Planta 146,281-286(1979)
P l a n t a 9 by Springer-Verlag 1979
Photoregulation of the Incorporation of Guaiacyl Units into Lignins* Claude Grand, Raoul Ranjeva, Alain M. Boudet**, and Gilbert Alibert Centre de Physiologie V6g+tale, Laboratoire Associ6 au C.N.R.S. N ~ 241, 118, Route de Narbonne, F-31077 Toulouse C+dex, France
Abstract. When fed with [14C] phenylalanine in the light, xylem tissues isolated from poplar stems were able to incorporate part of the radioactivity into both the guaiacyl and the syringyl residues of lignins. In the dark, only syringyl units were integrated into the polymer whereas the guaiacyl residues remained unlabeled. When a membrane perturber (isopropanol) was added to the incubation mixture, the label was incorporated into the guaiacyl units either in the light or in the dark. Conversely, a membrane stabilizer (CaC12) prevented the labeling of the guaiacyl units even when the tissues were illuminated. These results suggest that light acts through the modification of membrane permeabilities, altering specifically the synthesis and the transport or the polymerization of guaiacyl-type units during the process of lignification.
Key words: Guaiacyl units - Light effect - Lignification - Membrane permeability - Populus.
Introduction
Lignin, the phenolic cell-wall component, results from the polymerization of cinnamyl alcohols which arise from phenylalanine via a well-characterized sequence of enzymic reactions (for review see Grisebach, 1977). The actual structure of this complex polymer is not fully understood but the relative proportion of its monomeric building blocks is generally used to identify a particular lignin (Sarkanen and Hergert, 1971). It has been shown, for example, that the ratios * This is paper No. 4 in a series. For paper No. 3 see Grand and Ranjeva, in press ** To whom reprint requests should be addressed Abbreviations: S = syringyt residue; G = guaiacyl residue
of syringyl to guaiacyl residues (S/G ratios) may vary according to various factors including the taxonomic position of the plant and the developmental stage of a given tissue (Sarkanen and Hergert, 1971). In this way, Higuchi and his coworkers (Higuchi et al., 1977) have demonstrated that the O-methyltransferase from angiosperms is able to catalyze the O-methylation of both caffeate and 5-hydroxyferulate, whereas, the enzyme from gymnosperms catalyzes only the formation of ferulate from caffeate. Thus, the absence of S residues in gymnosperms may be explained by their inability to form sinapic acid. Moreover, the results obtained by our group (Grand and Ranjeva, in press) suggest that the changes in S/G ratios are significantly correlated to that of sinapate/ferulate CoA-ligase levels, the enzymes which activate the hydroxycinnamic acids to their corresponding CoA esters. Consequently, the nature and the proportions of monomers incorporated into lignins seem to depend upon the substrate specificity of some particular enzymes and their absolute or relative levels. Such systems may account for the regulation of the qualitative composition of lignins by means of the specific enzymic equipment of the tissues. Nevertheless, lignification involves not only the biosynthesis of the monomers but also their transport and their polymerization. In this context, the respective role of each individual sequence on the control of the overall process within the same tissue (or cell) remains unclear. Some years ago, Rubery and Northcote (1968) established that xylem fractions isolated from different plants are able to synthesize labeled lignin from [14C]phenylalanine. More recently, we have designed a method allowing the simultaneous measurements of the quantity and the radioactivity of the individual aldehydes obtained by nitrobenzene oxidation of the polymer (Alibert and Boudet, 1979). These tools were
0032-0935/79/0146/0281/$01.20
282
adapted to poplar use and to determine the minute changes in the lignin composition in response to various treatments. The present paper deals with the demonstration of a light effect on the monomeric composition of lignin polymer.
Materials and Methods
Chemicals The chemicals used were analytical grade when available. They were purchased respectively from: Pharmacia fine chemicals, Uppsala Sweden: Dextran T 40 M.W. 40,000; Commissariat fi l'Energie Atomique, Saclay France: [U-a~C]phenylalanine (1,7.10 l~ Bq mmol- 1); Merck, Darmstadt Germany other chemicals.
Plant Material Populus x. euramericana (Dode) c.v. " I 214" plants were cultured in a growth chamber and the xylem tissues were isolated from stems by dissection with a scalpel (for details see Grand and Ranjeva, in press).
Isotope Studies Incorporation by intact stems. Stems from 3-month-old poplars (15 nodes) were cut under water and introduced in small test tubes containing water. The water was sucked off and replaced by 0.2 ml [U-l*C]phenylalanine per stem (3.8.104 Bq). After 10 min, uptake of labeled solution was almost complete and water (1 ml) was added periodically. Half an hour later the plants were transferred to a beaker containing water and allowed to metabolize for 12 h, in the dark or in the light (under 4,000 lx) at 26~ C. Each run was performed in five replicates. After the period of metabolization, the xylem tissues were isolated (Grand and Ranjeva, in press), frozen in liquid nitrogen, and lyophilized. Incorporation by xylem tissues. The incubations of t h e xylem tissues were performed in stoppered Erlenmeyer flasks (25 ml) on a gyratory shaker (100 rev min- 1) during 30 h, under 4,000 lx illumination at 26~ C, in a standard medium containing: 100mM Tris-HC1 buffer pH7.8; 25g 1-1 Dextran T40; 0.1 mM mercaptoethanol; 9.5.104 Bq [14C]phenylalanine. The final volume was 5 ml for 300 mg of fresh tissues.
Oxygen Measurements The measurements of oxygen consumption were performed with a Gilson polarograph (K-1 CC type). At the end of the experiments, the tissues were frozen with liquid nitrogen and lyophilized.
Analysis of the Lignin Monomers The extraction of lignins, their oxidation by alkaline nitrobenzene and the automated analysis and measurements of the radioactivity of the liberated aldehydes were performed as described elsewhere (Alibert and Boudet, 1979).
C. Grand et al. : Influence of Light on Lignin Synthesis Results
When fed with [l'~C]phenylalanine, young poplar stems are able to synthesize labeled S and G residues of lignins (Alibert and Boudet, 1979). The extent of the incorporation of each of these units, in a given time, measures the rate of the respective metabolic fluxes involved in the synthesis of each monomer (Alibert and Boudet, 1979). Consequently, the measurement of the changes in the ratios of radioactive S/G units, in response to various factors, evaluates the minute changes in the relative importance of the fluxes and gives an indication of the dynamic state of the lignification process. Such a phenomenon is difficult to assess by the study of the net weight accumulation of the different monomers since the quantity actually synthesized during the time of the experiment is low with respect to the products already accumulated. On the other hand, the possible regulatory devices may be studied better with a simplified model whose phenolic metabolism is specifically oriented towards lignification. For this purpose, we used xylem tissues isolated from stems which were proven to be autonomous for lignin synthesis (Gross, 1977).
Effect of Light on the Synthesis of S and G Units by Isolated Xylem Tissues Conditions of the assays. Preliminary data led to the conclusions that the appropriate medium must include products : - inhibiting the browning due to chemical or enzymic oxidations of endogenous substances which prevent the absorption and metabolism of the amino acid, maintaining the external pH between 7 and 8. Therefore, the standard medium contained: mercaptoethanol, Tris buffer and, in addition, Dextran T 40 which is used as an osmoticum (Honda et al., 1966). Under these conditions, the isolated xylem tissues maintained an active respiration for at least 36 h, as judged by polarography. No autoxidation was recorded since malonic acid fully inhibited the oxygen consumption. For longer periods of incubation, the tissues became progressively brown, therefore, the maximum duration of the experiments was limited to 30 h. Effects of light. When placed in this appropriated medium, isolated tissues from poplar stems remained able to incorporate labeled phenylalanine into S and G units of their lignins in the light (Fig. 1 a). In the dark, the incorporation of S was not (or slightly) altered but that of G dropped dramatically (Fig. 1b).
c. Grand et al.: Influence of Light on Lignin Synthesis
283
J
->
~'
,/
r
i
g r i
Fig. l a and b. Radioelutogram of syringyl (S) and guaiacyl (G) residues from lignins synthesized from [z4C]phenylalanine by isolated xylem tissues, a In the light; b in the dark. The two tracings correspond to a record of the radioactivity at two different senstivities (ration 1/10) (same remark for Fig. 3)
As shown in Fig. 2, the ability of xylem to incorporate G units decreased from the younger part of the plant to the older ones. Conversely, the incorporation of S units was higher in the older tissues. These d a t a are consistent with the measured changes in the ratios of sinapate/ferulate CoA-ligases (Grand and Ranjeva, in press) and more generally with the increased accumulation of S units as a function of age (Sarkanen and Hergert, 1971). In these tissues of different ages, the dark conditions led to the almost complete and specific inhibition of the incorporation of G units into the lignins.
Possible mechanisms of light action on the incorporation of G units into lignins. At the m o m e n t of isolation of the tissues, the xylem fractions contain all the enzymes leading from phenylalanine to the cinnamyl alcohols (Grand and Ranjeva, in press). In addition, with whatever treatment used, the uptake of the labeled amino acid was the same in the light and in the dark. Thus, after 20 h of incubation, 97% ( + 3) of the labeled amino acid were absorbed by the tissues independent of the presence of different chemicals (0.1 m M actidione; 20 m M CaClz; 3.3 m M isopropa-
284
C. G r a n d et al. : Influence of Light on Lignin Synthesis
the incorporated G units decreased by at least 24% when the metabolization was performed in the dark. In contrast, darkness had a relatively small effect on the incorporation of S units into the polymer. Thus, it seems that even in intact plants, the integration of monomeric unit of the guaiacyl type into lignins is light dependent. Nevertheless, the responses are not as sharp as in the case of isolated xylem tissues, presumably due to differences in the environment.
200L
~3
loo-!
L
~
I
n_ I
II
IIl D i s c u s s i o n
Fig. 2. Radioactivity from [l~C]phenylalanine incorporated into S [ZZ]and G g/~ residues from lignins synthesized by xylem tissues at different stages, in the dark (D) and in the light (L). Stage I : apical zone ] Stage II : intermediary zone I 5 internodes each Stage III: basal zone
nol) in the medium. Therefore, the differences due to the dark conditions may be related to changes in the internal environment. These changes may affect either the enzyme capacities (inactivation, degradation), the compartmentation of reactants, or the transport of intermediary products. In order to test these hypotheses, compounds acting on different phenomena were added to the incubation media and their effects on the integration of G unit were studied: 1. The addition of actidione to tissues maintained in the dark or in the light did not modify at all their responses, whereas, the synthesis of proteins (as judged by [3H]leucine incorporation) was completely inhibited (unpublished data). These results do not support the involvement of a newly formed protein molecule responsible for the loss in enzyme capacities in the dark. 2. The addition of isopropanol, a membrane perturber (Bassim and Pecket, 1975) to dark incubating tissues gave rise to essentially the same responses as for tissue maintained in the light (Fig. 3 a). 3. In contrast, when CaCI2, a membrane stabilizer (Bassim and Pecket, 1975) is supplied to tissues placed in the light, then the incorporation of the G residues into lignins drops and the light stimulation is overcome (Fig. 3b). Obviously, the dark conditions affect the permeability of some membrane barriers.
Effect of light on the monomeric composition of lignins from intact poplar stems. The possible effect of light on the incorporation of G units into lignins formed in situ by intact stems was investigated. The data from distinct experiments (Table 1) show that
The results presented in this paper demonstrate that light may specifically control the integration of G units into the lignin polymer. Since changes in the relative proportion of the monomers may occur in short periods, the lignification appears as a true dynamic phenomenon and not as a simple accumulation process. It may be pointed out that the photoreceptor, which is to be indentified, remains associated with the isolated tissues and functional during the experiments. Whatever the developmental stages of the tissue considered, the dark treatment results in the decrease of the G unit incorporation whereas that of S residues is only slightly affected. From a more general point of view, it is known that light controls the metabolism of polyphenols (mainly the soluble compounds) in various ways. Thus, Zucker (1972) established that the regulation of chlorogenic acid synthesis in potato tubers is greatly dependent on light which regulates both the synthesis and the degradation of phenylalanine ammonia-lyase, the enzyme supplying cinnamic acid. Besides this long-term process, light may also control either the rapid activation of phenolic enzymes (Wolosiuk et al., 1978) or the availability of substrates and cofactors (Pecket and Bassim, 1974) and therefore the rate of a particular enzymic step. The formation of anthocyanins, from phenylalanine, may thus be stimulated by illumination without changes in enzyme capacity but only by an increase in the amino acid concentration at the subcellular site of its deamination (Pecket and Bassim, 1974; Leschem et al., 1976). The situation described in this paper appears to be particularly complex: S and G units are synthesized via common intermediates, only the incorporation of the G units into the polymer is light dependent. Our results suggest that light may control the permeability of membrane barriers and regulate specifically the diffusion of some precursors of coniferyl -
-
C. Grand et al. : Influence of Light on Lignin Synthesis
285
Fig. 3a and b. Effects of different treatments on the incorporation of [14C]phenylalanine into S and G residues from lignin (stage II). a Isopropanol on tissues incubating in the dark; b CaC12 on tissues incubating in the light
Table I. Effect of light on the incorporation of labeled phenylalanine in the lignin of the xylem tissues from " i n t a c t " stems Experimental conditions
Relative radioactivity incorporated as a percentage of the assay performed in the iight S
G
1
light dark
100 97
100 66
2
light dark
100 113
100 76
3~
light dark
100 96
100 45
a The plants were placed in the dark during 48 h before the incorporation of [14C]phenylalanine
alcohol and/or the transport of the alcohol to its site of polymerization. Interestingly, Zouaghi and Rollin (1976) have shown that the vesicle-mediated transport of/3-fructosidase from the cytosol to the cell wall is phytochrome dependent. To explain the results obtained in the case of lignin formation, changes in membrane properties could be involved at different levels: 1. The first possibility is that all or part of the reactions leading to the different monomers are duplicated in distinct subcellular particles, perhaps the "vesicles" as speculated by Stafford (1974), differing by their membrane properties. Thus, the activation of ferulate and sinapate which is catalyzed by different enzymes (Ranjeva et al., 1976) may occur in different
286
"vesicles" whose permeability to the precursors could be modulated by light. 2. Another possibility is that the permeability of the plasmalemma for coniferyl alcohol or its ability to fuse with the structures involved in the transport of these monomers is light dependent, so that the availability of the G type monomer at the site of polymerization is also variable. All these hypotheses support the occurrence of "vesicles" or membrane barriers whose role in the lignification was underlined by the work of PickettHeaps (1968) and Robards (1968) who speculated on the role of such organelles derived from the endoplasmic reticulum in the transport of materials from the cytoplasm to the cell wall. Nevertheless, the biochemical content of these vesicles is not yet known, and the poplar system may give a good opportunity for their identification and for the study of these properties of these putative organelles. Concerning the reliability of the results with the physiology of the intact plant, the action of light on xylem tissues located inside the stems is not unlikely. Effectively, green chloroplasts were detected in the phloem (Jupin et al., 1975) and the xylem (Catesson, unpublished) of young branches from different trees. Nevertheless, the physiological significance of the specific photoregulation of G units remains open. Thanks are due to Dr. V. S. Butt (Oxford, U.K.) and Prof. K. Hahlbrock (Freiburg, F.R.G.) for their helpful suggestions and careful reading of the manuscript. This work was supported by the "D616gation G6n6rale ~ la Recherqhe Scientifique" (Action Mat~riau Bois) and by the "Centre National de la Recherche Scientifique" (Laboratoire Associ+ N ~ 241).
References Alibert, G., Boudet, A.: La lignification chez le Peuplier - II Estimation des flux m6taboliques impliqu6s dans la biosynth~se des monom+res des lignines. Physiol. V+g. 17, (1979) Bassim, T.A.H., Pecker, R.C.: The effect of membrane stabilizers
C. Grand et al. : Influence of Light on Lignin Synthesis on phytochrome - controlled anthocyanin biosynthesis in Brassica oleraceae. Phytochemistry 14, 731-733 (1975)
Grand, C., Ranjeva, R.: La lignification chez le Peuplier - III Variation du niveau d'activit6 d'enzymes impliqu~es dans la biosynth~se des monomeres en fonction du degr~ de lignification et de la nature des tissus. Physiol. V6g. (in press) Grisebach, H. : Biochemistry of lignification. Naturwissenschaften 64, 619-625 (1977) Gross, G.G. : The structure, biosynthesis, and degradation of wood. Recent Adv. Phytochem. 11, 141-184 (1977) Higuchi, T., Shimada, M., Nakatsubo, F., Tauahashi, M. : Differences in biosyntheses of guaiacyl and syringyl lignins in woods. Wood Sci. Technol. 11, 153 167 (1977) Honda, S.I., Hongladarom, T., Laties, G.G. : A new isolation medium for plant organelles. J. Exp. Bot. 17, 460-472 (1966) Jupin, H., Catesson, A.M., Giraud, G., Hauswirth, N.: Chloroplastes fi empilements granaires anormaux, appauvris en photosyst~me I dans le phlo6me de Robinia pseudoacacia et de Aeerpseudoplatanus. Z. Pflanzenphysiol. 75, 95-106 (1975) Leshem, Y., Zieslin, N., Halevi, A., Spelgelstein, H.: Abstract. IXth International Conference on Plant growth substances, Lausanne (Switzerland), pp. 228-230, 1976 Pecket, R.C., Bassim, T.A.H. : Mechanism of phytochrome action in the control of biosynthesis of anthocyanin in Brassica oleraeea. Phytochemistry 13, 815-821 (1974) Pickett-Heaps, J.D. : Xylem wall deposition. Radioautographic investigations using lignin precursors. Protoplasma 65, 181-205 (1968) Ranjeva, R., Boudet, A.M., Faggion, R.: Phenolic metabolism in petunia tissues. IV. Properties of p-coumarate: coeuzyme A ligase isoenzymes. Biochimie 58, 1255-1262 (1976) Robards, A.W. : On the ultrastructure of differentiating secondary xylem in willow. Protoplasma 65, 449-464 (1968) Rubery, P.H., Northcote, D.H.: Site of phenylalanine ammonialyase activity and synthesis of lignin during xylem differentiation. Nature 219, 1230-1234 (1968) Sarkanen, K.V., Hergert, H.L.: Classification and distribution. In: Lignins Occurence, Formation, Structure and Reactions, pp. 43-94, Sarkanen, K.V., Ludwig, C.H., eds. New York, London, Sydney, Toronto: Wiley Interscience 1971 Stafford, H.A.: The metabolism of aromatic compounds. Annu. Rev. Plant Physiol. 25, 459-486 (1974) Wolosiuk, R.A., Nishizawa, A.N., Buchanan, B.B.: Regulation of chloroplast phenytalanine ammonia lyase by the ferredoxin/ thioredoxin system. Plant Physiol. Supplement 61, p. 97 (1978) Zouaghi, M., Rollin, P.: Phytochrome control of fl-fructosidase activity in radish. Phytochemistry 15, 897-901 (1976) Zucker, M.H.: Light and enzymes. Annu. Rev. Plant Physiol. 23, 133-156 (1972) -
Received 30 January; accepted 15 April 1979
P l a n t a 9 by Springer-Verlag 1979
Photoregulation of the Incorporation of Guaiacyl Units into Lignins* Claude Grand, Raoul Ranjeva, Alain M. Boudet**, and Gilbert Alibert Centre de Physiologie V6g+tale, Laboratoire Associ6 au C.N.R.S. N ~ 241, 118, Route de Narbonne, F-31077 Toulouse C+dex, France
Abstract. When fed with [14C] phenylalanine in the light, xylem tissues isolated from poplar stems were able to incorporate part of the radioactivity into both the guaiacyl and the syringyl residues of lignins. In the dark, only syringyl units were integrated into the polymer whereas the guaiacyl residues remained unlabeled. When a membrane perturber (isopropanol) was added to the incubation mixture, the label was incorporated into the guaiacyl units either in the light or in the dark. Conversely, a membrane stabilizer (CaC12) prevented the labeling of the guaiacyl units even when the tissues were illuminated. These results suggest that light acts through the modification of membrane permeabilities, altering specifically the synthesis and the transport or the polymerization of guaiacyl-type units during the process of lignification.
Key words: Guaiacyl units - Light effect - Lignification - Membrane permeability - Populus.
Introduction
Lignin, the phenolic cell-wall component, results from the polymerization of cinnamyl alcohols which arise from phenylalanine via a well-characterized sequence of enzymic reactions (for review see Grisebach, 1977). The actual structure of this complex polymer is not fully understood but the relative proportion of its monomeric building blocks is generally used to identify a particular lignin (Sarkanen and Hergert, 1971). It has been shown, for example, that the ratios * This is paper No. 4 in a series. For paper No. 3 see Grand and Ranjeva, in press ** To whom reprint requests should be addressed Abbreviations: S = syringyt residue; G = guaiacyl residue
of syringyl to guaiacyl residues (S/G ratios) may vary according to various factors including the taxonomic position of the plant and the developmental stage of a given tissue (Sarkanen and Hergert, 1971). In this way, Higuchi and his coworkers (Higuchi et al., 1977) have demonstrated that the O-methyltransferase from angiosperms is able to catalyze the O-methylation of both caffeate and 5-hydroxyferulate, whereas, the enzyme from gymnosperms catalyzes only the formation of ferulate from caffeate. Thus, the absence of S residues in gymnosperms may be explained by their inability to form sinapic acid. Moreover, the results obtained by our group (Grand and Ranjeva, in press) suggest that the changes in S/G ratios are significantly correlated to that of sinapate/ferulate CoA-ligase levels, the enzymes which activate the hydroxycinnamic acids to their corresponding CoA esters. Consequently, the nature and the proportions of monomers incorporated into lignins seem to depend upon the substrate specificity of some particular enzymes and their absolute or relative levels. Such systems may account for the regulation of the qualitative composition of lignins by means of the specific enzymic equipment of the tissues. Nevertheless, lignification involves not only the biosynthesis of the monomers but also their transport and their polymerization. In this context, the respective role of each individual sequence on the control of the overall process within the same tissue (or cell) remains unclear. Some years ago, Rubery and Northcote (1968) established that xylem fractions isolated from different plants are able to synthesize labeled lignin from [14C]phenylalanine. More recently, we have designed a method allowing the simultaneous measurements of the quantity and the radioactivity of the individual aldehydes obtained by nitrobenzene oxidation of the polymer (Alibert and Boudet, 1979). These tools were
0032-0935/79/0146/0281/$01.20
282
adapted to poplar use and to determine the minute changes in the lignin composition in response to various treatments. The present paper deals with the demonstration of a light effect on the monomeric composition of lignin polymer.
Materials and Methods
Chemicals The chemicals used were analytical grade when available. They were purchased respectively from: Pharmacia fine chemicals, Uppsala Sweden: Dextran T 40 M.W. 40,000; Commissariat fi l'Energie Atomique, Saclay France: [U-a~C]phenylalanine (1,7.10 l~ Bq mmol- 1); Merck, Darmstadt Germany other chemicals.
Plant Material Populus x. euramericana (Dode) c.v. " I 214" plants were cultured in a growth chamber and the xylem tissues were isolated from stems by dissection with a scalpel (for details see Grand and Ranjeva, in press).
Isotope Studies Incorporation by intact stems. Stems from 3-month-old poplars (15 nodes) were cut under water and introduced in small test tubes containing water. The water was sucked off and replaced by 0.2 ml [U-l*C]phenylalanine per stem (3.8.104 Bq). After 10 min, uptake of labeled solution was almost complete and water (1 ml) was added periodically. Half an hour later the plants were transferred to a beaker containing water and allowed to metabolize for 12 h, in the dark or in the light (under 4,000 lx) at 26~ C. Each run was performed in five replicates. After the period of metabolization, the xylem tissues were isolated (Grand and Ranjeva, in press), frozen in liquid nitrogen, and lyophilized. Incorporation by xylem tissues. The incubations of t h e xylem tissues were performed in stoppered Erlenmeyer flasks (25 ml) on a gyratory shaker (100 rev min- 1) during 30 h, under 4,000 lx illumination at 26~ C, in a standard medium containing: 100mM Tris-HC1 buffer pH7.8; 25g 1-1 Dextran T40; 0.1 mM mercaptoethanol; 9.5.104 Bq [14C]phenylalanine. The final volume was 5 ml for 300 mg of fresh tissues.
Oxygen Measurements The measurements of oxygen consumption were performed with a Gilson polarograph (K-1 CC type). At the end of the experiments, the tissues were frozen with liquid nitrogen and lyophilized.
Analysis of the Lignin Monomers The extraction of lignins, their oxidation by alkaline nitrobenzene and the automated analysis and measurements of the radioactivity of the liberated aldehydes were performed as described elsewhere (Alibert and Boudet, 1979).
C. Grand et al. : Influence of Light on Lignin Synthesis Results
When fed with [l'~C]phenylalanine, young poplar stems are able to synthesize labeled S and G residues of lignins (Alibert and Boudet, 1979). The extent of the incorporation of each of these units, in a given time, measures the rate of the respective metabolic fluxes involved in the synthesis of each monomer (Alibert and Boudet, 1979). Consequently, the measurement of the changes in the ratios of radioactive S/G units, in response to various factors, evaluates the minute changes in the relative importance of the fluxes and gives an indication of the dynamic state of the lignification process. Such a phenomenon is difficult to assess by the study of the net weight accumulation of the different monomers since the quantity actually synthesized during the time of the experiment is low with respect to the products already accumulated. On the other hand, the possible regulatory devices may be studied better with a simplified model whose phenolic metabolism is specifically oriented towards lignification. For this purpose, we used xylem tissues isolated from stems which were proven to be autonomous for lignin synthesis (Gross, 1977).
Effect of Light on the Synthesis of S and G Units by Isolated Xylem Tissues Conditions of the assays. Preliminary data led to the conclusions that the appropriate medium must include products : - inhibiting the browning due to chemical or enzymic oxidations of endogenous substances which prevent the absorption and metabolism of the amino acid, maintaining the external pH between 7 and 8. Therefore, the standard medium contained: mercaptoethanol, Tris buffer and, in addition, Dextran T 40 which is used as an osmoticum (Honda et al., 1966). Under these conditions, the isolated xylem tissues maintained an active respiration for at least 36 h, as judged by polarography. No autoxidation was recorded since malonic acid fully inhibited the oxygen consumption. For longer periods of incubation, the tissues became progressively brown, therefore, the maximum duration of the experiments was limited to 30 h. Effects of light. When placed in this appropriated medium, isolated tissues from poplar stems remained able to incorporate labeled phenylalanine into S and G units of their lignins in the light (Fig. 1 a). In the dark, the incorporation of S was not (or slightly) altered but that of G dropped dramatically (Fig. 1b).
c. Grand et al.: Influence of Light on Lignin Synthesis
283
J
->
~'
,/
r
i
g r i
Fig. l a and b. Radioelutogram of syringyl (S) and guaiacyl (G) residues from lignins synthesized from [z4C]phenylalanine by isolated xylem tissues, a In the light; b in the dark. The two tracings correspond to a record of the radioactivity at two different senstivities (ration 1/10) (same remark for Fig. 3)
As shown in Fig. 2, the ability of xylem to incorporate G units decreased from the younger part of the plant to the older ones. Conversely, the incorporation of S units was higher in the older tissues. These d a t a are consistent with the measured changes in the ratios of sinapate/ferulate CoA-ligases (Grand and Ranjeva, in press) and more generally with the increased accumulation of S units as a function of age (Sarkanen and Hergert, 1971). In these tissues of different ages, the dark conditions led to the almost complete and specific inhibition of the incorporation of G units into the lignins.
Possible mechanisms of light action on the incorporation of G units into lignins. At the m o m e n t of isolation of the tissues, the xylem fractions contain all the enzymes leading from phenylalanine to the cinnamyl alcohols (Grand and Ranjeva, in press). In addition, with whatever treatment used, the uptake of the labeled amino acid was the same in the light and in the dark. Thus, after 20 h of incubation, 97% ( + 3) of the labeled amino acid were absorbed by the tissues independent of the presence of different chemicals (0.1 m M actidione; 20 m M CaClz; 3.3 m M isopropa-
284
C. G r a n d et al. : Influence of Light on Lignin Synthesis
the incorporated G units decreased by at least 24% when the metabolization was performed in the dark. In contrast, darkness had a relatively small effect on the incorporation of S units into the polymer. Thus, it seems that even in intact plants, the integration of monomeric unit of the guaiacyl type into lignins is light dependent. Nevertheless, the responses are not as sharp as in the case of isolated xylem tissues, presumably due to differences in the environment.
200L
~3
loo-!
L
~
I
n_ I
II
IIl D i s c u s s i o n
Fig. 2. Radioactivity from [l~C]phenylalanine incorporated into S [ZZ]and G g/~ residues from lignins synthesized by xylem tissues at different stages, in the dark (D) and in the light (L). Stage I : apical zone ] Stage II : intermediary zone I 5 internodes each Stage III: basal zone
nol) in the medium. Therefore, the differences due to the dark conditions may be related to changes in the internal environment. These changes may affect either the enzyme capacities (inactivation, degradation), the compartmentation of reactants, or the transport of intermediary products. In order to test these hypotheses, compounds acting on different phenomena were added to the incubation media and their effects on the integration of G unit were studied: 1. The addition of actidione to tissues maintained in the dark or in the light did not modify at all their responses, whereas, the synthesis of proteins (as judged by [3H]leucine incorporation) was completely inhibited (unpublished data). These results do not support the involvement of a newly formed protein molecule responsible for the loss in enzyme capacities in the dark. 2. The addition of isopropanol, a membrane perturber (Bassim and Pecket, 1975) to dark incubating tissues gave rise to essentially the same responses as for tissue maintained in the light (Fig. 3 a). 3. In contrast, when CaCI2, a membrane stabilizer (Bassim and Pecket, 1975) is supplied to tissues placed in the light, then the incorporation of the G residues into lignins drops and the light stimulation is overcome (Fig. 3b). Obviously, the dark conditions affect the permeability of some membrane barriers.
Effect of light on the monomeric composition of lignins from intact poplar stems. The possible effect of light on the incorporation of G units into lignins formed in situ by intact stems was investigated. The data from distinct experiments (Table 1) show that
The results presented in this paper demonstrate that light may specifically control the integration of G units into the lignin polymer. Since changes in the relative proportion of the monomers may occur in short periods, the lignification appears as a true dynamic phenomenon and not as a simple accumulation process. It may be pointed out that the photoreceptor, which is to be indentified, remains associated with the isolated tissues and functional during the experiments. Whatever the developmental stages of the tissue considered, the dark treatment results in the decrease of the G unit incorporation whereas that of S residues is only slightly affected. From a more general point of view, it is known that light controls the metabolism of polyphenols (mainly the soluble compounds) in various ways. Thus, Zucker (1972) established that the regulation of chlorogenic acid synthesis in potato tubers is greatly dependent on light which regulates both the synthesis and the degradation of phenylalanine ammonia-lyase, the enzyme supplying cinnamic acid. Besides this long-term process, light may also control either the rapid activation of phenolic enzymes (Wolosiuk et al., 1978) or the availability of substrates and cofactors (Pecket and Bassim, 1974) and therefore the rate of a particular enzymic step. The formation of anthocyanins, from phenylalanine, may thus be stimulated by illumination without changes in enzyme capacity but only by an increase in the amino acid concentration at the subcellular site of its deamination (Pecket and Bassim, 1974; Leschem et al., 1976). The situation described in this paper appears to be particularly complex: S and G units are synthesized via common intermediates, only the incorporation of the G units into the polymer is light dependent. Our results suggest that light may control the permeability of membrane barriers and regulate specifically the diffusion of some precursors of coniferyl -
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C. Grand et al. : Influence of Light on Lignin Synthesis
285
Fig. 3a and b. Effects of different treatments on the incorporation of [14C]phenylalanine into S and G residues from lignin (stage II). a Isopropanol on tissues incubating in the dark; b CaC12 on tissues incubating in the light
Table I. Effect of light on the incorporation of labeled phenylalanine in the lignin of the xylem tissues from " i n t a c t " stems Experimental conditions
Relative radioactivity incorporated as a percentage of the assay performed in the iight S
G
1
light dark
100 97
100 66
2
light dark
100 113
100 76
3~
light dark
100 96
100 45
a The plants were placed in the dark during 48 h before the incorporation of [14C]phenylalanine
alcohol and/or the transport of the alcohol to its site of polymerization. Interestingly, Zouaghi and Rollin (1976) have shown that the vesicle-mediated transport of/3-fructosidase from the cytosol to the cell wall is phytochrome dependent. To explain the results obtained in the case of lignin formation, changes in membrane properties could be involved at different levels: 1. The first possibility is that all or part of the reactions leading to the different monomers are duplicated in distinct subcellular particles, perhaps the "vesicles" as speculated by Stafford (1974), differing by their membrane properties. Thus, the activation of ferulate and sinapate which is catalyzed by different enzymes (Ranjeva et al., 1976) may occur in different
286
"vesicles" whose permeability to the precursors could be modulated by light. 2. Another possibility is that the permeability of the plasmalemma for coniferyl alcohol or its ability to fuse with the structures involved in the transport of these monomers is light dependent, so that the availability of the G type monomer at the site of polymerization is also variable. All these hypotheses support the occurrence of "vesicles" or membrane barriers whose role in the lignification was underlined by the work of PickettHeaps (1968) and Robards (1968) who speculated on the role of such organelles derived from the endoplasmic reticulum in the transport of materials from the cytoplasm to the cell wall. Nevertheless, the biochemical content of these vesicles is not yet known, and the poplar system may give a good opportunity for their identification and for the study of these properties of these putative organelles. Concerning the reliability of the results with the physiology of the intact plant, the action of light on xylem tissues located inside the stems is not unlikely. Effectively, green chloroplasts were detected in the phloem (Jupin et al., 1975) and the xylem (Catesson, unpublished) of young branches from different trees. Nevertheless, the physiological significance of the specific photoregulation of G units remains open. Thanks are due to Dr. V. S. Butt (Oxford, U.K.) and Prof. K. Hahlbrock (Freiburg, F.R.G.) for their helpful suggestions and careful reading of the manuscript. This work was supported by the "D616gation G6n6rale ~ la Recherqhe Scientifique" (Action Mat~riau Bois) and by the "Centre National de la Recherche Scientifique" (Laboratoire Associ+ N ~ 241).
References Alibert, G., Boudet, A.: La lignification chez le Peuplier - II Estimation des flux m6taboliques impliqu6s dans la biosynth~se des monom+res des lignines. Physiol. V+g. 17, (1979) Bassim, T.A.H., Pecker, R.C.: The effect of membrane stabilizers
C. Grand et al. : Influence of Light on Lignin Synthesis on phytochrome - controlled anthocyanin biosynthesis in Brassica oleraceae. Phytochemistry 14, 731-733 (1975)
Grand, C., Ranjeva, R.: La lignification chez le Peuplier - III Variation du niveau d'activit6 d'enzymes impliqu~es dans la biosynth~se des monomeres en fonction du degr~ de lignification et de la nature des tissus. Physiol. V6g. (in press) Grisebach, H. : Biochemistry of lignification. Naturwissenschaften 64, 619-625 (1977) Gross, G.G. : The structure, biosynthesis, and degradation of wood. Recent Adv. Phytochem. 11, 141-184 (1977) Higuchi, T., Shimada, M., Nakatsubo, F., Tauahashi, M. : Differences in biosyntheses of guaiacyl and syringyl lignins in woods. Wood Sci. Technol. 11, 153 167 (1977) Honda, S.I., Hongladarom, T., Laties, G.G. : A new isolation medium for plant organelles. J. Exp. Bot. 17, 460-472 (1966) Jupin, H., Catesson, A.M., Giraud, G., Hauswirth, N.: Chloroplastes fi empilements granaires anormaux, appauvris en photosyst~me I dans le phlo6me de Robinia pseudoacacia et de Aeerpseudoplatanus. Z. Pflanzenphysiol. 75, 95-106 (1975) Leshem, Y., Zieslin, N., Halevi, A., Spelgelstein, H.: Abstract. IXth International Conference on Plant growth substances, Lausanne (Switzerland), pp. 228-230, 1976 Pecket, R.C., Bassim, T.A.H. : Mechanism of phytochrome action in the control of biosynthesis of anthocyanin in Brassica oleraeea. Phytochemistry 13, 815-821 (1974) Pickett-Heaps, J.D. : Xylem wall deposition. Radioautographic investigations using lignin precursors. Protoplasma 65, 181-205 (1968) Ranjeva, R., Boudet, A.M., Faggion, R.: Phenolic metabolism in petunia tissues. IV. Properties of p-coumarate: coeuzyme A ligase isoenzymes. Biochimie 58, 1255-1262 (1976) Robards, A.W. : On the ultrastructure of differentiating secondary xylem in willow. Protoplasma 65, 449-464 (1968) Rubery, P.H., Northcote, D.H.: Site of phenylalanine ammonialyase activity and synthesis of lignin during xylem differentiation. Nature 219, 1230-1234 (1968) Sarkanen, K.V., Hergert, H.L.: Classification and distribution. In: Lignins Occurence, Formation, Structure and Reactions, pp. 43-94, Sarkanen, K.V., Ludwig, C.H., eds. New York, London, Sydney, Toronto: Wiley Interscience 1971 Stafford, H.A.: The metabolism of aromatic compounds. Annu. Rev. Plant Physiol. 25, 459-486 (1974) Wolosiuk, R.A., Nishizawa, A.N., Buchanan, B.B.: Regulation of chloroplast phenytalanine ammonia lyase by the ferredoxin/ thioredoxin system. Plant Physiol. Supplement 61, p. 97 (1978) Zouaghi, M., Rollin, P.: Phytochrome control of fl-fructosidase activity in radish. Phytochemistry 15, 897-901 (1976) Zucker, M.H.: Light and enzymes. Annu. Rev. Plant Physiol. 23, 133-156 (1972) -
Received 30 January; accepted 15 April 1979
