The salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae family and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Despite their important role in plant defense, their biosynthetic pathway is not known, although recent work has suggested that benzyl benzoate acts as a possible biosynthetic intermediate. We identified three candidate genes encoding BAHD-type acyltransferases that are predicted to produce benzylated secondary metabolites, named PtACT47, PtACT49, and PtACT54.
Expression of PtACT47 and PtACT49 generally correlated with PG content in a variety of tissues and organs of wild type hybrid poplar plants. This correlation was also found in transgenic hybrid poplar where PG content varied with the level of expression of the condensed tannin regulator MYB134 transcript. In these plants, a suppression of PtACT47 and PtACT49 expression was correlated with lower PG content. In contrast, PtACT54 exhibited very low expression in all tissues tested, and this level of expression was not affected in MYB134 plants.
In order to better understand their possible biochemical functions, cDNA cloning, heterologous expression, and in vitro functional characterization was performed on these three BAHD acyltransferases. Recombinant PtACT47 exhibited a low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8 nM-1 sec-1) and lowest Km values (45.1 µM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA:salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, specifically benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8 nM-1 sec-1) and lowest Km (55.3 µM) was observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA:benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters at trace levels, for example hexenyl benzoate. Recombinant PtACT54 shares low sequence identity with PtSABT (52.3%) and PtBEBT (52.5%) and exhibited only moderate BEBT-like properties. PtSABT and PtBEBT appear to be paralogs based on their high sequence identity (90.6%) and closely related yet distinct biochemical functions. They likely arose from gene duplication and subsequent functional diversification possibly by neofunctionalization.
Wounding experiments showed that abiotic damage stimulated the synthesis of specific PGs, notably salicin and salicortin within 24-48hrs. This was accompanied by a proportional increase in the expression of PtSABT and PtBEBT. Furthermore, experiments using transgenic RNAi lines with knock-down suppression of PtBEBT, and PtSABT, and both genes simultaneously, provided the first direct evidence that BAHD acyltransferases are important in PG production. PtSABT suppression, both individually and in the double knock-down suppression, significantly lowered salicortin content, particularly in mature leaves. However, a reduced level of PtBEBT expression did not have a significant effect on the PGs measured. This could indicate that BEBT-like activity may be a shared function among closely related BAHDs. The suppression of multiple BEBT-like genes may be necessary to further delineate their functions. / Graduate / rjchedgy@uvic.ca
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/6008 |
| Date | 24 April 2015 |
| Creators | Chedgy, Russell James |
| Contributors | Constabel, Carsten Peter |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web, http://creativecommons.org/licenses/by-nc-nd/2.5/ca/ |
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