INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

4-Coumarate:coenzyme A (CoA) ligase (4CL) mediates activation of hydroxycinnamic acids 4-(p)-coumaric acid (PA), caffeic acid (CA), ferulic acid (FA), 5-hydroxyferulic acid (5HFA), and sinapic acid (SA) into the high-energy intermediates used for biosynthesis of lignin, flavonoids, and various other protective, attractant, and signaling metabolites (Hahlbrock and Scheel, 1989; Dixon and Paiva, 1995; Higuchi, 1997; Whetten et al., 1998). Multiple 4CL isoforms with differential in vitro substrate specificities have been reported in several species (Knobloch and Hahlbrock, 1975; Ehlting et al., 1999), including aspen (Populus tremuloides Michx.; Hu et al., 1998), and these isoforms have been proposed to control the relative abundance of flavonoids and various lignin precursors (monolignols) during structural, protective, and reproductive development (Ranjeva et al., 1976; Knobloch and Hahlbrock, 1977; Grand et al., 1983). Wounding, UV light, and elicitors increase transcript abundance of different 4CL isoforms (Uhlmann and Ebel, 1993; Ehlting et al., 1999), reinforcing the model that there are distinct associations between isoforms and specific metabolic activities. However, all 4CLs subjected to in vitro kinetic analysis so far exhibit similar broad substrate specificity, generally favoring PA, followed by FA and CA (Ranjeva et al., 1976; Grand et al., 1983; Uhlmann and Ebel, 1993; Hu et al., 1998; Ehlting et al., 1999). Site-directed mutagenesis has recently been applied to elucidate the mechanism and evolution of 4CL catalysis (Stuible et al., 2000). Questions remain as to why certain species appear to possess multiple, apparently similar, 4CL isoforms, whereas others such as potato (Solanum tuberosum) and pine (Pinus taeda) do not (Becker-André et al., 1991; Voo et al., 1995; Zhang and Chiang, 1997). If the possibility is accepted that isoforms expressed in specialized tissues of certain species have specific and distinct roles, then it is also possible that those isoforms are more specialized and differ more strikingly and with greater consequences in vivo than would be concluded from the in vitro studies to date.

Analysis of transgenic tobacco (Nicotiana tabacum) and aspen plants revealed strikingly unequal effects of 4CL down-regulation on in vivo utilization of its three most preferred substrates, PA, CA, and FA (Kajita et al., 1997; Hu et al., 1999). In particular, antisense suppression of Pt4CL1 in aspen led to sharp increases in wall-bound PA and FA, but not CA in xylem tissue where Pt4CL1 predominates (Hu et al., 1998, 1999). A number of factors could contribute to this, but one that we were in a position to investigate using an in vitro approach was the possibility that CA is the preferred in vivo substrate of 4CL1 in lignifying tissue due to substrate interactions. In non-lignifying tissues, such as leaves, where 4CL2 is expressed (Hu et al., 1998), down-regulation of Pt4CL1 reduced wall-bound PA and FA, but not CA (S.A. Harding, V.L. Chiang, and C.-J. Tsai, unpublished data), suggesting distinct regulation of hydroxycinnamic activation in xylem and leaves. Therefore, we conducted the present work focusing on mixed-substrate enzyme kinetics of Pt4CL1 and Pt4CL2 proteins and their mRNA localization patterns in developing tissues of quaking aspen. 4CL from a progenitor gymnosperm, loblolly pine, was included in enzyme assay experiments to determine whether 4CLs from species like pine with only a single, apparently all-purpose 4CL, differ catalytically under mixed-substrate conditions from 4CLs of species expressing distinct, and perhaps metabolically more specialized, 4CL proteins.

Our results indicate that CA acts as a strong competitive inhibitor with respect to other hydroxycinnamic acids, including PA, and thus can direct the activity of aspen 4CL1 toward lignin biosynthesis in maturing xylem. By a similar competitive mechanism, PA is the preferred substrate of aspen 4CL2, the predominantly expressed isoform in epidermis and expanding leaves. Both isoforms are coexpressed in rapidly dividing cells of pre-expanding leaves. We also report a kinetic distinction between aspen and pine 4CL and we discuss the significance of this finding in terms of developmental and adaptive features that distinguish woody gymnosperms and angiosperms.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Kinetic Analysis of Recombinant Aspen 4CL Proteins

To identify 4CL product hydroxycinnamoyl-CoA esters unequivocally, and to develop a means for measuring 4CL substrate preference in assays of mixed phenolic substrates, we used HPLC-UV/mass spectrometry (MS) to quantify product CoA esters directly from aqueous reaction mixtures. The approach represents an improvement over UV spectrophotometric procedures (Stöckigt and Zenk, 1975), which cannot be used to discriminate between these esters in mixed-substrate assays. In agreement with our UV spectrophotometric results (Hu et al., 1998), V_max of recombinant Pt4CL1 and Pt4CL2 was highest when PA was used as the substrate, as shown in Table I. Further kinetic analysis revealed that Pt4CL1 activated CA and PA with similar catalytic efficiency (V_max/K_m; Table I). This is consistent with characteristics of lignin-related 4CLs in other species with multiple, kinetically distinct isoforms (Knobloch and Hahlbrock, 1975; Ehlting et al., 1999). Pt4CL2 exhibited a much higher catalytic efficiency for PA than for CA, due primarily to a very low K_m for PA (Table I). Other 4CLs with low apparent K_m for PA are strongly induced by UV light treatments that also stimulate the biosynthesis of UV-absorbing flavonoids (Knobloch and Hahlbrock, 1977; Ehlting et al., 1999). FA appears to be a relatively poor substrate for recombinant Pt4CL1 and Pt4CL2.

Kinetic data from single-substrate assays can illustrate apparent catalytic preferences of 4CLs, but they do not consider multi-substrate interactions that could affect activity. We conducted mixed-substrate 4CL assays to better reflect 4CL activity in a variable cellular milieu of several cinnamic acid (CiA) derivatives. Preliminary enzyme assays with mixtures of six (CiA + PA + CA + FA + 5HFA + SA), five (PA + CA + FA + 5HFA + SA), or three (PA + CA + FA) substrates revealed distinct utilization and inhibition profiles for the two aspen 4CL isoforms. CiA, 5HFA, and SA yielded no product in mixed-substrate reactions and were not used further. In all mixed-substrate assays, CA-CoA was the predominant product of Pt4CL1, as PA-CoA was of Pt4CL2 (data not shown). We then conducted experiments to analyze two-substrate interactions between PA/CA, CA/FA, and PA/FA. With respect to both Pt4CL isoforms, all two-substrate interactions were of the competitive inhibition type, as illustrated in Figure 1 and summarized in Table II. CA, the preferred substrate of Pt4CL1, inhibited utilization of PA and FA with a K_i of approximately 4 µM, whereas PA and FA were weakly inhibitory with respect to CA (K_i  120 µM). Together, the single- and mixed-substrate kinetic data indicated that Pt4CL1 can convert PA and CA with equal catalytic efficiency, but that it discriminates against PA in the presence of CA.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Representative plots of inhibition kinetics of recombinant Pt4CL1 and Pt4CL2. Lineweaver-Burk plots of 1/V versus 1/[S] in the presence of different fixed inhibitor concentrations as indicated. A, Competitive inhibition effects of CA on 4CL1 activation of PA in mixed-substrate assays. B, Competitive inhibition effects of PA on 4CL2 activation of CA. Insets show replots of apparent Km (Km app) versus the corresponding inhibitor concentration used to calculate Ki values summarized in Table II. Ki is determined as the negative intercept on the x axis.

In mixed-substrate assays with Pt4CL2, PA was a strong competitive inhibitor with respect to CA (K_i = 4.3 µM), whereas K_i of CA with respect to PA, though low at 12.4 µM, was still nearly four times K_m PA (Tables I and II). PA and CA strongly inhibited Pt4CL2 utilization of FA with K_i of 2.8 and 6.6 µM, respectively (Table II). Thus, in mixed- as in single-substrate assays, FA was a relatively poor 4CL substrate. These results indicate a mechanism by which different 4CL proteins with similar broad substrate specificities have the potential to become strongly and differentially directed as a function of substrate pool composition. Variation in relative abundance of these kinetically complimentary isoforms could additionally modulate hydroxycinnamate activation for spatially or temporally localized metabolic needs. To better evaluate this possibility, we analyzed the distribution of Pt4CL1 and Pt4CL2 within developing tissues by measuring extractable 4CL activity, in situ hybridization patterns, and promoter::-glucuronidase (GUS) activity.

4CL Activities in Aspen Tissue Extracts

4CL activity was assayed using three-substrate mixtures, and as was the case for recombinant proteins, tissue extractable 4CLs exhibited signature, highly discriminate substrate utilization patterns. Under subsaturating, mixed-substrate concentrations of 10 or 25 µM CA, PA, and FA, Pt4CL1 exhibited the expected preference for CA over PA and FA, whereas Pt4CL2 exhibited the expected preference for PA, and did not convert FA at all (Fig. 2, A and B). Aspen xylem extracts exhibited a strong preference for CA (Fig. 2D), whereas leaf and apex extracts favored PA (Fig. 2E; data not shown). Aspen xylem extracts yielded a CA:PA:FA product ratio of 1:0.24:0.22 and 1:0.16:0.12 with 10 and 25 µM substrate mixtures, respectively (Fig. 2D), compared with 1:0.24:0.10 and 1:0.18:0.05 for Pt4CL1 (Fig. 2A; n = 3 assays for each ratio; all ratios were normalized with respect to CA-CoA = 1). The result from xylem extracts was interpreted to indicate that 4CL1 synthesized in planta and known to predominate in xylem (Hu et al., 1998) behaves like recombinant 4CL1 catalytically, and, therefore, was not kinetically modified by post-translational or other mechanisms. Analysis of extracts from furled, 2nd internode leaves in the presence of 10 and 25 µM substrates resulted in ratios of CA:PA products of 1:14.1 and 1:8.5, respectively (Fig. 2E), compared with 1:7 for Pt4CL2. The data appeared to indicate that 4CL2 was the predominant isoform expressed in young leaves. The trend was similar for apical bud extracts (not shown), although the proportion of PA product was lower than in leaves (1:4.7 and 1:3.3 with 10 or 25 µM substrates, respectively).

View larger version (32K): [in this window] [in a new window]   Figure 2.   Representative chromatograms of CoA products from mixed-substrate 4CL assays. CoA thioester products from enzymatic reactions using substrate mixtures of equal molar PA, CA, and FA were separated by HPLC. Shown are the extracted ion chromatograms (m/z 930 for CA-CoA, m/z 914 for PA-CoA, and m/z 944 for FA-CoA) resulting from assays containing 10 µM (solid lines) or 25 µM (dashed lines) substrate mixtures. Enzyme assays were conducted using recombinant aspen 4CL1 (A), recombinant aspen 4CL2 (B), recombinant loblolly pine 4CL (C), aspen xylem crude extracts (D), aspen leaf crude extracts (E), or loblolly pine xylem proteins (F).

Loblolly pine 4CL was characterized because it is considered a progenitor of angiosperm 4CLs and it represents the only known 4CL activity in pine (Voo et al., 1995; Zhang and Chiang, 1997). Unlike aspen 4CLs, recombinant pine 4CL efficiently utilized all three substrates with similar preference in mixed-substrate assays, yielding a CA:PA:FA product ratio of 1:1.4:1 and 1:1.1:0.9 with 10 and 25 µM substrates, respectively (Fig. 2C). The trend was very similar when assays were conducted with individual substrates at 10 and 25 µM (1:1.4:1 and 1:1.2:0.9, respectively; data not shown). 4CL in pine xylem extracts exhibited similar substrate preference in mixed-substrate assays as well (1:1.4:1.6; Fig. 2F). All experiments were repeated with similar results using independently prepared recombinant or plant proteins.

In Situ Hybridization Analysis

Although we concluded from the mixed-substrate assays that 4CL1 and 4CL2 predominated in xylem and shoot apex/leaves, respectively, that conclusion is preliminary in part because it depends on the assumption that plant extractable 4CLs are kinetically equivalent to the recombinant proteins. Protein modification or competing activities can interfere with measurement of enzyme activity in tissue extracts, potentially causing isoform distribution patterns based on similarity with recombinant enzyme activity to be misleading. In the case of transcriptionally regulated genes like 4CL (Douglas, 1996; Ehlting et al., 1999), transcript distribution would be expected to correlate with isoform activity pattern. A high resolution in situ analysis of these genes in developing tissues was performed to confirm the general distribution of 4CL isoforms derived from enzyme activity, as well as to reveal any discrepancies between enzyme activity and gene expression patterns. Several digoxygenin (DIG)-labeled RNA probes and hybridization conditions were tested to visualize gene-specific expression patterns for the 4CL transcripts. During preliminary in situ hybridization experiments, hydrolyzed probes corresponding to the full-length coding sequence, and nonhydrolyzed probes corresponding to 250- to 350-bp sequences of the 3'- or 5'-coding or -noncoding regions of Pt4CL1 and Pt4CL2 tended to produce no signal or indistinguishable patterns, depending on hybridization stringency. Nonhydrolyzed probes corresponding to the full-length coding sequence of the respective genes were successfully used to analyze the expression patterns of Pt4CL1 and Pt4CL2 in various developing organs of 20-week-old aspen trees. The full-length antisense probes hybridized specifically and exclusively with their respective sense transcripts in northern-blot experiments conducted under in situ hybridization conditions (not shown). Within the apical cylinder, 4CL1 was highly expressed in protoxylem, followed by protophloem and procambium, whereas 4CL2 is poorly expressed in those cells (Fig. 3, A and B). 4CL1 also exhibited strong expression in adaxial cells (arrow) and protoxylem of bud scales to the right of the apical cylinder (Fig. 3A). In the midvein of leaves emerging at the 2nd internode, 4CL1 was primarily localized to xylem, with very little expression seen in cambium and phloem, whereas 4CL2 was expressed primarily in the cambial zone (Fig. 3, C and D). Expression of both genes was strong throughout lamina of newly emerged leaves at the apex (not shown) and remained strong in the furled lamina of the 2nd internode leaves (Fig. 3, E and F), but diminished in unfurled lamina near the midvein (Fig. 3, C and D, arrows). Note the expression of 4CL1 in xylem vessel elements of fine veins (Fig. 3E). Despite the observation that 4CL1 was apparently significantly expressed in leaf and apex tissues, very little 4CL1 product, CA-CoA, was detected in the enzyme assays (Fig. 2E). Instead, PA-CoA comprised 75% to 80% of the Co-A product of apex assays (not shown) and >90% of the product seen in leaf assays. It is possible that competing or CoA-utilizing activities were present in leaf extracts. However, tests of the effect of active and boiled leaf extracts on PA-, CA-, and FA-CoA compounds did not reveal any differential stability of these esters in leaf extracts (not shown).

View larger version (174K): [in this window] [in a new window]   Figure 3.   In situ localization of Pt4CL1 and Pt4CL2 mRNAs in aspen shoot tips. Transverse shoot tip sections (10-µm thickness) were hybridized with DIG-labeled antisense 4CL1 (A, C, and E) or 4CL2 (B, D, and F) RNA probes and were photographed in bright field. Shown are basal section of shoot apex (A and B), midvein (C and D), and curled lamina of leaf emerging at the 2nd internode (E and F). The arrow in A indicates lignifying adaxial cells, and in C and D, it indicates leaf lamina near the midvein. Insets in E and F are negative controls hybridized with DIG-labeled sense 4CL1 or 4CL2 probes, respectively. Scale bar = 200 µm (A-F). bs, Bud scales; cz, cambial zone; p, phloem; x, xylem.

Stem cross-sections were used to analyze 4CL1 and 4CL2 expression during differentiation of secondary vascular structures between internodes 3 and 10 (Fig. 4). At the 3rd internode, vascular elements appeared as discrete clusters containing a few small xylem vessels. 4CL1, but not 4CL2, exhibited very strong localized expression in the metaxylem, whereas both genes were well expressed in the cambial zone, with expression tapering off in developing phloem (Fig. 4, A and B). 4CL2 was most conspicuous in epidermal cells of the upper (3rd-6th) internodes (Fig. 4, B and D), whereas both genes were moderately well expressed in rapidly dividing cortical cells, giving rise to epidermal protrusions that would later develop into lenticels (Fig. 4, C and D, arrows). At internode 10, 4CL1 became strongly expressed in phloem fibers while it remained highly expressed in ray parenchyma and lignifying cells of maturing xylem, as well as the cambial zone (Fig. 4E). Expression of 4CL2 remained evident in the cambium, with weak expression in some areas of the xylem and phloem, excluding lignifying cells where 4CL1 was strongly expressed (Fig. 4F). These expression patterns were sustained through the 20th internode (not shown).

View larger version (180K): [in this window] [in a new window]   Figure 4.   In situ localization of Pt4CL1 and Pt4CL2 mRNAs in aspen stem. Transverse stem sections (10-µm thickness) were hybridized with DIG-labeled antisense 4CL1 (A, C, and E) or 4CL2 (B, D, and F) RNA probes and were photographed in bright field. Shown are the 3rd internode (A and B), 6th internode (C and D), and 10th internode (E and F). Scale bar = 100 µm (A-F). cz, Cambial zone; e, epidermis; p, phloem; pf, phloem fiber; x, xylem.

Promoter Activity

The in situ hybridization results revealed certain overlaps in the patterns of 4CL1 and 4CL2 gene expression not predicted by expressing Pt4CL promoter::GUS constructs in tobacco (Hu et al., 1998). Although 4CL1 and 4CL2 promoter-driven GUS staining was strictly localized to tobacco xylem and epidermis, respectively (Hu et al., 1998), 4CL1 and 4CL2 transcripts were expressed together in the cambial zones, phloem, and epidermis of aspen stem (Fig. 4). In part to determine whether the apparent discrepancy reveals aspen-specific developmental controls, the 1-kb Pt4CL1and 1.2-kb Pt4CL2 promoters used in the tobacco study were used to measure promoter activity in aspen stems (Fig. 5). The 1-kb 4CL1 promoter drove GUS expression mainly in developing xylem of aspen, but faint GUS staining was also found in the cambial zone and in certain areas of the phloem, phloem fibers, and epidermis (Fig. 5, A-C). In contrast to 4CL1, the 1.2-kb 4CL2 promoter regulated GUS activity following a steep developmental gradient. At the 3rd internode, 4CL2 promoter activity was conspicuous in the epidermis, cortex, phloem, and cambial zone (Fig. 5D), but activity in the phloem and cambial zone diminished at the 5th internode, becoming limited to the interfascicular regions (Fig. 5E). In maturing internodes with more advanced secondary development, the 4CL2 promoter was active in the epidermis, with weak cambial zone and phloem activity (Fig. 5F). It is interesting that tobacco plants expressing Pt4CL1 or Pt4CL2 promoter::GUS constructs exhibited no detectable GUS activity in the phloem or cambial zones (Hu et al., 1998), a possible indication of differential transcriptional regulation of 4CL genes in woody and herbaceous species. Discrepancies of promoter activity in tobacco and aspen have also been observed with the caffeate O-methyltransferase (COMT, also known as 5-hydroxyconiferyl aldehyde O-methyltransferase [AldOMT], C.J. Tsai and V.L. Chiang, unpublished data) and Phe ammonia-lyase promoters (Gray-Mitsumune et al., 1999).

View larger version (105K): [in this window] [in a new window]   Figure 5.   Histochemical localization of GUS activity driven by Pt4CL1 and Pt4CL2 promoters. GUS activity under the control of Pt4CL1 (A, B, and C) or Pt4CL2 (D, E, and F) promoters were analyzed using free-hand sections from the 3rd (A and D), 5th (B and E), and 10th (C and F) internodes of 5-month-old transgenic aspen. Scale bar = 200 µm. cz, Cambial zone; e, epidermis; if, interfascicular region; p, phloem; pf, phloem fibers; x, xylem. A similar pattern was observed in several independent transgenic lines.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Pine and Aspen 4CLs Exhibit Differential Selectivity in Substrate Mixtures

We report that two kinetically distinct 4CLs are constitutively expressed at readily detectable levels in aspen. The aspen isoforms activated several hydroxycinnamate substrates (Table I), a common characteristic among 4CL proteins (Hu et al., 1998 and refs. therein), but we were able to show in addition that their metabolic specificity was sharply and differentially modulated by substrate-substrate interactions at the active site (competitive inhibition; Fig. 1; Table II). These isoforms possess complimentary substrate-preference and kinetic properties (Tables I and II) that could modulate the balance of PA-derived and CA-derived intermediates in rapidly dividing or undifferentiated cells where they are coexpressed (developing leaves and stem lenticels and vascular cambium). When expressed as the only isoform as in certain differentiated cells, each 4CL may become, on the basis of our in vitro assay data, a channel for utilization of only one substrate. This could favor exclusive support of a single phenylpropanoid branchway, despite the presence of several 4CL substrates. PA-derived flavonoid biosynthesis may be an important activity in epidermal cells where 4CL2 is predominantly expressed. CA-derived monolignol biosynthesis appears to be supported by xylem-expressed 4CL1 (Table II; Fig. 2A), a hypothesis that is consistent with our recent findings regarding the S lignin biosynthetic pathway (see below).

Previous results from single-substrate kinetics (Lee and Douglas, 1996; Allina et al., 1998; Hu et al., 1998; Ehlting et al., 1999) offered no models for how a single or multiple 4CLs would control hydroxycinnamate distribution in a mixed-substrate environment. The present data suggest that exposure of 4CL(s) to different substrate mixtures could constitute a simple mechanism for CoA activation and distribution of hydroxycinnamates. It is interesting that the 4CL of pine, a gymnosperm progenitor of angiosperms, lacks the sensitivity to substrate pool composition exhibited by each aspen isoform. One example of the physiological relevance of this may be the response of pine to everyday mechanical stresses like wind and leaning. Compression wood is formed in conifers during the response to such stresses and involves the synthesis of significant amounts of p-hydroxyphenyl (H) lignin subunits derived from PA (see Timell, 1986). Therefore, it is possible that the ability of pine 4CL to equally utilize PA, CA, and FA facilitates this adaptive response within lignifying tissues. It is also possible that pine 4CL possesses such metabolic flexibility to compensate for the absence of additional 4CL isoforms (Voo et al., 1995; Zhang et al., 1997; pine expressed sequence tag database http://www.cbc.umn.edu/ResearchProjects/Pine/DOE.pine/index.html). We have conducted in situ hybridization experiments with the pine 4CL showing strong expression in lignifying xylem cells and around tannin storage cells and resin ducts in xylem and phloem (S.A. Harding and C.J. Tsai, unpublished data). Tannins are derived from flavonoids, indicating that 4CL expressed near tannin-storage cells may utilize PA. In contrast, Pt4CL1 expression in xylem and phloem of aspen is more localized near lignifying cells. Taken together, the kinetic and in situ data indicate that the broad substrate specificity of the single 4CL of pine is associated with a broad range of functions in the stem. At the same time, we may ask whether the lignification process of aspen, or of hardwoods in general, requires a 4CL with special substrate utilizing properties.

Pt4CL1 Becomes Recruited for S-G Lignin Biosynthesis: A Hypothesis

Lignin of gymnosperms like pine is largely composed of G subunits, whereas lignin of most angiosperms, including aspen, contains significant levels of S subunits (50%) as well (see Higuchi, 1997). During aspen stem development, G lignin is deposited within the primary growth internodes, whereas S-G lignin is deposited in maturing xylem and phloem fibers of secondary growth internodes (Osakabe et al., 1996; Li et al., 2001). Based on our kinetic, in situ, and promoter analyses, aspen 4CL1 is strictly coupled with lignification during secondary vascular development. According to the model we will discuss, 4CL1 is catalytically predisposed to become part of an efficient scavenging mechanism to support S-G lignification at the expense of other pathways competing for hydroxycinnamic acids. New findings have revealed a pathway for S lignin biosynthesis in angiosperms that branches away from the pathway for G lignin biosynthesis at coniferaldehyde, and that operates parallel to and independently of the G lignin pathway (Fig. 6; Osakabe et al., 1999; Li et al., 2000, 2001). It has also been concluded from in vitro experiments that intermediates of the S lignin pathway can modulate the upstream methylation and hydroxylation of CA and FA (Fig. 6, shown in gray; Osakabe et al., 1999; Li et al., 2000). We now suggest that 4CL activity also is modulated (indirectly) by S pathway activity. During S lignin biosynthesis, methylation of CA to FA by AldOMT (COMT) can be blocked by competitive inhibition from 5-hydroxyconiferaldehyde, an S lignin precursor (Fig. 6; Li et al., 2000). Our in vitro analysis of 4CL argues that any increase in levels of CA due to a partial block of its methylation to FA would competitively inhibit 4CL1's utilization of PA (Fig. 1; Table II). This negative feedback mechanism is not associated with the biosynthesis of G units and would not be expected to occur in pine or in the primary growth internodes of angiosperms where G lignin predominates. In maturing angiosperm xylem, the result is a 4CL1-mediated pathway for the biosynthesis of S and G monolignols derived exclusively from CA (Fig. 6) instead of from PA, CA, FA, and perhaps 5HFA and SA, as traditionally described (see Higuchi, 1997).

View larger version (23K): [in this window] [in a new window]   Figure 6.   Proposed biosynthetic pathway for the formation of monolignols and flavonoids in angiosperms. PAL, Phe ammonia-lyase; C4H, cinnamate 4-hydroxylase; C3H, 4-coumarate 3-hydroxylase; COMT, caffeate O-methyltransferase; F5H, ferulate 5-hydroxylase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAld5H, coniferyl aldehyde 5-hydroxylase; AldOMT, 5-hydroxyconiferyl aldehyde O-methyltransferase; CAD, cinnamyl alcohol dehydrogenase; SAD, sinapyl alcohol dehydrogenase. 4CL substrates and products no longer considered to be involved in monolignol biosynthesis are shown in gray.

Furthermore, 4CL1 activation of PA for flavonoid biosynthesis would become restricted as would the synthesis of FA used in competing pathways for cell wall cross-linking and for conversion to 5HFA and SA. One interesting suggestion from this is that aspen 4CL1 is a specialized isoform able to reinforce a mechanism that may be specific to angiosperm trees, in part, for conserving metabolic capitol for S-G lignin biosynthesis. S subunits possess one additional methyl group than G subunits and they are bioenergetically more costly to synthesize. Timely metabolic conservation through the diversion of CA to lignin synthesis may be important in regions of maturing phloem and xylem where, over a short period, certain structural and conducting cells undergo autolysis during and immediately following crucial secondary wall lignification (Groover and Jones, 1999).

4CL Activities in Lignifying Versus Non-Lignifying Tissues

Although associated with lignifying cells in vascular tissues (Hu et al., 1998), 4CL1 is also expressed in leaf lamina where little lignification occurs (Fig. 3E). To gain insight related to a possible role for 4CL1 in leaf lamina, free PA, CA, and FA in methanolic extracts of leaf and xylem tissue were analyzed by HPLC-UV/MS. Our analysis revealed free PA (approximately 140 µM), as well as readily detectable levels of the flavonoid precursor, naringenin (based on retention time, MS, and UV signatures), in leaf but not in xylem tissue. Free PA at a concentration of 140 µM in developing leaves is enough to saturate leaf 4CL1 or 4CL2 activity, regardless of CA (see K_i values in Table II). 4CL1, as well as 4CL2, thus may function to support flavonoid biosynthesis in developing leaf tissue. It is also possible that 4CL1 expressed in leaves is important for esterification of PA into cell walls. The predominant phenolic acid found esterified into cell walls in developing leaves was PA, at 7 µmol g¹ (S.A. Harding, V.L. Chiang, and C.J. Tsai, unpublished data). In contrast, PA is not the predominant phenolic acid esterified into cell walls of stem xylem (0.2 µmol g¹; Hu et al., 1999). In support of the involvement of 4CL in leaf tissue, antisense down-regulation of 4CL1 in aspen (Hu et al., 1999) resulted in a >50% reduction in the levels of wall-bound PA in leaves (S.A. Harding, V.L. Chiang, and C.J. Tsai, unpublished data). The involvement of 4CL1 extends our previous conclusion based on northern and promoter analysis that it is primarily Pt4CL2 that regulates flavonoid biosynthesis in developing tissues (Hu et al., 1998). It is possible, however, that 4CL2 regulates flavonoid biosynthesis, whereas 4CL1 channels PA toward wall esterification. It is interesting that 4CL1 is expressed in adaxial cells of apical bud scales (modified leaves), but 4CL2 is not (Fig. 3A, arrow). These cells become lignified as revealed by UV fluorescence microscopy (not shown). We have only observed coexpression of 4CL1 and 4CL2 in non-lignifying cells, and perhaps it is the coexpression of 4CL2 in non-lignifying cells that prevents 4CL1 from mediating lignin biosynthesis.

We have noted that the 4CL1 product CA-CoA, predominant in in vitro assays of xylem extracts, was a relatively minor product in assays of leaf extracts (Fig. 2, D and E). According to the in situ hybridization results, 4CL1 and 4CL2 were abundantly expressed in the leaf tissues used in the enzyme assays, but the product profile did not show the expected accumulation of 4CL1 product, CA-CoA. Why 4CL1 in leaf extracts activates CA poorly under mixed-substrate assay conditions where CA was present at >K_i levels with respect to PA raises interesting questions. When purified recombinant 4CL1 and 4CL2 were mixed for assay, the product profile reflected the signature activities of both enzymes (not shown). Although the areas of the lamina exhibiting strong coexpression of these genes were not separated from other parts of the leaf for enzyme assay, the overall expression of either 4CL was not strong in other parts of these leaves, and thus, only a dilutive effect should have been observed. As mentioned in the "Results," we found no activity in leaf extracts that differentially modified/degraded the various CoA products of 4CL under our assay conditions. More difficult to eliminate is the possibility that 4CL substrates were differentially converted by competing activities such as thioesterases. However, many candidate cosubstrates for such activities were removed by desalting, somewhat limiting the potential impact of competing activities on 4CL activity. Although we cannot exclude the possibility that other as-yet-unidentified 4CL isoforms could contribute to the result we observed, our data also argues that 4CL1 in leaf extracts is somehow altered in terms of its substrate preference, perhaps through a mechanism involving coexpressed 4CL2.

Overall, our data indicate that the high concentration of free PA in developing leaf lamina recruited 4CL primarily for processes other than lignin biosynthesis, such as flavonoid biosynthesis and cell wall esterification. The high level of free PA in apical leaves would not favor activation of CA, and this is in line with the low demand of lignification in leaves. Interesting questions remain with regard to the role of 4CL2 in leaf lamina of aspen. Because Pt4CL2 is most abundant in tissues such as developing leaves where it is coexpressed with Pt4CL1, determination of isoform roles during growth and defense in those tissues will be challenging. We presently are investigating the effects of 4CL1 down-regulation on leaf 4CL activity to address some of these issues. In woody tissues where 4CL1 is predominantly expressed, mechanisms of adaptive growth leading to the formation of reaction wood to counteract mechanical stresses contrast sharply between conifer and hardwood trees. This may be used to gain insight about the role of 4CL during adaptive growth, a process that greatly affects timber quality and utilization.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Preparation of Protein Extracts

Purification of Escherichia coli expressed 6×-His-tagged recombinant aspen (Populus tremuloides Michx.) Pt4CL1 and Pt4CL2 and loblolly pine (Pinus taeda) 4CL proteins was carried out as described (Hu et al., 1998). For recombinant expression of pine 4CL, coding sequence of the cDNA (Zhang and Chiang, 1997) was amplified using PCR primers with introduced BamHI and KpnI sites immediately upstream of the start and stop codons, respectively. The PCR product was cloned into the BamHI and KpnI sites of pQE-30 (Qiagen, Valencia, CA) and the vector was transformed into E. coli host strain M15 for expression. Plant crude proteins were extracted from apices and subapical (2nd-3rd internodes) leaves of greenhouse-grown aspen and from developing xylem of field-grown aspen and loblolly pine trees. Tissues were ground in liquid nitrogen, extracted in three volumes of buffer (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 25 mM ascorbate, 1 mM dithiothreitol, 2 µg mL¹ leupeptin, 5% [w/v] polyvinylpolypyrrolidone, and 15% [v/v] ethylene glycol), filtered through two layers of Miracloth, and clarified at 10,000g for 15 min. Purified recombinant proteins and clarified plant crude proteins were centrifuged (500g for 3 min) through 10 bed volumes of Sephadex G-25 equilibrated with 100 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 0.5 mM dithiothreitol, 2.5 µg mL¹ leupeptin, and 15% (v/v) ethylene glycol, aliquoted in small volumes, snap frozen in liquid nitrogen, and stored at 80°C until use. Protein concentrations were determined by the Bradford (1976) method using bovine serum albumin as a standard.

Enzyme Assays

4CL activity was measured in a 200-µL assay buffer containing 100 mM Tris-HCl (pH 7.5), 2.5 mM MgCl₂, and 2.5 mM ATP. Substrate concentrations were varied as indicated below, and in table and figure legends. Reactions were initiated with 0.2 mM CoA, incubated at 30°C for 3 min, terminated by boiling for 2 min, and centrifuged to precipitate denatured proteins. Clarified reaction mixtures with 1 µg of phenyl acetyl-coenzyme A added as internal standard were analyzed directly by HPLC-UV/MS. For the measurement of K_m and V_max, 100 ng of Pt4CL1 or 200 ng of Pt4CL2 recombinant proteins were diluted in 100 mM Tris-HCl, pH 7.5, 1 mg mL¹ bovine serum albumin, and 15% (v/v) ethylene glycol and were assayed as outlined above using substrate concentrations ranging from 1 to 400 µM for PA and CA, and from 10 to 2,000 µM for FA. Assays to evaluate two-substrate interactions contained one of the three combinations: PA + CA, PA + FA, or CA + FA. At least four substrate levels of PA and CA and three substrate levels of FA were used in these pair-wise combinations to determine the mechanism of inhibition. Assays used to calculate kinetic constants K_m, V_max, and K_i were repeated three times. Three-substrate assays containing 10 or 25 µM each of PA, CA, and FA were conducted using 200 ng of recombinant proteins (Pt4CL1, Pt4CL2, and loblolly pine 4CL) or 100 µg of plant crude proteins.

HPLC-UV/MS Analysis

HPLC-UV/MS analysis of 4CL products was performed using a Hewlett-Packard 1100 Series (Agilent Technologies, Palo Alto, CA). Fifty microliters of the reaction mixture was loaded onto a C₁₈ Discovery column (2.1 mm × 15 cm × 5 µm; Supelco, Bellefonte, PA) at 50°C and 0.250 mL min¹. The CoA thioesters were separated by a gradient elution from 11.8% to 15% (v/v) acetonitrile/10 mM formic acid (pH 7.0) over 5 min, followed by a 5-min hold at 15% (v/v) acetonitrile/10 mM formic acid (pH 7.0). The thioesters were detected by UV absorbance at 260 and 350 (approximate  max's for thioesters of substituted cinnamates at pH 7.0; Stöckigt and Zenk, 1975). MS detection was attained in atmospheric pressure ionization-electrospray-positive mode with a fragmentor voltage of 70V. Selected ion monitoring mode was set to identify m/z 914, PA-CoA; m/z 930, CA-CoA; m/z 944, FA-CoA; m/z 960, 5HFA-CoA; m/z 974; SA-CoA, and m/z 886, phenyl acetyl-CoA, which correspond to the M_r of each thioester and their base peak when run in the scanning mode. Calibration curves using authentic CoA thioesters synthesized as previously described (Li et al., 1997) were used to quantify reaction products.

In Situ Hybridization

In situ hybridization protocol was based on that of Jackson (1992) with modifications. Vacuum-infiltrated tissue sections were fixed for 15 h at 4°C in freshly prepared 4% (w/v) paraformaldehyde buffered with phosphate-buffered saline (PBS, pH 7.2). Fixed tissues were dehydrated in a graded ethanol:tert-butanol series and were impregnated with Paraplast-plus embedding medium (Polysciences, Warrington, PA). Thin sections (10 µm) were affixed to Superfrost Plus slides (Fisher, Chicago), dewaxed, rehydrated, and heated in a humid chamber at 70°C for 30 min. Sections were proteolyzed by treatment with proteinase K (10 µg mL¹ in PBS) for 1 h at room temp, post-fixed for 10 min with 4% (w/v) PBS-buffered paraformaldehyde, rinsed, and acetylated using 0.1 M triethanolamine (pH 8.1) and 0.25% (v/v) acetic anhydride. Sections were equilibrated with ethanol and dried prior to hybridization. DIG-labeled sense and antisense RNA probes of 4CL1 and 4CL2 were prepared using an in vitro transcription kit from Roche (Indianapolis). Full-length probes at a concentration of 1.5 ng µL¹ in 5× SSC, 50% (v/v) formamide, 1% (w/v) nucleic acid blocking reagent (Roche), 0.5 mg mL¹ tRNA, 0.4 mg mL¹ heparin, and 0.2% (w/v) SDS were hybridized with the sections for 12 h at 53°C. Anti-DIG Fab conjugated with alkaline phosphatase (Roche) was used at a dilution of 1:750. Color development using nitroblue tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate in 100 mM Tris (pH 9.5), 100 mM NaCl, 20 mM MgCl₂, and 10% (w/v) polyvinylalcohol (DeBlock and Debrouwer, 1993) was complete after 8 h. Images were recorded using a fluorescence microscope (E-400; Nikon, Tokyo) equipped with a digital imaging system.

Promoter Analysis

Aspen 4CL1 and 4CL2 promoter::GUS fusion constructs (Hu et al., 1998) were used to generate transgenic aspen using Agrobacterium tumefaciens-mediated transformation as previously described (Tsai et al., 1994). Histochemical staining of GUS activity in stem hand-sections was conducted according to Hawkins et al. (1997). After clearing in ethanol series to remove chlorophyll, sections were photographed using a microscope and digital imaging system (both Nikon).