The phenylpropanoid pathway was investigated with a focus on lignin biosynthesis by DGE profiling in B. napus genotypes, Express617, which has high lignin content and V8, with a moderate level of lignin. Mapman assisted the visualization of differential expression of phenylpropanoid pathway genes during whole seed development period in these B. napus winter genotypes at seven developing time points. Over view of genes expression at selected time points showed that at early stages of seed development most of genes have similar expression level in both genotypes. The highest up-regulation of genes were observed at 42 and 70 dap which is in fact seed maturation period (Fig 3.2 a-g).
Phenylalanine ammonia-lyase (PAL) catalyzes the first step of the phenylpropanoid pathway, and is responsible for the deamination of phenylalanine to yield trans-cinnamic acid and ammonia. PAL1, PAL2 and PAL4 genes were found in UGs which shows low level of expression during early seed developmental points and highest expression was observed at 70 dap in both genotypes while these genes show down regulation in Express617 relative to V8 at 84dap (Fig 3.2 a-g). Three hydroxylation steps in lignin biosynthesis are controlled by cytochrome P450s and they also play their role to determine the contribution of guaiacyl and syringyl monomers. Three P450s Cytochromes are Cinnamate 4-hydroxylase (C4H), p- Coumarate 3-hydroxylase (C3H) and Ferulate 5-hydroxylase (F5H). C4H is responsible for the conversion of cinnamate into p-coumarate and C3H catalyzes the hydroxylation of p- coumaroyl shikimate to yield caffeoyl shikimate. Expression level of C4H and C3H was higher in late developing time points while F5H show high expression in early time points. C4H and C3H are up-regulated in Express617 compare to the V8 from 28 to 70 dap and down-regulated at 84dap. F5H is the third P450cytochrome, catalyzing the NADPH- and O2-
dependent hydroxylation of both coniferaldehyde and coniferyl alcohol to yield 5-hydroxy- coniferaldehyde and 5-hydroxyconiferyl alcohol, respectively which are required for S-lignin production. F5H showed a high expression during the whole developmental period and relatively shows up-regulation in Express617 at 42dap (Fig 3.2 a-g).
4-coumarate: CoA ligase (4CL) catalyzes the formation of CoA esters of p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid, and sinapic acid. Seven 4CL putative genes were detected in UGs dataset which shows up-regulation in Express617 at 42 and 70dap (Fig 3.2 a-g). HCT catalyzes two steps in phenylpropanoid metabolism. First is its involvement to
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convert p-coumaroyl CoA to coumaroyl shikimate/quinate and secondly it catalyze caffeoyl shikimate/quinate conversion to caffeoyl CoA. Single HCT gene in known in Arabidopsis and B. napus six unigenes were annotated as HCT and show lower to higher expression level from early to late development points of seeds in both genotypes. HCT was up-regulated in Express617 at 28 and 42 dap and down-regulated at 84dap.
Two necessary methylation steps invoved in biosynthesis of monolignols are catalyzed by CCoAOMT and COMT. CCoAOMT catalyzes the methylation of caffeoyl-CoA to feruloyl- CoA and 5-hydroxyferuloyl-CoA to sinapoyl-CoA and is, together with COMT, responsible for the methylation of the monolignol precursors. COMT has a dominating role for
methylation of 5-hydroxyconiferaldehyde and/or 5-hydroxyconiferyl alcohol to
sinapaldehyde and/or sinapyl alcohol, respectively. Single COMT and four CCoAOMT genes were found in UGs dataset. CCoAOMT1 (side block in Fig 3.2 a-g) is mainly recognized to involve in methylation step of monolignols precursors and showed higher expression in Express617 relative to V8 during seed development. COMT shows almost constant level of expression in both genotypes during seed development; only slight down-regulation at 42dap and higher expression at 84dap in Express617 was observed (Fig 3.2 a-g).
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Fig 3.2 a. Phenylpropanoid pathway for lignin biosynthesis visualized in Mapman.
Expression of genes at 2nd dap in B. napus genotypes, Express617 relative to V8. Red color show higher expression of genes and blue means down-regulation of genes expression in Express617.
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Fig 3.2 b. Phenylpropanoid pathway for lignin biosynthesis visualized in Mapman.
Expression of genes at 14dap in B. napus genotypes, Express617 relative to V8. Red color show higher expression of genes and blue means down-regulation of genes‘ expression in Express617.
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Fig 3.2 c. Phenylpropanoid pathway for lignin biosynthesis visualized in Mapman.
Expression of genes at 28 dap in B. napus genotypes, Express617 relative to V8. Red color show higher expression of genes and blue means down-regulation of genes expression in Express617
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Fig 3.2 d. Phenylpropanoid pathway for lignin biosynthesis visualized in Mapman.
Expression of genes at 42 dap in B. napus genotypes, Express617 relative to V8. Red color indicates higher expression of genes and blue means down-regulation of genes expression in Express617
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Fig 3.2 e. Phenylpropanoid pathway for lignin biosynthesis visualized in Mapman.
Expression of genes at 56 dap in B. napus genotypes, Express617 relative to V8. Red color show higher expression of genes and blue means down-regulation of genes‘ expression in Express617.
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Fig. 3.2 f. Phenylpropanoid pathway for lignin biosynthesis visualized in Mapman.
Expression of genes at 70 dap in B. napus genotypes, Express617 relative to V8. Red color show higher expression of genes and blue means down-regulation of genes expression in Express617
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Fig. 3.2 g. Phenylpropanoid pathway for lignin biosynthesis visualized in Mapman.
Expression of genes at 84 dap in B. napus genotypes, Express617 relative to V8. Red color show higher expression of genes and blue means down-regulation of genes‘ expression in Express617.
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CCR is the first enzyme of the monolignol specific part of the lignin biosynthetic pathway which catalyzes the conversion of cinnamoyl-CoA esters to their respective cinnamaldehydes. Three CCR related genes along with CCR1 and CCR2 genes were found in UGs set. CCR1 show high expression in both genotypes during seed development and higher expression in Express617 compared to V8. Expression of CCR2 was not detected in both genotypes from 28-56dap while down-regulation was observed in Express617 at 84dap. CAD genes are catalyzing the last step in monolignol biosynthesis, and are responsible for final reduction of cinnamyl aldehydes into their corresponding alcohols which are final precursors of monolignols. Six CAD genes were found in whole UGs set. Expression of CAD1 was not detected and CAD5 appeared only at 84dap in V8. CAD4 showed higher level of expression in Express617 and in V8 no expression was found at 42 and 84dap. CAD7 and CAD9 genes expression was detected only until 28dap in V8 while in Express617 CAD9 expression was detected until 56dap. In V8, at 42dap no expression was detected from all CAD genes (Fig. 3.3).
Fig.3.3 Expression of CAD genes in Express617 (Exp) and V8 during seed development
from 2nd day after pollination (dap) to 84 dap
0 2 4 6 8 10
2dap 14dap 28dap 42dap 56dap 70dap 84dap
CAD1 CAD4 CAD5 CAD7 CAD8 CAD9 Exp 0 2 4 6 8 10
2dap 14dap 28dap 42dap 56dap 70dap 84dap
CAD1 CAD4 CAD5 CAD7 CAD8 CAD9 V8 E xpre ssi on va lues E xpre ssi on va lues
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