Elisabeth Grenet

Effect of modification of the O-methyltransferase activity on cell wall composition, ultrastructure and degradability of transgenic tobacco

The effect of O-methyltransferase (OMT) cDNA modulation on cell wall composition, ultrastructure and rumen degradability was measured on transgenic tobacco (Nicotiana tabacum). The expression of OMT cDNA in antisense orientation (AS) inhibited OMT activity by 92% whereas expression of sense constructs led to plants either co-suppressed (CS, 98% inhibition) or overexpressing OMT activity. The cell wall residues of stems were analysed for lignin content, products of nitrobenzene oxidation (NBO) and polysaccharide content. Degradability was determined by a cellulase method. Sections of stem were stained by acid phloroglucinol and Maule reactant. Stem samples were incubated in the rumen for 8, 24 and 48 h and observed by scanning electron microscopy (SEM). Compared to controls, OMT-depleted stems showed decreased hemicellulose content but unchanged lignin content. In contrast, syringyl units decreased by 40 and 90% in AS and CS samples respectively and NBO content followed a similar trend. Dry matter cellulase degradability was significantly improved by 3.5 and 5.6 percentage units in AS and CS samples respectively. SEM showed a greater bacterial colonisation in these samples and indicated a higher rate of rumen degradability in CS tissues than in controls. Overexpressing plants had a composition and a degradability similar to that of controls. For all the plants studied, the improvements in dry matter degradability were closely linked to the syringyl to guaiacyl ratio or to the NBO content. The modifications observed in down-regulated tobacco were similar to those produced by bm3 maize mutation, but without lignin decrease. Genetic modifications should therefore be considered for improving forage digestibility.

Rumen anaerobic fungi and plant substrate colonization as affected by diet composition

Abstract The influence of diet composition on rumen fungal population was studied by offering successively to a rumen fistulated cow 11 different diets rich in fibre, starch or soluble carbohydrates. At the same time, the colonization of four different plant substrates introduced into the rumen in nylon bags (soya bean teguments, lucerne stems, maize stalks and beet pulps) was investigated. The population of rumen anaerobic fungi, particularly abundant with lignocellulose-rich diets, decreased with starch or soluble-carbohydrate-rich diets; the fungi selectively colonized the plant tissues with thick or lignified cell walls of soya bean tegument, lucerne or maize stalks, but seldom the beet pulp. The development of rumen fungi depended both on the substrate on which they became attached and on the ruminal medium. The fungi, which were normally present in the duodenum, caecum and faeces, were eliminated by the unfavourable diet but rapidly proliferated once the feed ingredients became more favourable.

Colonization of thick-walled plant tissues by anaerobic fungi

Abstract An abundant colonization of thick-walled tissues, lignified in the case of wheat straw and maize stem, or non-lignified in the case of soya-bean hulls, by anaerobic rumen fungi was observed during an ultrastructural study of the degradation of plant cell walls in the rumen. It is suggested that fungi are able to digest cellulose and hemicellulose even when those carbohydrates are present in lignified cell walls.

Immunogold labelling of xylans and arabinoxylans in the plant cell walls of maize stem

Summary— Polyclonal antibodies against 4‐O‐methyl‐glucuronoxylan and α L‐1‐3 arabinofuranosyl poly‐β‐d‐1‐4‐xylopyranosyl were raised from rabbits. An immunocytochemical technique was used to localize xylans and arabinoxylans in the plant cell walls of the apical internode of two maize lines of different digestibility. The sclerenchyma, fibres and xylem (lignified tissues) and the parenchyma (non‐lignified tissue) were studied. The arabinoxylans were more heavily labelled than the xylans in the lignified tissues of the less digestible maize whereas in the more digestible line the labelling of the two polysaccharides was similar. The xylans and arabinoxylans were localized in the secondary cell wall. In both maize lines, labelling increased from the base upwards of the apical internode, reflecting the changes in growth stage.

Degradation of wheat straw and maize stem by a monocentric and a polycentric rumen fungi, alone or in association with rumen cellulolytic bacteria

Abstract Two rumen anaerobic fungi, Neocallimastix frontalis MCH3 and Orpinomyces (Neocallimastix) joyonii TP 90-9 were cultured on wheat straw or maize stem alone or in association with one of two rumen cellulolytic bacteria, Ruminococcus flavefaciens 007 and Fibrobacter succinogenes S85. Observations by scanning electron microscopy showed that the behaviour of the different microorganisms was qualitatively similar. The phloem and inner parenchyma of the wheat straw and maize stem were completely degraded after 48 h in both the monocultures and cocultures. Quantitatively, the two fungal species degraded comparable amounts of wheat straw and maize stem as the two cellulolytic bacteria. In the coculture of N. frontalis or O. joyonii with F. succinogenes , wheat straw or maize stem were digested to the same extent as in the fungal monocultures. The associations of N. frontalis with R. flavefaciens on the two substrates and of O. joyonii with R. flavefaciens on wheat straw were less efficient than the fungal monocultures. R. flavefaciens exerted an inhibitory effect on the cellulolytic activity of N. frontalis and O. joyonii while F. succinogenes did not interact with the two fungal species.

Immunocytochemical localisation of para-coumaric acid and feruloyl-arabinose in the cell walls of maize stem

Two phenolic compounds, p-coumaric acid and feruloyl-arabinose, were localised by immunocytochemistry in the cell walls of the apical internode of two lines of maize (Co125 and W401) of different digestibility. The compounds were detected at two stages of cell maturity in the lignified tissues (sclerenchyma, fibres and xylem) and in the medullary parenchyma, which, in the samples studied, was not lignified. p-Coumaric acid is a phenolic acid associated with lignins, which confer resistance on plant cell walls to microbial degradation in the rumen. Feruloyl-arabinose is a compound associated with xylans, the principal hemicelluloses in Gramineae, which are potentially degradable. Labelling of p-coumaric acid decreased in both maize lines with cell age and as the cell walls became lignified. The mass of lignin deposited in the cell walls masked p-coumaric acid, thereby making it less accessible to the antibodies. There was an inverse relationship in the labelling of p-coumaric acid and feruloyl-arabinose. Feruloyl-arabinose was more heavily labelled as the plant cell walls matured in all the lignified tissues of both maize lines and in the parenchyma of the less digestible line. All tissues except the parenchyma were more heavily labelled with both sera in Co125, the more digestible line.

Immunogold labelling of arabinoxylans in the plant cell walls of normal and bm3 mutant maize

Summary— Polyclonal antibodies directed against α, l‐1.2‐arabinofuranosyl poly‐β,d‐1.4‐xylopyranosyl (degree of polymerization 130) have been raised from rabbits. The immunogold labelling in transmission electron microscopy (TEM) evidenced the arabinoxylans of the plant cell walls. Comparison between the stems of normal and mutant bm3 maize demonstrated a greater accessibility of arabinoxylans in the walls of the mutant maize. The method, specific and swift, allows us to specify the repartition in the different parts of the stem: sclerenchyma, fibers, parenchyma.

Rice straw degradation and biomass synthesis by rumen micro-organisms in continuous culture in response to ammonia treatment and legume extract supplementation

Abstract The effects of ammonia treatment and fresh legume extract supplementation on the metabolism of rumen micro-organisms maintained on rice straw ( Oryza sativa , variety Thaibonnet) in dual outflow fermenteurs were investigated. Alfalfa ( Medicago sativa L.) was chosen as a model legume, similar in biochemical composition to cowpea ( Vigna unguiculata ). Diets provided comparable amounts of readily available N. The interaction between ammonia treatment and legume extract supplementation was generally negligible. The relative productions of VFA (in moles/100 moles of hexoses fermented) were oriented to an increased proportion of C 4 to the detriment of C 2 by ammonia pre-treatment (+100% versus −28% at the intermediate extract input level), which also lowered the relative production of CH 4 by 36%. The true degradability of organic matter (OM) was 27% higher with treated rice straw (TRS) than with untreated rice straw (URS). The degradabilities of ash-free neutral detergent fibre (NDF) and acid detergent fibre (ADF) ranged from 30 to 50% and from 26 to 48%, respectively. At low levels of extract supplementation, the degradabilities of ash-free NDF and ADF tended to be lower with TRS than with URS (30% versus 46% in the case of ADF), while at high levels of the alfalfa extract they were not modified by straw pre-treatment. The outflow of non-ammonia nitrogen (NAN) was higher with TRS (+41% at the intermediate legume input rate). This increased NAN outflow was essentially of microbial origin. Alfalfa extract supplementation also increased net production of microbial protein. The efficiency of microbial protein synthesis was strongly increased by ammonia pre-treatment and by extract supplementation, in a linear manner. Both treatments were beneficial to rumen microbial metabolism in vitro, while acting independently on microbial functions. Ammonia treatment induced the most significant changes, especially in favouring extensive biomass synthesis.

Immunocytochemical localization of arabinoxylans in the cell wall of maize apical internode after microbial degradation in the rumen

Summary— Arabinoxylans were localised by immunocytochemistry using polyclonal antibodies in the cell walls of the apical internode of maize after degradation in the rumen. In order to understand the significance of arabinoxylan in digestibility property, two lines of maize differing in digestibility were used. Wide variations in the intensity of labelling were observed in the four tissues studied (sclerenchyma, fibres, xylem and parenchyma) from the first hours of incubation in the rumen. Incubation time in the rumen greatly influences the intensity of labelling.

Microbial degradation of the apical internode of Co125 and W401 maize in the rumen

Abstract The internodes of maize lines Co125 and W401, harvested five days after anthesis were cut into three fragments of equal length. In the internodes of Gramineae there is a gradient of maturity from the base upwards. Measurements of digestibility made in situ with the nylon bag method showed that the bases of the internodes were more digestible than their tops and that line Co125 was more digestible than line W401. Lignin-specific histological stains showed clear differences in staining between the sclerenchyma, fibres and xylem. The parenchyma was never stained. Lignification of tissues occurred first in the xylem and then in the fibres and sclerenchyma. There were great differences in the percentage of area stained by acid phloroglucinol, in relation to total stem area, in the bases, which were of comparable digestibility. Conversely, the percentage of area stained in the tops, which were not of comparable digestibility, differed little. SEM and TEM observations showed that the cell walls of lignified tissues were thicker in the tops than in the bases in both maize lines. They also showed that the most intensely stained tissues were the most resistant to microbial degradation. The parenchyma was degraded very rapidly but not at the same rate in all samples (base Co125 > top Co125 > base W401 > top W401) whereas the xylem was never degraded. The most extensively degraded tissues in all samples were the fibres and the parenchyma. The bases of the two maize lines were more degraded than the tops. Of the cell walls that reacted positively to lignin-specific stains only the secondary walls of the fibres and sclerenchyma were sometimes degraded. However, the sclerenchyma secondary wall was not always degraded in the top of maize W401. The phenolic compounds seemed to have different characteristics in the bases and tops of the two maize lines. This study shows the suitability of the internode as a model for observing the growth and lignification of cell walls and for determining the effects on digestibility.