Alfalfa Stem Tissues: Cell-wall Development and Lignification

Alfalfa stems contain a variety of tissues with different patternsof cell-wall development. Development of alfalfa cell wallswas investigated after histochemical staining and with polarizedlight using light microscopy and scanning electron microscopy.Samples of the seventh internode, from the base of stems grownon cut stems, were harvested at five defined stages of developmentfrom early internode elongation through to late maturity. Internodeseven was elongating up to the third sample harvest and internodediameter increased throughout the entire sampling period. Chlorenchyma,cambium, secondary phloem, primary xylem parenchyma and pithparenchyma stem tissues all had thin primary cell walls. Pithparenchyma underwent a small amount of cell-wall thickeningand lignification during maturation. Collenchyma and primaryphloem tissues developed partially thickened primary walls.In contrast to a recent report, the formation of a ring shaped,lignified portion of the primary wall in a number of cells inthe exterior part of the primary phloem was found to precedethe deposition of a thick, non-lignified secondary wall whichwas degradable by rumen microbes. In numerous xylem fibres fromthe fourth harvest date onwards, an additional highly degradablesecondary wall layer was deposited against a previously depositedlignified and undegradable secondary wall. The pattern of lignificationobserved in alfalfa stem tissues suggests that polymerizationof monolignols by peroxidases at the luminal border of the primarycell wall creates an impermeable zone which restricts lignificationof the middle lamella region of tissues with thick primary walls.Copyright1998 Annals of Botany Company Alfalfa,Medicago sativaL., stem tissue, cell wall, development, lignification, degradation.     

Microscopic investigation of changes in histology and digestibility in the rumen of a forage grass and a forage legume during the first growth stage.

An Italian "Dalita" ryegrass (Lolium italicum) and a European lucerne (Medicago sativa) were harvested at 5 different growth stages to determine the anatomical factors limiting their digestibility and in particular the effects of lignification of the tissues. In vitro digestibility, cell wall contents of the whole plant and stem of lucerne and of the whole plant, stem and leaf blade of ryegrass were determined. The rate and the extent of degradation in the rumen of the different tissues were observed by scanning electron microscopy. This degradation occurred very rapidly with the lucerne stems; the xylem of lucerne was the only undegradable tissue whatever the stage. The collenchyma was degraded in the rumen although with acid phloroglucinol it stained positive for the presence of phenolic compounds. Ryegrass stems were digested more slowly than lucerne stems, and the sclerenchyma and xylem of ryegrass were indigestible whatever the stage. The parenchyma located close to the sclerenchyma became indigestible as the cell walls lignified progressively from the third stage. These results contribute to the understanding of the decrease in digestibility over the first growth stage and the variation in rate of digestion of lucerne and ryegrass in the rumen.     

Degradation of lignified secondary cell walls of lucerne (Medicago sativa L.) by rumen fungi growing in methanogenic co-culture

Aims: To compare the abilities of the monocentric rumen fungi Neocallimastix frontalis, Piromyces communis and Caecomyces communis, growing in coculture with Methanobrevibacter smithii, to colonize and degrade lignified secondary cell walls of lucerne (alfalfa) hay. Methods and Results: The cell walls of xylem cylinders isolated from stems of lucerne contained mostly xylans, cellulose and lignin together with a small proportion of pectic polysaccharides. All of these major components were removed during incubation with the three fungi, and differing cell wall polysaccharides were degraded to different extents. The greatest dry weight loss was found with N. frontalis and least with C. communis, and scanning electron microscopy revealed that these extensively colonized different cell types. C. communis specifically colonized secondary xylem fibres and showed much less degradation than N. frontalis and P. communis. Conclusions: Neocallimastix frontalis and P. communis were efficient degraders of the cell walls of lucerne xylem cylinders. Degradation occurred of pectic polysaccharides, xylan and cellulose. Loss of lignin from the xylem cylinders probably resulted from the cleavage of xylan releasing xylan–lignin complexes. Significance and Impact of the Study: Unlike rumen bacteria, the rumen fungi N. frontalis, P. communis and C. communis are able to degrade lignified secondary walls in lucerne stems. These fungi could improve forage utilization by ruminants and may have potential in the degradation of lignocellulosic biomass in the production of biofuels.     

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.     

A barrier covering lignified cell walls of barley straw that restricts access by rumen microorganisms

In barley straw, all stem tissues except the chlorenchyma are lignified. Electronmicroscopic investigations employing staining for cell-wall constituents and replica techniques revealed the presence of a tertiary wall covering the secondary wall and a warty layer deposited on the tertiary wall. The tertiary wall was composed of cellulose fibrils orientated in all directions, which were embedded in matrix material. The warty layer was comprised of granules and globules that were in many instances associated with a thin, flat, continuous layer. Though the tertiary wall could be degraded, the warty layer was resistant to degradation by rumen microorganisms. Only where the warty layer was mechanically disrupted could underlying cell-wall material be degraded. Bacteria could also burrow beneath these layers from exposed cell walls at the cut edges of the plant material. The warty layer forms a barrier to cell-wall-degrading bacteria and limits their colonization of straw stem.     

Contrasting Strategies of Alfalfa Stem Elongation in Response to Fall Dormancy in Early Growth Stage: The Tradeoff between Internode Length and Internode Number

Fall dormancy (FD) in alfalfa (Medicago sativa L.) can be described using 11 FD ratings, is widely used as an important indicator of stress resistance, productive performance and spring growth. However, the contrasting growth strategies in internode length and internode number in alfalfa cultivars with different FD rating are poorly understood. Here, a growth chamber study was conducted to investigate the effect of FD on plant height, aboveground biomass, internode length, and internode number in alfalfa individuals in the early growth stages. In order to simulate the alfalfa growth environment in the early stage, 11 alfalfa cultivars with FD ratings from one to 11 were chosen and seeded at the greenhouse, and then were transplanted into an artificial growth chamber. The experimental design was a randomized complete block in a split-plot arrangement with three replicates. Plant height, above-ground biomass, internode length, and internode number were measured in early growth stage in all individuals. Our findings showed that plant height and the aboveground biomass of alfalfa did not significantly differ among 11 different FD rated cultivars. Also, internode length and internode number positively affected plant height and the aboveground biomass of alfalfa individuals and the average internode length significantly increased with increasing FD rating. However, internode number tended to sharply decline when the FD rating increased. Moreover, there were no correlations, slightly negative correlations, and strongly negative correlations between internode length and internode number in alfalfa individuals among the three scales, including within-FD ratings, within-FD categories and inter-FD ratings, respectively. Therefore, our results highlighted that contrasting growth strategies in stem elongation were adopted by alfalfa with different FD ratings in the early growth stage. Alfalfa cultivars with a high FD rating have longer internodes, whereas more dormant alfalfa cultivars have a larger number of internodes. There were tradeoffs between internode length and internode number in response to FD in alfalfa, which reflected certain scale-dependence.     

Degradation of maize stem by two rumen fungal species, Piromyces communis and Caecomyces communis, in pure cultures or in association with cellulolytic bacteria.

Two species of rumen fungi, Piromyces (Piromonas) communis FL and Caecomyces (Sphaeromonas) communis FG10, were cultured alone or in association with the cellulolytic bacteria Ruminococcus flavefaciens or Fibrobacter succinogenes on maize stem. A kinetic study of the degradation of the substrate was then made. After 48 h of culture, all non-lignified tissues observed by scanning electron microscopy disappeared with P communis and degradation was as complete as that observed in the rumen. In contrast, C communis degraded little of the plant cell walls. The ability of P communis to more rapidly degrade maize stem was probably due to the presence of filamentous rhizoids. The extent of dry matter loss after 8 days of incubation was practically the same in all the monocultures and in the 4 cocultures. However, the rate of degradation was faster in the bacterial than in the fungal monocultures and the co-cultures. No metabolic interaction was observed.     

Rumen bacterial and fungal degradation of Digitaria pentzii grown with or without sulfur.

Sheep fed the forage Digitaria pentzii fertilized with sulfur were compared with those fed unfertilized forage for the rumen microbial population involved with fiber degradation. No differences were detected in the bacterial population as determined by anaerobic cultures on a habitat-simulating medium, xylan, or pectin, by 35S labeling techniques for microbial protein, or by transmission electron microscopic studies of bacterium-fiber interactions. Rumen volume and water flow from the rumen were not different for sheep fed each of the forages. Rumen fungi were prevalent in sheep fed sulfur-fertilized D. pentzii as shown by sporangia adhering to forage fiber and by colonies developing from zoospores in roll tubes with cellobiose plus streptomycin and penicillin. Fungi were absent or in extremely small numbers in sheep fed unfertilized forage. Nylon bag digestibility studies showed that the fungi preferentially colonized the lignified cells of blade sclerenchyma by 6 h and caused extensive degradation by 24 h. In the absence of bacteria in in vitro studies, extensive hyphal development occurred; other lignified tissues in blades (i.e., mestome sheath and xylem) were attacked, resulting in a residue with partially degraded and weakened cell walls. Nonlignified tissues were also degraded. Breaking force tests of leaf blades incubated in vitro with penicillin and streptomycin and rumen fluid from sheep fed sulfur-fertilized forage or within nylon bags in such sheep showed a residue at least twice as fragile as that from sheep fed unfertilized forage. In vitro tests for dry matter loss showed that rumen fungi, in the absence of actively growing bacteria, could remove about 62% of the forage material. The response of rumen fungi in sheep fed sulfur-fertilized D. pentzii afforded a useful in vivo test to study the role of these microbes in fiber degradation. Our data establish that rumen fungi can be significant degraders of fiber and further establish a unique role for them in attacking and weakening lignocellulosic tissues. The more fragile residues resulting from attack by fungi could explain the greater intake consistently observed by sheep eating sulfur-fertilized compared with unfertilized D. pentzii forage.     

The Rumen Ciliate Epidinium in Primary Degradation of Plant Tissues

Pieces of lucerne stem suspended in a sheep rumen in nylon bags were removed after different time intervals and examined by scanning electron microscopy. By 15 min large numbers of the ciliate protozoan Epidinium Crawley were attached to damaged areas of the stem, although a complex protozoal fauna was present in the rumen contents. Highest concentrations were on cortex and phloem tissues, with densely packed protozoa forming a complete ring around the transversely cut end of the stem between the epidermis and the vascular cylinder. Within 2 h there was extensive degradation of thin-walled tissues, as indicated by the amount of exposed vascular cylinder. Epidermis was not degraded but slid down the side of the stem as the underlying tissues were removed. This rapid degradation of plant tissue is explained by direct ingestion of tissues by the protozoa on the plant fragments. Many of the epidinia attached to the stem pieces were ingesting phloem elements and chlorophyllous tissue. The rumen protozoa have not previously been shown to participate on this scale in the physical degradation of plant material.     

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.     

The contribution of internode and mesocotyl tissues to root-to-shoot signalling of abscisic acid.

The xylem of first internode of runner bean and of previously etiolated maize mesocotyl segments was perfused with media containing abscisic acid (ABA) or abscisic acid glucose ester (ABA-GE) in concentrations as they occur under stress conditions. ABA-GE passed through the internode and mesocotyl segments unchanged. Within 10 min the concentration of ABA-GE(xyl) rose to a level similar to that in the external perfusion medium. By contrast, 30-40 min passed before the concentration of free ABA in the xylem sap [ABA(xyl)] reached the level in the external medium. When ABA-free media were used, ABA was released from the xylem parenchyma to the xylem vessels resulting in an [ABA(xyl)] of 13-23 nM (runner bean internode) or 1-6 nM (maize mesocotyl). The total perimeter and, hence surface area, of the xylem elements was measured microscopically and from these measurements it was estimated that, in both bean internodes and maize hpyocotyls, the flux of ABA to the xylem was 1 pmol m(-2) s(-1). The ABA efflux from the stem and mesocotyl parenchyma into the xylem could be increased when the tissues were treated with tetcyclacis, an inhibitor of ABA degradation, but also by changing the pH from its normal value of about pH 5.8 to pH 7.0 and by adding 100 mM NaCl to the perfusion medium. If 100 nM ABA was added to the perfusion medium the above treatments had only small effects on the release of ABA from the tissues into the xylem.     

Localisation and characterisation of cell wall mannan polysaccharides in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Polysaccharides containing -1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.Abbreviations BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - PACE polysaccharide analysis by carbohydrate gel electrophoresis     

Vertical and radial profiles in tracheid characteristics along the trunk of Douglas-fir trees with implications for water transport

The main stems of three young Douglas-fir (Pseudotsuga menziesii var. menziesii (Mirbel) Franco) trees were dissected to obtain samples of secondary xylem from internodes axially along the trunk and radially within each internode. From these samples, measurements were obtained of tracheid diameter, length, the number of inter-tracheid pits per tracheid, and the diameter of the pit membranes. In addition, samples were obtained along the trunks of three old growth trees and also a small sample of roots for measurement of tracheid diameter. A gradient was apparent in all measured anatomical characters vertically along a sequence among the outer growth rings. These gradients arose not because of a gradient vertically along the internodes, but because of the strong gradients present at each internode among growth rings out from the pith. Tracheid characteristics were correlated: wider and longer tracheids had more numerous pits and wider pits, such that total pit area was about 6% of tracheid wall area independent of tracheid size. A stem model combining growth rings in parallel and internodes in series allowed for estimates of whole trunk conductance as a function of tree age. Conductance of the stem (xylem area specific conductivity) declined during the early growth of the trees, but appeared to approach a stable value as the trees aged.     

Tracheid production in response to indole-3-acetic acid varies with internode age in Pinus sylvestris stems

Summary Different concentrations of indole-3-acetic acid (IAA) in lanolin were applied to the cambial region of approximately 10- and 34-year-old internodes in the main stem of Pinus sylvestris (L.) trees during the tracheid production period. After 5 weeks of treatment, the radial width of xylem produced in both ages of internode was positively related to exogenous IAA concentration measured at 0, 1 and 3 cm directly below the application site. Tracheid production in response to exogenous IAA in the 34-year-old internode was approximately one-half of that in the 10-year-old internode. The endogenous IAA level in the 7-, 17- and approximately 34-year-old internodes of similar trees was measured by radioimmunoassay, using gas chromatography-selected ion monitoring-mass spectrometry for validation. No consistent relationship was found between xylem radial width and IAA concentration. The data indicate that the cambium's ability to respond to exogenous IAA is qualitatively the same in 1-year-old shoots and older internodes. However, as the internode ages, there is a decrease in the extent of the response and in the optimal IAA level for inducing tracheid production.     

Invertase Activity, Carbohydrate Metabolism and Cell Expansion in the Stem of Phaseolus vulgaris L.

In the stem of Phaseolus vulgaris L. the specific activity ofacid invertase was highest in the most rapidly elongating internode.Activity of the enzyme was very low in internodes which hadcompleted their elongation, in young internodes before the onsetof rapid elongation, and in the apical bud. From shortly afterits emergence from the apical bud the elongation of internode3 was attributable mainly to cell expansion. Total and specificactivities of acid invertase in this internode rose to a maximumat the time of most rapid elongation and then declined. Transferof plants to complete darkness, or treatment of plants withgibberellic acid (GA₃), increased the rate of internode elongationand final internode length by stimulating cell expansion. Bothtreatments rapidly increased the total and specific activitiesof acid invertase in the responding internodes; peak activitiesof the enzyme occurred at the time of most rapid cell expansion. In light-grown plants, including those treated with GA₃, rapidcell and internode elongation and high specific activities ofacid invertase were associated with high concentrations of hexosesugar and low concentrations of sucrose. As cell growth ratesand invertase activities declined, the concentration of hexosefell and that of sucrose rose. In plants transferred to darkness,stimulated cell elongation was accompanied by a rapid decreasein hexose concentration and the disappearance of sucrose, indicatingrapid utilization of hexose. No sucrose was detected in theapical tissues of light-grown plants. The results are discussed in relation to the role of acid invertasein the provision of carbon substrates for cell growth. Key words: Cell expansion, Acid invertase, Hexose, Sucrose, Phaseolus     

Compressive/Tensile Stresses and Lignified Cells as Resistance Components in Joints between Cladodes of Opuntia laevis (Cactaceae)

The Cactaceae are a diverse group of plants with a wide variety of morphologies. Many species of Opuntia have segmented stems in which terminal cladodes may be separated from main-stem cladodes with varying amounts of resistance. From a geometric approach, derivations were used to calculate normal (axial and bending) and shear (transverse force and torque) stresses at joints due to the weight of the cladodes. Normal and shear stresses act perpendicular and parallel to (along) the cross sections of joints, respectively. Normal stress caused by bending was >10 times that of the mean value of any other stress. Analyses were performed to determine the relationship between maximum normal stress and the amount of lignified xylem cells. Such cells had thicker cell walls compared with the various other cells of stem joints that had thin cell walls and that thus would provide the most resistance to normal stresses. An analogy was made between cactus joints and a composite beam with reinforcing rods. In such joints, thin-walled parenchyma cells might be analogous to concrete that has little resistance to tensile stress, while the thick-walled, lignified xylem cells would be analogous to reinforcing rods. There were statistically significant relationships between normal stresses (from bending and axial loads) and mean percentage of lignified xylem cells (r=0.73) and between normal stresses and total areas of lignified xylem cells (r=0.65) (more stress, more reinforcing xylem cells). Tensile portions of cactus joints had 23% lignified xylem cells, while compressive portions had only 10% lignified xylem cells in joint areas (more tension, more reinforcing xylem cells). In addition, tensile joint tissues had two to three times more thick-walled, lignified xylem cells in the outer 30% of the radius compared with other joint tissues types (more reinforcing near the surface). To our knowledge, this is the first publication to present mechanical stresses at stem joints of cacti and the first to relate these stresses to characteristics of resisting tissues in the joints of a cactus.     

Ultrastructure of rigid and lignified forage tissue degradation by a filamentous rumen microorganism.

A small (less than 1 mum)-filamentous, branching microorganism was observed in Gram-stained smears of the rumen microflora and was found to degrade tissues in forage samples incubated in vitro and in vivo with rumen fluid and observed by scanning and transmission electron microscopy. The microbe had prokaryotic cytoplasmic features and a gram-positive type of cell wall structure. Round to oval bodies apparently attached to hyphae resembled the sporulation pattern reported for Micromonospora. Filaments and rod and coccal forms of the microbe degraded rigid forage cell walls and lignified, thick-walled sclerenchymal cells. Location of the microbe at a slight distance from the degraded zones suggested the action of extracellular enzymes. The presence of a microbe with the capability of degrading lignified tissue represents an important and unique function in the rumen ecosystem.     

Physical Degradation of Lignified Stem Tissues by Ruminal Fungi

Ruminal bacteria or fungi were selected by the addition of cycloheximide or streptomycin and penicillin, respectively, to ruminal fluid, and the weakening and degradation of lignified tissues in alfalfa and Bermuda grass stems by these treatments and whole ruminal fluid were evaluated in vitro. Dry weight loss in alfalfa was similar for whole ruminal fluid and streptomycin-penicillin treatment, whereas that with streptomycin-penicillin treatment was significantly higher (P ≤ 0.05) than that with cycloheximide treatment. In Bermuda grass, dry weight loss was significantly higher with streptomycin-penicillin than that with whole ruminal fluid and cycloheximide treatment, which were equal. Both peak load (Newtons) and peak stress were less (P ≤ 0.05) for streptomycin-penicillin treatment than with other treatments in both forages. Fungi colonized the lignified ring in alfalfa and tended to reduce the width of cell walls in this tissue, but a large number of fungal penetrations through cell walls was not observed. In contrast, fungal rhizoids frequently penetrated into and through cell walls in the lignified ring of Bermuda grass, often expanding the pit fields between the cells. Ruminal fungi disrupt lignified tissues in stems, and their activity results in a weakened residue more amendable to physical degradation. This weakening may allow plant digesta to be more easily broken apart during animal's rumination and thus facilitate digesta flow and fiber utilization.     

Differentiation Between Tissues from Carnation (Dianthus caryophyllus) Stems by Pyrolysis-Mass Spectrometry

Small pieces of different tissues from stems of young and oldcarnation plants were analyzed for lignification (lignin/celluloseratios) and lignin composition by means of pyrolysis-(gas chromatography)-massspectrometry. The epidermis and phloem of young and old stemswere essentially non-lignified. Pith parenchyma was only lignifiedin mature and senescing tissues. The type of lignin in sclerenchymadiffered from that in xylem and pith. Lignification in the xylemof very young tissues was a mainly guaiacyl-type lignin, whichgradually changed into a mixed guaiacyl-syringyl lignin in oldertissues. In mature tissues, the sclerenchyma was more highlylignified than the xylem. All tissues yielded comparatively large amounts of dihydroferulicacid, a compound which may be specific for carnation. Carnation, Dianthus caryophyllus, epidermis, cortex, sclerenchyma, phloem, xylem, pith, lignification, aging, dihydroferulic acid, pyrolysis-(gas chromatography)-mass spectrometry     

The role of the leaves in the regulation of internode elongation in Phaseolus vulgaris

Time-course patterns of leaf and internode elongation were studied in bean plants. Each leaf started its main elongation period when the leaf below reached half of its final length. The onset of leaf unfolding was nearly synchronous with the initiation of the elongation of the subjacent internode. Excision of young leaves increased the rate of stem elongation as a result of an earlier unfolding of the next upper leaves and the concomitant advancement in the elongation of their subjacent internodes. IAA or NAA (1% in lanolin) suppressed the enhancement effects of leaf excision on leaf and internode elongation. The excision of a young leaf increased the final length of internodes located below it, and at the same time decreased the final length of the internodes located above the excised leaf. The reduction was greater the younger the internode. Differences in internode elongation after leaf excision were related to changes during internode ontogenesis in their relative response to the availability of assimilates on the one hand, and on the other hand to hormonal factors transported acropetally from the young leaves to the growing internodes.