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

Two genomic sequences encoding 4-coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) in loblolly pine (Pinus taeda L.) were cloned. Both sequence s contained three introns and four exons with identical coding sequenc es predicting 537 amino acids. Two of the three introns in these two c lones were different both in sequence and in length. Sequences of both ICL clones were found in all nine megagametophyte DNAs tested, provid ing genetic evidence that these two 4CL genomic sequences are nonallel ic genes. Our analyses suggest that there are at least two distinct, i ntron-containing 4CL genes, at least one of which is transcribed into 4CL mRNA in developing xylem tissue of loblolly pine. The levels of 4C L gene transcription in xylem were influenced by compressional stress, resulting in an elevated 4CL enzyme activity with 4-coumaric acid. 4C L enzyme activity with ferulic acid remained unchanged, whereas with c affeic acid it was significantly inhibited. Exogenously applied trans- cinnamic acid in the protein extracts from normal wood xylem caused in hibition of 4CL activity toward caffeic acid similar to that under com pressional stress. The implications of this cinnamic acid-modulated ef fect on 4CL enzyme activities toward different substrates in regulatin g monolignol synthesis in xylem under compressional stress are discuss ed.