Transcriptional Control of Monolignol Biosynthesis in Pinus taeda

Abstract

Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/ lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mM potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mM) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly ratelimiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in monolignol biosynthesis.

Reconstitution of the Entry Point of Plant Phenylpropanoid Metabolism in Yeast (Saccharomyces cerevisiae)

“…a low K m value (3.9 M) and 10 -100 times higher turnover number than animal P450s for detoxification (41), C4H is expected to be a key driver for high throughput metabolic flux at the entry point of phenylpropanoid pathway. This conclusion is consistent with the finding that carbon allocation into monolignol biosynthesis is controlled by C4H expression levels, as determined by expression profile study of lignin-biosynthesis associated genes in Pinus taeda (42) and by the identification of the AtMYB4 transcription factor that exclusively regulates C4H transcription in Arabidopsis (43). This is also consistent with the apparent roles of other plant P450s as important gatekeeper enzymes in the control of flux into specific pathways of secondary metabolism, such as those downstream in the lignin biosynthetic pathway (42, 44 -46) or in the biosynthesis of the maize defense compound DIMBOA (47).…”

Formation of two methylenedioxy bridges by a Sesamum CYP81Q protein yielding a furofuran lignan, (+)-sesamin

“…5A). Interestingly, the microsomal fraction also catalyzed the transformation of (ϩ)-pinoresinol (RT of 8.6 min) into two lignan products eluted at RTs of 12.7 and 16.9 min, identical to the RTs of (29). ger, germinating seeds treated with H 2 O for 2 days; l, leaves; p, petals; st, stems; sp, seed pods.…”

“…It also was expressed in leaves but not in germinating seeds, petals, or stems, whereas the S. indicum p-coumarate 3-hydroxylase (C3H, CYP98A20) gene, which is involved in phenylpropanoid pathway, was expressed ubiquitously (Fig. 2B) (29).…”

A Novel Mg2+-dependent O-Methyltransferase in the Phenylpropanoid Metabolism of Mesembryanthemum crystallinum

“…To 10 ml of 1 mM caffeic acid in KP i buffer (pH 7.0), 10 g of highly purified SGT preparation and UDP-glucose (10 mM final concentration) were added. After incubation for 30 min the reaction was stopped by adding acetonitrile to a final concentration of 20% and the mixture was concentrated by RP 18 solid-phase cartridge extraction (Waters). The concentrated mixture of caffeic acid and caffeoylglucose was further purified by a semipreparative reverse phase-HPLC on Nucleosil-RP 18 (10-mm inner diameter, 25 cm length) (Machery & Nagel) with a 30-min gradient of increasing acetonitrile concentration in 1% acetic acid (5-50% in 30 min, flow rate 2 ml min Ϫ1 ) and evaporated to dryness.…”

“…16 and 17). However, studies on transcriptional profiling of monolignol biosynthesis in P. taeda cell cultures question any such difference of methylating enzymes between angiosperms and gymnosperms, and confirm that methylation of guaiacyl residues is the exclusive role of class I CCoAOMTs (18). Other phenylpropanoid esters or flavonoids have never been described as substrates for any of these enzymes (16).…”