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Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium.

Cass CL, Peraldi A, Dowd PF, Mottiar Y, Santoro N, Karlen SD, Bukhman YV, Foster CE, Thrower N, Bruno LC, Moskvin OV, Johnson ET, Willhoit ME, Phutane M, Ralph J, Mansfield SD, Nicholson P, Sedbrook JC - J. Exp. Bot. (2015)

Bottom Line: The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion.RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions.The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Illinois State University, Normal, IL 61790 USA US Department of Energy Great Lakes Bioenergy Research Center, Madison, WI 53706, USA.

No MeSH data available.


Related in: MedlinePlus

BdPAL RNAi cell walls have altered lignin composition and reduced FA and pCA content. (A) Phloroglucinol staining of stem sections. Lighter staining is consistent with less lignin. Note that vascular bundles (marked by an arrow) appear normal (e.g. no collapsed cells) in both RNAi lines. (B) Amounts of acid-insoluble and acid-soluble lignin, as well as lignin S:G units ratios, in senesced stems plus leaf sheaths. (C) The amounts of ferulate (FA) and p-coumarate (pCA) released from lignin (pCA) and hemicelluloses (FA and pCA) upon 2M NaOH treatment. Vertical bars represent the SD. For (B), each column represents a biological rep with three technical reps averaged; significant differences can be found in Supplementary Table S3 at JXB online. For (C), n=3 biological reps and n=2 technical reps. Asterisks denote Student’s t-test significant differences (P<0.01) between each PAL knocked-down line and its corresponding control grown concurrently (BdPAL RNAi1-1 versus WT; BdPAL RNAi1-3 versus CTL).
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Figure 3: BdPAL RNAi cell walls have altered lignin composition and reduced FA and pCA content. (A) Phloroglucinol staining of stem sections. Lighter staining is consistent with less lignin. Note that vascular bundles (marked by an arrow) appear normal (e.g. no collapsed cells) in both RNAi lines. (B) Amounts of acid-insoluble and acid-soluble lignin, as well as lignin S:G units ratios, in senesced stems plus leaf sheaths. (C) The amounts of ferulate (FA) and p-coumarate (pCA) released from lignin (pCA) and hemicelluloses (FA and pCA) upon 2M NaOH treatment. Vertical bars represent the SD. For (B), each column represents a biological rep with three technical reps averaged; significant differences can be found in Supplementary Table S3 at JXB online. For (C), n=3 biological reps and n=2 technical reps. Asterisks denote Student’s t-test significant differences (P<0.01) between each PAL knocked-down line and its corresponding control grown concurrently (BdPAL RNAi1-1 versus WT; BdPAL RNAi1-3 versus CTL).

Mentions: Brachypodium transformants harbouring the BdPAL RNAi construct were generated and screened for lignin-related phenotypes. It was hypothesized that the stem biomass of reduced-lignin plants would stain a lighter red with the phloroglucinol lignin stain and would release relatively larger amounts of cell wall polysaccharide-derived sugars upon saccharification, compared with the WT. This was, in fact, observed in plants from seven independent transgenic lines, with the relative strengths of these phenotypes mirroring the relative amounts of BdPAL gene knockdown (Figs 1B, 2, 3, and data not shown). T2 generation plants from two independent lines (BdPAL RNAi1 lines 1 and 3) were characterized in detail and found to have stem biomass with substantially higher digestibility compared with that of both WT and empty vector control (CTL) plants (Fig. 2). A more encompassing evaluation of biomass digestibility showed that stem biomass from both lines had significantly higher release of glucose and pentose sugars associated with a variety of chemical pre-treatments including hot water, weak acid, and weak base followed by partial enzymatic hydrolysis. With all pre-treatments, BdPAL RNAi1 line 1 senesced stem biomass had the greatest amounts of sugars released, in most cases being twice the amount released from the WT (Fig. 2) under the chosen assay (Santoro et al., 2010).


Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium.

Cass CL, Peraldi A, Dowd PF, Mottiar Y, Santoro N, Karlen SD, Bukhman YV, Foster CE, Thrower N, Bruno LC, Moskvin OV, Johnson ET, Willhoit ME, Phutane M, Ralph J, Mansfield SD, Nicholson P, Sedbrook JC - J. Exp. Bot. (2015)

BdPAL RNAi cell walls have altered lignin composition and reduced FA and pCA content. (A) Phloroglucinol staining of stem sections. Lighter staining is consistent with less lignin. Note that vascular bundles (marked by an arrow) appear normal (e.g. no collapsed cells) in both RNAi lines. (B) Amounts of acid-insoluble and acid-soluble lignin, as well as lignin S:G units ratios, in senesced stems plus leaf sheaths. (C) The amounts of ferulate (FA) and p-coumarate (pCA) released from lignin (pCA) and hemicelluloses (FA and pCA) upon 2M NaOH treatment. Vertical bars represent the SD. For (B), each column represents a biological rep with three technical reps averaged; significant differences can be found in Supplementary Table S3 at JXB online. For (C), n=3 biological reps and n=2 technical reps. Asterisks denote Student’s t-test significant differences (P<0.01) between each PAL knocked-down line and its corresponding control grown concurrently (BdPAL RNAi1-1 versus WT; BdPAL RNAi1-3 versus CTL).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493789&req=5

Figure 3: BdPAL RNAi cell walls have altered lignin composition and reduced FA and pCA content. (A) Phloroglucinol staining of stem sections. Lighter staining is consistent with less lignin. Note that vascular bundles (marked by an arrow) appear normal (e.g. no collapsed cells) in both RNAi lines. (B) Amounts of acid-insoluble and acid-soluble lignin, as well as lignin S:G units ratios, in senesced stems plus leaf sheaths. (C) The amounts of ferulate (FA) and p-coumarate (pCA) released from lignin (pCA) and hemicelluloses (FA and pCA) upon 2M NaOH treatment. Vertical bars represent the SD. For (B), each column represents a biological rep with three technical reps averaged; significant differences can be found in Supplementary Table S3 at JXB online. For (C), n=3 biological reps and n=2 technical reps. Asterisks denote Student’s t-test significant differences (P<0.01) between each PAL knocked-down line and its corresponding control grown concurrently (BdPAL RNAi1-1 versus WT; BdPAL RNAi1-3 versus CTL).
Mentions: Brachypodium transformants harbouring the BdPAL RNAi construct were generated and screened for lignin-related phenotypes. It was hypothesized that the stem biomass of reduced-lignin plants would stain a lighter red with the phloroglucinol lignin stain and would release relatively larger amounts of cell wall polysaccharide-derived sugars upon saccharification, compared with the WT. This was, in fact, observed in plants from seven independent transgenic lines, with the relative strengths of these phenotypes mirroring the relative amounts of BdPAL gene knockdown (Figs 1B, 2, 3, and data not shown). T2 generation plants from two independent lines (BdPAL RNAi1 lines 1 and 3) were characterized in detail and found to have stem biomass with substantially higher digestibility compared with that of both WT and empty vector control (CTL) plants (Fig. 2). A more encompassing evaluation of biomass digestibility showed that stem biomass from both lines had significantly higher release of glucose and pentose sugars associated with a variety of chemical pre-treatments including hot water, weak acid, and weak base followed by partial enzymatic hydrolysis. With all pre-treatments, BdPAL RNAi1 line 1 senesced stem biomass had the greatest amounts of sugars released, in most cases being twice the amount released from the WT (Fig. 2) under the chosen assay (Santoro et al., 2010).

Bottom Line: The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion.RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions.The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Illinois State University, Normal, IL 61790 USA US Department of Energy Great Lakes Bioenergy Research Center, Madison, WI 53706, USA.

No MeSH data available.


Related in: MedlinePlus