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

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Abstract
Two genomic sequences encoding 4-coumarate:coenzyme A ligase (4CL;EC 6.2.1.12) in loblolly pine (Pinus taeda L.) 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.