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 rate-limiting, 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.
Highlights
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
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 indicates that these steps are not truly ratelimiting, because they are modulated according to metabolic demand
The others, phenylalanine ammonia lyase (PAL) [32], 4-coumarate:CoA ligase (4CL) [33], caffeoyl-CoA O-methyltransferase (CCOMT) [34], cinnamyl alcohol dehydrogenase (CAD) [35], and a proposed caffeate O-methyltransferase (COMT), known as AEOMT [36], had been reported previously. These studies had claimed that PAL [32], 4CL [37], and CAD [35] were regulatory enzymes in loblolly pine on the basis of their relative positions in the phenylpropanoid pathway (PAL is the entry point, 4CL and CCOMT are putative branch points, and CAD is the last step) and correlation of their expression with lignification
Summary
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). After incubation at 42 °C for 50 min and at 65 °C for 15 min, the resulting cDNA (1 l of the 20-l reverse transcription reaction) was used as a template to amplify P. taeda genes encoding PAL, C3H, 4CL, hydroxycinnamic acid/hydroxycinnamoyl-CoA ester O-methyltransferase (AEOMT), CCOMT, and CAD using gene-specific primers (Tables I and II) designed from sequences submitted to the GenBankTM or, in the case of C3H, from the newly determined sequence of the 5Ј and 3Ј ends.
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