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Phenylpropanoid metabolism in poplar : characterization of the 4-coumarate: CoA ligase gene family and a putative hydroxycinnamoyl-CoA thioesterase Allina, Sandra Mary

Abstract

The work presented in this thesis is part of a larger study designed to examine the possibility of altering lignin content and/or composition by genetic engineering. Hybrid poplar was used as a model tree system. Lignin precursors (cinnamyl alcohols) are derived from phenylpropanoid metabolism. 4-coumarate:CoA ligase (4CL) activity is required for the activation of hydroxycinnamic acids prior to their being fed into the lignin specific branch pathway. The purpose of this research was to test the hypothesis that multiple genes encode 4CL isoforms that are differentially expressed and preferentially utilize one or more methoxylated hydroxycinnamic acid substrate. 4CL is encoded by a gene family in hybrid poplar. Two full-length cDNA clones (4CL-216 and 4CL-9) representing two different genes (4CL1 and 4CL2) were isolated from a young leaf library and characterized. Southern blot analysis demonstrated that other 4CL genes exist in the poplar genome. Northern blot analysis demonstrated that 4CL1 and 4CL2 are differentially expressed. 4CL1 is preferentially expressed in old leaves, while 4CL2 is preferentially expressed in young leaves. Neither gene is expressed at high levels in developing xylem and they may not play a major role in lignification of this tissue. The 4CL cDNA clones were expressed in a insect cell/baculovirus system. Partially purified recombinant 4CL proteins were tested for substrate preference. Both of the recombinant 4CL proteins used hydroxycinnamic acid substrates in a similar manner. The substrate preferences were similar to those found for each of the partially purified native 4CL isoforms (Allina et al., 1998). This suggests that 4CL isoforms with large differences in their substrate utilization profiles do not, in fact, exist in poplar. The recombinant 4CL proteins use cinnamic acid while the native proteins do not. It was hypothesized that a cellular factor was responsible for modifying native 4CL such that it could no longer use cinnamic acid as a substrate. However, the results of this study strongly suggest that 4CL activity is not being modified but rather, that a thioesterase activity is responsible for the observed phenomena. The thioesterase is able to use all hydroxycinammoyl-CoA thioesters as substrates. This was previously masked by the fact that recombinant 4CL could convert most hydroxycinnamic acids to hydroxycinnamoyl-CoA thioesters faster than the thioesterase could perform the reverse reaction. The possible role of a hydroxycinnamoyl-CoA thioesterase in phenylpropanoid metabolism is discussed.

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