Molecular Cloning of 4-Coumarate:Coenzyme A Ligase in Loblolly Pine and the Roles of This Enzyme in the Biosynthesis of Lignin in Compression Wood

Abstract

Two genomic sequences encoding 4-coumarate:coenzyme A ligase (4CL; EC 6.2.1.1 2) in loblolly pine (Pinus faeda 1.) were cloned. Both sequences contained three introns and four exons with identical coding sequences predicting 537 amino acids. Two of the three introns in these two clones were different both in sequence and in length. Sequences of both 4CL clones were found in all nine megagametophyte DNAs tested, providing genetic evidence that these two 4CL genomic sequences are nonallelic genes. Our analyses suggest that there are at least two distinct, intron-containing 4CL genes, at least one of which is transcribed into 4CL mRNA in developing xylem tissue of loblolly pine. The levels of 4CL gene transcription in xylem were influenced by compressional stress, resulting in an elevated 4CL enzyme activity with 4-coumaric acid. 4CL enzyme activity with ferulic acid remained unchanged, whereas with caffeic acid it was significantly inhibited. Exogenously applied trans-cinnamic acid in the protein extracts from normal wood xylem caused inhibition of 4CL activity toward caffeic acid similar to that under compressional stress. The implications of this cinnamic acid-modulated effect on 4CL enzyme activities toward different substrates in regulating monolignol synthesis in xylem under compressional stress are discussed.

4-Coumarate:Coenzyme A Ligase in Hybrid Poplar1

“…A similar activity capable of specifically inhibiting the cinnamate-utilizing activities of recombinant 4CL-216 and 4CL-9 proteins seems to exist in poplar xylem protein extracts (S. Allina, B. Ellis, and C. Douglas, unpublished data). It is interesting to note that the substrate-utilization profile of the pine 4CL enzyme is altered during formation of compression wood (Zhang and Chiang, 1997). These results could be explained by posttranslational modification(s) to 4CL that affect its activity.…”

“…The absence of activity against sinapic acid, coupled with the apparent absence of catalytically distinct 4CL isoforms in poplar and other plants (Lozoya et al, 1988;Voo et al, 1995;Lee and Douglas, 1996), makes it unlikely that the differential expression of 4CL gene family members encoding enzymes with different substrateutilization profiles is a mechanism used in poplar, or most other plants, to partition carbon into guaiacyl and syringyl lignin, or into other phenylpropanoid end products. However, there is evidence from tobacco (Lee and Douglas, 1996), pine (Zhang and Chiang, 1997), and now poplar that modification of 4CL enzyme activity, resulting in changes in its substrate-utilization profile, could be important in carbon partitioning. The availability of abundant, catalytically active recombinant 4CL will facilitate future work on the nature of such possible modifications.…”

Differential Substrate Inhibition Couples Kinetically Distinct 4-Coumarate:Coenzyme A Ligases with Spatially Distinct Metabolic Roles in Quaking Aspen

“…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 mixedsubstrate assays, yielding a CA:PA:FA product ratio of 1:1.4:1 and 1:1.1:0.9 with 10 and 25 m substrates, respectively (Fig.…”

“…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.…”

“…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 (BeckerAndré 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.…”