascorbate via dehydroascorbate reductase (E.C. 1.8.5.1). Laccases (E.C. 1.10.3.2) were dismissed as lignin polymerases following studies by Nakamura (1967) that showed laccases were unable to form lignins in vitro. Harkin and Obst (1973) then provided evidence favoring peroxidases and thus laccases were ignored. Recently, Sterjiades, Dean and Eriksson (1992) provided supporting evidence for Freudenberg et al. (1958) findings of laccase-like activity in gymnosperm cambium that polymerizes lignols without the addition of peroxide but uses oxygen as the acceptor instead. Savidge and Udayama-Randeriya (1992) located a coniferyl alcohol oxidase activity that was specific to lignification and distinct from peroxidases in conifers. They suggested that lignification cannot occur without 0 2 oxidation. Support for this argument was found in the low oxygen availability in vivo. Siegal, Rosen and Rewick (1962) found that plants grown in low oxygen environments had reduced lignin content but increased peroxidase activity and rate of desiccation. The initiation of 33 lignification in spring coincides with high tissue 0 2 levels. Thus, oxygen availability may be the rate limiting step in the process. At the same time, Sterjiades, Dean and Eriksson (1992) isolated a laccase from Acer pseudoplatans that could polymerize monolignols. Davin et al. (1992) provided more evidence by finding laccase activity in cell wall preparations. Antibody (Driouch et al., 1992) and histochemical (Bao et al., 1993) studies confirmed the relative specificity of laccase in lignification of differentiating xylem in pine. Peroxidases were also present. Similar laccase/lignification correlations have since been shown in bean (Chabanet et al., 1994) and Zinnia (Liu et al., (1994). Badiani, DeBiasi and Felici (1990) postulated that the laccase in the middle lamella may increase in activity as lignin concentration increases causing hydrophobicity to eliminate water and peroxide leaving oxygen as possibly the only remaining oxidant. This role may also account for the increased condensation of lignins polmerized early in cell development. Sterjiades et al. (1993), based on observed differential in vitro abilities of laccase and peroxidases, postulated that laccases may polymerize monolignols to oligolignols while peroxidases may polymerize the oligolignols to lignins. McDougall, Stewart and Morrison (1994a; b) provides support for the hypothesis in finding that oxidation of coniferyl alcohol continues in tobacco cell walls in the presence of peroxide blockers. Activity has been difficult to assign to a specific laccase enzyme and it is hard to separate laccases from other oxidases (Boudet, Lapierre and Grima-Pettenati, 1995). 34 1.8 Soluble Phenolics Cvikrova et al. (1988) examined soluble phenolics in tobacco cultures and found that 40-70% were esterified, 20-35% were bound as glycosides and 5-20% were free.The numbers varied depending on age. Porter, Banwort and Hassett (1986) in soybean compared soluble phenolic acids and flavonoids. In roots, p-hydroxy benzoic, vanillic, ferulic, gentisic, syringic, and protocatechuic acids, the isoflavones genistein and daidzein and coumesterol were found. In leaves, salicylic, p-hydroxybenzoic, vanillic, p-coumaric, ferulic, caffeic, gentisic, and syringic acids as well as naringenin, quercetin, genistein and daidzein were found. Bokern et al. (1991) examined soluble phenolics in Chenopodium rubrum and found that most consisted of glycoside and amide conjugates. In insoluble pools, primarily wall bound HCAs were found. Fertilization of beech with ammonium nitrate (Balsberg-Pahlsson, 1992) led to a dramatic decrease in leaf phenolics which may explain an increase in susceptibility to parasites. There is also an increase in various amino acids. The results suggest that a lack of available carbon leads to reduced amounts of phenolics. This also suggests that evolutionary pressures in rich environments stress plant-plant competition over herbivore and pathogenic factors. Niemann et al. (1992) compared relative growth rate (RGR) and cell wall compostion in various species and found that plants with low RGR had increased cell wall components, including lignin and cell wall proteins, whereas high RGR plants contained increased cytoplasmic features. Etiolated pea seedlings exposed to light increased lignification by a factor of ten in 24 hours. 35 This supports the theory that certain types of secondary metabolism may act as a spillway in times of sudden carbohydrate increase. 1.9 Polyamines Goldberg and Perdrizet (1984) showed that extracellular polyamines become covalently bound to the cell wall in a developmentally regulated manner. Martin-Tanguy et al. (1978) noted that polyamines are often conjugated to phenolics that resemble monolignols. Conjugates play a role in the differentiation of certain tissues, especially floral tissues (Evans and Malmberg, 1989; Wyss-Benz, Streit and Ebel, 1990). Dean and Eriksson (1992) speculate that these conjugates might provide for the cotransport of stoichiometric amounts of polyamines and phenolic residues to the apoplasm during lignification. 2.0 General Viral Symptoms Interactions between viruses and plant cells in viral infection of plant cells can result in a number of symptoms. While many symptoms are not readily visible to the naked eye, some that are noticeable are: necrosis, chlorosis, and wilting. The interactions of the plant and virus often result in "non-host" responses (Culver, Lindbeck and Dawson, 1991) which block replication and/or movement of the virus within the plant. Some interactions, however, lead to systemic host infections that spread the virus throughout the plant and can dramatically affect or even kill the host plant. The effects of viruses on plant cells are difficult to summarize. Nicholson and Hammerschmidt (1992, p. 370) emphasize " the importance of understanding 36 each disease interaction as a unique phenomenon that rarely can be considered, in a generalized sense, as representative of host responses that occur in diverse plant species. " Compounding the difficulty of generalization is the usual practice of distinguishing between plant interactions with fungi, bacteria and viruses. However, Rohm et al. (1993), suggest that "Although resistance interactions of plant viruses are often considered separately from those of bacteria and fungi, there are several reasons for considering all of these interactions as part of an integrated defense system in plants against pathological attack." One of the reasons given to support their argument is that resistance to pathogens is often controlled by dominant resistance genes such as Rx in potato or N ' in tobacco (Culver, Lindbeck and Dawson, 1991). The dominant genes recognize a component of the pathogen. In fungi and bacteria, this component is called the avirulence determinant. In the two examples cited above, the coat protein of Potato Virus X (PVX) and Tobacco Mosaic Virus (TMV) act in a similar manner to avirulence determinants. There is also at least superficial similarity in mechanistic responses to the pathogen in the form of HR (hypersensitive response). The biochemical similarity and also the induction of similar Pathogenesis Related (PR) proteins certainly suggest some overlap within the system. Gene for gene resistance to the tobamovirus group in tobacco, which involves pathogen recognition, is mediated by two genes. The N ' gene responds in association with the viral coat protein of some tobamoviruses. The N gene (not allelic with N') provides resistance to virtually all tobamoviruses (Culver, 37 Lindbeck arid Dawson, 1991). However, exceptions do occur (Sanfacon et al., 1993). The recognition of the N gene does not reside with the coat protein based on mutagenic studies (Padgett and Beachy, 1993). They suggest that the viral 126 kD replicase is required for recognition and HR response. 2.1 Chlorosis The two symptoms to be examined in the proposed study, chlorosis and necrosis, are probably the best known. Chlorosis is often associated with systemic responses. Symptoms of chlorosis include yellowed areas in leaves which expand prior to infection or a mosaic pattern of dark and light green, yellow, and possibly white, in leaves that expand after infection (Culver, Lindbeck and Dawson, 1991). These symptoms result from general or localized loss of chlorophyll. Mature, yellowed tissue often appears to be cytologically, physiologically and biochemically similar to senescing tissues in uninfected plants, (van Loon, 1987). The dark green regions of mosaics are often virtually virus free. During initial infection, viruses are unable to cross the cell wall in a healthy plant; some form of wounding is necessary e.g., insect feeding or mechanical damage (Goodman, Kiraly and Wood, 1986). Presence of the virus inside the cell can lead to significant alterations in the cell wall and cell organelles. For example, Gao and Nasuth (1992) studied the effects of systemic Wheat Streak Mosaic Virus (WSMV) in wheat using light microscopy. They observed the formation of cylindrical inclusions in the cytoplasm of epidermal cells. In the mesophyll cells, the nuclei and chloroplasts degraded over time. The cell wall 38 composition also changed which included the formation of deposits that were most likely phenolic compounds including lignin. The mesophyll cells collapsed at later stages, a symptom not seen in senesced virus free mesophyll or infected epidermal cells. The cell wall changes were examined by staining with toluidine blue O, phloroglucinol, Hoepfher-Vorsatz reagents, vanillin-HCl and 2% FeCl. . Unfortuately, staining alone provides only preliminary evidence for the presence of lignins (Lewis and Yamamoto, 1990). 2.2 Hypersensitive Response In contrast, necrosis is a process that may be associated with localization of the viral attack. When the plant specifically recognizes the pathogen and undergoes a rapid defense response that consists of localized cell necrosis, which usually restricts the virus, the process is called a hypersensitive response (HR) (Fritig et al., 1987). A slower forming local necrosis is also possible. The second form tends to result in a "race" between viral replication and movement and the localizing necrosis (Culver, Lindbeck and Dawson, 1991). White and Sehgal (1993) examined the HR of lima bean leaves to Tobacco Ring Spot Virus (TRSV). The lesions in this case were very large. The infection proceeded in two phases. The first phase consisted of the formation of inclusion bodies in mesophyll cells. The cells maintained metabolic activity and structural integrity. The second phase was the degradative phase wherein the cell organelles and membranes degenerated, the cell wall collapsed and formed folds and loops around the shrunken cytoplasm. There was no observed cell wall thickening. Owing to the 39 large size of the lesion, White and Sehgal (1993) termed the response "inefficient hypersensitivity". There were islands of healthy cells within the affected zones which suggested that some paths were successfully blocked while others remained open. The reaction above suggests that lignification may work as a delaying tactic against viral expansion while other defense mechanisms are developing. The HR is considered a universal function in the plant kingdom, and is effective against viral, bacterial and fungal pathogens. The sequence of events in HR is host cell death, necrosis, accumulation of toxic phenols, cell wall modification and finally phytoalexin synthesis (Nicholson and Hammerschmidt, 1992). Prusky (1988) calls the HR the "last line of defense" as it occurs once intimate contact between the pathogen and host cells has occured. Fritig et al. (1987) propose a model for the HR to fungal infections whereby oligosaccharides from the fungus are bound by a receptor, possibly a lectin (Callow, 1977), triggering a cascade of events leading to HR. They also suggest that the production of chitinase and glucanases as pathogenesis-related proteins in response to viruses, which are unlikely to be affected by them, may serve to produce host cell oligosaccharides that act in a similar manner. Fritig et al. (1987) compared TMV infection in Samsun NN and nn and found that over thirty hours the rate of virus increase was similar. From 33-36 hours necrosis occured in NN tobacco, yet viral levels were still comparable. From this point however, virus levels begin to diverge increasingly over time. Some support for their model (Fritig et al., 1987) comes from findings that many resistance genes that induce 40 HR are non-functional in protoplasts while resistance genes that cause a direct effect on viral multiplication are able to maintain resistance (Larkin et al., 1991). The potential importance of communication between the cell wall and cytoplasm is underscored by the finding that PAL levels surged at times of active fungal penetration in barley (Shirashi et al., 1995). Along the same line, Mutaftaschiev et al. (1993) found that cell wall fragments increase growth, which supports findings by MacDougall and Fry (1990) that oligosaccharides can act as pseudo-auxins although they are less effective. Using carrot, Messiaen et al.(1993) and Messiaen and Van Cutsem (1994), showed that oligogalacturonides could stimulate calcium release in the cytoplasm leading to increasing PAL activity. 2.2.1 Early Events Characteristic early events in HR include ion fluxes such as K 7 H + exchange and a loss of cellular compartmentation (Atkinson et al., 1990). The discovery of mutants that spontaneously undergo necrosis suggests that HR is under genetic control (Greenberg et al., 1994; Dietrich et al., 1994). These plants show characteristics of induced resistance constitutively. Dunigan and Madlener (1995) found that activation of serine/threonine protein phosphatase correlated with temperature dependent induction of the death program. Blockage of the enzyme inhibits or delays HR. Lipoxygenases (LOX)-catalyzed oxidation of polyunsaturated fatty acids from membranes that are released by phospholipases. There is an increase in 41 activity in response to elicitation (Esquerre-Tugaye et al., 1993). Slusarenko et al., (1993) apply three possible roles for LOX in pathogenesis: as a signal molecule, a component in membrane damage and in production of toxic metabolites to inhibit the pathogen. Beckman and Ingram (1994) have shown that cytokinins (CKs) inhibit hypersensitive responses in potato tubers infected with Phytopthora infestans. The application of exogenous CK's resulted in decreased cell death and phytoalexin accumulation and an increase in fungal growth. Conversely, Liu and Bushnell (1986) found that CK addition to barley infected with Erysiphe graminis increased HR. The application of CK to barley increased senescence which is the opposite of situations in most plants. 2.2.2 Oxidative Burst An oxidative burst can occur within five minutes of elicitation and thus may comprise a first line of defense. The release of active oxygen destroys the auxin IAA (Apostal, Heinstein and Low, 1989). Addition of exogenous peroxide stimulates phytoalexin synthesis while the addition of active oxygen scavengers inhibits elicitation. Devlin and Gustine (1992) correlated oxidative burst in clover with exposure to the incompatible bacterium Pseudomonas corrugata. They applied HgCl 2, which can oxidize sulphydryl groups in membrane proteins, and were able to mimic elicitation without the presence of peroxide. Thus a possible target of oxidative burst was elucidated. Levine et al. (1994) suggest that H 2 0 2 drives cross-linking of cell wall proteins and acts as the trigger for cell death. 42 Peroxide can also act as a diffusible signal to produce glutathione -S- transferases and glutathione peroxidase in neighbouring cells. Oxidative burst does not appear to be the primary signal for activation of defense genes such as PR proteins or phytoalexin and lignin biosynthesis genes. In vivo studies in potato (Miura, Yoshioke and Doke, 1995) provided evidence implicating calcium ions, calmodulin and protein kinases in the stimulation of active oxygen. Conversely, the same group (Doke and Miura, 1995), found no calcium dependence for in vitro studies on an NADPH dependent active oxygen producing system in a plasma membrane fraction. In soybean, Legendre et al. (1993) found that an increase in inositol phosphate preceded peroxide increase. Addition of peroxide to tobacco led to a transient increase in Ca 2 + ions but a second application produced no effect. Different treatments such as cold shock and touching could still release Ca 2 + , thus suggesting that different pools of Ca 2 + were involved. In trench bean, elicitation led to an increase in 0 2 use followed by oxidative burst. At the peak of oxygen uptake, the ATP and NADH/NAD ratio fell suggesting stress in oxidative metabolism (Robertson et al., 1995). The fall parallels the peak in active oxygen production. Cells recover and begin to produce ATP immediately upon cessation of peroxide production. The cells also produce alcohol dehydrogenase which may indicate transient compromising of the respiratory status. 43 2.2.3 Structural Proteins In the case of protein increases in the cell wall corresponding to pathogen effects, the result is probably the formation of a more impenetrable cell wall (Showalter, 1993). Keller (1993) found that HRGPs (Hydroxyproline rich proteins) played a role in HR responses to fungal infection in bean. HRGPs increased faster in HR responding cultivars. The increasingly cross-linked proteins provide additional bonding strength to the wall slowing the action of pathogenic enzymes in wall breakdown. In soybean, the peroxide mediated cross linking of cell wall proteins inhibits the spread of fungus in incompatible reactions whereas this does not occur in compatible reactions (Brisson, Tenhaken and Lamb, 1994). By cross linking proteins, the wall may become more resistant to viral movement between cells. 2.2.4 Polyamines Martin-Tanguy and coworkers (1985) have studied HCA-polyamine conjugates in tobacco focusing particularly on their role in viral pathogenesis. Conjugates include caffeoyl, p-coumaroyl and feruloyl derivatives of putrescine, tyramine and octopamine (Martin-Tanguy, 1985). 2-Hydroxyputrescine amides of PCA and FA were also found in rust resistant wheat (Stoessl, Rohringer, and Samborski, 1969; Samborski and Rohringer, 1970). More recently, Hohlfield (1995) has shown using potato that elicitor induces the activity of tyramine hydroxycinnamoyl transferase to produce feruloyl tyramine. Mono- and diferuloyl putrescine have been implicated in hypersensitive responses to TMV in tobacco 44 (Martin-Tanguy, Martin and Gallet, 1973). Martin-Tanguy et al. (1976) also found antiviral properties in vitro with the conjugates. One interesting component of TMV infection in the resistant Xanthi n.c. cultivar is that at 32°C the HR does not occur allowing the virus to spread systemically (Martin and Gallet, 1966 a; b). At 20°C, the plants form large quantities of feruloyl putrescine, diferuloyl putrescine and feruloyl tyramine. When a hybrid between Nicotiana rustica and N. tabacum var. Maryland Mammoth, which was unable to form polyamines, was infected, the necrotic lesions appeared but the virus was able to spread through the plant leading to necrosis throughout the plant (Martin and Martin-Tanguy, 1981). Studies on amide biosynthesis (Negrel, Vallee and Martin, 1984) showed that ornithine decarboxylase (ODC) increased 20 fold in infected Xanthi n.c. at 20° whereas at 32° this did not occur. When 32°C tobacco was placed at 20°, ODC activity increased rapidly leading to collapse of infected cells in 7-9 hours. The activity of ODC parallels findings with PAL (Paynot, Martin and Giraud, 1971). Interestingly, the activity of feruloyl CoA-tyramine N-feruloyl CoA transferase involved in the production of feruloyl tyramine was maximal 24 hours after the death of the cells (Negrel and Martin, 1984). The feruloyl tyramine produced in HR appears to bind to the cell wall (Negrel, 1984 as cited in Martin-Tanguy, 1985). Negrel and Smith (1984) found that the amides were good substrates for peroxidases in vitro and may be converted to a polymer that acts to restrict viral movement. 45 2.2.5 Ethylene Ethylene is an important hormonal regulator and is associated with lignin biosynthesis and polyamine metabolism (Yang and Hoffman, 1984; Mattoo and Suttle, 1991 and Dean and Mattoo, 1992). Ethylene increases lignification in many plants, e.g., winter rye (Ievinsh and Romanovskaya, 1991). One of ethylene's important precursors, S-adenosyl methionine (SAM) is also important for phenylpropanoid methylation. Ethylene also regulates key polyamine synthesis enzymes, e.g., SAM synthetase and when SAM acts as the propyl amine donor in polyamine synthesis (Slocum, Kaur-Sawhney and Galston, 1984). Additional work by Greppin, Penal and Gaspar (1986) showed that ethylene leads to increased peroxidase activity. However, Pennazio and Roggero (1990) found that while ethylene is produced in TNV infection of soybean, inhibition of ethylene has no effect on necrosis. Similarly Silverman et al. (1993) showed that SA accumulation was not impeded by blocking ethylene biosynthesis. Ethylene increase likely plays an important role in enhanced senescence of infected leaves but not a systemic role (Knoester et al., 1995). 2.2.6 Phenolic Accumulation Phenolic accumulation in response to infection has been shown by numerous studies (Nicholson and Hammerschmidt, 1992). The phenolics involved include benzoic acids and hydroxycinnamic acids (Kurosaki, Tashino and Nishi, 1986). Esterification of the phenolic components to the cell wall is a common theme in resistance (Friend 1981; Fry 1986; 1987). Polymer accumulation is also 46 appearent (Bolwell, Robbins and Dixon, 1985). Matern and Kneusel (1988) found that FA esterification was especially common and proposed that the increase in esterification slows initial pathogen growth until other induced secondary systems can be applied to more effectively restrict the pathogen. Examination of phenolic involvement presents a problem since there is little information in the literature involving viral attack where soluble phenolics or cell wall changes have been directly measured. One study (Tanguy and Martin, 1972) found that Xanthi n.c. tobacco innoculated with TMV accumulated flavonol glycosides, caffeoyl, feruloyl and p-coumaroyl quinic acids as well as glucose esters and glycosides of cinnamic and benzoic acids. Owing to the paucity of previous work in the viral area, it is useful to examine phenolic response to fungal and bacterial challenge. Histochemical staining has shown phenol accumulation to occur within three hours of rust infection in wheat (Bruzzese and Hasan, 1983). The activity of PAL was shown to increase at the same time. The area inside the lesion was determined not to be the region of maximal PAL induction (Cuypers, Schmelzer and Hahlbrock, 1988). Instead the cells neighbouring the lesion were maximally induced. The results show that PAL mRNA decreases after six hours in incompatible responses whereas PAL continues to increase over time in compatible responses. Matern and coworkers have shown that parsley esterifies phenylpropanoids to the cell wall especially FA. (Matern and Kneusel, 1988; Matern et al., 1988). Prior to production of coumarin phyoalexins, the cells 47 produce caffeoyl-CoA from p-coumaroyl CoA via an unusual hydroxylase (discussed in more detail in 1.6 Biosynthesis) In alfalfa, Hrubcova et al. (1992) showed an increase in ester bound p-hydroxybenzoic, vanillic, p-coumaric and ferulic acids in response to fungal elicitation. Cvikrova et al. (1993) compared resistant and sensitive cultivars. The sensitive cultivars showed the highest increase in free and bound phenolics with a corresponding decrease in glycosylated forms. The resistant cultivars showed an overall increase in all forms of phenolics examined. The level of phenolics also increased at an earlier time in resistant varieties. Glazener (1982) found an increase in PCA and FA after inoculation of tomato with Botrytis cinerea. A lignin-like polymer was obtained by nitrobenzene oxidation of the cell walls. In maize infected with Helminthosporium maydis three phenomena were noted (Lyons et al., 1993). The first is accumulation of two caffeic acid esters at sixteen hours post infection that are likely a component of tissue browning. Next PCA and FA are esterified to the cell wall in higher amounts in resistant cultivars. Finally lignin synthesis occurs with a high S component despite the lack of sinapic acid in maize (Hagerman and Nicholson, 1982) which suggest 5-hydroxy ferulic acid may be the immediate precursor with O-methylation occuring during formation of the monolignol. In tomato, Beiman, Witte and Barz (1992), found accumulation of chlorogenic acid, rutin, and wall bound cinnamic acids (PCA, FA and especially caffeic acid) in response to the bacterium Clavibacter micheganense. Interestingly, 48 unlike fungal pathogens, the microorganism did not induce formation of cinnamoyl tyramines or p-hydroxybenzaldehyde and vanillin in the cell wall. Friend (1981) notes that simple phenols accumulate early in both compatible and incompatible responses, which may suggest a general metabolic increase in phenolic molecules to act as precursor pools. The accumulation of HCA esters in virally challenged tobacco occurs after necrosis suggesting that these molecules are not an important factor in HR initiation (Fritig, Legrand and Hirth, 1972; Tanguy and Martin, 1972; Martin -Tanguy, 1985 ). Lignins 2.2.7 Cell wall modifications are often pronounced in HR interactions (Goodman, Kiraly and Wood, 1986). Kimmins and Wuddah (1977) determined that there was an increased lignin content in TNV infected dwarf bean leaves. There was also a lesser increase in lignin in abraded leaves. Again, few studies have examined lignification in virally challenged plants. Plant-fungal interactions with regard to lignin formation are better studied. Lignification in four wheat strains differing in resistance to Septoria nodurum was studied (Bird and Ride, 1981). Fungal penetration of the cell led to rapid collapse of the cell and increased lignification. Resistant cells showed lignified papillae and haloes of lignification around the cells. Southerton and Deverall (1990a; b) showed that virulent and avirulent strains of leaf rust led to increased levels of PCA, FA and SinA in both bound and unbound forms in cultivars with the Lr28 49 resistance gene. In Lr20 resistance gene cultivars, a decrease in phenolics was observed. Yet both genes were associated with HR at sites of infection. Southerton and Deverall (1990a; b) also attempted, without success, to use inhibitors against PAL to alter resistance expression. However, Moerschbacher et al. (1990) were successful; using PAL and CAD inhibitors, they were able to decrease lignification and lower resistance of wheat to stem rust. The CAD inhibitors were the most effective. Carver et al. (1992) studied wheat, barley and oat interactions with Erysiphe graminus strains specific to each species. When a strain specific to one of the other species was inoculated, an "inappropriate" or non-host response occured. The use of PAL inhibitors did not affect resistance results but did decrease the amount of cells undergoing necrosis. These results suggest that lignification can have an effect on pathogen resistance. The timing of the lignification response is also important. Lignification does not appear to be a primary response to infection (Garrod et al., 1982). The addition of high S content lignins appears to be a late process. However, lignification does occur more rapidly in resistant plants than in susceptible ones (Hammerschmidt, Lamport and Muldoon, 1984). Campbell and Ellis (1992a; b) have shown that elicited cell cultures of Pinus banksiana showed increased lignification within twelve hours of elicitation. 2.3 Phytoalexins A large number of phenolics are induced by infection processes in plants. Many are defined as phytoalexins (Nicholson and Hammerschmidt, 1992). 50 Phytoalexins are generally viewed as a dicot response to pathogens, although, they have been found in oat (Mayama et al., 1981 a; b), rice (Cartwright et al., 1981; Kono et al., 1985 ), sugarcane (Brinkler and Seiger, 1991) and in Sorghum (Hipskind et al., 1990; Nicholson et al., 1987). Although the rice phytoalexin is not a phenolic derivative, some eighty percent of phytoalexins are phenolics (Zaprometov, 1993). Phytoalexins function by means of a direct action on the pathogen. An example of this process can be seen in Sorghum where after the formation of an appressorium by the fungus, the plant cell immediately below will fill with vesicle-like inclusions containing 3-deoxyanthocyanidin-type flavonoids. The vesicles burst killing the cell and release the phytoalexins to diffuse to the fungus, killing it (Mayama and Tani, 1982). 2.4 Systemic Acquired Resistance Systemic acquired resistance (SAR) occurs when a plant is exposed to a pathogen or some other stress such as UV light or ozone (Yalpani et al., 1994) and responds in a defensive manner. Subsequent challenge with a pathogen results in reduced damage to the plant. For example, using cucumber, Hammerschmidt and Kuc (1982) showed that innoculation of a leaf with Colletotrichum lagnerium led to SAR against the same fungus or Cladosporium cucumerinum via faster and more extensive lignification. Such resistance can be important agriculturally in protecting crops against severe pathogenic effects by exposing plants to less harmful ones, which is analogous to immunization in animals. SAR is primarily 51 associated with the hypersensitive response resulting in the formation of a lesion that limits the spread of the pathogen. Different pathogens provide different levels of protection. For example, in tobacco cv. Havana 425, an NN tobacco, producing lesions from TMV and TNV led to SAR against Erysiphe cichoranearum with TMV giving a stronger response (Marte, Buonaurio and Delia Torre, 1993). The use of Ethephon, an ethylene releasing compound, gave less resistance than the viruses. Lesions caused by point freezing sections of the leaf gave no protection. Not all plant-pathogen reactions induce SAR, e.g., in soybean challenged with TNV no induced resistance was found against further exposure to TNV or other viruses (Pennazio and Roggero, 1992). 2.5 Pathogenesis Related Proteins Pathogenesis related (PR) proteins are induced by the plant in response to infection. The best known occur in tobacco. There are five classes of pathogenesis related (PR) proteins. Classes 1 and 4 have an unknown function while classes 2,3, and 5 comprise glucanases, chitinases and an antifungal component respectively (Lindhorst, 1991, and Woloshuk et al., 1991). Classes 1,2,3 and 5 comprise acidic extracellular forms and basic intracellular forms. Salicylic acid induced acidic forms whereas wounding and ethylene induce basic forms. The basic forms are constitutive in roots and older leaves (Brederode, Lindhorst and Bol, 1991). Other PR associated proteins in tobacco include two hydrolases, two inhibitors of microbial proteases, two peroxidases and two proteins of unknown function. 52 2.6 Salicylic Acid Salicylic acid (SA) concentration increases twenty-fold in TMV inoculated tobacco (Xanthi n.c.) leaves and five-fold in the systemic leaves. The addition of SA also induces PR genes (Malamy et al., 1990). Silverman et al. (1993) showed that 96 hours after TMV infection Xanthi n.c.(NN) tobacco had accumulated twenty eight times the SA of TMV infected Xanthi (nn). Metraux et al. (1990) showed induction of SAR against TNV and fungal challenge by Colletotrichum lagenarium. The necrotic spot formed faster in the virally challenged rather than the fungally challenged plant. Yalpani et al. (1991) showed that the addition of SA at 32°C induced PR-1 despite the fact that TMV resistance is temperature sensitive. Salicylic acid concentration is highest around lesions where it occurs primarily as the O-B-D glucosyl conjugate (Enyedi et al., 1992). The addition of SA to hydroponically grown tobacco led to an increase in SA in virus-free leaves and induced SAR. A hybrid between Nicotiana glutinosa and N. debneyi gives high SA levels resulting in constitutive PR levels. Addition of exogenous SA reveals no changes (Yalpani, Shulaev and Raskin, 1993). The hybrid is also capable of accumulation of SA at higher temperatures at which resistance is normally inhibited. Chen, Ricigliano and Klessig (1993) determined that SA bound to a catalase enzyme inhibiting the breakdown of peroxidase which may act to enable peroxide accumulation to reach a threshold of six millimolar where death rather than anti-oxidant stimulation occurs. Similar results were obtained with 53 Arabidopsis (Sanchez-Casas and Klessig 1994). Klessig and Malamy (1994) question the role for the SA responding catalase as the oxidative burst is much more rapid than the accumulation of SA, thus the catalase may not be able to accumulates quickly enough to affect peroxide accumulation. The removal of Ca 2 + ions by chelators inhibits the induction of chitinase by SA (Kurosaki and Nishi, 1994). Pretreatment of parsley suspension cultures with SA led to increased formation of phenylpropanoids including esterified FA, PCA, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillin and coumarins in response to ellicitation(Kurosaki and Nishi, 1994). Salicylic acid appears to be produced by B-oxidation of trans-cinnamic acid (Yalpani et al., 1993) as addition of benzoic acid to tobacco cells leads to SA while o-coumaric acid addition does not. Leon et al. (1993) found that benzoic acid 2-hydroxylase which converts benzoic acid to SA increased significantly in tobacco following TMV infection. Seo, Ishizuka and Ohashi (1995) located a SA P-glucosidase in SA treated cells that was not present in untreated cells. The application of exogenous SA to barley led to induced SAR against Erysiphe graminis. Resistance was most pronounced from one to two days after spraying but continued to be effective for up to twelve days (Walters et al., 1994). Scott and Yamamoto (1994) showed that SA levels in rice were extremely high, 61.7 + 2 u.g/g, while barley levels were much lower 0.21 ± 0.01 ug/g. Silverman et al. (1995) found using rice that SA levels did not respond to fungal infection. However in terms of general resistance, varieties with higher natural SA levels 54 were more resistant. . Most of the endogenous SA was present as the free acid but if exogenous SA was added to the rice plant, it was glucosylated. Gaffney et al. (1993) inserted a bacterial hydrolase, which converts SA to catechol in a transgenic tobacco plant, leading to inhibition of SAR. Vernooij et al. (1994), using the same enzyme, showed that SA is required for SAR by grafting transgenic tobacco plants with normal plants. Infection of a stock plant able to accumulate SA could not induce SAR in a scion unable to accumulate SA. Infection of a stock plant unable to accumulate SA grafted to a scion that could accumulate SA showed that SAR could still occur due to some diffusable element. Thus SA does not appear to be the signal molecule for SAR. 3.0 The Viruses In this study, the viruses that were utilized were Tobacco Necrosis Virus (TNV), Barley Stripe Mosaic Virus (BSMV), Potato Virus X (PVX) and Tobacco Mosaic Virus (TMV). 3.1 Tobacco Necrosis Virus TNV is a small, icosahedral necrovirus with an RNA genome of 3759 nucleotides (Coutts et al., 1991). The virus consists of 5 open reading frames (ORFs), one of which contains an amber codon allowing the production of a much larger transcript. TNV is transmitted naturally by a soil fungus, Olpidium brassicae. There are several serotypes. The A serotype is capable of supporting a satellite virus. TNV A and D share only limited homology (Coutts et al., 1991). TNV induces a hypersensitive reaction resulting in the formation of a "dead spot" 55 on the leaf. The infection is contained within these necrotic spots and does not spread systemically. TNV infection in White Burley tobacco was examined by D'Agostino and Pennazio (1985) and found to form non-limiting lesions with a central necrotic area surrounded by a halo of yellowed cells where the mitochondria and chloroplasts are closely associated. The lesions continued to expand slowly. 3.2 Barley Stripe Mosaic Virus BSMV has a large genome consisting of three RNAs that code for 7 polypeptides. Al l three RNAs are required for systemic infection. Two of the polypeptides code for a replicase, while others encode a coat protein and two movement proteins (Donald and Jackson, 1993) One of the other polypeptides codes for an unknown product important in systemic movement and in control of the viral B-strand (Donald and Jackson, 1994). Jackson et al. (1991) has developed a new strain with a point mutation that results in a single amino acid change that alters the phenotype of the virus from chlorotic to necrotic. The altered protein is important but not critical for virus movement. There is no specific indication of whether the necrotic response is systemic, but judging from Jackson's other results with mutated BSMV, lack of systemic infection leads to necrotic infection instead of chlorotic effects. In some cases necrotic spots may appear within chlorotic spots, which may be a phenomenon caused by viral stress rather than necrosis due to HR, as seen with TNV. Overall, the symptoms of virus infection are somewhat intermediate between necrotic and chlorotic. 56 3.3 Tobacco Mosaic Virus T M V is a rod shaped, +sense R N A virus belonging to the tobamovirus group. The virus encodes four proteins, two for viral replication, a movement protein and a coat protein.(Dawson, 1991). T M V symptoms consist of a dark and light green mosaic generally dependent on the stage of leaf development. Dark green regions are virus free and appear to be resistant (Culver, Lindbeck and Dawson, 1991). Cultured tobacco protoplasts from the dark green regions were regenerated and remained resistant to T M V for some time. Upon infection, T M V viral particles are uncoated by cytoplasmic ribosomes and the genome is translated (Shaw, Plaskitt, and Wilson, 1986). The first protein produced is the replicase which leads to multiplication of the viral genome in the cytoplasm. The uncoating process can occur within three minutes of entering the cell (Wu, X u and Shaw, 1994). Aid ing the viral uncoating process in the cell is the presence of carboxylate groups in glutamate and aspartate residues in the T M V viral coat protein. The carboxylate group provide electrostatic repulsion during disassembly of virus particles (Culver et al., 1995). 3.4 Viral Movement Cel l to cell movement is crucial in determining whether the infection wi l l be localized or systemic. This movement often occurs via the action o f specific viral proteins. Amino acid sequences suggest that viral movement proteins (MPs) of all + strand R N A viruses, s s D N A geminiviruses, d s D N A pararetroviruses and - strand tospoviruses may be representative of a vast superfamily of proteins 57 (Mushegian and Koonin, 1993). All viruses that move cell to cell are believed to have a movement protein (Atabekov and Taliansky, 1990). MPs are thought to function in two areas; modification of plasmodesmatal size exclusion limit (SEL) and in nucleic acid binding. In transgenic tobacco containing the 30 kD MP of TMV, a 9.4 kD FITC (fluorescein isothiocyanate) labelled dextran could be moved through plasmodesmata (Wolf et al., 1991). The TMV MP binds ssRNA and DNA cooperatively and nonspecifically (Citovsky et al., 1990). Citovsky et al. (1990) propose a model MP that binds RNA and unfolds it into a narrow complex. This binding does not appear to be sequence specific. The MPs from TMV, Alfalfa Mosaic Virus, Cauliflower Mosaic Virus and Red Clover Mosaic Virus have all been located in and around plasmodesmata (Linstead et al., 1988; Maule, 1991). Viral movement is classed into two types; the tobamotype, for which TMV is the model, and the comotype based on Cowpea Mosaic Virus (CPMV) (Van Lent et al., 1991). The tobamotype modifies plasmodesmata enabling the virus to move through the cell wall, while the comotype movement requires both MP and coat protein to make a tubular structure that crosses the cell wall. For viruses of the Cowpea Mosaic Virus -type movement may occur via the formation of secondary plasmodesmata without the apressed ends. The potexviruses, including PVX, carlaviruses and hordeiviruses, including BSMV, encode a triple gene block (Koonin and Dolja, 1993) of two small MPs and an RNA helicase that are all required for movement (Petty and 58 Jackson, 1990). Carmoviruses and necroviruses, including TNV, contain two small proteins that may represent a truncated triple block (Hacker et al., 1992). Plasmodesmata were originally believed to be simple cytoplasmic bridges. The new model (Lucas, Ding and Vander Schoot, 1993) expands their roles to a supramolecular complex of membranes and proteins controlling not only the size exclusion limit but also potentiating movement of macromolecules such as proteins and nucleic acids. Wolf and Lucas (1995) suggest that plants should now be considered as supracellular rather than multicellular. There are two types of plasmodesmata (PDs). The first or primary plasmodesmata, consist of a compressed cylinder of endoplasmic reticulum(ER) that allows the ER to be continuous between cells. Around the apressed ER is the cytoplasmic annulus or sleeve. While the ends of the plasmodesmata are compressed, the middle is open, somewhat like a hard candy wrapper with twisted ends. The primary PDs are formed during cytokinesis when a portion of the ER is positioned perpendicular to the plane of the new cell plate so that vesicles cannot fuse in a manner that would allow complete separation of the two daughter cells. The second type of PD, secondary plasmodesmata, are formed along non divisional walls after cytokinesis. These plasmodestmata consist of multi-cytoplasmic strands interconnected in the middle lamella through holes "drilled" by cellwall degrading enzymes. Plant virus MPs have provided a valuable tool for probing PD function as well as providing information about viral movement. In examining transgenic 59 tobacco containing the TMV MP, Lucas, Ding and Vander Schoot (1993) found an increase in SEL and also an increase in sucrose, glucose, fructose and starch which is postulated to be an effect on carbon source-sink relationships within the young leaf. SEL adjustment and sealing likely occurs by means of callose, a p 1-3 glucose polymer (Oleson and Robards, 1990). Ding et al. (1993) showed that the blockage of secondary plasmodesmata induced senescense. The increase of acid invertase seems to play a role in this blockage, leading to increased free sugar levels (Ding et al., 1993). Injected TMV MP can increase SEL to enable movement of a 20kD dextran (Waigmann et al., 1993). This also affects cells away from the injection site either by crossing plasmodesmata or by triggering release of a diffusible element. 60 4.0 Materials and Methods Plant material: Triticum aestivum cv. Katepwa was grown for seven days in a greenhouse with sixteen hours light prior to infection at the one leaf stage. Nicotiana tabacum cv. White Burley, and Xanthi n.c. were grown for six weeks in the greenhouse prior to infection of the two largest leaves (leaves two and three). Inoculum: Viral inoculum consisted of fresh tobacco plants infected for four-five days with TNV, TMV, and PVX and wheat infected for seven or more days with BSMV which were ground in distilled water. Plants were coated with fine carborundum then mechanically inoculated on initial leaves with virus or, in the case of control plants, with distilled water(mock inoculated)to control for wounding as an aspect of the result. Harvest Scheme: For each treatment (TNV, BSMV and control) in all five trials, three pots of approximately twenty-five wheat plants per pot were harvested 1, 3, 5, 7, 10 and 15 days after infection. The effects of systemic acquired resistance were determined in second leaves (uninoculated) on days 7 and 15. With tobacco, three plants were harvested for each treatment (TMV, TNV, PVX and control) (one trial) on days 1, 2, 3, 5, 8 and 11. Effects of SAR were examined in younger expanded leaves on days 3,5,8, 11. Methanol Extraction: The extraction method was initially based on that of van Sumere (1989) but has undergone substantial simplification and modification. Freeze dried leaves were weighed (300-700 mg) and ground in a blender at room temperature in 75 ml of MeOH and lOOpl of 1 g/ml o-anisic acid which was added 61 as an internal standard. (Meuwly and Metraux, 1993). The ground material was stirred in a 200ml flask using a magnetic stirrer for one hour and vaccuumfiltered through a fine porosity 500 ml sintered glass funnel. The residue (cell walls) was washed with approximately 75 ml of MeOH and the filtrate was added to the initial filtrate. The residue was resuspended in 100 ml of MeOH and stirred for six hours and filtered. The above process was repeated and the suspension left to stir overnight. The combined methanol extracts were evaporated to dryness under vacuum. The dry sample was redissolved in 2 ml MeOH to which 15 ml boiling water was added. The sample was swirled then poured into a centrifuge tube. The flask was rinsed with 1 ml of MeOH followed by 10 ml boiling water. After swirling, the contents were added to the centrifuge tube. The combined material was centrifuged at 15,000 rpm for 30 min. The supernatant was decanted into a flask and the green pellet discarded. The aqueous/MeOH fraction was extracted with two x 20 ml of ethyl acetate. The upper ethyl acetate fraction containing the free phenolic acids was removed and dried with anhydrous Na 2 S0 4 , evaporated to dryness under vacuum and the residue redissolved in 1 ml HPLC grade MeOH. To protect the HPLC column, the MeOH was then passed through a 1 ml Chromosep (Chromatographic Specialties) or a 1 ml Sep-Pak (Waters) C-18 column. The column was washed with 0.5 ml MeOH. The sample was collected in a 1.8 ml Eppendorf and placed in the refrigerator or freezer until HPLC analysis. 62 The remaining aqueous phase containing phenolic glucosides and phenolic esters was made up to 30 ml with distilled water and divided into two equal portions. One portion was hydrolysed with B-glucosidase (Sigma) in 15 ml acetate buffer (pH 4.3) at 37°C overnight and extracted with two x 20 ml ethyl acetate. The other portion was hydrolysed in 2 M NaOH at room temperature overnight in a nitrogen atomosphere. After adjusting to pH 2 with HC1, the alkali hydrolyzed fraction was washed with two x 20 ml ethyl acetate. The ethyl acetate for each fraction was prepared for HPLC analysis as above. The aqueous hydrolysates were then each exracted with two x 15 ml butanol. The butanol was evaporated to dryness under vacuum, and the residue redissolved in 1 ml methanol and prepared for HPLC as above. Alkali samples were washed with methanol to remove salt. The methanol was then dried and the residue resuspended in 1 ml MeOH and prepared for HPLC. Cell Wall Extraction: Lyophilized methanol extracted cell walls (100 mg) were hydrolysed with 2 M NaOH in a nitrogen atmosphere for 48 hours. The samples were adjusted to pH 2, extracted with two x 20 ml ethyl acetate and prepared for HPLC as above. The cell wall residue was then lyophilized for lignin analysis. Lignin Analysis: Lignin was determined by the acetyl bromide method (Morrison, 1972) using an absorptivity value for lignin of 23.7 L/(g cm). Five millilitres of 25% (v/v) acetyl bromide in glacial acetic acid was added to 14mmxl50mm test tubes containing 10 mg of dried alkali extracted cell walls. 63 Each tube was capped with a marble and digested by shaking for 30 min. at 70°C in a shaking water bath. The samples were vigorously mixed by hand (holding the marble in place) every ten minutes during shaking. The samples were allowed to cool to room temperature and transferred to 100ml volumetric flasks containing 1.8 ml two N NaOH and ten ml glacial acetic acid. The residual contents of the test tube were washed into the volumetric flask with approximately 3 ml acetic acid, which was vigorously mixed. Next, 3.2 ml of 0.5 M NH 2OH*HCl was added and the flask brought to volume with glacial acetic acid. The mixture was shaken and allowed to stand one hour. Control blanks were prepared in the same manner without the addition of sample. The absorption was read at 280 nm on a spectrophotometer. The amount of lignin was determined from the equation (Morrison, 1972) (As-Ab)x0.\lx\00 %[ =-^— — 23.7xW where A s is the sample absorbance, A b is the blank absorbance, 0.1/ is the sample volume in litres and W is the sample weight in grams. Systemic Acquired Resistance: Preliminary determination of SAR in wheat was determined by rechallenging the second leaf with TNV after lesions had appeared on the initial leaf (four-five days post infection (DPI)). The innoculum was again crude ground plant material. Comparison was made between lesion number and size of lesion in infected and control plants that had previously been inoculated with water. 64 HPLC: Initial separations were carried out on a C-18 reverse phase u-Bondapak 3.9 x 300mm (Waters) column using a gradient of MeOH and one percent acetic acid. The gradient was MeOH: 1% acetic acid (25: 75) initially increasing to 100% MeOH over 30 minutes, the methanol was maintained for five minutes and the system was returned to the initial conditions over the next five minutes. The initial conditions were maintained for 20 minutes to flush and equilibrate the column. Because of problems with the separation of the major compounds a new method was developed using a Waters 3.9x150mm novaPak C-18 column and a gradient of acetonitrile and 0.1% trifluroacetic acid. For all fractions except the butanol fraction, the gradient was initially MeCN: 0.1%TFA (10:90) at a flow of 1 ml/min. The solvent ratio was gradually increased to 20:80 over the first 15 minutes and 40:60 over the following 25 minutes. From 40 to 45 minutes the MeCN was increased to 100%, held there for five minutes and returned to initial conditions by 55 minutes into the run. The column was equilibrated for ten minutes at the initial conditions at this point to make up a total run time of 65 minutes. All adjustments to the gradients were conducted using the slightly concave gradient curve seven (Waters). For the butanol fraction the gradient was initially MeCN:0.1% TFA (5:95) increasing linearly tol0:90 over ten minutes and 15:85 over a further ten minutes. From 20 to 30 minutes the gradient increased linearly to MeCN: TFA (25:75) and over the next five minutes was increased to 100% MeCN, held for five minutes and returned to initial conditions 65 over five minutes. The column was equilibrated at this point for 15 minutes to make up a run time of one hour. The UV absorption results from the HPLC were used in two ways. Initially the instrument output was used to establish 95% confidence limits. Due to high variation between trials, the data was converted to a ratio based on the value of the control samples. Again 95% confidence intervals were determined along with an F test for mean differential (in Appendix 1). 66 5.0 Results 5.1 General Pathology 5.1.1 Wheat The TNV infected wheat developed necrotic lesions 5 days after infection. The lesions were generally long and narrow, usually in a ratio of 3-4:1. The lesions were yellow-brown and slightly overlapped the ribs of the leaf. The extreme tips of the TNV infected leaves were occasionally prone to desiccation. The BSMV plants did not develop streaks of chlorosis until 7 days after infection. These streaks were longer than the TNV lesions and were more yellow in colour. Mock plants remained similar to unhandled plants although occasionally the tips of the treated leaves turned brown. 5.1.2 Tobacco The induction of HR was much faster with TMV than TNV in the Xanthi n.c. cultivar. With TMV infections, the lesions developed at 1.5-2.5 days, 24-36 hours prior to those of TNV. The TMV lesions were larger, with a defined border and displayed complete tissue collapse within their areas, whereas the TNV lesions were more like a pin-prick of necrosis surrounded by a halo of yellowed cells. The White Burley cultivar response to TMV was obviously different as it did not contain the NN genes for TMV resistance. TMV could therefore move systemically causing vein clearing, which meant that the veins in the leaf became more visible. Eventually the leaves developed a mosaic of light and dark green spots in the leaves with older leaves containing the most dark green area. The 67 response of the White Burley cultivar to TNV also differed. The lesions were produced after a similar time to those in Xanthi n.c. but the lesions were larger, darker in colour and had a diffuse boundary. The TNV did not move systemically throughout the plant. Interestingly, studies in rice (Singh et al., personal comm.) infected with TNV also displayed lesion darkening. 5.2 Systemic Acquired Resistance in Wheat. Preliminary studies to determine the presence of an SAR response in the second leaf of wheat to a second challenge (first challenge on the first leaf) with TNV displayed only slightly fewer lesions, thirteen for a second challenge and fifteen for a first occurence (average of twenty plants). The lesions in the second challenge plants appeared to be smaller than primary lesions from plants exposed to the virus for the first time, especially in regard to diameter. Secondary lesions were about two-thirds the size. The systemic (uninoculated) leaves of TNV inoculated plants remained visually indistinguishable from mock infected plants. The BSMV second leaf contained more yellow tissue than green and was visibly stunted. The presence of virus in this tissue was shown by inoculating healthy wheat plants with homogenized second leaves from the infected plants. At maturity the systemic BSMV leaves contained many distinct stripes on each leaf. 68 5.3 Analysis of Phenolic Compunds Induced by Infection. The structures of the identified compounds are shown in Figure 19 and 20. 5.3.1 Free Phenolics from Methanol Extracts. 5.3.1.1 Wheat. No compounds were identified. 5.3.1.2 Tobacco In the free phenolic methanol extracts of mock samples of tobacco (Figure 3), protocatechuic acid was identified. The infected tobacco did not display any compounds identifiable with the library. If protocathechuic acid were present in these samples, its concentration was too low to readily identify. 5.3.2 fi-Glucosidase Treated Methanol Extracts 5.3.2.1 Wheat To determine which compounds were present in the samples, single samples, representing each fraction, were examined with the new protocol. In the wheat samples the only component identified was ferulic acid in BSMV and TNV samples (Figure 4) but not in mock samples. 5.3.2.2 Tobacco In the B-glucosidase-hydrolysed samples the Xanthi n.c. tobacco mock sample showed very low concentrations of phenolic acids making identification difficult. The TMV samples showed (Figure 5) a mixed peak of vanillic and caffeic acids as well as peaks for salicylic acid, ferulic acid (FA) and a coumarin that was identified as scopoletin from its retention time on the HPLC and UV 69 70 72 spectrum. There was also a compound that matched retention time and UV spectrum (i.e. absoption at 260 nm) with a compound produced as a major product in a mixture of FA and PCA (p-coumaric acid) exposed to UV to form truxinic and truxillic acids. The standard compound was not purified or determined in any manner other than general UV spectra which suggested a truxinic or truxillic acid (Rajaonarivony, personal comm.) In the TNV sample (Figure 6) the same peaks were found and in addition 4-hydroxybenzoic acid was identified. 5.3.3 Alkaline Hydrolysis of Methanol Extracts 5.3.3.1 Wheat In alkaline hydrosylates of wheat treated with BSMV (Figure 7) samples showed vanillic acid, anthranilic acid, FA, PCA and a mixed peak that appeared to include salicylic acid. Mock samples (Figure 8) contained vanillic acid in a peak contaminated with a compound which showed spectral similarity to p-hydroxy phenylacetic acid. They also contained FA and PCA. TNV samples were similar to mock samples. 5.3.3.2 Tobacco The alkali hydrolysed extracts of tobacco showed low concentrations of phenolic acids in the mock-infected sample. TMV challenged plants (Figure 9) contained vanillic acid, caffeic acid, vanillin, p-coumaric acid, benzoic acid, and salicylic acid. The TNV sample showed a similar apparent profile (Figure 10) except for the lack of a salicylic acid peak. The peaks were of much lower 73 74 o o o o o .g > 0 0 O O p x o s O T X X T x n a ^ / O T U T x r u ^ pXOB DTXXOXXBS p x o e o x a B u m o o - d -SpxOB OXXITUBail^UB ' O X X X T I 1 B A I I I I I I I I I I I 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I in <D a -H o o o CN o -o VO CN o CD VO CN O CO VO CN o CN CN CN CN H H H rH o o o O o O O O O O O O O O o o o o o nv x <u u » B -a <D 1 _g M s eg o c Cu, o C + H O o o 4) O U o at • CD CU i_ bo S fl .Si0'55 75 76 77 78 concentration, however, thus it was only possible to identify benzoic acid by UV spectra. 5.3.4 Butanol Extracts of Methanol Extracts 5.3.4.1 Wheat No compounds were identified. 5.3.4.2 Tobacco No compounds were identified. 5.4 Cell Wall Associated Phenolics. 5.4.1 Alkaline Hydrolysis of Cell Walls 5.4.1.1 Wheat The alkali hydrolysed wheat cell wall samples were the only samples run and replicated using the improved HPLC protocol (Figure 11). For all samples (BSMV, TNV and mock-infected plants), no new compounds were found to be produced exclusively in response to TNV and BSMV challenge. The identified compounds included vanillic acid (Figure 12), p-hydroxybenzoic acid (Figure 13), vanillin (Figure 14), PCA (Figure 15), FA (Figure 16), 5,5 diferulic acid (DFA)(Figure 17). The only significant findings were an increase in p-hydroxybenzoic acid on day 15 in both virally challenged treatments (Figure 13). The BSMV sample also showed a significant increase in the second (systemically-infected) leaf at day 15. For the ferulic acid peak (Figure 16) significant decreases were shown by 79 80 "D CZ CO H- _co If 00 >, CQ co ^ CD 1:1 3 CD CO > ~Bo O CD O Q. £= co — CD •4—* J*' CD o _ CD o o "D "D CD y= CO C >» O O o . -j= -^o CD CD CO "co c CD O ~Zi CO co CD -Q -- a.* co co o O <B § co~ o > c o O D O c co ^ 0 3 m ~ CM CO »-i - CD c a> +- co ^ CD E E co 81 •o c CO > Z H CO Cd •| « > o T 5 - Q B E -§ 5T O "O O CD C C S'g> -C .> 5 O CD O Q_ « c5 "CO >" | o 0) BJ o « T3 £ <j> CD c 2 CD T3 O C SZ CD = "D CO '•tz . CO o C so CD — o -a to c o ® o o « 59 CD X o o CO O o © 0 ID CD c o o o o E o CD o CO CO 2> E - | CO CO CD i i . E co 82 83 8 4 LI E 03 86 BSMV treated samples. The diferulic acid peak showed significant decreases at day 10 and 15 in both viral treatments compared to mock treated plants. SAR There was no change in phenolic compounds in response to TNV infection in wheat. 5.4.1.2 Tobacco. The cell walls of tobacco samples showed PCA in mock challenged samples . In the TNV-infected sample p-hydroxybenzaldehyde and vanillin were found in addition to the PCA . TMV infected samples were similar to the TNV infected samples with the addition of caffeic acid to the other peaks (Figure 18). 5.4.2 Lignin analysis. 5.4.2.1 Wheat Lignin concentration remained constant in wheat plants that were mock inoculated and in plants that were uninoculated except for a slight increase in wounded plants at day 3 (Figure not shown). TNV plants showed a sharp increase between day 3 and day 5 resulting in increased lignin contents in infected plants at day five compared to controls. The lignin level in TNV plants was 4.5% and the level in control leaves was 3.5%. The lignin measurement was not replicated. BSMV effects on lignin were not determined. 8 7 88 COOH COOH CHO OH OH Benzoic acid p-Hydroxybenzoic acid p-Hydroxybenzaldehyde COOH OCH3 OH Vanillic acid CHO OCH3 OH Vanillin CHO OH OCH3 Isovanillin COOH OH Salicylic acid COOH / O H OH Protocatechuic acid Figure 19. Structure of benzoic acid derivatives found in inoculated tobacco. 89 X O O H COOH OH p-Hydroxyphenyl acetic acid NIL, Anthranilic acid H O ^ ^ ^ Scopoletin O ^ ^ O COOH COOH 1 J 1 \ C H 3 0 y y OCH3 OH OH Diferulic acid Figure 20. Structures of other compounds from inoculated wheat and tobacco 90 6.0 Discussion The results of the alkaline extracted cell wall fractions show that most phenolics, including ferulic and diferulic acids, that are ester bound to the cell wall are decreasing in concentration over the course of viral infection. Only late in the process (Days 10, 15) does the decrease become statistically significant (Appendix 1). This finding is similar to that of Chigrin et al. (1984) in wheat infected with rust who found that in general 44-57% of phenolic acids in wheat were in esterified form. Upon infection with rust, esterified phenolic acids in the walls decreased 63% in incompatible (non-host) relationships, 29% in resistant cultivars and only 8% in compatible (host) varieties. Chigrin et al. (1984) showed an increase in fungal esterase activity which could account for this response. However, it is also possible that an increase in oxidizing enzymes such as laccases or peroxidases, which are also common responses to pathogenesis, may increase the number of alkali-stable ether and carbon-carbon bonds between ester bound compounds and the wall. The net result would be to make the wall more resistant to degradation, inhibiting the release of esterified compounds either sterically or by direct bonding, thus lowering extraction efficiency. The high variability between trials during the first seven days could be attributed to a number of factors. The conditions in the greenhouse may vary enough to allow substantial variation in cell wall phenolics. The rate of pathogenesis may also vary between trials thus shifting trends in phenolic addition to different days and leading to scatter in the data that cannot be resolved 91 statistically. Another possibility is that in weighing out the samples to 100 mg instead of using all of the tissue a bias may have been created between vascular and fiber tissue, and other leaf tissues. A report by Malinovskii et al. (1994) working with Xanthi n.c. (NN) tobacco showed an increase in resistance in TMV infected plants to cell wall maceration enzymes. This corresponded to infected plants that exhibited a reduction in number of lesions, viral production and viral movement. A similar mechanism may occur in wheat, thus inhibiting release of esterified components from the cell wall. Additional support for the enhanced retention of esterified groups in the cell wall comes from the lignin analysis which suggests a slight increase in lignin in virally challenged wheat implying an increase in phenolic residues in the cell wall. To determine whether lignin increase is represented by monolignol addition, the cell wall material could have been extracted with alkali at high temperature (170°C) which cleaves ether bonds and would release much more of the FA and DFA contained in the wall (Hartley and Ford, 1989). The reduction of dimer (DFA) release between younger (Day 7) and older (Day 15) leaves in wheat also provides support for the hypothesis that ether and other bonds (C-C) hold esterified phenolics in the wall. The changes in the phenolic esters in the cell wall do not appear to correlate with inhibition of viral movement. The significant changes occur in both the systemic and localized virus and the changes occur after the virus has become 92 systemic. If there is any effect it is likely masked by the high variability between trials. The role of senescense in viral infections also appears to be important. The infection of primary leaves leads to more rapid yellowing and desiccation of the infected leaf than in control plants. In addition, healthy older leaves are more difficult to infect than younger leaves. For example, a substantial reduction in lesion formation was found when wheat plants were infected with the virus two days later than the plants used in this study. The inhibition of infection may simply be caused by increased lignification in the cell wall resulting in fewer opportunities to enter wounded cells during mechanical infection. It is also possible that other effects occur during maturation that generally inhibit viral infection such as increased production of anti-viral proteins and other compounds. The initial leaf in wheat has a higher tensile strength compared to later leaves at a similar age suggesting that the cell wall structure may be more lignified. The fact that the second leaf contains far more esterified phenolics at day 15 (leaf is ten days old) than are present in comparably aged day 3 initial leaves suggests the possibility of increased cross linking in the initial leaves. A potentially important difference that is not readily shown by the data is the bonding pattern in the cell wall. Initial cell wall responses that lead to increased lignification due to HR initially increase lignin content. However, as healthy and systemically infected plants age, they also increase lignin levels until they match (healthy) or may surpass (systemically infected) the hypersensitively 93 responding TNV plant. Unfortunately, the data available in this study provides only limited information as to the construction of the phenolic network. Thus the wall could contain equivalent overall levels of phenolic compounds but in significantly different bonding patterns or even monomer compositions. Mitchell, Hall and Barber (1994) found that fungal elicitation of wheat induced only sinapoyl alcohol dehydrogenase and not other CAD forms suggesting specificity in the type of lignin added is important. Other studies have shown an increase in S lignins (Garrod et al., 1982; Lyons et al., 1993). Ding et al. (1993) noted that blockage of secondary plasmodesmata led to increased senescence. If lignification, either in the form of monolignols or HCAs, can restrict SEL of plasmodesmata, this characteristic could apply to the increased senescence rate in the TNV and BSMV infected leaves. The induction of SAR which leads to a decrease in lesion size especially in length in wheat may mean that SAR targets primary plasmodesmata in some manner, restricting viral movement. Kehlenbeck et al. (1994) showed that barley undergoing SAR had an increased yield in comparison to uninfected plants. They suggest that this is due to an alteration in source-sink relationships within the plant. Lucas, Ding and Vander Schoot (1993) also point to an alteration in source-sink relationships in transgenic tobacco containing the TMV movement protein. They attribute the change to the induction of an acid invertase which increases sugar levels in the cell and seems to play a role in blockage of secondary plasmodesmata. In barley infected with 94 powdery mildew an increase in the activity of acid invertase was noted (Scholes et al., 1994). In comparing wheat cultivars resistant and sensitive to fungal infection, Heisteruben, Schulte and Moerschbacher (1994) found that initial acid invertase levels increased faster in resistant plants. Over time however, a fungally encoded invertase led to increased enzyme activity in the sensitive cultivar. It is interesting that a mechanism that apparently is important in defensive response by the plant, would also have the potential to aid the pathogen. The invertase likely provides energy (via sugar metabolism) for various defensive processes, such as HR, in resistant plants prior to the pathogen becoming strong enough to benefit as well. In the tobacco extractions that were completed, an interesting effect was the reduction of protocatechuic acid in TNV and TMV plants. This effect was similar to the response involving chlorogenic acid in Lycopersicon peruvianum to fungal elicitors.(Bieman, Witte, and Burz, 1992). This study also found many of the compounds isolated from the wheat cell walls including p-hydroxybenzaldehyde, vanillin, PCA, and FA. p-Coumaroyl and feruloyl tyramines were also present. If phenolic amides were present in the samples, they would likely be present in the butanol fractions (Negrel et al., 1993). Martin, Martin-Tanguy and coworkers have shown (Martin-Tanguy, 1985) phenolic amides may have important roles in HR to TMV in Xanthi n.c. These studies have been largely ignored outside of their own group probably due to the language of publication. 95 The production of scopoletin by TMV challenged NN tobacco is supported by observations by Fritig, Legrand and Hirth (1972). In the present study, scopoletin was also induced by TNV. It would be interesting to know whether an nn tobacco like Xanthi nn or White Burley i.e. cultivars lacking the N gene for restistance, also produces scopoletin in response to TNV infection. The increase of phenolics ester-bound to the cell wall at the time of symptom expression is also interesting. The addition of the esters does not lead to formation of a lesion nor does it restrict the virus from moving systemically through the plant as the virus is already present in systemic leaves. The burst of extractable esters may function in the protection of the "green islands" between the chlorotic regions. If the pathogen differentiates between the two types of plasmodesmata using one for rapid systemic movement through cells to the vascular system while utilizing the other for slower cell to cell movement within the leaf this burst may help to contain this slower movement. The fact that length and width of lesions in grasses are disparate is obvious even from the names of the many cereal viruses e.g. barley stripe mosiac virus and wheat streak mosaic virus. This effect may be a product of leaf morphology in grasses with their parallel venation providing an impenetrable boundary to viral spread across the leaf. Another possibility for the burst of esterified cell wall phenolics in BSMV infected tissues may be that as viral pathology continues the wall is weakened briefly, perhaps by release of H + ions, while an attempt to maintain normal metabolic functions occurs. Extraction at this phase may allow increased 96 accessibility to wall phenolics increasing extractability. Later, the increase in senescence rate causes the wall to re-form via induction of peroxidases or other enzymes. The accumulation of ester bound phenolics is widespread in plant pathology, e.g. fungal elicitation of carnation led to accumulations of benzoic acid, p-hydroxybenzoic acid, vanillic acid, PCA, cis and trans FA, dihydroferulic acid and anthranilic amides of benzoic, salicylic and p-coumaric acids. Similar results occur in fungally challenged maize (Lyons et al., 1993). The fact that the present study found a decrease in release of esterified compounds may point to an interesting difference in response to viral rather than fungal infection. Another possibility is that this response is wheat specific. The induction of salicylic acid in BSMV-infected samples that do not undergo HR and the observation that SA does not accumulate in necrotically responding TNV samples is interesting. It may mean that SA levels in BSMV samples do not reach high enough concentration to have any effect. It may also suggest that SA has limited effect in wheat. As exogenous SA has been used in barley to induce SAR it would be useful to run a trial spraying SA on wheat to determine if SAR can be induced. Studies in corn (Yalpani pers. comm.) suggest that SA does accumulate in response to viral challenge in the manner described in tobacco. Another interesting point is that dicots seem to favor caffeic acid and caffeic conjugates to a much greater extent than the Gramineae in pathogen 97 responses (Martin-Tanguy, 1985). It is possible that some of the unknown compounds, especially those in the butanol fractions, may include some of these caffeic conjugates. However, the only supporting evidence in the literature is the maize study of Lyons et al. (1993). They postulate that the caffeic acid esters they isolated play a role in tissue browning. Interestingly, the aqueous components of the hydrolysed cell wall samples of TNV and BSMV become brown as time progresses. The TNV samples show darker coloration from day 3-5 in comparison to mock and BSMV samples. By day 7 the BSMV samples were similar in colour to TNV samples and are substantially darker than TNV samples by day 10 and 15. The brown colour was present equally in both fractions in the ethyl acetate/aqueous extractions and may slightly favour the butanol fraction in the butanol/aqueous extractions. The number of changes in response to HR outlined in the introduction indicates the complexity of the response to viral infection. A mixture of new components and production of altered levels of other compounds comprise this response. 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APPENDIX 1 F values for ANOVA of absorption mean based on ratio to mock vanillic acid F p-coumaric acid F Day1 0.1575376 Day1 1.6864278 Day3 0.3427061 Day3 0.5293547 Day5 2.1522179 Day5 2.7323206 Day7 0.9229853 Day7 0.8436892 Day10 0.2757927 Day 10 1.1524109 Day15 0.8371323 Day15 0.6543792 Day7leaf2 0.2888608 Day7leaf2 2.4850114 Day15leaf2 0.3774107 Day15leaf2 3.0525319 p-hydroxybenzoic acid F ferulic acid F Day1 0.000803 Day1 0.8931896 Day3 1.0210747 Day3 0.656566 Day5 1.9084009 Day5 7.3457407 Day7 1.7332202 Day7 0.8337712 Day10 4.2036299 Day 10 15.274535 Day15 6.7133801 * Day15 18.770062 Day7leaf2 1.7717924 Day7leaf2 1.4476039 Day15leaf2 9.5804146 * Day15leaf2 0.5988214 vanillin F diferulic acid F Day1 0.39091 Day1 0.7925636 Day3 0.0367516 Day3 0.2402162 Day5 0.4075305 Day5 5.5974785 Day7 0.4856659 Day7 1.2137094 Day10 0.5774331 Day10 12.172224 Day15 0.0625242 Day15 21.282066 Day7leaf2 1.3748419 Day7leaf2 2.9169835 Day15leaf2 0.172284 Day15leaf2 0.7077011 F(0.05),2,9= 5.71 *=significant 

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