Search

Reduced lignin contents in brown midrib (bmr) mutants of sorghum (Sorghum bicolorL. Moench.) are reported to improve the digestibility of this forage. The objective of this study was to evaluate microscopically leaf blades of normal‐(N) 12 and bmr‐12 sorghum for variations in anatomy, histological reactions of tissues for lignin, and specific tissue digestion to elucidate the contribution of the bmr‐ 12 mutation to improved forage quality. No differences in anatomy or histology of lignified tissues was observed between N‐12 and bmr‐ 12 plants. Midribs of N and bmr plants, examined by scanning electron microscopy after a 48 to 72 h incubation with rumen fluid were partially degraded, with a tendency for parenchyma in bmr leaf blades to be more degraded than that in N leaf blades. Other tissues, i.e., epidermis and xylem, were not degraded in either plant type. In the non‐midrib portion of blades, the parenchyma bundle sheaths and adaxial sclerenchyma areas were degraded to a greater extent in bmr plants by mixed rumen microorganisms and by a pure culture of the cell wall‐degrading rumen bacterium Ruminococcus flavefaciensFD‐1. Epidermis and mesophyll at times more rapidly degraded in bmr plants. In leaf sheaths, no marked differences in digestion between N and bmr plants were found with parenchyma totally degraded in both plant types. Xylem and other tissues normally highly resistant to microbial degradation were not degraded in plant parts of either N or bmr types. The bmr‐12 mutation did not result in changes in leaf blade anatomy or histological reactions for lignin and did not result in susceptibility to microbial degradation of the most resistant tissues. The bmr‐12 mutation did allow an improved microbial degradation of marginally digestible tissue, i.e., those tissues usually only poorly degraded.

Marginally digested tissues in the brown midrib‐12 (bmr) sorghum (Sorghum bicolor(L.) Moench.) mutant are more readily degraded than similar tissues in the normal (N) line. Reduced lignin concentrations have been reported to be a factor affecting digestibility of bmr genotypes, but lignin type could also influence digestibility. The objectives of this work were to (i) examine N‐12 and bmr‐12 plants for variations in chemical composition, including phenolic acids and phenolic structure, using solid‐state nuclear magnetic resonance (NMR) spectroscopy; and (ii) evaluate by microscopy the effect of permanganate delignification. Leaf blades of bmr plants were significantly (Ρ≤ 0.05) lower in neutral detergent fiber (NDF) and permanganate lignin (PML), and higher in in vitro dry matter digestibility; leaf sheaths contained significantly less PML. Solid‐state NMR spectroscopy suggested that bmr leaf blades had lower amounts of bound syringyl moieties. The predominant alkali‐soluble (1.0 M NaOH) phenolic acids in both plant types were ρ‐coumaric and ferulic acids, and ρ‐coumaric acid was significantly lower (Ρ ≤ 0.05) in concentration in bmr leaves. Treatment of intact blade sections with permanganate to oxidize lignin compounds resulted in a reduced staining intensity for lignin identified by both the acid phloroglucinol and chlorine‐sulfite tests in all but midrib sclerenchyma; lignin staining intensity did not differ between plant types. Degradation of delignified midribs by rumen microbes indicated that the tissues were more degradable in bmr than in N plant types. Results suggest that reduced concentrations of phenolic acids (especially ρ‐coumaric acid) and reduced levels of etherated syringyl moieties in conjunction with reduced concentrations of lignin are factors in the improved quality of bmr‐12 sorghum.

The Panicumgenus has C3–C4 species with characteristics between those of C3and C4species, and digestibility of C4or C3–C4species might be improved by hybridizing with more digestible C3plants. Leaves of greenhouse‐grown. P. spathellosumDoell (C3–C4), P. bolivienseHack. (C3), and their F1hybrid were evaluated for characteristics related to forage quality. Leaves were digested in rumen fluid to determinine in vitro dry matter digestion (IVDMD) and degradation of specific leaf tissues. The hybrid was intermedite and differed (P< 0.05) from its parents in leaf IVDMD with values of 642 g kg−1compared to 630 and 709 g kg−1for the C3–C4and C3species, respectively. The hybrid was also intermediate (P< 0.05) to its parents in the percentage of sclerenchyma, lignified bundle tissue, phloem, abaxial epidermis, and mesophyll. The hybrid had 18.8% of its leaf cross‐section as parenchyma bundle sheath compared 15.0% for P. bolivienseand 16.5% for P. spathellosum. Neutral detergent fiber was 583 g kg−1for the hybrid compared to 511 and 568 g kg−1for the C3and C3–C4parents, respectively. Acid detergent fiber was 61 g kg−1greater than for the C3parent. The hybrid showed greater loss of mesophyall and epidermal tissues than P. spathellosumin top leaves, but only for abaxial epidermis in bottom leaves. Hybridization affected leaf anatomy and fiber characteristics related to digestibility. However, degradation of specific tissues did not follow consistent trends, suggesting that intrinsic factors within cell walls can override anatomical changes.

Ozone has been reported to reduce the lignin concentration in forages and to improve cell wall digestibility. The objective of this work was to evaluate the effect of ozonolysis on cell wall structure of various lignified tissue types in forages. In vitro digestibility was evaluated gravimetrically and by scanning electron microscopy for leaf blades and stems of ‘Coastal’ bermudagrass [Cynodon dactylon(L.) Pers.] and for leaf blades of ‘Kentucky‐31’ tall fescue (Festuca arundinaceaSchreb.) and ‘Booneo’ orchardgrass (Dactylis glomerataL.). Ozonolysis of bermudagrass increased the in vitro digestibility by 12.5 percentage units over control material, and further showed that the epidermis, parenchyma bundle sheath, and lignified xylem cells of the vascular bundles in leaf blades were made more available for microbial degradation. Ozonated bermudagrass stems showed greater degradation of the parenchyma tissue, and cell wall widths of the more recalcitrant xylem and sclerenchyma ring were reduced in width. Ozone affected the most resistant tissues, i.e., xylem cell walls, and increased their susceptibility to microbial degradation at the surface of cut ends of sections. Ozonolysis of leaf and stem sections cut at 20‐, 10‐, and 5‐mm lengths usually did not significantly (P> 0.05) increase in vitro digestibility of various lengths of leaves and stems of forages, indicating the inability to penetrate and improve tissue digestibility within intact sections. The ability of O3to oxidize lignin without substantial losses of tissues may provide a means to elucidate the phenolic‐carbohydrate interaction in cell walls.

Effects of ingestive mastication on physical degradation of Coastal bermudagrass hay (CBH), grazed Coastal bermudagrass (GCB) and grazed ryegrass (GRG) were studied macro-scopically, microscopically and histochemically. The masticate of each forage was collected from cattle fitted with 5-cm id esophageal cannulae and separated into fragments retained on sieves of 20 to 1,600-µm. Masticated particles passing a l,000-µm sieve made up 15.5, 51.9 and 25.8% (by weight) of the consumed CBH, GCB and GRG, respectively. Particles retained on sieves of 500, 800, 1,000 and 1,600 µm appeared as irregular sized particles of leaf and stem characterized by ends formed from totally crushed vascular bundles (VB) and edges defined by cleavage between VB. The central areas of these fragments contained many sites of rupture of the cuticle, partial fracturing of the VB and exposure of the epidermis and mesophyll. Observed only in GRG were large isolated sheets of cuticle with attached and readily exposed mesophyll or epidermal cells. Particles of 100 to 500 µm appeared to be derived largely by further reduction in VB length and longitudinal fracturing between the VB. Particles <100 µm (<10% of the masticate) were represented by epidermis, cuticle, layers of parenchyma bundle sheath cells and isolated VB. Based on their anatomy and histochemistry, they appeared higher in indigestible tissue (lignin staining) components than most larger particles. Mastication decreased particle size and thereby exposed more surface area. Additionally, the crimping and crushing action of the mastication process exposed more area of digestible tissue within a given particle size. In view of the protective nature of intact plant structures, ingestive mastication appears important in disrupting these anatomical barriers to subsequently allow increased microbial enzyme accessibility.

Two bermudagrass (Cynodon dactylonL. Pers.) hybrids, ‘Tifton 78’ (Tilt‐78) and ‘Coastal’ (CBG), differ in dry matter digestibility. The objective of this study was to distinguish factors at the ultra‐structural level which may contribute to the differences in digestibility. Percent tissue types and histological reactions for lignin were similar for both cultivars. Scanning electron microscopy (SEM) leaf blades incubated in rumen fluid or cellulase revealed that non‐lignified tissues were digested to a greater extent in Tift‐78 than in CBG. Pretreatment with 0.1 MHCI or acid‐pepsin augmented cellulase digestion of the more slowly.degraded tissues. Transmission electron microscopy showed that Tift‐78 had a higher number of attached bacteria during incubation with rumen fluid. The ratio of attached Bacteroidesto Ruminococcusmorphotypes also varied between cultivars. After 24 h, Tift‐78 supported a population of predominantly Bacteroides(74%), whereas CBG had only Bacteroides. By 72 h, the attached population of Ruminococcusand Bacteroideshad shifted to 59% and 17%, respectively, for Tift‐78 and 60% and 0%, respectively, for CBG. A SEM‐energy dispersive X‐ray spectroscopic analysis of the elemental composition of parenchymal bundle sheath and epidermal cell walls revealed that CI was significantly higher in untreated tissues of CBG than Tift‐78 but higher in Tift‐78 after cellulase incubation. The higher concentration of CI in CBG may prevent initial colonization of cell walls by specific bacterial morphotypes and reduce the number of attaching bacteria, thus reducing digestibility. Silicon, which has been described as an antiquality factor, did not differ between cultivars. Responses to enzymes indicated that structural carbohydrates in specific cell walls varied in digestibility between bermudagrass cultivars, and differences in cell walls further influenced the adherence of fiber‐digesting bacteria.

Digestion is lower for grasses adapted to tropical regions than for species from temperate areas. These differences in digestibility have been attributed to anatomical structure and to effects of temperature. This research compared the degradation of tissue types in leaf blades of 29 Panicumspecies of tropical origin having diverse anatomies and representing C3, C4, and intermediate (C3/C4) photosynthetic pathways and grown in identical environments. Blades were incubated in rumen fluid, and tissue degradation was studied by light and scanning electron microscopy. Mesophyll was rapidly degraded in most C4and C3species and estimated to be 86 and 80%, respectively, after 20 hours incubation; a few species of each type (e.g., P. effusumand P. laxum) exhibited slower mesophyll degradation than other grasses. However, the mesophyll digestibility in the C3/C4species was unexpectedly low, averaging only 72% after incubation for 20 hours and only 50% for P. milioides. Epidermal tissues were readily degraded in most species and the average digestibility of the adaxial epidermis tended to be higher (5 to 8 percentage units) than that the abaxial for all photosynthetic types. Parenchyma bundle sheaths were about 90 to 100% digested both in C3and C3/C4species, but those of many C4species were poorly degraded with an average for all species of only 50% after incubation for 20 hours. The outer portion of the sheath cell wall was particularly low in degradation and remained in many C4species after 48 hours incubation. The low degradation of parenchyma bundle sheath appeared to be a major influence on blade digestibility in C4species. Vascular tissues were either not degraded or only partially degraded. These results suggest that improved forage quality could result from plant breeding to increase the degradation of limiting tissues, e.g., the parenchyma bundle sheaths of C4species, without alterations in the basic leaf anatomical structure. Improvement in quality may also be possible by breeding to increase the proportion of mesophyll content, but such a change may not always improve digestibility since the mesophyll of some species was more slowly degraded than that of others.

Samples of forage fragments were obtained from the upper (RUS) and lower (RLS) strata of the reticulorumen and feces (F) of four Brahman × Jersey steers grazing Coastal bermudagrass (CB) of two maturities with dry matter digestibilities (DMD) of 54.8 and 64.3%. Forage fragments were separated by particle size and evaluated histochemically for tissue type and fragmentation pattern. Fragmentation pattern was similar to that previously observed due to ingestive mastication. There was longitudinal separation of vascular bundles (VB) and severing at VB ends. Microscopically, similar size fragments from RUS were indistinguishable from those of RLS. The major difference between RUS and RLS was the distribution of different size particles. Larger particles were associated with the RUS in cattle consuming immature and mature CB. More large particles were associated with mature compared with the immature CB in the RUS and RLS. The distribution of different size particles in the F was similar for both maturities, suggesting that similar particle size reduction was required regardless of maturity. Smaller particles in the rumen and F appeared to contain more lignin (determined histochemically) and were composed of indigestible fragments of cuticle and lignified vascular tissue. Cattle grazing mature CB had higher ruminal fills (2.40 vs 2.02 kg dry matter/100 kg body weight), reduced rates of passage and lower voluntary intake (2.50 vs 3.14 kg DM/100 kg body weight). Lower intake of mature CB may have resulted from a reduced rate of particle size reduction. Similarities in fragmentation patterns due to ingestive and ruminative mastication were interpreted to indicate that mastication was responsible for most of the particle size reduction of CB and that mastication facilitated digestion of potentially digestible tissues.

High-resolution solid-state 13C NMR and NIR diffuse reflectance spectra were obtained on microcrystalline and “noncrystalline” celluloses. Particle sizes and relative crystallinity were confirmed by scanning electron microscopy and MIR transmission spectroscopy, respectively. The results showed that NMR is more sensitive to order changes and less sensitive to particle size. NIR reflectance, on the other hand, is very sensitive to particle size changes and essentially insensitive to differences in order.

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.

Conclusion: The discovery of anaerobic fungi has added a new member to the indigenous microorganisms that inhabit the rumen ecosystem. Anaerobic fungi do not appear essential for the survival of ruminants due to their presence in very low numbers, and sometimes absence, in ruminants fed low fiber diets, but their presence may likely be very important in the digestion of fibrous diets. The anaerobic fungi have adapted well to the rumen environment. They are able to ferment a large array of soluble carbohydrates and can synthesize cellular components in an anaerobic environment. The fungi posses hydrogenosomes for the removal of reducing equivalents in the form of molecular hydrogen and the removal of trace oxygen is a accomplished via removal by NADH oxidase. Their positive synergistic interaction with methanogenic bacteria eludes to their highly evolved role in the rumen environment. The fungi also produce resistant sporangia that allows for transfer of species to a new host in an oxygen environment. The anaerobic fungi posses a highly active array of polysaccharide degrading enzymes that may provide an advantage in the highly competitive rumen ecosystem. The production of specific enzymes that hydrolyze the lignocellulosic fraction of plant walls is unique in rumen microorganisms and allows for their attachment and growth on fibrous plant particles that are not available to the rumen bacteria.

Factors that limit forage quality are complex and interactive. Structural characteristics that limit forage breakdown are primarily highly lignified support tissues like sclerenchyma and xylem. However, the presence of chemical barriers (likely the low molecular weight phenolic compounds) within “living” cell walls, such as the parenchyma bundle sheath and epidermis, prevents fiber degradation in these tissues. The presence of high proportions of these tissues in leaves of warm‐season grasses plus growth in hotter climates, which tends to speed maturity, combine to limit breakdown of warm‐season grass leaves compared to cool‐season species. Legume leaflets in warm‐ and cool‐season species can be readily degraded due to a high proportion of mesophyll. However, leaflets of certain species or cultivars with high tannin concentrations are poorly degraded, with mesophyll and vascular bundles comprising the residue. Grass stems have a more rigid structure than leaves, with the non‐digestible epidermis, sclerenchyma ring, and vascular tissue occupying about 30% of the tissues in the cross sections. In addition, mature grass stems have a poorly degraded parenchyma that contributes to a rigid structure in the residue. Parenchyma in legume stems is totally degraded, leaving a residue of a hollow cylinder surrounded by a uniformly lignified ring. Possibly, variations between structures of mature grass and legume stems contribute to observed differences in forage quality for these forage types. Rumen bacteria and fungi both physically associate with the more recalcitrant forage tissues. Fungi are better able to colonize the lignocellulosic tissues and can partially degrade the most resistant xylem tissues. Research avenues for improving forage quality are: (i) to enhance the ability of anaerobic fungi to weaken lignocellulosic barriers, (ii) to reduce the amount of non‐degradable barriers, particularly in stems, by plant breeding, and (iii) to improve by plant breeding the rate and extent of slowly or partially degraded tissues in leaves.

Increasing growth temperature lowers digestibility of tall fescue (Festuca arundinaceaSchreb.) cell walls, but specific tissue responses have not been fully elucidated. The objectives of our research were to determine the effect of increasing growth temperature on leaf anatomy, histological reactions for lignin, alkali‐extractable phenolic acids, and the digestion of different tissue types by rumen microorganisms after 4, 10, 24, or 48 h. Plant material was grown in a controlled environment in which temperature was changed sequentially from 13/10°C (T1), to 20/18°C (T2), to 30/27°C (T3), and back to 13/10°C (T4). Two consecutive harvests of uppermost leaf blades following 28 days growth were made at each temperature, and analyses were performed on leaf blades. Histological reactions for lignin and leaf anatomy were similar for all treatments but with higher amounts of lignified vascular tissue in T3. At incubation times less than 48 h, leaf tissues from plants grown at T1 or T4 were degraded, as determined by scanning electron microscopy, to a greater extent than those at T2 or T3. However, after 48 h, tissues from plants grown at T2 were clearly the most degraded, and the order for all digestible tissues was as follows: T2 > T4 > T1 > T3. From 786 to 854 g kg−1of the phenolic acids extracted from forage with 1 MNaOH were trans‐p‐coumaric and trans‐ferulic acids. Increases in growth temperature tended to increase levels of these and other phenolic acids, with T3 resulting in the highest level; amounts at T1 were lower (P≤10.05) than those at T2 through T4. Increased temperature resulted in a lower digestibility of easily degraded tissues without causing a marked change in leaf anatomy or histological reaction for lignin. Increased levels of p‐coumaric and ferulic acids up to T3 suggest that these compounds may contribute to limiting the digestibility of leaf blade tissues at higher temperatures.

Abstract: Oil and wax contents of confectionery (non-oil) and oilseed, open-pollinated and hybrid sunflower seed and their hulls, testae, and kernels were determined. Wax content inversely correlated with hull content, and the degree to which the seeds were compressed corre-lated with hull thickness. Light and electron microscopy showed increased connective tissue in the hybrid seed. This combination of higher wax content in the dehulled hybrids and increased connective tissue between hull and kernel could explain the reports of high wax content of the oil from newly developed hybrid seed.

This paper presents a parameter optimization technique for deciding the force distribution on a payload being transported along a predetermined trajectory using two planar manipulator arms. The methodology begins by transforming the singular dynamics of two-arm transport to an ordinary set of differential equations and then proceeds to obtain a relation between the torques exerted by each arm. This relation is then used in a quadratic torque cost which is subsequently minimized to yield an optimal torque distribution. Significant savings in energy were found to occur when the arms were allowed to interact by transmission of forces through the payload. Even more significant are the savings found over one-arm transport of payloads where the arm torques are fixed by the prescribed trajectory.

We previously reported that both the nuclear import rate of large karyophilic gold particles and the functional size of the pores are significantly greater in simian virus 40-transformed fibroblasts (the SV-T2 cell line) than in nontransformed BALB/c 3T3 cells. In this study, we found that cytosolic fractions obtained from SV-T2 cultures can increase nuclear transport capacity (both import rate and pore size) when microinjected into BALB/c 3T3 cells. The transport-enhancing function of the extracts can be abolished by the protein kinase inhibitors staurosporine and K252a as well as 5'-p-fluorosulfonylbenzoyladenosine and protein phosphatase 2A, which, although less specific, also interfere with kinase activity. Increases in transport capacity of the same magnitude as that produced by the SV-T2 extracts were obtained by microinjecting protein kinase A or C or recombinant mitogen-activated protein kinase. These data provide further support for the interpretation that the enhancer is a protein kinase. From experiments performed with specific kinase inhibitor peptides, it appears likely that protein kinase C is the active factor in the SV-T2 cytosolic fractions; however, this will require further verification. It was also determined, by using gold particles coated with bovine serum albumin conjugated to synthetic nuclear localization signal peptides that lacked phosphorylation sites, that the enhancer affects the transport machinery rather than the activity of the nuclear localization signals.

Myeloperoxidase is a major component of specialized lysosomes known as azurophil granules in polymorphonuclear leukocytes or neutrophils. The processing of myeloperoxidase in human HL-60 promyelocytic leukemia cells was studied by pulse-labeling cells in culture with [35S]methionine followed by immunoprecipitation and identification of myeloperoxidase polypeptides from cell fractions after various chase intervals. These studies revealed the presence of a previously unidentified intermediate with Mr 74,000 which kinetically followed the appearance of a larger Mr 81,000 intermediate. Using an in vitro lysosomal preparation the newly identified Mr 74,000 intermediate was directly converted within protected granules to mature forms of myeloperoxidase (Mr 63,000 and 60,000). This conversion occurred optimally at pH 7.5 and was not inhibited by lysosomotropic agents (chloroquine, NH4Cl) or protonophores (monensin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Furthermore, the uptake of radiolabeled amines indicated a neutral intragranular environment (pH 7.35-7.67) which remained unchanged in the presence and absence of 1 mM ATP or 2.5 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone. We conclude that, in contrast to other lysosomal pathways, the final proteolytic cleavage of myeloperoxidase does not require an acidic environment.

The time of Microsporum gypseum macroconidial development, with particular regard to spore ornamentation, was studied with the scanning electron microscope. The roughness of the spores was seen to be due to rounded projections (averaging 0·8 μ in diameter) which we have termed polyps; they were formed 2-3 h after macroconidial development.Thin sections of macroconidia revealed that the polyps were contiguous with the outer layer of the trilaminar cell wall.