Differential substrate inhibition couples kinetically distinct 4-coumarate:coenzyme a ligases with spatially distinct metabolic roles in quaking aspen

Abstract

4-Coumarate:coenzyme A ligase (4CL) activates hydroxycinnamates for entry into phenylpropanoid branchways that support various metabolic activities, including lignification and flavonoid biosynthesis. However, it is not clear whether and how 4CL proteins with their broad substrate specificities fulfill the specific hydroxycinnamate requirements of the branchways they supply. Two tissue-specific 4CLs, Pt4CL1 and Pt4CL2, have previously been cloned from quaking aspen (Populus tremuloides Michx.), but whether they are catalytically adapted for the distinctive metabolic roles they are thought to support is not apparent from published biochemical data. Therefore, single- and mixed-substrate assays were conducted to determine whether the 4CLs from aspen exhibit clear catalytic identities under certain metabolic circumstances. Recombinant Pt4CL1 and Pt4CL2 exhibited the expected preference for p-coumarate in single-substrate assays, but strong competitive inhibition favored utilization of caffeate and p-coumarate, respectively, in mixed-substrate assays. The Pt4CL1 product, caffeoyl-CoA, predominated in mixed-substrate assays with xylem extract, and this was consistent with the near absence of Pt4CL2 expression in xylem tissue as determined by in situ hybridization. It is interesting that the Pt4CL2 product p-coumaroyl-CoA predominated in assays with developing leaf extract, although in situ hybridization revealed that both genes were coexpressed. The xylem extract and recombinant 4CL1 data allow us to advance a mechanism by which 4CL1 can selectively utilize caffeate for the support of monolignol biosynthesis in maturing xylem and phloem fibers. Loblolly pine (Pinus taeda), in contrast, possesses a single 4CL protein exhibiting broad substrate specificity in mixed-substrate assays. We discuss these 4CL differences in terms of the contrasts in lignification between angiosperm trees and their gymnosperm progenitors.

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 K_m (K_m app) versus the corresponding inhibitor concentration used to calculate K_i values summarized in Table II. K_i is determined as the negative intercept on the x axis.

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).

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.

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.

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.

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.