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a USDA-ARS Plant Science Res. Unit and U.S. Dairy Forage Res. Center Cluster, Dep. of Agronomy and Plant Genetics, 411 Borlaug Hall, 1991 Upper Buford Circle, Univ. of Minnesota, St. Paul, MN 55108
b Dep. of Plant Sciences, P.O. Box 341, Wageningen Univ., Wageningen, The Netherlands
* Corresponding author (jungx002{at}tc.umn.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The tools of molecular biology offer the potential of altering the development of alfalfa stems in very precise ways to improve forage quality. It has long been assumed that lignin limits cell wall degradability because lignin concentration of stems increases during maturation and degradability declines (Jung and Deetz, 1993). Shifting the composition of lignin toward a lower syringyl:guaiacyl monolignol ratio is another possible route to improving alfalfa stem quality because this type of lignin has been associated with more degradable cell walls (Cherney, 1990). Therefore, modifying lignin biosynthesis is a popular strategy for improving the cell wall degradability of alfalfa and other forages (Bernard Vailhe et al., 1996; Baucher et al., 1999). Another strategy for potentially improving the quality of alfalfa stems might be to increase pectin content of the cell wall because this polysaccharide is rapidly and extensively degraded in the rumen (Chesson and Monro, 1982; Hatfield and Weimer, 1995).
While these cell wall traits may represent reasonable targets for improving alfalfa stem quality, nontissue-specific genetic transformations of lignin biosynthesis to reduce plant lignin content have generally resulted in agronomically nonviable plants (Piquemal et al., 1998; Tamagnone et al., 1998). This negative result is not surprising, given the diversity of functions (mechanical support, water transport, disease resistance) ascribed to lignin (Higuchi, 1990). While almost every tissue in grasses will lignify to some extent (Engels and Schuurmans, 1992), lignification of alfalfa stems occurs in a limited number of tissues (Wilson, 1993; Vallet et al., 1996; Engels and Jung, 1998). Similarly, cell wall polysaccharide content of alfalfa stem tissues vary. There is limited information concerning differences among forage tissues in cell wall concentration, composition, and development (Chesson et al., 1997; Grabber et al., 1991; Hatfield et al., 1999). Successful implementation of molecular biology methods to improve alfalfa quality will require a more refined understanding of cell wall structure and degradability for the diversity of tissues that comprise the stem.
Our objectives were to describe the developmental pattern of cell walls in the various tissues that comprise the alfalfa stem, characterize the shifts in cell wall concentration and composition resulting from differences in tissue proliferation and development during maturation, and explain the pattern of declining cell wall degradability associated with plant maturation based on differences in proliferation and development of stem tissues. A single, specific internode of developing alfalfa stems was chosen as a model for stem tissue development. Three alfalfa genotypes were included to provide variation in cell wall development and/or degradability. We combined microscopic examination of individual tissues with bulk chemical analysis of whole internodes to address these objectives. A description of the development and lignification of alfalfa stem tissues based on microscopic evaluation from this study has been published (Engels and Jung, 1998).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The number of stem shoots collected from individual plots at each sampling date ranged from 24 to 98, with more stems being collected at the earlier maturities in an attempt to obtain sufficient internodes for subsequent analyses. For every sampling date, 10 fresh stem shoots from each plot were immediately placed in 50% ethanol:water (vol/vol) for preservation until microscopic examinations were conducted. Internode 7 was excised from the remaining shoots. These internodes were then counted, frozen, and lyophilized for cell wall chemical and in vitro degradability analyses. After weighing, the lyophilized internode samples were ground in a shaking ballmill for 15 min. This grinding treatment results in particle size reduction such that 98% of alfalfa stem samples pass through a 106 µm screen (Jung et al., 2000).
Chemical Analysis
Concentration and composition of cell walls in the internodes was determined on the dried samples using the Uppsala dietary fiber method (Theander et al., 1995). Briefly, after removal of simple sugars and starch using enzymes and 80% ethanol:water (vol/vol) the cell wall residues were hydrolyzed with sulfuric acid in a two-stage procedure. Klason lignin was measured gravimetrically as the ash-free, nonhydrolyzed residue and the neutral sugar components of the cell wall polysaccharides were determined as alditol-acetate derivatives by gas chromatography. Uronic acid polysaccharide components were measured colorimetrically in an aliquot from the first step of the acid-hydrolysis procedure using galacturonic acid as the calibration standard (Ahmed and Labavitch, 1977). Total cell wall concentration was calculated as the sum of Klason lignin, glucose, xylose, arabinose, galactose, mannose, rhamnose, fucose, and uronic acid residues. On the basis of the known general composition of alfalfa polysaccharides (Hatfield, 1991, 1993), cellulose concentration was estimated as the glucose residue content, hemicellulose as the sum of xylose, mannose, and fucose residues, and pectin as the sum of uronics, arabinose, galactose, and rhamnose residues. All data were corrected to an organic matter basis by determining 100 °C dry matter content overnight and subsequent ashing at 450 °C for 6 h.
Composition of the lignin in the alfalfa internodes was determined by pyrolysis-GC-MS analysis (Ralph and Hatfield, 1991). The syringyl:guaiacyl ratio of the monolignol components in lignin was calculated using data normalized to the guaiacol content of each sample (Jung and Buxton, 1994).
In Vitro Degradability
Degradability of the cell walls in the alfalfa internodes was determined using rumen fluid for in vitro incubations. For the dried samples, the donor animal was a fistulated lactating Holstein cow fed a total mixed ration containing alfalfa hay, maize (Zea mays L.) silage, and a concentrate supplement. Rumen fluid was collected 
12 h post-feeding. A 20% rumen fluid:McDougall's buffer (vol/vol) mixture was added to screwcap centrifuge tubes containing 100 mg of sample and were incubated at 39 °C for 12 and 96 h (Jung and Buxton, 1994). At the end of the incubation periods, the entire contents of the in vitro tubes were frozen and subsequently lyophilized. The fermentation residues were analyzed for cell wall components using the Uppsala dietary fiber method as described previously. Empty centrifuge tubes were inoculated, incubated, and analyzed to correct for cell wall material contributed by the rumen fluid.
Thin sections prepared from the ethanol preserved internode samples (see following section for details) from 1996 were fermented with rumen fluid using the method of Engels and Brice (1985). Rumen fluid was collected 12 h after feeding from a fistulated Holstein steer fed maize stover and a small amount of concentrate supplement. Sections were mounted on microscopic slides with double-sided tape and placed in a 250-mL fermentation vessel fitted with a Bunsen valve, including 0.5 g of ground maize stover to maintain a normal fermentation. The fermentation vessel was inoculated with a 20% rumen fluid:bicarbonate buffer (vol/vol) mixture and incubated at 39 °C for 48 h. At the conclusion of the fermentation the slides were gently washed with tap water and placed in 50% ethanol for preservation until they were examined under the microscope.
Microscopic Analysis
Two randomly chosen stem shoots from each of the ethanol-preserved samples were used for measurement of physical dimensions and microscopic analysis. The length of the stem shoots and number of internodes were determined. Length of Internode 7 was measured and its diameter was determined with calipers. After excising Internode 7 from the shoots, 15 serial sections 100 µm in thickness were made from the middle of each internode piece. Three randomly chosen sections from each internode of each sample were then mounted on a slide with double-sided tape for examination by light microscopy. Replicate slides with three random sections from each internode of each sample in 1996 were mounted on slides for the in vitro fermentations described above.
Nonfermented control sections were first examined under a microscope with normal light. The diameter of the sections was measured using a calibrated ocular eyepiece and the approximate width of the xylem tissue ring was similarly determined. The individual tissues comprising the stem cross section (epidermis, collenchyma, chlorenchyma, phloem, cambium, xylem, and parenchyma) were examined to estimate the degree of cell wall thickening with advancing maturity across sampling dates. Polarized light was used to visualize thickened secondary cell walls rich in cellulose microfibrils. After examination with normal and polarized light, sections were stained with phloroglucinol (Jensen, 1962) to determine which tissues were lignified and when during development the lignification occurred. Additional replicate sections from each sample were stained with ruthenium red (Jensen, 1962) to visualize tissues with pectin-rich cell walls.
The fermented internode cross sections were examined under normal light to estimate the degree of cell wall degradation that had taken place during the 48-h in vitro incubation with rumen microbes. Degradation was estimated using the following categories: 0, undegraded; 1, <50% of the walls degraded for thin-walled tissues or partial thinning of thick walls; 2, >50% of the walls degraded, but not complete degradation, for thin-walled tissues or extensive thinning of thick walls; and 3, complete degradation of the wall. For each individual tissue, an average extent of degradation score was assigned based on examination of the three replicate sections. Degradation scores were averaged for the two internodes for every sample. After this scoring, the sections were stained with phloroglucinol to determine if the residual cell walls of the nondegraded tissues were lignified.
Statistical Analysis
All chemical and degradability analyses were done in duplicate and the results averaged. The data for 1996 were subjected to an analysis of variance as a randomized complete block with three replications and maturity stage (sampling date) as a subunit in a split-plot design. Alfalfa clone was the main unit and it was tested for significance with the clone x replicate interaction term. Data for the third and fourth maturity stages from 1996 and 1997 were combined into an analysis of variance using a randomized complete block design with three replications in a split-split-plot. Year was the main unit, alfalfa clone the subunit, and maturity stage the subsubunit. Each unit of the split-split-plot design was tested for significance with the appropriate error term. Year was considered a fixed effect because the perennial nature of alfalfa required use of sequential years. Clone and maturity were also treated as fixed effects because the experimental alfalfa clones and maturity stages were selected for specific reasons. The P < 0.05 probability level was used to determine significance. For those model parameters that had a significant F-test, means were compared using the least-significant difference method. All statistical analyses were done using PC-SAS (SAS Institute, 1985).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Associated with the increase in cell wall concentration during maturation of the internode, pectin concentration declined sharply and cellulose, hemicellulose, and lignin concentrations increased (Fig. 6, 7) . No significant changes in cell wall composition were found between the first and second sampling dates when the internode was still rapidly elongating. The greatest changes in cell wall composition occurred between the second and third sampling dates when internode development shifted from elongation to radial growth plus secondary cell wall thickening. Tissues that developed thickened secondary walls were the only alfalfa internode tissues to deposit lignin as the cells matured, but the pattern of lignification was different in each tissue (Engels and Jung, 1998). Primary xylem vessels were all lignified at the youngest maturity stage sampled. This is presumably because primary xylem vessels are the first water-conducting tissue that develops in the internode. Xylem tissues deposited lignin in both primary and secondary wall regions, and deposition of lignin and thickening of the secondary wall appeared to be almost simultaneous events in xylem tissue. Some xylem fibers developed an additional secondary wall layer by the last sampling date that was only slightly lignified, as determined by visual appraisal of phloroglucinol staining. Lignin was also deposited in pith parenchyma cell walls, beginning with cells closest to the xylem tissue and progressing toward the center of the internode with advancing maturity. In both pith parenchyma and xylem tissues, lignification began in the original primary wall and progressed into the secondary wall (Engels and Jung, 1998). Phloem fiber (mature primary phloem tissue) had a unique lignification pattern in that lignification also began in the primary wall, but for those individual cells with thickened primary walls, lignification occurred at the lumen edge of the primary wall and did not progress throughout the thick primary wall (Engels and Jung, 1998). In phloem fiber, the thick secondary wall never deposited lignin during maturation, contrary to the conclusion of Vallet et al. (1996). The final result was that phloem fiber developed lignified ring structures that only included a portion of the primary wall and did not include secondary wall.
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Microscopic observations showed that xylem was the predominant tissue type that proliferated through cambial activity after elongation of alfalfa internodes ended and that xylem tissues had thin primary cell walls and thick secondary walls (Engels and Jung, 1998). Histochemical staining indicated the presence of a small amount of pectin in the primary walls and high concentrations of lignin in both primary and secondary walls of secondary xylem tissues. Because secondary xylem was the only alfalfa stem tissue that proliferated after elongation ended, it can be inferred from our data that the cell walls of secondary xylem contained more xylan and less mannan, and have a more syringyl-rich lignin than the other tissues in the alfalfa stem. We estimate that alfalfa secondary xylem has a cell wall concentration of 750 g kg-1 organic matter with a composition of 400 g cellulose, 200 g hemicellulose, 200 g pectin, and 200 g lignin kg-1 cell wall. These estimates are based on the fact that cell wall concentration and composition of Internode 7 did not change between the fourth and fifth sampling dates, even though secondary xylem proliferation increased the xylem proportion of the internode cross sections by 50% (Fig. 3). However, it must be noted that a portion of the cellulose, and presumably other polysaccharides, deposited in cell walls formed during internode maturation derive from the development of the thick, nonlignified secondary wall in the phloem fiber, although this tissue did not proliferate through the addition of new cells after internode elongation ceased.
Tissue and Cell Wall Degradability
As Internode 7 of alfalfa stems became more mature, the amount of tissue remaining undegraded after a 48-h in vitro incubation with rumen microorganisms increased markedly (Fig. 5). Only the lignified, spiral ring structure of primary xylem vessels (Engels and Jung, 1998) could be identified as a relatively intact tissue after degradation of the youngest sample of Internode 7 (Fig. 5f). Primary xylem was the only lignified tissue at the first sampling date and all other stem tissues appeared to have been completely degraded by the rumen microbes. The same result was seen for the second sampling date (Fig. 5g), although a few individual internode sections from the second sampling date contained some lignified secondary xylem and this tissue also remained as a fermentation residue after incubation with rumen microbes (data not shown). Tissues with only nonlignified, primary walls (collenchyma, chlorenchyma, secondary phloem, cambium, and primary xylem parenchyma) remained completely degradable at all stages of stem development and maturation (Fig. 5). In contrast, the xylem, phloem fiber, and pith parenchyma tissues all became less degradable during internode maturation. Waxy cuticle, secondary xylem vessels, and xylem fiber residues remained after degradation of the third sampling date internodes (Fig. 5h). A thin-walled residue of the phloem fiber, xylem tissues, and some pith parenchyma on the periphery remained after degradation of internodes from the fourth sampling date (Fig. 5i). By the last sampling date, all pith parenchyma cells remained undegraded (Fig. 5j). The effect of maturity on degradability of these tissues is quantified in Table 3.
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Primary phloem was completely degradable in most Internode 7 sections from the first through the third sampling dates. By the fourth sampling date, primary phloem had begun to mature into phloem fiber by deposition of a secondary cell wall and lignification of its primary wall (Engels and Jung, 1998). Lignification of phloem fiber coincided with the appearance of a thin ring structure in the in vitro degradation residues (Fig. 5d, i). This residue was the unique lignified ring structure that developed in the primary wall of alfalfa phloem fiber tissue (Engels and Jung, 1998). Nonlignified portions of the primary wall from phloem fiber remained completely degradable with further maturation. Also, the secondary wall of phloem fiber, which did not lignify (Engels and Jung, 1998), remained completely degradable at both the fourth and fifth sampling dates (Fig. 5d, e, i, j).
Pith parenchyma underwent limited cell wall thickening during internode maturation and ultimately became lignified in 1996 (Fig. 5). These changes in cell wall development of pith parenchyma were associated with a progressive decline in degradability of the tissue, from completely degradable to entirely undegradable (Fig. 5). The decrease in pith parenchyma degradability began at the end of elongation and was first observed along the outer edge of the pith, next to xylem fibers (data not shown). This change from degradable to undegradable pith parenchyma cells progressed inward to the center of the pith region as maturity of the internode advanced. This pattern for changing pith parenchyma degradability mirrored the pattern for lignin deposition in this tissue (Engels and Jung, 1998).
Reduced degradability of alfalfa stem tissues was always associated with the deposition of lignin in cell walls of these tissues, except for the epidermis which did not lignify but did exhibit a decline in degradability. The epidermis was completely degradable in the youngest internode samples, but a thin sheet of residue from the epidermis remained from the third sampling date onwards (Fig. 5h). It appeared that the only part of the epidermal tissue remaining after in vitro degradation was the waxy cuticle layer on the outside surface of the epidermis. Apparently the entire polysaccharide cell wall matrix was degraded.
On the basis of visual appraisal of degradation extent, differences were detected among the three alfalfa clones for degradability of the primary wall of phloem fiber and pith parenchyma (Table 3). Clone 718 was the least degradable genotype, while Clone 403 was intermediate between Clones 143 and 718 in degradability of these tissues. The least degradable tissues (xylem vessels and fiber) did not appear to differ in degradability among the clones.
Because of insufficient amounts of sample, the in vitro degradability of cell wall polysaccharides of the dried internodes from the youngest maturity stage sampled in 1996 could not be determined. Figure 8 illustrates the patterns for cellulose, hemicellulose, and pectin degradation across the remaining four maturity stages. After 12-h incubations, pectin was almost completely degraded from the cell walls of internodes collected on the second sampling date (Fig. 8a). The pectin degraded during the 12-h incubations probably originated mainly from the cell walls of epidermis, collenchyma, and chlorenchyma because these tissues were previously shown to be completely degraded after only 8 h of microbial activity (Jung and Engels, 2001). Degradability of the pectin declined with advancing maturity, but remained >600 g kg-1 for even the most mature internode cell walls. The small decline in pectin degradation observed with maturation was partially due to inclusion of 4-O-methylglucuronic acid residues from alfalfa hemicellulose in the pectin fraction. Lengthening the time of fermentation to 96 h had no effect on extent of pectin degradability (Fig. 8b), indicating that the potentially degradable pectin in the cell walls of alfalfa internodes was very rapidly degraded. Degradability of cellulose and hemicellulose were the same after 12 h of fermentation, except for the most mature samples (Fig. 8a). Increasing the incubations to 96 h in length resulted in marked increases in cellulose and hemicellulose degradation (Fig. 8b), unlike the lack of response for pectin degradation to longer incubation times. Also, extended fermentations caused greater degradabilities for cellulose than hemicellulose at all maturity stages, except the second sampling date. At this youngest maturity stage, the degradabilities of cellulose and hemicellulose were not different and almost as great as observed for pectin.
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Isolated alfalfa pectins were very rapidly degraded by rumen microbes in vitro (Hatfield and Weimer, 1995). The pectic fraction of both alfalfa and red clover (Trifolium pratense L.) was also rapidly degraded from ground forage samples (Chesson and Monro, 1982; Hatfield and Weimer, 1995). Using thin sections, Jung and Engels (2001) demonstrated that pectin-rich tissues in alfalfa (chlorenchyma, collenchyma, epidermis, and the primary wall of phloem fiber) were completely degraded in vitro within 8 h by rumen microbes. Therefore, it was not surprising that increasing time available for microbial activity from 12 to 96 h did not alter the extent of pectin degradation in the current study. As shown in Fig. 5, the pectin-rich alfalfa tissues were completely degradable from all maturity stages of Internode 7. The data from the first two sampling dates of our experiment, when only primary xylem vessels were lignified while all other tissues consisted only of nonlignified primary walls, indicated that primary cell walls in alfalfa contained approximately twice as much pectin as did thick secondary, lignified xylem cell walls (Fig. 6). It appears that most pectin in alfalfa is located in tissues that do not lignify during maturation, and therefore remains highly degradable. The rapid and high degradability of pectin, even from mature alfalfa internodes, is a reflection of both the intrinsically high degradability of pectin and its distinctive distribution pattern among alfalfa stem tissues.
Degradation rate of isolated cellulose by rumen bacteria ranges from 0.05 to 0.08 h-1 (Weimer, 1996). Hemicellulose degradation rates range across a wider interval (0.07 to 0.26 h-1), presumably because of the diversity of structures (sugar residues and linkages) found among different hemicelluloses (Hespell and Cotta, 1995). These rates of cellulose and hemicellulose degradation are substantially lower than observed for isolated pectins (0.30 to 0.50 h-1) from alfalfa and other sources (Hatfield and Weimer, 1995). Degradation rates of cell wall polysaccharides from forage cell walls are similar to those observed for the isolated polysaccharides (Mertens, 1993), and the slow degradation rates for cellulose and hemicellulose explain why increasing length of the in vitro incubation increased observed extent of degradation for these polysaccharides in the current study (Fig. 8). Cellulose and hemicellulose typically have much lower potential extents of degradation than pectin, even when time for degradation is not limited (Buxton, 1991). This indicates that because most of the alfalfa stem cellulose and hemicellulose is concentrated in lignified xylem tissues, lignification is responsible for their limited potential degradability. Obviously, the presence of lignin in alfalfa cell walls is critical to the potential degradation of cell wall polysaccharides, but unfortunately, lignin concentration alone does not explain variability of forage cell wall degradation in alfalfa (Jung and Deetz, 1993; Jung et al., 2000). It would appear that reducing lignification of secondary xylem tissues may increase cell wall polysaccharide degradability; however, the modification should probably be limited to xylem fiber because reduced lignification of xylem vessels caused vessel collapse in transgenic tobacco (Nicotiana tabacum L.) (Piquemal et al., 1998). The presence of lignin in primary xylem vessels very early in alfalfa development may signal its importance to water transport and normal plant development (Engels and Jung, 1998).
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication April 30, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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