Non-lignified Walls Research Articles

Imaging Study by Mass Spectrometry of the Spatial Variation of Cellulose and Hemicellulose Structures in Corn Stalks.

The study used mass spectrometry imaging (MSI) to map the distribution of enzymatically degraded cell wall polysaccharides in maize stems for two genotypes and at several stages of development. The context was the production of biofuels, and the overall objective was to better describe the structural determinants of recalcitrance of grasses in bioconversion. The selected genotypes showed contrasting characteristics in bioconversion assays as well as in their lignin deposition pattern. We compared the pattern of cell wall polysaccharide degradation observed by MSI following the enzymatic degradation of tissues with that of lignin deposition. Several enzymes targeting the main families of wall polysaccharides were used. In the early stages of development, cellulose and mixed-linked β-glucans appeared as the main polysaccharides degraded from the walls, while heteroxylan products were barely detected, suggesting subsequent deposition of heteroxylans in the walls. At all stages and for both genotypes, enzymatic degradation occurred preferentially in nonlignified walls for all structural families of polysaccharides studied here. However, our results showed heterogeneity in the distribution of heteroxylan products according to their chemical structure: arabinosylated products were mostly represented in the pith center, while glucuronylated products were found at the pith periphery. The conclusions of our work are in agreement with those of previous studies. The MSI approach presented here is unique and attractive for addressing the histological and biochemical aspects of biomass recalcitrance to conversion, as it allows for a simultaneous interpretation of cell wall degradation and lignification patterns at the scale of an entire stem section.

Developmental changes in collenchyma cell-wall polysaccharides in celery (Apium graveolens L.) petioles

BackgroundCollenchyma cells occur widely in eudicotyledons and provide mechanical support for growing organs. At maturity, the cells are elongated and have thick, non-lignified walls, which in celery contain cellulose and pectic polysaccharides, together with xyloglucans and heteroxylans and heteromannans. A previous study suggested that at least some of the collenchyma cell wall in celery is laid down after expansion has stopped and is thus secondary. In the present study, we re-examined this. We used chemical analysis and immunomicroscopy to determine changes in the polysaccharide compositions of these walls during development. Additionally, solid-state NMR spectroscopy was used to examine changes in polysaccharide mobilities during development.ResultsWe showed the collenchyma walls are deposited only during cell expansion, i.e. they are primary walls. During cell-wall development, analytical and immunomicroscopy studies showed that within the pectic polysaccharides there were no overall changes in the proportions of homogalacturonans, but there was a decrease in their methyl esterification. There was also a decrease in the proportions of the (1 → 5)-α-l-arabinan and (1 → 4)-β-d-galactan side chains of rhamnogalacturonan I. The proportions of cellulose increased, and to a lesser extent those of xyloglucans and heteroxylans. Immunomicroscopy showed the homogalacturonans occurred throughout the walls and were most abundant in the middle lamellae and middle lamella junctions. Although the (1 → 4)-β-d-galactans occurred only in the rest of the walls, some of the (1 → 5)-α-l-arabinans also occurred in the middle lamellae and middle lamella junctions. During development, the location of the xyloglucans changed, being confined to the middle lamellae and middle lamella junctions early on, but later occurred throughout the walls. The location of the heteroxylans also changed, occurring mostly in the outer walls in young cells, but were more widely distributed in mature cells. Solid-state NMR spectroscopy showed that particularly cellulose, but also homogalacturonans, decreased in mobility during development.ConclusionsOur studies showed that celery collenchyma cell walls are primary and that during their development the polysaccharides undergo dynamic changes. Changes in the mobilities of cellulose and homogalacturonans were consistent with the cell walls becoming stiffer as expansion ceases.

Distribution of Fucosylated Xyloglucans among the Walls of Different Cell Types in Monocotyledons Determined by Immunofluorescence Microscopy

Xyloglucans in the non-lignified primary cell walls of different species of monocotyledons have diverse structures, with widely varying proportions of oligosaccharide units that contain fucosylated side chains (F side chains). To determine whether fucosylated xyloglucans occur in all non-lignified walls in a range of monocotyledon species, we used immunofluorescence microscopy with the monoclonal antibody CCRC-M1. The epitope of this antibody, α-L-Fucp-(1→2)-β-D-Galp, occurs in F side chains. In most non-commelinid monocotyledons, the epitope was found in all non-lignified walls. A similar distribution was found in the palm Phoenix canariensis, which is a member of the basal commelinid order Arecales. However, in the other commelinid orders Zingiberales, Commelinales, and Poales, the occurrence of the epitope was restricted, sometimes occurring in only the phloem walls, but often also in walls of other cell types including stomatal guard and subsidiary cells and raphide idioblasts. No epitope was found in the walls of the commelinids Tradescantia virginiana (Commelinaceae, Commelinales) and Zea mays (Poaceae, Poales), but it occurred in the phloem walls of two other Poaceae species, Lolium multiflorum and L. perenne. The distribution of the epitope is discussed in relation to xyloglucan structures in the different taxa. However, the functional significance of the restricted distributions is unknown.

(1→3),(1→4)‐ß‐d‐Glucans in the cell walls of the Poales (sensu lato): an immunogold labeling study using a monoclonal antibody

(1→3),(1→4)-ß-Glucans had previously been detected in nonlignified cell wall preparations of only the Poaceae and five other families in the graminoid clade of the Poales (s.l.). Cell walls of vegetative organs of 12 species in nine families of the Poales (s.l.) were examined by immunogold labeling using a monoclonal antibody to (1→3),(1→4)-ß-glucans. Three types of wall-labeling patterns were identified depending on the density of labeling of the nonlignified walls of epidermal and parenchyma cells and the lignified walls of sclerenchyma fibers and xylem tracheary elements: type 1 in Poaceae and Flagellariaceae, type 2 in Restionaceae and Xyridaceae, and type 3 in Cyperaceae and Juncaceae. Type 1 had the heaviest labeling of nonlignified walls and type 2 the heaviest labeling of lignified walls. Type 3 had the least wall labeling, with only very light labeling of nonlignified and lignified walls. No labeling was found over walls of Typhaceae, Sparganiaceae, or Bromeliaceae. The results are discussed in relation to Poales phylogeny.

Polysaccharide compositions of leaf cell walls of forage chicory ( Cichorium intybus L.)

The polysaccharide compositions of cell walls isolated from the laminae and midribs of chicory (cv. Grasslands Puna II) leaves were investigated. The walls, which, except for the walls of xylem tracheary elements in vascular bundles, were non-lignified, were fractionated progressively with 50 mM CDTA, 50 mM Na 2CO 3, 1 M KOH, 4 M KOH, 4 M KOH + 3.5% H 3BO 3, and hot water. Monosaccharide and linkage analyses of the polysaccharides in the walls and wall fractions indicated that the polysaccharides were similar to those in the non-lignified walls of other dicotyledons. However, the proportions of pectic polysaccharides were particularly high (67% of the total wall polysaccharides in the laminae). These included homogalacturonans (50% of the total wall polysaccharides in the laminae) and rhamnogalacturonan-I (RG-I). In contrast, the proportions of cellulose, xyloglucans, heteroxylans and glucomannans were particularly low. The xyloglucans were probably fucogalactoxyloglucans. The wall polysaccharides from the leaf laminae differed from those from the midribs in having higher proportions of homogalacturonans, lower proportions of non-pectic polysaccharides, and RG-I with a higher ratio of galactan to arabinan.

Cyto- and histochemical demonstration of lignins in plant cell walls: an evaluation of the chlorine water/ethanolamine-silver nitrate method of Coppick and Fowler

The chlorine water/ethanolamine-silver nitrate method introduced by Coppick and Fowler for the detection of lignins was evaluated for cyto- and histochemical work using different reagents and fixatives for specimens embedded in epoxy resin. Fixation schedules tested included ethanol, glutaraldehyde, and glutaraldehyde followed by OsO4 as a post-fixative. Chlorine water, sodium hypochlorite, and calcium hypochlorite were the oxidising agents evaluated for their efficacy as part of the Coppick and Fowler procedure. The Coppick and Fowler method was tested against stem woody tissue ofLophomyrtus obcordata, and haustorial xylem tissue of the sucker of its attached dwarf mistletoeKorthalsella lindsayi. The presence of lignins in walls of these cells was indicated in thin sections for transmission electron microscopy by fine electron-dense deposits. Post-staining thin sections did not affect the lignin reaction, but tended to mask its effect due to increased wall contrast. In histological preparations lignified walls stained orange/brown. Counter-staining in methylene blue/azur B caused lignified walls to appear dark green/brown and non-lignified walls blue. Fixation in either ethanol or glutaraldehyde produced identical staining for lignins. Penetration by chlorine water was sometimes irregular, more so with glutaraldehyde fixation, with parts of tissues consequently not responding to the lignin reaction. Post-fixation in osmium tetroxide following primary fixation in glutaraldehyde slightly improved penetration of chlorine water. However, osmium caused greater amounts of extraneous stain deposits compared with other fixative regimes. Chlorine water was confirmed as the most effective oxidising agent for reacting with groups in lignins to produce reducing residues in the Coppick and Fowler method. Sodium hypochlorite caused no reaction. Calcium hypochlorite exhibited limited oxidative capacity resulting in slight staining for lignins. The Coppick and Fowler procedure was concluded to be a suitable method for demonstrating lignins in cyto- and histochemical preparations using material fixed in either ethanol or glutaraldehyde, and with embedding in epoxy resin.

P-Hydroxyphenyl, Guaiacyl, and Syringyl Lignins Have Similar Inhibitory Effects on Wall Degradability

Studies with normal, mutant, and transgenic plants have not clearly established whether the proportion of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units in lignin directly affects the degradability of cell walls by hydrolytic enzymes. Dehydrogenation polymer−cell wall complexes containing varying ratios of H, G, and S lignins were formed by peroxidase/H2O2-mediated polymerization of p-coumaryl, coniferyl, and sinapyl alcohols into nonlignified walls isolated from cell suspensions of maize (Zea mays L). Lignification substantially reduced the degradability of cell walls by fungal hydrolases, but degradability was not affected by lignin composition. On the basis of these results, we propose that improvements in wall degradability, previously attributed to changes in lignin composition, were in fact due to other associated changes in wall chemistry or ultrastructure. Keywords: Gramineae; Zea mays; cell wall; brown midrib; transgenic; O-methyltransferase; hydroxycinnamyl alcohols; lignin; cellulase; degradability

WOOD ANATOMY AND RELATIONSHIPS OF LACTORIDACEAE

Mature wood of Lactoris, not previously available for study, reveals ten distinctive characters: vessels with simple perforation plates; vessels in pore multiples; vessel‐to‐axial parenchyma pits scalariform or transitional, vessel‐to‐vessel pits alternate; fiber‐tracheids with vestigial pits; fiber‐tracheids, vessels, and axial parenchyma storied; axial parenchyma vasicentric scanty; axial parenchyma either not subdivided or, if subdivided, with thin nonlignified walls between the cells (like the septa in septate fibers); rays wide and tall, little altered during ontogeny; ray cells upright; and ray cells taller adjacent to fascicular areas. All of these features occur in woods of Piper and other Piperaceae. The systematic position of Lactoris is therefore reassessed. Evidence available to date is consonant with placement of Lactoridaceae in Piperales, in which it would be more primitive than Piperaceae or Saururaceae. Features cited as evidence for alternative placements of Lactoridaceae are reviewed.

Wood Anatomy and Relationships of Lactoridaceae

Mature wood of Lactoris, not previously available for study, reveals ten distinctive characters: vessels with simple perforation plates; vessels in pore multiples; vessel-to-axial parenchyma pits scalariform or transitional, vessel-to-vessel pits alternate; fiber-tracheids with vestigial pits; fiber-tracheids, vessels, and axial parenchyma storied; axial parenchyma vasicentric scanty; axial parenchyma either not subdivided or, if subdivided, with thin nonlignified walls between the cells (like the septa in septate fibers); rays wide and tall, little altered during ontogeny; ray cells upright; and ray cells taller adjacent to fascicular areas. All of these features occur in woods of Piper and other Piperaceae. The systematic position of Lactoris is therefore reassessed. Evidence available to date is consonant with placement of Lactoridaceae in Piperales, in which it would be more primitive than Piperaceae or Saururaceae. Features cited as evidence for alternative placements of Lactoridaceae are reviewed.

The Structure of the Column in Stylidium

In the genus Stylidium, the style and filaments of the flower are fused into a single column. In most species the column, when stimulated mechanically, undergoes a fast firing movement followed by a slow resetting movement. This movement is produced by changes in shape of a normally curved region of the column, the bend. In a wide range of species, the bend has a specialised anatomy and consists essentially of a longitudinal central layer of cells with two distinctive multi-celled layers of thick-walled cells on either side. The thick-walled cells are rich in cytoplasm with amyloplasts and vacuoles of varying sizes, and have non-lignified walls whose cellulose fibrils are arranged approximately transversely. Within the bend, the phloem occurs as discrete small groups of cells separated by some distance from the xylem. In species from the subgenus Centridium both the morphology and the internal structure of the bend differ somewhat from those in most species of Stylidium, and in two species of Stylidium with nonmoving columns, the characteristic cellular anatomy of the bend is entirely absent. The specialised anatomy of the cells and tissues in the bend are clearly associated with the movement of the column. Changes of shape and size of these cells are almost certainly responsible for the change in shape of the bend.

The effect of induced lignification on the resistance of wheat cell walls to fungal degradation

Lignified papillae and haloes from Botrytis cinerea inoculated wheat leaves were highly resistant to in vitro degradation by fourteen fungal species. Wheat pathogens were generally no more capable of degrading the structures than non-pathogens. Papillae and haloes from leaves taken as early as 12 h after inoculation showed substantial resistance indicating the possible importance of relatively small amounts of lignin. Quantitative measurements of the release of carbohydrate from lignified and non-lignified walls by fungal culture filtrates supported these visual observations. The activities of laccase and p-coumaryl esterase in culture filtrates did not correlate with pathogenicity. The significance of the results is discussed in relation to the proposed role of lignification in the resistance of wheat to fungi.

Simple method for differential staining of paraffin embedded plant material using toluidine blue o.

Differential staining of paraffin embedded plant material is accomplished without removing the paraffin. Material fixed in 3% glutaraldehyde in 0.02 M phosphate buffer is dehydrated, embedded, sectioned, and mounted on glass slides using conventional methods. The slides are placed in 0.05% toluidine blue O in distilled water for 2–30 min, rinsed in water for 1 min, and allowed to air dry. Paraffin is removed with 2 changes of xylene and the cover slip mounted with resin. Lignified tissue, suberized tissue, and some tannins are stained blue-green, while nonlignified walls are stained red-purple. An advantage of the method is the short time required for staining and mounting. Also, critical counterstaining is not required.

Plant Water Deficits and Physiological Processes

The plant component of the soil-plant-atmosphere system is an organized body or structure permeated by water. When we speak of a plant as an organization we refer to evidence of the integrated unity that is apparent throughout its entire phenologic cycle ( 186) . In the view of Needham (1 12) the plant (as a micro-universe) can be con­ sidered as having a number of levels of organization and complexity from the atomic to the whole organism. Laws applying to one level may not apply to other levels. Each level requires its own concepts. Any study of more than one level at a time must involve an attempt to determine the intrinsic rela­ tionships between one level of complexity and the others, and, finally, the organizations perceived at each level of complexity must be integrated. These same considerations should govern our concepts of the relation between the plant and the soil and atmospheric environmental factors affect­ ing its life, with water recognized as a vital factor that is both intrinsic and extrinsic. Water is essential to the life and growth of plants, being a major constituent of living green tissues and often comprising as much as 90 to 95 per cent of the fresh weight. Water provides plants with their mechanical strength through the mechanism of cell turgor, since most vegetative tissues are composed of cells with non-lignified walls. Such plastic bodies become mechanically stable under the influence of the hydrostatic pressure developed by water inside the cell. Water is a raw material in metabolism and in the synthesis reactions that occur in living plants, even though the amounts involved here are rather small compared to the total amount of water that passes through the plant. Large amounts of water move into the roots, through the stem, into the leaves, and out into the atmosphere in the form of vapor, in what is known as the transpiration stream. Growth itself is an expression of cell enlargement brought about by the action of water. Many of the metabolic reactions of plants are conditioned by the degree of hydra­ tion of certain of the reactants or enzymes (23, 185). To understand this complex system we must approach it with the realiza­ tion that it is highly dynamic and composed of many interlocking and inter­ dependent phenomena. One of the most difficult problems encountered in understanding the behavior of water in the soil-plant system arises because observed reactions involving water have to be integrated both over time and through volume (141) .