expression of genes from the lignin synthesis pathway in...

Universidade de São Paulo

2012

Expression of genes from the lignin synthesis

pathway in guineagrass genotypes differing in

cell-wall digestibility GRASS AND FORAGE SCIENCE, MALDEN, v. 67, n. 1, supl. 1, Part 4, pp. 43-54, MAR, 2012http://www.producao.usp.br/handle/BDPI/34593

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http://www.producao.usp.br/handle/BDPI/34593

Expression of genes from the lignin synthesispathway in guineagrass genotypes differing incell-wall digestibility

S. S. Stabile*, A. P. Bodini*, L. Jank†, F. P. Renno*, M. V. Santos* and L. F. P. Silva*

*Department of Animal Nutrition and Production, Universidade de Sao Paulo, Pirassununga, Brazil, and

†Embrapa-Beef Cattle, Campo Grande, MS, Brazil

Abstract

Rapid decline in cell-wall digestibility hinders efficient

use of warm-season grasses. The objective of this study

was to identify genes whose expressions are related to the

slope of decline in cell-wall digestibility. Eleven guinea-

grass genotypes were harvested at three ages and classi-

fied according to fibre digestibility. Extreme genotypes

were separated into groups with either FAST or SLOW

decline in fibre digestibility. Expression of transcripts

from six genes from the lignin synthesis pathway was

quantified by real-time PCR. Fast decline in fibre digest-

ibility was associated with higher DM yield after 90 d of

regrowth. Apart from lower fibre digestibility and higher

lignin content for the FAST group, there were no other

differences between the two groups for the chemical

composition of stems and leaves. Maturity affected

differently the expression of two of the six genes,

cinnamate 4-hydroxylase and caffeoyl-CoA O-methyl-

transferase (C4H and CCoAOMT). Genotypes with fast

decline in fibre digestibility had greater increase in the

expression of C4H and CCoAOMT from 30 to 60 d of

regrowth, than genotypes with slower decline. Expres-

sion of C4H and CCoAOMT appears to be related to the

decline in cell-wall digestibility with advance in maturity

of guineagrass.

Keywords: CCoAOMT, C4H, lignin, Panicum maximum,

tropical forages

Introduction

Tropical grasses, such as guineagrass (Panicum maximum

Jacq.), have a huge potential for dry matter production,

leading to high stocking rates during the summer. On

the other hand, tropical grasses have rapid elongation of

stems and rapid decline in forage quality with advance

in maturity (Nelson and Moser, 1994). Given the

importance of forage digestibility on livestock perfor-

mance, efforts have been made to genetically improve

in vitro digestibility of perennial forage crops, with great

success (Casler and Vogel, 1999).

For tropical grasses, however, genetic improvement

in forage quality has been relatively slow, mainly

because of the lack of sexual plants in the collections,

which limits the progress of the breeding programme

(Araujo et al., 2005). In guineagrass, leaf yield and leaf-

to-stem ratio (LSR) have been used as the most

important traits when selecting for forage quality (Muir

and Jank, 2004), with very little information available

about fibre digestibility.

Forage maturity is frequently associated with less

leafiness and lower LSR, and stems are usually consid-

ered as lower-quality components than leaves. How-

ever, this is not always true. Alfalfa (Medicago sativa L.)

and many other legume species use the stem as

structural components (lower quality) and the leaves

as metabolic organs (higher quality). In contrast, grasses

use leaves both for structure, through the lignified

midrib, and as metabolic organs. Thus, the nutritive

value of alfalfa leaves will be maintained during the

ageing process, whereas grass leaves will decrease in

quality (Van Soest, 1994). Conversely, in some grasses,

the stem is considered to be a reserve organ, and this

will lead to the stems having higher nutritive value

than leaves. For example, timothy (Phleum pratense L.)

and sugarcane (Saccharum spp.) utilize the stem as a

reserve organ (Van Soest, 1994).

In young guineagrass genotypes, fibre digestibility of

stems was higher than that of leaves, but decreased

Correspondence to: L. F. P. Silva, School of Veterinary

Medicine and Animal Science, USP Av. Duque de Caxias

Norte, 225, 13635-900 – Pirassununga, Sao Paulo, Brazil.

E-mail: [email protected]

Received 27 October 2010; revised 21 June 2011

doi: 10.1111/j.1365-2494.2011.00817.x � 2011 Blackwell Publishing Ltd. Grass and Forage Science, 67, 43–54 43

Grass and Forage Science The Journal of the British Grassland Society The Official Journal of the European Grassland Federation

rapidly with advance in maturity, which suggests that

stem nutritive value should be addressed as a quality

trait of tropical forages (Stabile et al., 2010).

Lignin is a component of cell walls and is recognized

as the main factor limiting digestion of cell-wall

polysaccharides in the rumen. Lignin seems to exert

its negative effect on cell-wall digestibility by shielding

the polysaccharides from enzymatic hydrolysis (Jung

and Deetz, 1993). Expression of genes encoding

enzymes from the phenylpropanoid pathway has been

shown to modulate rate of lignin synthesis in grasses

and legumes (Ralph et al., 2004). Given the high degree

of lignin heterogeneity among species and among

tissues within a plant, the regulation and nature of

the pathway may differ among cell types and among

species (Campbell and Sederoff, 1996).

The specific differences in lignification of each tissue

can be responsible for the low correlation between

lignin content and cell-wall digestibility, in forage

samples harvested at similar maturity stages (Jung and

Casler, 2006). Similarly, different enzymes can be

responsible for controlling lignifications at different

plant tissues and at different forage species. The iden-

tification of genes controlling lignin biosynthesis during

tropical forage development would enhance our com-

prehension of the lignification process and facilitate the

development of better cultivars. Studies in maize have

demonstrated that several genes in the lignin pathway

are simultaneously under-expressed in line with higher

cell-wall degradability (Barriere et al., 2009).

It was hypothesized that the decline in stem nutritive

value is related to differential expression of specific

genes from the lignin synthesis pathway, and therefore,

the objective of this study was to quantify the expres-

sion of six genes in guineagrass genotypes phenotypi-

cally classified in divergent groups according to stem

fibre digestibility.

Materials and methods

Plant material

The characteristics and composition of these samples

are fully described by Stabile et al. (2010). In brief,

established plots of eleven guineagrass genotypes were

grown at Embrapa Beef Cattle experimental station

(Campo Grande, Brazil, 20�26¢S lat; 54�43¢W long;

530Æ7 m above sea level). After an initial cut approx-

imately 20 cm above the soil on 29 December 2005, the

plots were fertilized with 100 kg ha)1 of N (urea),

100 kg ha)1 of P2O5 (single superphosphate) and

100 kg ha)1 of K2O (potassium chloride) and harvested

after 30, 60 or 90 d of regrowth.

The climate over the experimental period was in

accordance with the average for the season, without

prolonged water deficit (Table 1). Each genotype was

replicated in three plots of six rows width, spaced by

0Æ5 m, and 4 m length in a randomized block design. At

each harvest date, two rows were cut with electric

clippers approximately 20 cm above the soil level. A

subsample of 2 kg was taken and separated into leaf

blades, stems (stem plus leaf sheaths) and senescent

material and stored in plastic bags at )20�C for chemical

analysis and in vitro incubations. A portion of the stems

was cut with scissors, put in 15-mL Falcon tubes, frozen

in liquid nitrogen and stored at )80�C for subsequent

analysis. Total green dry matter production was calcu-

lated by subtracting the senescent material from the leaf

and stem fractions.

Chemical analysis and in vitro digestibility

The leaf and stem components were analysed for dry

matter (DM), ash and crude protein (CP) according to

AOAC (1997). Neutral detergent fibre (NDF) contents

were determined in g per g of dry matter (DM),

according to the method described by Van Soest et al.

(1991), without addition of a-amylase and sodium

sulphite. Acid detergent fibre (ADF) and acid detergent

lignin (ADL) were determined according to the meth-

ods described by Goering and Van Soest (1970).

DM and NDF digestibilities of leaf and stem samples

were determined by an in vitro procedure, using 30 h of

incubation time (Tilley and Terry, 1963 as modified by

Goering and Van Soest, 1970). Rumen fluid was collected

from three fistulated non-lactating Holstein cows that

were kept on pasture receiving mineral supplementa-

tion. Approximately 2 L of rumen fluid was collected

from each cow into a pre-warmed thermos and imme-

diately transported to the laboratory. Samples were

incubated in duplicate, in three series, one for each

block, to minimize assay-to-assay variation. Ten millili-

tres of filtered rumen fluid was added to each flask

containing the feed, medium and reducing solution. The

flasks were connected to a CO2 manifold and incubated

at 39�C for 30 h. Flasks were then removed, 20 mL of

Table 1 Weather conditions for the duration of the experiment.

Period 12 ⁄ 29 ⁄ 2005–02 ⁄ 01 ⁄ 2006 02 ⁄ 02 ⁄ 2006–03 ⁄ 01 ⁄ 2006 03 ⁄ 02 ⁄ 2006–04 ⁄ 01 ⁄ 2006

Mean temperature (�C) 25Æ5 24Æ9 25Æ1

Rainfall (mm) 149Æ3 178Æ3 141Æ2

44 S. S. Stabile et al.

� 2011 Blackwell Publishing Ltd. Grass and Forage Science, 67, 43–54

neutral detergent solution was added and the flasks were

immediately frozen to stop fermentation. The contents of

each flask were then transferred into a 600-mL beaker

that contained 80 mL of neutral detergent solution and

were refluxed for 1 h. The contents of the beaker were

then transferred to a Gooch crucible and filtered under

vacuum to isolate the fibre residue. The residual fibre was

rinsed with hot water and acetone, dried at 105�C for

24 h and weighed.

The eleven guineagrass genotypes were separated

according to the slope of decline in stem NDF digest-

ibility with advance in maturity. Three genotypes

(Milenio, Mombaca and Tanzania) with fast decline in

NDF digestibility (FAST) and three genotypes (PM39,

PM47 and Massai) with slow decline in NDF digestibil-

ity (SLOW) were selected for gene-expression analysis.

Quantitative Real-Time PCR

Total RNA from tissue samples was isolated using

TRIzol� Reagent (Invitrogen, Carlsbad, CA, USA)

according to Chomczynski and Sacchi (1987) and

included a DNase I (Invitrogen) treatment. The quality

of isolated RNA was determined by measuring the

absorbance at 260 and 280 nm, and its integrity was

verified as mainly 18S and 28S rRNA by electrophoresis

in 1Æ5% (w ⁄ v) agarose gel. Three lg of total RNA from

each tissue sample was used for cDNA synthesis. After

denaturing at 70�C for 10 min, half of the sample

(1Æ5 lg) was reverse-transcribed into cDNA with 0Æ5 lg

of oligo thymidine and 200 units of Superscript II

reverse transcriptase (RT) (Invitrogen) in a final volume

of 20 lL, for 60 min at 42�C. The other half was

incubated without reverse transcriptase and used as a

negative control in polymerase chain reaction (PCR) to

confirm the absence of residual genomic DNA contam-

ination.

Primer pairs specific for six genes from the monolig-

nols biosynthesis pathway: cinnamate-4-hydroxylase

(C4H), 4-coumarate-CoA ligase (4CL), cinnamoyl-CoA-

reductase (CCR), caffeoyl-CoA O-methyltransferase

(CCoAOMT), cinnamyl alcohol dehydrogenase (CAD),

phenylalanine ammonia lyase (PAL) and glyceralde-

hyde 3-phosphate dehydrogenase (GAPDH) were

designed based on conserved regions of maize and rice.

After PCR amplification and sequencing of the ampli-

cons, new primers were designed for real-time PCR

quantification of gene expression (Table 2).

Studies in other grasses, such as maize, have dem-

onstrated that the genes from the lignin biosynthesis

pathway are usually present as members of small

multigene families (Barriere et al., 2009). Two C4H

genes have been described in maize (Guillaumie et al.,

2007; Barriere et al., 2009), and based on the amplicon

sequence obtained with our guineagrass primers (Gen-

Bank EU741932), we are probably detecting the C4H1

gene (95% similarity) and not the C4H2 gene (74%

similarity). Five classes of 4CL genes have been

described in maize (Guillaumie et al., 2007; Barriere

et al., 2009), and based on the guineagrass amplicon

Table 2 Oligonucleotide primer pairs designed for use in real-time polymerase chain reaction (PCR) amplification.

Genes* Oligonucleotide primers: 5¢ fi 3¢GenBank accession

number

PCR insert

size (bp)

4CL F: (T ⁄ A)GAACACCATCGAC(T ⁄ G)AGGAC EU741933† 336

R: TGGATTTCGTGAAGAAGACC

C4H F: TCGCAGAGCTTCGAGTACA EU741932† 230

R: AGGACGTTGTCGTGGTTGAT

CAD F: ACATGGGCGTGAAGGT(G ⁄ A)GC 2239257‡ 230

R: CTT(G ⁄ C)CCGTCCAGCTTCAG

CCR F: CTGGTACTGCTACGGGAAGG EU741931† 394

R: CATCTTGTACTCCTGCTTCC

CCoAOMT F: AAGAGCGACGACCTGTACCA EU741935† 211

R: GGAGGGAGTAGCCGGTGTAG

PAL F: AGGTCAAATCCGTGAACGAC EU741934† 347

R: GAGTTTCACGTCCTGGTTGT

GAPDH F: GTTCGTTGTTGGTGTCAACC 24415113‡ 247

R: TCCAGTGCTGCTGGGAATGA

*4CL, 4-coumarate-CoA ligase; C4H, cinnamate-4-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; CCR, cinnamoyl-CoA-

reductase; CCoAOMT, caffeoyl-CoA O-methyltransferase; PAL, phenylalanine ammonia lyase.†Primers were designed based on the sequences of guineagrass PCR amplicons obtained in this study that were deposited in

GenBank.‡Primers were designed based on previously deposited maize sequences.

Expression of genes from the lignin synthesis pathway 45


sequence (EU741933), our primers are specific for the

class I genes, with 93% similarity with the maize

sequence, which is a putative orthologue to 4CL2 from

Arabidopsis and poplar (GenBank AY566301.1). Seven

CCR genes have been described in maize (Guillaumie

et al., 2007; Barriere et al., 2009), and our guineagrass

sequence (GenBank EU741931) has high similarity

with class 1 genes (89% similarity with CCR1 and

72% similarity with CCR2).

One COMT gene and five CCoAOMT genes have

been described in maize (Guillaumie et al., 2007; Bar-

riere et al., 2009), and based on sequence homology

with the amplified guineagrass amplicon (EU741935),

we are detecting the class 1 genes, with 95% similarity

to maize CCoAOMT1 and CCoAOMT2. There are seven

genes codifying for CAD in maize (Guillaumie et al.,

2007; Barriere et al., 2009), and our primers are able to

amplify the two genes from the class 1 (GenBank

Y13733 and Contig 2405118.2.2), but not the genes

from the other classes of CAD. For PAL, our guineagrass

amplicon sequence (EU741934) has high similarity

with class I genes (88% similarity with PAL3 –

GenBank NM_001111864).

Quantification of gene expression was performed

using the StepOne Real-Time PCR System (Applied

Biosystems, Foster City, CA, USA). The PCR reactions

were incubated at 95�C for 10 min, followed by forty

cycles of 95�C for 10 s and 60�C for 30 s. Each reaction

contained 10 lL of SYBR� Green PCR Master Mix reagent,

1Æ0 lL of template cDNA, 1Æ25 lL of each primer

(10 lMM) and 7Æ5 lL of nuclease-free water. All reactions

were performed in triplicate wells.

Glyceraldehyde 3-phosphate dehydrogenase was

used as housekeeping gene, as it has been shown

before to be stable in maturing grass internodes

(Iskandar et al., 2004), and its expression did not vary

more than onefold from the mean under the conditions

of this experiment.

Changes in gene expression were calculated by

relative quantification using the DDCt method (Livak

and Schmittgen, 2001), where Ct is the cycle number at

which the fluorescence signal of the product crosses an

arbitrary threshold set with exponential phase of the

PCR and DDCt = (Cttarget gene unknown sample ) CtGAPDH

unknown sample) ) (Cttarget gene calibrator sample ) tGAPDH

calibrator sample). Average abundance of target genes at

30 d of regrowth was considered as the calibrator. Fold

changes in gene expression were calculated as 2)DDCt

after testing for efficiency of amplification not different

than 100%.

Statistical analysis

All statistical analyses were conducted using SAS,

version 9.1.2 for Windows (SAS Institute Inc., Cary,

NC, USA). Data for in vitro NDF digestibility (IVNDFD)

of the stem were analysed as a randomized block design

in a split-plot arrangement. Genotype was considered

the plot, maturity the subplots and genotype

(treatment) as blocks. Analysis of variance was per-

formed using the MIXED procedure of SAS according to

the model: Y = l + genotypes + maturity + geno-

types · maturity + block + block · maturity + e, where

the terms block and block · maturity were considered

as random. Because there was a significant geno-

types · maturity effect (P < 0Æ05) on stem NDF digest-

ibility, the slope of decline in NDF digestibility was

calculated by linear contrast and compared by t-tests

adjusted for multiple comparisons (Gill, 1978).

After separation of the genotypes in two groups (fast

and slow), data for DM production, morphological

components, chemical composition, in vitro digestibility

and gene expression were analysed as a randomized

block design in a split-plot arrangement. Treatment was

considered the plot, maturity the subplots and genotype

(treatment) as blocks. Analysis of variance was per-

formed using the MIXED procedure of SAS according to

the model: Y = l + treatment + maturity + treat-

ment · maturity + genotype (treatment) + genotype

(treatment) · maturity + e, where the terms genotype

(treatment) and genotype (treatment) · maturity were

considered as random. When there was a significant

treatment · maturity effect, the average of the treat-

ments in each maturity was compared by contrast using

the SLICE option of SAS. The P-values were repre-

sented as * for P < 0Æ05, ** for P < 0Æ01, *** for

P < 0Æ001 and NS for not significant.

Results

Eleven guineagrass genotypes were classified according

to the slope of decline in stem NDF digestibility with

advanced maturity and separated into two groups with

three genotypes each: FAST or SLOW decline in NDF

digestibility (Table 3). For better comprehension of the

text, these two groups of cultivars will be addressed as

treatments in this study. The accession PM45, although

it had a small difference in IVNDFD from 30 to 90 d,

was not selected as part of the SLOW group because of

the abnormal behaviour (Table 3). For this accession,

there was a large increase in stem IVNDFD from 30 to

60 d, followed by a great decline from 60 to 90 d.

Therefore, the accession PM39 was included in the

SLOW group, instead of the accession PM45.

Dry matter production

In our study, there was no treatment effect (P = 0Æ12) or

treatment · age interaction (P = 0Æ13) on total green

dry matter production (Table 4). However, the contrast



analysis demonstrated that the FAST group had higher

(P < 0Æ05) DM production with 90 d of regrowth

(Table 4). The error term for testing the main effect of

treatment on green DM production was genotype

(treatment), and there was large variation in average

production among the three genotypes within treat-

ments (CV = 16%), which could explain the lack of

treatment effect on DM production. Average green DM

productions were 6541, 7192 and 8249 kg ha)1 for the

genotypes on the SLOW group and 7312, 9906 and

10 409 kg ha)1 for the genotypes in the FAST group.

Plant morphology

There was no difference between the two treatments for

height, percentage of leaves or percentage of senescent

material (Table 4). Also, there was no difference for

leaf ⁄ stem ratio between treatments (Table 4).

Chemical composition

As expected, there was a significant treatment · age

interaction for stems IVNDFD (P < 0Æ05), with the FAST

group having lower (P < 0Æ05) IVNDFD with 90 d of

regrowth (Table 5). Apart from IVNDFD, there were

few differences between the two treatments for the

chemical composition of stems and leaves (Table 5).

There was a tendency (P = 0Æ10) for overall treatment

effect on stem lignin content, expressed as per cent (%)

of NDF, with the FAST group having greater average

lignin content (7Æ5 ± 0Æ3 vs. 6Æ7 ± 0Æ3 for the FAST and

SLOW groups, respectively). There was no effect of

treatment, age or treatment · age interaction on

IVNDFD or chemical composition of leaves (P > 0Æ10,

Table 5).

Table 3 Comparison of the slope of decline in stem in vitro

NDF digestibility of 11 guineagrass genotypes harvested at

three ages.

Genotypes

IVNDFD* (% NDF)Linear contrast†

(30–90 d)30 d‡ 60 d 90 d

Milenio 48Æ2 40Æ5 27Æ5 21Æ0a

Mombaca 44Æ5 47Æ3 25Æ8 18Æ8ab

Tanzania 47Æ2 44Æ7 28Æ6 18Æ6ab

PM46 47Æ8 34Æ3 30Æ7 17Æ1abc

PM40 44Æ3 51Æ3 31Æ2 13Æ1abc

PM44 40Æ4 40Æ0 29Æ1 11Æ4abc

PM41 45Æ4 48Æ8 34Æ1 11Æ3abc

PM39 50Æ0 47Æ9 38Æ8 11Æ2bc

PM47 49Æ1 48Æ1 39Æ6 9Æ4c

PM45 38Æ2 46Æ0 32Æ1 6Æ4c

Massai 39Æ7 43Æ4 34Æ0 5Æ8c

s.e.m.§ 2Æ6 2Æ7 3Æ2 3Æ8

*IVDNDF: In vitro neutral detergent fibre digestibility.†Values in the same column with different lowercase super-

script letters are significantly different by adjusted t-tests at

P < 0Æ05.‡Days of growth after levelling cut at 29 ⁄ 12 ⁄ 2005.§ s.e.m., Standard error of mean.

Table 4 Effect of treatment and maturity on mean herbage production, height and proportion of leaf, stem and dead material in

two groups of guineagrass genotypes separated according to the slope of decline in stem NDF digestibility with advance in maturity.

Variables

Treatment

s.e.m.‡

Significance (P)SLOW FAST

30 d† 60 d 90 d 30 d 60 d 90 d Trt§ Age T · A–

GDMP†† (kg ha)1) 2851 5726 13405b 3900 5060 18667a 1397 NS *** NS

Height (cm) 48 84 132 62 97 138 12 NS *** NS

Stem (%) 8 12 27 9 17 39 4 NS *** NS

Dead (%) – 6Æ9 15Æ7 – 6Æ8 11Æ6 2Æ8 NS * NS

LSR‡‡ 19Æ6 12Æ2 2Æ7 22Æ7 7Æ0 1Æ4 4Æ9 NS ** NS

†Days of growth after levelling cut at 29 ⁄ 12 ⁄ 2005.‡Standard error of the mean.§Treatment.–Interaction treatment · age.††Green dry matter production (total production – dead material).‡‡Leaf ⁄ stem ratio. Values in the same row with different lowercase superscript letters are significantly different by t-test at P < 0Æ05.

*P < 0.05; **P < 0.01; ***P < 0.001; NS, Non-significant; FAST, fast decline in NDF digestibility; SLOW, slow decline in NDF

digestibility.



Gene expression

Before comparing gene expression, it is necessary to

adjust for unequal efficiencies of cDNA amplification

(Yuan et al., 2006). In our study, all efficiencies were

similar (lcon · gene with P = 0Æ93) and not different

than two (all confidence intervals for slopes included

the number )1); therefore, an efficiency of two (100%)

was assumed for all genes (Table 6). Expression of the

housekeeping gene, GAPDH, was not affected by age

(P > 0Æ10) or by treatment (P > 0Æ10), demonstrating its

validity as a reference gene in this study (Table 7).

We were most interested in existing treatment · age

interactions for gene expression, which would indicate

that gene expression was differentially altered by

maturity between the two groups. There was a signif-

Table 5 Effect of treatment and maturity on chemical composition and in vitro NDF digestibility of stem and leaf tissue in two

groups of guineagrass genotypes separated according to the slope of decline in stem NDF digestibility with advance in maturity.

Variables†

Treatment

s.e.m.§


30 d‡ 60 d 90 d 30 d 60 d 90 d Trt– Age T · A††

Stem

CP, %DM 4Æ4 5Æ8a 3Æ5 4Æ7 4Æ7b 2Æ8 0Æ22 NS *** NS

NDF, %DM 77Æ9 80Æ9 84Æ4 76Æ2 79Æ5 83Æ1 0Æ65 NS *** NS

Lignin, %NDF 6Æ03 5Æ84 8Æ16b 6Æ25 6Æ44 9Æ86a 0Æ28 0Æ10 *** NS

IVNDFD, %NDF 46Æ3 46Æ5 37Æ5a 46Æ3 44Æ2 27Æ3b 1Æ6 NS *** **

Intercept 52Æ2 58Æ7 2Æ9 NS

Linear slope )4Æ4a )9Æ7b 1Æ3 *** **

Leaf

CP, %DM 9Æ0 9Æ2 6Æ9 9Æ4 9Æ4 6Æ9 0Æ4 NS *** NS

NDF, %DM 75Æ4 76Æ8 74Æ5 74Æ3 76Æ7 74Æ2 1Æ0 NS * NS

Lignin, %NDF 5Æ17 5Æ87 6Æ70 5Æ04 5Æ78 6Æ21 0Æ28 NS *** NS

IVNDFD, %NDF 37Æ0 34Æ5 33Æ4 39Æ0 37Æ3 33Æ8 3Æ0 NS NS NS

†CP, Crude protein, NDF, Neutral detergent fibre, IVDNDF, In vitro neutral detergent fibre digestibility; *P < 0.05; **P < 0.01;

***P < 0.001; NS, Non-significant; FAST, fast decline in NDF digestibility; SLOW, slow decline in NDF digestibility.‡Days of growth after levelling cut at 29 ⁄ 12 ⁄ 2005.§Standard error of the mean.–Treatment.††Interaction treatment · age. Values in the same row with different lowercase superscript letters are significantly different by t-test

at P < 0Æ05.

Table 6 Efficiency of gene amplification using real-time polymerase chain reaction.

Genes*

Slope

coefficient†

95% confidence interval Amplification

efficiency

(E = 2()1 ⁄ slope))

Percentage

amplification

efficiencyMinimum Maximum

GAPDH )1Æ1 )1Æ2 )0Æ9 1Æ9 0Æ95

4CL )1Æ2 )1Æ9 )0Æ5 1Æ8 0Æ84

C4H )1Æ1 )1Æ7 )0Æ4 1Æ9 0Æ93

CAD )1Æ0 )1Æ4 )0Æ6 2Æ0 1Æ01

CCR )1Æ0 )1Æ4 )0Æ5 2Æ1 1Æ05

CCoAOMT )1Æ1 )1Æ3 )0Æ9 1Æ9 0Æ91

PAL )1Æ0 )1Æ2 )0Æ9 2Æ0 0Æ98

*GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 4CL, 4-coumarate-CoA ligase; C4H, cinnamate-4-hydroxylase; CAD,

cinnamyl alcohol dehydrogenase; CCR, cinnamoyl-CoA-reductase; CCoAOMT, caffeoyl-CoA O-methyltransferase; PAL, phenyl-

alanine ammonia lyase.†Linear slope between the Log2 of the cDNA concentration and threshold cycle.



icant treatment · age interaction for the expression of

the genes C4H and CCoAOMT (Table 8).

The decomposition of the interaction by the SLICE

option of SAS revealed that expression of CCoAOMT

was altered by age only at the FAST group, with no

change in the expression of CCoAOMT with advance in

maturity at the SLOW group (Table 8, Figure 1). There

was a great increase in CCoAOMT expression from 30

to 60 d in the FAST group, followed by a decline in

expression from 60 to 90 d (Figure 1).

For the expression of C4H, the decomposition of the

interaction demonstrated that there was a steady

increase in C4H expression with maturity in the SLOW

group, C4H expression at 60 d was similar to expression

at 30 or 90 d and expression at 90 d was higher than

expression at 30 d of regrowth (Figure 1). In the FAST

group, there was a great increase in C4H expression

from 30 to 60 d of regrowth, with expression at 60 d

being higher than that at 30 d and similar to 90 d of

regrowth (Figure 1).

The expression of 4CL and CCR was altered by age

(P < 0Æ05), but there was no effect of treatment or of

treatment · age interaction (Table 8). Expression of 4CL

increased with age at both treatments, while expression

of CCR decreased from 30 to 60 d and increased from

60 to 90 d in both groups (Table 8).

Table 7 Threshold cycle for real-time PCR detection (CT) of the control gene GAPDH.

Age

Treatment


30 d* 60 d 90 d 30 d 60 d 90 d Trt† Age T · A‡

CT-GAPDH§ 21Æ2 21Æ8 22Æ1 22Æ6 22Æ0 21Æ3 NS NS NS

*Days of growth after levelling cut at 29 ⁄ 12 ⁄ 2005.†Treatment.‡Interaction treatment · age.§GAPDH=glyceraldehyde 3-phosphate dehydrogenase.

NS, Non-significant; FAST, fast decline in NDF digestibility; SLOW, slow decline in NDF digestibility.

Table 8 Probability of main effects and interaction on expression of genes from the lignin synthesis pathway in two groups of

guineagrass genotypes separated according to the slope of decline in stem NDF digestibility with advance in maturity.

Genes†

Treatments

s.e.m.§

Significance

Slice

SLOW FAST Age (Trt)–

30 d‡ 60 d 90 d 30 d 60 d 90 d Trt# Age Trt · Age†† SLOW FAST

DDCt‡‡ P

4CL 0Æ0 )0Æ3 )1Æ7 0Æ0 )0Æ5 0Æ3 1Æ5 NS ** NS

C4H 0Æ0 )0Æ5B )1Æ8 0Æ0 )1Æ7A )0Æ9 0Æ5 NS ** * ** *

CAD 0Æ0 0Æ6 0Æ5 0Æ0 )0Æ4 )0Æ2 0Æ6 NS NS NS

CCR 0Æ0 0Æ7 )0Æ7 0Æ0 1Æ2 )1Æ0 0Æ8 NS * NS

CCoAOMT 0Æ0 1Æ6B 0Æ5 0Æ0 )2Æ6A )1Æ3 1Æ3 NS NS ** NS *

PAL 0Æ0 1Æ0 1Æ5 0Æ0 )1Æ1 0Æ8 1Æ2 NS NS NS

†4CL, 4-coumarate-CoA ligase; C4H, cinnamate-4-hydroxylase; CAD, cinnamyl alcohol dehydrogenase; CCR, cinnamoyl-CoA-

reductase; CCoAOMT, caffeoyl-CoA O-methyltransferase; PAL, phenylalanine ammonia lyase.‡Days of growth after levelling cut at 29 ⁄ 12 ⁄ 2005.§Standard error of the mean.–Decomposition of the Trt · age interaction through the SLICE option of SAS, testing for the effect of Age within Trt.#Treatment.††Interaction treatment · age.‡‡DDCt = (Cttarget gene unknown sample ) CtGAPDH unknown sample) ) (Cttarget gene calibrator sample ) CtGAPDH calibrator sample). Values in the

same row with different upper case superscript letters are significantly different by t-test at P < 0Æ05.

*P < 0.05; **P < 0.01; NS, Non-significant; FAST, fast decline in NDF digestibility; SLOW, slow decline in NDF digestibility.



There was no effect of age, treatment or of treat-

ment · age interaction on the expression of the genes

CAD and PAL (Table 8).

Discussion

Warm-season grasses produce more edible dry matter

but are typically low in digestibility (Reid et al., 1988).

The lower forage nutritive value at a given stage of

maturity in tropical forages is mainly attributed to a

relatively low leaf-to-stem ratio and rapid rates of

maturation (Jones, 1985). The major effect of this rapid

maturity rate is a fast increase in NDF content concom-

itantly with an increase in lignification of the cell wall,

which leads to lower DM digestibility and also lower

NDF digestibility. The experiment tested the hypothesis

that the rate of decline in stem fibre digestibility with

advance in maturity is controlled by differential expres-

sion of genes from the lignin biosynthesis pathway. The

results indicate that expression of two genes,

CCoAOMT and C4H, is important in determining the

decline in fibre digestibility with maturity.

Dry matter production

Greater DM production is commonly associated with

fast decline in stem digestibility, and genotypes with

faster stem elongation are usually the ones with higher

DM production (Bregard et al., 2001). Also, rapid

decline in digestibility of tropical grasses is often

associated with stem elongation and higher percentage

of stem in the total dry matter (Cherney et al., 1993).

Our results agreed in part with this idea, because the

genotypes with fast decline in stem digestibility had

greater DM production after 90 d of regrowth. How-

ever, there was no overall difference between treat-

ments when the three ages were considered.

It has been reported that the cultivars Tanzania and

Mombaca, which have rapid stem elongation, usually

reach 95% of light interception (critical leaf area index)

between 90 and 100 cm of height (Carnevalli et al.,

2006), which in our study corresponded to the age of

60 d. These cultivars with the capacity of rapid stem

elongation will also be capable of maintaining greater

green dry matter production after a long period of

growth, as seen in our study.

Most management or environmental factors that

increase total DM production, such as temperature

and maturity at harvest, usually decrease plant nutri-

tive value (Neel et al., 2008; Nordheim-Viken et al.,

2009). However, it is possible to breed, or to select

forages, for higher DM digestibility without affecting

DM production (Jank et al., 1994; Casler and Vogel,

1999). The modern cultivars of guineagrass, such as

Tanzania and Mombaca, were selected for commercial

release among other accessions in the population,

because of their higher DM production, higher leaf

production and better nutritive value than older culti-

vars (Jank et al., 1994). The development of ‘Tifton 78’

also demonstrated that it is feasible to improve both

yield and dry matter digestibility in bermudagrass

(Burton and Monson, 1988; Hill et al., 1993).

Plant morphology

The leaf ⁄ stem ratio is the most common trait used to

evaluate tropical forage nutritive value in a breeding

programme, because voluntary feed intake of foraging

(b)

(a)

Figure 1 Data reported as least-squares mean ± s.e.m. (a)

Alterations in mRNA abundance of caffeoyl-CoA O-methyl-

transferase in the stems of Panicum maximum genotypes

separated into two groups with either slow or fast decline in

NDF digestibility with advance in maturity. Tissues were

obtained with 30, 60 and 90 d after a levelling cut. Data were

analysed by the 2)DDCt method with 30 d as the reference

expression point. (b) Alterations in mRNA abundance of

cinnamate-4-hydroxylase (C4H) in the stems of P. maximum

genotypes separated into two groups with either slow or fast

decline in NDF digestibility with advance in maturity. Significant

differences (P < 0.05) among days within the SLOW treatment

are represented by different capital letters, and significant

differences (P < 0.05) among days within the FAST treatment

are represented by different lowercase letters.



cattle is usually directly related to percentage of leaves

on the pasture; foraging cattle eat more leaves than

stems (Minson, 1990). This happens because leaves

have usually higher nutritive value than stems (Allison,

1985); moreover, leaves loose nutritive value less

intensely with maturity (Hacker and Minson, 1981;

Stabile et al., 2010). However, immature stems are high

in nutritive value, sometimes even higher than leaves

on the same date (Minson, 1990; Stabile et al., 2010).

Our results suggest that the rate of decline in stem

IVNDFD of guineagrass genotypes is not related to

leaf ⁄ stem ratio. Similarly, in timothy (Phleum pratense

L.), selection for lower ratio of acid detergent lignin

(ADL) to cellulose (ADL ⁄ CEL) reduced ADL and NDF

concentrations and increased DM digestibility of stems,

but did not change leaf ⁄ stem ratio or other aspects of

plant morphology, nor did it change any leaf charac-

teristics (Claessens et al., 2005).

Chemical composition

When comparing different genotypes of tropical for-

ages, it is common to encounter greater differences for

stem than for leaf nutritive value (Buxton and Redf-

earn, 1997). Likewise, maturity has a much greater

effect on stem than on leaf nutritive value (Griffin and

Jung, 1983). This could be clearly demonstrated in our

study. The genotypes with higher lignin and lower fibre

digestibility of the stems did not differ for leaf compo-

sition. Therefore, these results support the idea that

breeding or selecting for forage quality should focus also

on stem, rather than only on leaf nutritive value (Casler

and Carpenter, 1989).

Maturity has a great effect on stem digestibility. After

the plant reaches 95% of light interception, there is a

rapid decline in nutritive value (Carnevalli et al., 2006),

and therefore, farmers should plan to graze or harvest

tropical forages at young ages. However, even in those

farms that adopt rotational grazing with electric fences,

it is not always feasible to adjust stock density according

to forage availability, because of the great variability of

forage growth rate during the summer. Consequently,

farmers would greatly benefit from the development of

cultivar that maintained high nutritive value during

longer periods.

Gene expression

To minimize undesired side effects and improve success

of genetic modifications, it is important to identify the

points of regulation of the lignin biosynthesis pathway.

As in other metabolic pathways, not all enzymes are

transcriptionally regulated when there is a change in

monolignol synthesis in the plant. It was our hypothesis

that the decline in stem nutritive value is related to

differential expression of specific genes from the lignin

synthesis pathway. Among the six genes studied, only

the expression of CCoAOMT and C4H was differentially

affected by age between the two treatments.

Caffeoyl-CoA O-methyltransferase catalyses the

methylation of caffeoyl-CoA to feruloyl-CoA and 5-

hydroxyferuloyl-CoA to sinapoyl-CoA and is believed

to occupy a pivotal position in the lignin biosynthetic

pathway (Pincon et al., 2001) and probably also cross-

linking in grasses (Ralph et al., 2004). Cinnamate 4-

hydroxylase enzymes, which belong to the cytochrome

P450 enzyme family, catalyse the first hydroxylation

step in the phenylpropanoid pathway with the produc-

tion of p-coumaric acid from cinnamic acid (Riboulet

et al., 2009).

Our results indicated that the greater increase in the

expression of C4H and CCoAOMT in the FAST group

with 60 d of regrowth, when compared with the SLOW

group, could indicate greater rate of monolignol syn-

thesis during this period. For the SLOW group, there

was no effect of age on the expression of CCoAOMT

over the 90 d studied, a striking difference from the

sharp increase in CCoAOMT expression after 60 d of

regrowth in the FAST group. This increase in monolig-

nol synthesis around 60 d of regrowth could be

responsible for the greater decline in IVNDFD from 60

to 90 d observed in the FAST group.

In other grasses, such as maize, most of the genes

involved in monolignol biosynthesis belong to small

multigene families (Barriere et al., 2009). Barriere et al.

(2009) identified five CCoAOMT genes, two C4H genes,

six PAL genes, five 4CL genes, seven CCR genes and six

CAD genes in the maize genome. Heath et al. (1998)

identified three COMT-like cDNA homologues from

perennial ryegrass (LpOMT1, LpOMT2 and LpOMT3),

which are differentially expressed in young or mature

stems. Also working with perennial ryegrass, Tu et al.

(2010) observed that an upregulation in OMT expres-

sion was correlated with lignin deposition during the

reproductive stage; however, this effect was specific for

the LpOMT1 gene.

In maize, quantification of expression of CCoAOMT

genes in different tissues during development suggests

that CCoAOMT3 and CCoAOMT4 are of little impor-

tance for stem lignification (Riboulet et al., 2009), while

CCoAOMT2 is highly expressed at the stem (Guillaumie

et al., 2007). The different members of the CCoAOMT

multigene family have not been described in guinea-

grass; however, based on sequence similarity with

maize, the observed different expression of CCoAOMT

in our study probably refers to CCoAOMT1 and

CCoAOMT2.

Expression of C4H was also correlated with lower

fibre digestibility of the stems in our study. For the

genotypes in the SLOW group, with a lesser decline in



IVNDFD with advance in maturity, there was a linear

increase in C4H expression with age, with the expres-

sion being greater at 90 d of regrowth than at 30 d,

while expression at 60 d was intermediate. The greater

expression of C4H at 90 d in the SLOW group, in

contrast to the greater expression of C4H at 60 d for the

FAST group, suggests that there may be a delay in

monolignol biosynthesis in this group, which is related

to the slower rate of lignifications of the stem.

A similar pattern of C4H expression was reported in

the ear internode in maize (Riboulet et al., 2009), where

C4H had increased expression from silking to 8 d after

silking, followed by a decline in expression at 15 d after

silking. Data from Guillaumie et al. (2007) demonstrate

that C4H2 has a tendency to be more highly expressed

in all organs of young maize plants, while C4H1 is by far

the predominant gene during late stem development.

Sequence similarity suggests that our primers are

detecting C4H1 expression in guineagrass.

Studies with genetically engineered plants have

clearly demonstrated the importance of these two

genes: CCoAOMT and C4H in monolignol biosynthesis.

Downregulation of C4H in alfalfa decreases lignin

deposition and reduces or eliminates the needs for

chemical pre-treatment in the production of ferment-

able sugars (Chen and Dixon, 2007). In tobacco,

downregulation of C4H also reduced lignin deposition

(Sewalt et al., 1997). In maize, expression of C4H and

other genes from the lignin synthesis pathway was

much more expressed in younger than in older already

lignified internodes (Guillaumie et al., 2008).

Downregulation of COMT-like genes in other grasses,

such as maize, wheat and ryegrass, has shown decrease

in lignin concentration and increase in IVNDFD (Pique-

mal et al., 2002; Ma and Xu, 2008; Tu et al., 2010).

Downregulation of CCoAOMT in alfalfa and Arabidop-

sis reduced lignin content, with a significant reduction

in guaiacyl (G) units and almost no effect on syringyl

(S) unit yield, leading to an increased S ⁄ G ratio (Chen

et al., 2006; Do et al., 2007).

Lignin biosynthesis can also be modulated by other

genes. It has been demonstrated that downregulation of

CAD in Festuca arundinacea decreases lignin content and

increases DM digestibility 7Æ2–9Æ5% (Chen et al., 2003).

Also, a greater decrease in lignin content (50%) was

reported for transgenic tobacco plants with downregu-

lation of CAD and CCR expression (Chabannes et al.,

2001). In our study, we did not see an association

between expression of CAD, CCR, 4CL and PAL with

the rate of decline in stem fibre digestibility of guinea-

grass genotypes. As mentioned before, because of the

existence of multiple genes coding for these proteins, it

is possible that some members of the multigene families

not amplified with our primers have different profiles of

expression than described here.

Rapid decrease in fibre digestibility limits the nutri-

tional value of tropical forages. Increase in mRNA

expression of enzymes from the lignin synthesis path-

way may be responsible for the decrease in fibre

digestibility. This study demonstrates that genotypes

with fast decrease in NDF digestibility of the stems with

advance in maturity also have different patterns of C4H

and CCoAOMT expression, with earlier increase in the

expression of these two genes than genotypes with low

decrease in NDF digestibility. The results from this study

have possible implication for forage breeding or devel-

opment of transgenic technologies in forage plants.

Acknowledgments

The study was funded by FAPESP (Fundacao de

Amparo a Pesquisa do Estado de Sao Paulo).

References

ALL ISONLLISON C.D. (1985) Factors affecting forage intake by

range ruminants: a review. Journal of Range Management,

38, 305–311.

AOAC. (1997) Official methods of analysis, 16th edn. Gai-

thersburg: Association of Official Analytical Chemists.

ARAUJOR AUJO A.C.G., NO BREGAOBRE GA J.M., POZZO BONOZZOBON M.T. and

CARNEIROAR NE IRO V.T.C. (2005) Evidence of sexuality in

induced tetraploids of Brachiaria brizantha (Poaceae).

Euphytica, 144, 39–50.

BARR IER EARRI E RE Y., ME CHI NECHIN V., LAFAR GUETTEAFARGUETTE F., MANI CACC IANICACCI

D., GUI LLO NUILLON F., WANGANG H., LAURESSERGUESAURESSERGUES D., PICHONI CH ON

M., BOSIOO SI O M. and TATOUTATOUT C. (2009) Toward the discovery

of maize cell wall genes involved in silage maize quality

and capacity to biofuel production. Maydica, 54, 161–198.

BREGARDRE GARD A., BELANGERELANGER G., MICHAUDICHAUD R. and TRE MBLAYREMB LAY

F. (2001) Biomass partitioning, forage nutritive value,

and yield of contrasting genotypes of timothy. Crop

Science, 41, 1212–1219.

BURTONURTON G.W. and MONSONO NSON W.G. (1988) Registration of

‘Tifton 78’ bermudagrass. Crop Science, 28, 187–188.

BUXTONUXTON D.R. and REDFEARNEDFEARN D.D. (1997) Plant limitations

to fiber digestion and utilization. Journal of Nutrition, 127,

814S–818S.

CAM PB ELLAMPBELL M.M. and SEDE ROFFEDEROFF R.R. (1996) Variation in

lignin content and composition. Plant Physiology, 110,

3–13.

CARNEVALLIARNEVALL I R.A., DAA SI LVAILVA S.C., BUENOUENO A.A.O., UEBE LEE BELE

M.C., BUE NOUENO F.O., HO DG SONODGSON J., SI LVAILVA G.N. and

MORAISORAIS J.P.G. (2006) Herbage production and grazing

losses in Panicum maximum cv. Mombaca under four

grazing managements. Tropical Grasslands, 40, 165–176.

CASLERASLE R M.D. and CAR PE NTE RARPENTER J.A. (1989) Morphological

and chemical responses to selection for in vitro dry

matter digestibility in smooth bromegrass. Crop Science,

29, 924–928.

CASLERASLE R M.D. and VOGELOGE L K.P. (1999) Accomplishments

and impact from breeding for increased forage nutritional

value. Crop Science, 39, 12–20.



CHAB ANNESHABANNE S M., BARAKATEARAKATE A., LAPI ERREAPIE RRE C., MAR ITAARITA

J.M., RALPHALPH J., PE ANEAN M., DANOUNANOUN S., HALP INALP IN C.,

GRIM AR IMA-PETTENATIETTE NATI J. and BOUDE TO UD ET A.M. (2001) Strong

decrease in lignin content without significant alteration

of plant development is induced by simultaneous down-

regulation of cinnamoyl CoA reductase (CCR) and

cinnamyl alcohol dehydrogenase (CAD) in tobacco

plants. Plant Journal, 28, 257–270.

CHENHE N F. and DIXONI XON R.A. (2007) Lignin modification

improves fermentable sugar yields for biofuel production.

Nature Biotechnology, 25, 759–761.

CHENHE N L., AUHUH C.K., DOWLINGOWLI NG P., BE LLELL J., CHENHEN F.,

HOPKINSO PK I NS A., DI XONIXON R. and WANGANG Z.Y. (2003) Improved

forage digestibility of tall fescue (Festuca arundinacea) by

transgenic down-regulation of cinnamyl alcohol

dehydrogenase. Plant Biotechnology Journal, 1, 1–13.

CHENHE N F., REDDYEDDY M.S.S., TEMPLEEM PLE S., JACKSONACKSON L., SHADLEHADLE

G. and DI XONIXON R.A. (2006) Multi-site genetic modulation

of monolignol biosynthesis suggests new routes for

formation of syringyl lignin and wall-bound ferulic acid

in alfalfa (Medicago sativa L.). Plant Journal, 48, 113–124.

CHERNEYHE RNEY D.J.R., CHERNEYHERNEY J.H. and LUCE YUCEY R.F. (1993) In

vitro digestion kinetics and quality of perennial grasses as

influenced by forage maturity. Journal of Dairy Science, 76,

790–797.

CHOMCZYNSKIHOM CZYNSKI P. and SACC HIACCHI N. (1987) Single-step

method of RNA isolation by acid guanidinium

thiocyanate-phenol-chloroform extraction. Analytical

Biochemistry, 162, 156–159.

CLAESSENSLAE SSE NS A., MICHAUDICHAUD R., BELANGERELANGER G. and MATHERATHER

D. (2005) Leaf and stem characteristics of timothy plants

divergently selected for the ratio of lignin to cellulose.

Crop Science, 45, 2425–2429.

DOO C.T., POLLETOLLE T B., THEVENI NHEVENIN J., SIBOUTIB OUT R., DENOUEENOUE D.,

BARR IER EARRIE RE Y., LAPIE RREAPI ERRE C. and JOUANINOUANIN L. (2007) Both

caffeoyl Coenzyme A 3-O-methyltransferase 1 and

caffeic acid O-methyltransferase 1 are involved in

redundant functions for lignin, flavonoids and sinapoyl

malate biosynthesis in Arabidopsis. Planta, 226, 1117–

1129.

GI LLILL J.L. (1978) Design and analysis of experiments in the

animal and medical sciences. Ames, IA: Iowa State Univ.

Press.

GO ERI NGOE RING H.K. and VANAN SOESTOE ST P.J. (1970) Forage fiber

analysis (apparatus, reagents, procedures, and some applica-

tions). Washington, DC: USDA Agric. Handb. 379. U.S.

Gov. Print. Office.

GR IFF INRIFF IN J.L. and JUNGUNG G.A. (1983) Leaf and stem forage

quality of big bluestem and switchgrass. Agronomy

Journal, 75, 723–726.

GUILLAUM IEUILLAUMIE S., SANAN-CLEMENTELEMENTE H., DESWARTEESWARTE C.,

MARTINEZARTI NE Z Y., LAPIE RREAPIER RE C., MURIGNE UXURIGNEUX A., BARRIEARRI E ‘RERE

Y., PI CH ONICHON M. and GOFFNEROFFNER D. (2007) MAIZEWALL.

Database and developmental gene expression profiling of

cell wall biosynthesis and assembly in maize. Plant

Physiology, 143, 339–363.

GUILLAUM IEUILLAUMIE S., GOFFNEROFFNER D., BARBIE RARB IER O., MARTINANTARTI NANT

J.P., PICHONI CH ON M. and BARRIE REARRI ERE Y. (2008) Expression of

cell wall related genes in basal and ear internodes of

silking brown-midrib-3, caffeic acid O-methyltransferase

(COMT) down-regulated and normal maize plants. BMC

Plant Biology, 8, 71–87.

HACKERACKE R J.B. and MINSONINSON D.J. (1981) The digestibility of

plant parts. Herbage Abstracts, 51, 459–482.

HEATHEATH R., HUXLE YUXLEY H., STONETONE B. and SPANGE NBERGPANGENBE RG G.

(1998) cDNA cloning and differential expression of three

caffeic acid O-methyltransferase homologues from

perennial ryegrass (Lolium perenne). Journal of Plant

Physiology, 153, 649–657.

HI LLILL G.M., GATESATES R.N. and BURTONURTON G.W. (1993) Forage

quality and grazing steer performance from Tifton 85 and

Tifton 78 bermudagrass pastures. Journal of Animal

Science, 71, 3219–3225.

ISKANDARSKANDAR H.M., SI MP SO NIMPSON R.S., CASUASU R.E., BO NN ETTONNE TT

G.D., MACLE ANACLEAN D.J. and MANNERSANNERS J.M. (2004)

Comparison of reference genes for quantitative real-

time polymerase chain reaction analysis of gene

expression in sugarcane. Plant Molecular Biology Reports,

22, 325–337.

JANKANK L., SAVIDANAVIDAN Y.H., SOUZAO UZA M.T.C. and COSTAO STA J.C.G.

(1994) Avaliacao do germoplasma de Panicum maximum

introduzida da Africa. I: Producao forrageira. Brazilian

Journal of Animal Science, 23, 433–440.

JONESONE S C.A. (1985) C4 grasses and cereals, growth, development

and stress response. New York: John Wiley & Sons.

JUNGUNG H.G. and CASLE RASLER M.D. (2006) Maize stem tissues:

cell wall concentration and composition during

development. Crop Science, 46, 793–1800.

JUNGUNG H.G. and DE E TZEE TZ D.A. (1993) Cell wall lignification

and degradability. In: Jung H.G. (Ed) Forage cell wall

structure and digestibility, pp. 315–346. Madison, WI, USA:

ASA, CSSA, SSSA.

LIVAKI VAK K.J. and SCHM ITTGENCHMITTGEN T.D. (2001) Analysis of

relative gene expression data using real-time quantitative

PCR and the 2)DDCT method. Methods, 25, 402–408.

MAA Q.H. and XUU Y. (2008) Characterization of a caffeic acid

3-O-methyltransferase from wheat and its function in

lignin biosynthesis. Biochimie, 90, 15–524.

MINSONINSON D.J. (1990) Forage in ruminant nutrition. San Diego,

CA: Academic Press.

MUIRUI R J.P. and JANKANK L. (2004) Guineagrass. In: Moser L.E.

(Ed) Warm-season (C4) grasses, pp. 589–621. Madison, WI,

USA: ASA, CSSA, SSSA.

NE ELEE L J.P.S., FELDHAKEELDHAKE C.M. and BELE SKYELESKY D.P. (2008)

Influence of solar radiation on the productivity and

nutritive value of herbage of cool-season species of an

understorey sward in a mature conifer woodland. Grass

Forage Science, 63, 38–47.

NELSONELSON C.J. and MOSE ROSER L.E. (1994) Plant factors affecting

forage quality. In: Fahey G.C. Jr (Ed.) Forage quality,

evaluation and utilization, pp. 115–154. Madison, WI, USA:

ASA, CSSA and SSSA.

NORDHEIMORDHE IM-VI KE NIKEN H., VO LD E NOLDE N H. and JO RGE N SE NORGENSEN M.

(2009) Effects of maturity stage, temperature and

photoperiod on growth and nutritive value of timothy

(Phleum pratense L.). Animal Feed Science and Technology,

152, 204–218.

PINCONI NCON G., MAURYAURY S., HOFFM ANNOFFMANN L., GEOFFROYEO FFROY P.,

LAPIE RREAPI ERRE C., POLLETOLLET B. and LEGRANDEGRAND M. (2001)

Repression of O-methyltransferase genes in transgenic



tobacco affects lignin synthesis and plant growth.

Phytochemistry, 57, 1167–1176.

PIQUE MALI QU EMAL J., CHAMAYOUHAMAY OU S., NADAUDADAUD I., BECKE RTECKERT M.,

BARRI EREARRI E RE Y., MI LAILA I., LAPIE RREAPIE RRE C., RIGAUI GA U J.,

PUI GDOME NECHUIGDOMENECH P., JAUNEAUAUNEAU A., DIGONNE TIGONNET C., BOUDE TOUDET

A.M., GOFFNE ROFFNER D. and PICHONICHON M. (2002)

Downregulation of caffeic acid O-methyltransferase in

maize revisited using a transgenic approach. Plant

Physiology, 130, 1675–1685.

RALPHALPH J., LUNDQUISTUNDQUIST K., BR UNOWRUNO W G., LUU F., KIMI M H.,

SCHATZCHATZ P.F., MARITAAR ITA J.M., HATFIELDATFIELD R.D., RALPHALPH S.A.,

CHRI STE NSE NHRISTENSEN J.H. and BOERJANOE RJAN W. (2004) Lignins:

natural polymers from oxidative coupling of 4-

hydroxyphenylpropanoids. Phytochemistry, 3, 29–60.

RE IDE ID R.L., JUNGUNG G.A. and THAYNEHAY NE W.V. (1988)

Relationships between nutritive quality and fiber

components of cool season and warm season forages: a

retrospective study. Journal of Animal Science, 66, 1275–

1291.

RIBO ULE TIBOULET C., GUILLAUM IEUI LLAUMIE S., MECHINECHIN V., BOSIOOSIO M.,

PICHONICHON M., GOFFNEROFFNER D., LAPIE RREAPIER RE C., POLLETOLLE T B.,

LEFEV REE FE VRE B., MARTINANTARTINANT J.P. and BARRI EREARRI E RE Y. (2009)

Kinetics of phenylpropanoid gene expression in maize

growing internodes: relationships with cell wall

deposition. Crop Science, 49, 211–223.

SEWALTEWALT V.J.H., NII W.T., BLOUNTLO UNT J.W., JUNGUNG H.G.,

MASOUDASOUD S.A., HOWLESO WLE S P.A., LAM BAMB C. and DI XONIXO N R.A.

(1997) Reduced lignin content and altered lignin

composition in transgenic tobacco down-regulated in

expression of L-phenylalanine ammonia-lyase or

cinnamate 4-hydroxylase. Plant Physiology, 115, 41–50.

STABI LETABILE S.S., SALAZARALAZAR D.R., JANKANK L., RENNOENNO F.P. and

SI LVAILVA L.F.P. (2010) Characteristics of nutritional quality

and production of genotypes of guineagrass harvested in

three maturity stages. Brazilian Journal of Animal Science,

39, 1418–1428.

TI LLEYILLEY J.M. and TERRYE RRY R.A. (1963) A two-stage technique

for the in vitro digestion of forage crops. Journal of the

British Grassland Society, 18, 104–111.

TUU Y., ROCHFORTO CHFOR T S., LIUI U Z., RANAN Y., GRI FFI THRIFF ITH M., BADE N-ADEN-

HO RSTHORST P., LOUIEOUI E G.V., BOWMANO WM AN M.E., SM ITHMITH K.F., NOELOE L

J.P., MOURADOVOURADOV A. and SPANGE NBERGPANGENBERG G. (2010)

Functional analyses of caffeic acid O-methyltransferase

and cinnamoyl-CoA-reductase genes from perennial

ryegrass (Lolium perenne). Plant Cell, 22, 3357–3373.

VANAN SOE STOEST P.J. (1994) Nutritional ecology of the ruminant.

Ithaca: Cornell University Press.

VANAN SOE STOEST P.J., ROBERTSONOBER TSON J.B. and LEWISEWIS B.A. (1991)

Methods for dietary fiber, neutral detergent fiber, and

nonstarch polysaccharides in relation to animal

nutrition. Journal of Dairy Science, 74, 3583–3597.

YUANUAN J.S., RE EDEE D A., CHENHEN F. and STEWARDTEWARD C.N. JRR (2006)

Statistical analysis of real-time PCR data. BMC

Bioinformatics, 7, 85–97.



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