Cellulose Content in Fibers of Cottons which Differ in their Lint Lengths and Extent of Fuzz

Three cottons differing in their extent of fuzz fibers (linters) and final length of lint fibers were analyzed for amount of fiber cell walls and fiber cellulose at various times postanthesis. Cellulose determinations were performed directly on whole fibers and on fiber cell wall preparations. The data suggest that the presence of fuzz fibers does not account for a rise, followed by a plateau, followed by a rise, in cellulose content expressed as a percentage of cell wall material. It is concluded that: (1) under our greenhouse conditions, all fuzz fibers are initiated by day eight after anthesis; (2) weight per mm length of all fibers increases up to the point of secondary wall deposition and increases even more rapidly after that; (3) deposition of secondary wall cellulose in fuzz fibers probably does not begin until after similar deposition begins in lint fibers; (4) the actual amount of cellulose in primary walls of all elongating fibers (fuzz and lint) is a constant value, about 1 × 10^?16 mg/mm; and (5) secondary wall cellulose deposition in lint fibers begins very sharply, in advance of cessation of elongation, at a time closely related to final lint fiber length. It is speculated that: (1) cell wall preparation procedures may remove significant amounts of noncellulosic wall material, thus making it difficult to define all functional constituents on the basis of what is left in a cell wall residue; and/or (2) primary walls may lose to the cytoplasm some of their constituents in advance of secondary wall deposition, the extent of loss varying due to developmental age of the elongating fibers.     

DEVELOPMENTAL MORPHOLOGY OF COTTON FLOWERS AND SEED AS SEEN WITH THE SCANNING ELECTRON MICROSCOPE

This report assembles and pictorially presents observations on the timing of relatively uniform and well-defined developmental events in the cotton flower and its component parts. The first floral bud occurs on the 7–9th node approximately 35–40 days postemergence; 20–25 additional days elapse until anthesis. Floral parts are morphologically well defined by two weeks preanthesis. In about 85 % of the flowers the basal, abaxial surface of two of the three bracts contains an outer involucral nectary; occasionally, none, one, or three nectaries are found. The maximum rate of increase in floral bud length occurs during the 24 hrs preceding anthesis. Flower opening occurs at about daylight, although light is not required. Multipored pollen grains germinate in about ½ hr after deposition on the stigmatic hairs. Fertilization is accomplished, for most ovules, by the end of the first day postanthesis. Stomata are abundant, particularly at the chalazal ends of ovules. Fiber initials (epidermal cells of the ovule) begin their elongation phase on the morning of anthesis and are bounded by a thin primary wall. Areas of contrast (spots) observed through the scanning electron microscope are speculated to be organelles “seen through” the relatively amorphous fiber wall, which lacks extensive fibrillar orientation of cellulose. Fiber elongation ceases by about 24–28 days postanthesis, and by 50–70 days postanthesis fibers are mature and exhibit a thickened secondary wall and spiral twisting. Concomitant with the time of fiber maturity, the ovary wall splits and opens along locular suture lines.     

Changes in cell wall polysaccharides in the internodes of submerged floating rice

In excised stem segments of floating rice (Oryza sativa L.), as well as in intact plants, submergence greatly stimulates the elongation of internodes. The differences in the composition of cell wall polysaccharides along the highest internodes of submerged and air-grown stem segments were examined. The newly elongated parts of internodes that had been submerged for two days contained considerably less cellulosic and noncellulosic polysaccharides than air-grown internodes, an indication that the cell walls of the newly elongated parts of submerged internodes are extremely thin. In the young parts of both air-grown and submerged internodes, the relative amounts of noncellulosic polysaccharides were equal to those of -cellulose, whereas the relative amounts of -cellulose were higher than those of noncellulosic polysaccharides in the upper, old parts. In the cell-elongation zones of both air-grown and submerged internodes, glucose was predominant among the noncellulosic neutral sugars of cell wall. The relative amount of glucose in noncellulosic neutral sugars decreased toward the upper, old parts of internodes, whereas that of xylose increased.     

Changes in cell-wall polysaccharide composition of developingNitella internodes

Changes in the uronide, neutral-polysacharide, and cellulose composition of the cell wall ofNitella axillaris Braun were followed throughout development of the internodes and correlated with changes in growth rate. As the cells increased in length from 4 to 80 mm during development, the relative growth rate decreased. Cell wall thickness, as measured by wall density, increased in direct proportion to diameter, indicating that cell-wall stress did not change during elogation. Cell-wall analyses were adapted to allow determination of the composition of the wall of single cells. The total amounts of uronides, neutral sugars and cellulose all increased during development. However, as the growth rate decreased, the relative proportions of uronides and neutral sugars, expressed as percent of the dry weight of the wall, decreased, while the proportion of cellulose increased. The neutral sugars liberated upon hydrolysis ofNitella walls are qualitatively similar to those found in hydrolysates of higher plant cell walls: glucose, xylose, mannose, galactose, arabinose fucose and rhamnose. Only the percentage of galactose was found to increase in walls of mature cells, while the percentage of all other sugars decreased. The rate of apposition (g of wall material deposited per unit wall surface area per hour) of neutral polysaccharides decreased rapidly with decreasing growth rate during the early stages of development. The rate of apposition of uronides decreased more steadily throughout development, while that of cellulose, after an early decline, remained constant until dropping off at the end of the elongation period. These correlations between decreasing growth rate and decreasing rate of apposition of neutral sugars and uronides indicate that synthesis of these cell-wall components could be involved in the regulation of the rate of cell elongation inNitella.     

Changes in expansin activity and cell wall susceptibility to expansin action during cessation of internodal elongation in floating rice

We investigated the involvement of expansin action in determining the growth rate of internodes of floating rice (Oryza sativa L.). Floating rice stem segments in which rapid internodal elongation had been induced by submergence for 2 days were exposed to air or kept in submergence for 2 more days. Both treatments reduced the elongation rate of the internodes, and the degree of reduction was much greater in air-exposed stem segments than in continually submerged segments. These rates of internodal elongation were correlated with acid-induced cell wall extensibility and cell wall susceptibility to expansins in the cell elongation zone of the internodes, but not with extractable expansin activity. These results suggest that the reduced growth rate of internodes must be due, at least in part, to the decrease in acid-induced cell wall extensibility, which can be modulated through changes in the cell wall susceptibility to expansins rather than through expansin activity. Analysis of the cell wall composition of the internodes showed that the cellulosic and noncellulosic polysaccharide contents increased in response to exposure to air, but they remained almost constant under continued submergence although the cell wall susceptibility to expansins gradually declined even under continued submergence. The content of xylose in noncellulosic neutral sugars in the cell walls of internodes was closely and negatively correlated with changes in the susceptibility of the walls to expansins. These results suggest that the deposition of xylose-rich polysaccharides into the cell walls may be related to a decrease in acid-induced cell wall extensibility in floating rice internodes through the modulation of cell wall susceptibility to expansins.     

Changes in the composition of cotton fibre cell walls during development

Purified cell walls, prepared from cotton fibres (Gossypium arboreum L.) at different growth stages, were subjected to successive extractions to give pectic, hemicellulosic, and -cellulosic fractions. The protein content and sugars obtained after hydrolysis of the total cell walls and of the various fractions were quantitatively estimated. The amount of protein in the fibre cell walls from one ovule reached a maximum value at the end of the elongation growth, decreased, and then reached a second maximum at the end of the secondary wall deposition. The absolute amounts of fucose, galactose, mannose, rhamnose, arabinose, uronic acid, and non-cellulosic glucose residues all reached a maximum at the end of the primary wall formation or at the beginning of the secondary wall formation. Only the absolute amounts of xylose and of the cellulosic glucose residues increased until the end of the fibre development. Most conspicuous was the decrease in the absolute amounts of non-cellulosic glucose and of arabinose residues during the secondary wall formation, possibly indicating a turnover of at least some of the hemicellulosic wall material.Abbreviations DPA days post anthesis - TLC thin layer chromatography - SDS sodium dodecyl sulphate     

Soluble Cell Wall Polysaccharides Released from Pea Stems by Centrifugation : II. EFFECT OF ETHYLENE

The effect of ethylene on cell wall metabolism in sections excised from etiolated pea stems was studied. Ethylene causes an inhibition of elongation and a pronounced radial expansion of pea internodes as shown by an increase in the fresh weight of excised, 1-cm sections. Cell wall metabolism was studied using centrifugation to remove the cell wall solution from sections. The principal neutral sugars in the cell wall solution extracted with H₂O are arabinose, xylose, galactose, and glucose. Both xylose and glucose decline relative to controls in air within 1 hour of exposure to ethylene. Arabinose and galactose levels are not altered by ethylene until 8 hours of treatment, whereupon they decline in controls in air relative to ethylene treatment. When alcohol-insoluble polymers are fractionated into neutral and acidic polysaccharides, xylose and glucose predominate in the neutral fraction and arabinose and galactose in the acidic fraction. Ethylene depresses the levels of xylose and glucose in the neutral fraction and elevates arabinose and galactose in the acidic fraction. Ethylene treatment does not affect the level of uronic acids extracted with H₂O; however, the level of hydroxyproline-rich proteins in this water-extracted cell wall solution is increased by ethylene. Extraction of sections with CaCl₂ results in an increase in the levels of neutral sugars particularly arabinose. Ethylene depresses the yield of arabinose in calcium-extracted solution relative to controls in air. Similarly, extraction with CaCl₂ increases the yield of extracted hydroxyproline in ethanol-insoluble polymers and ethylene depresses its level relative to controls. Metabolism of uronic acids and neutral sugars and growth in response to ethylene treatment contrast markedly with auxin-induced polysaccharide metabolism and growth. With auxin, sections increase mostly in length not radius, and this growth form is associated with an increase in the levels of xylose, glucose, and uronic acids. With ethylene, on the other hand, stem elongation is suppressed and expansion is promoted, and this growth pattern is associated with a decrease in xylose and glucose in the ethanol-insoluble polysaccharides.     

Utilization of nucleoside diphosphate glucoses in developing cotton fibers

The capacity for biosynthesis of hot alkali-insoluble products using uridine diphosphate (UDP)-glucose and guanosine diphosphate (GDP)-glucose as substrate has been studied in isolated cotton fibers harvested at various stages of development following anthesis. During the period of rapid elongation and primary wall synthesis (7-14 days postanthesis), incorporation of radioactivity from GDP-¹⁴C-glucose into hot alkali-insoluble product is high. This activity gradually declines and is not demonstrated in older fibers undergoing active deposition of secondary wall. With respect to all characteristics examined, the product from GDP-glucose resembles cellulose. Incorporation of UDP-¹⁴C-glucose into hot alkali-insoluble product was low in young fibers but increased to high levels in older fibers. This product was shown to be soluble in chloroform-methanol, and when chromatographed in lipid solvents it was separated into three components. Activity for the production of two of these three presumed glucolipids increased with increasing age of fibers.     

Auxin-induced Changes in Avena Coleoptile Cell Wall Composition

Sugar and uronic acid residues were derived from wall polysaccharides of oat (Avena sativa, var. Victory) coleoptiles by means of 2 N trifluoroacetic acid, 72% sulfuric acid, or enzymic hydrolysis. The products of hydrolysis were reduced and acetylated to form alditol acetates which were analyzed using gas chromatography. Time-course studies of auxin-promoted changes in various wall fractions indicate that when exogenous glucose was available, increases in certain wall constituents paralleled increases in length. However, under conditions where exogenous glucose was not available, and where wall synthesis was limited, such correlations with growth were not apparent. Under these latter conditions total wall weight initially increased slightly, then decreased. These changes in weight were the net of increases in cellulose and some noncellulosic constituents and a decrease of over 75% in noncellulosic glucose. When coleoptile sections were preincubated without exogenous glucose for 8 hours to deplete endogenous wall precursors and subsequently treated with auxin, there were no detectable increases in wall weight. There was instead an auxin-promoted decrease in wall weight, and this decrease paralleled a decrease in noncellulosic glucose. There were no significant changes in other wall components. The auxin-promoted decreases in noncellulosic glucose are interpreted as a possible step in the mechanism of growth.     

ULTRASTRUCTURAL ASPECTS OF EARLY STAGES IN COTTON FIBER ELONGATION

Fine structural alterations associated with early stages of cotton fiber elongation in Gossypium hirsutum L. var. dunn 56 C occur rapidly following anthesis and appear to be correlated with the formation of the central vacuole, plasma membrane, and primary cell wall as well as with increased protein synthesis necessary for cell elongation. Association of dilated cisternae of the endoplasmic reticulum with the tonoplast suggests that the endoplasmic reticulum is involved in the formation of the central vacuole. Dictyosome involvement in both plasma membrane and primary cell wall formation was suggested from observations of similarities between dictyosome associated vesicles, containing fibrils appearing similar in morphology to fibrils found in the primary cell wall, and plasma membrane associated vesicles. The single nucleolus found in cotton fibers enlarges following anthesis, shows segregation of granular and fibrillar components by 1 day postanthesis, develops a large “vacuole,” thus appearing ring-shaped, and occupies much of the nuclear volume by 2 days postanthesis. Prominent nucleoli were not observed in nuclei after 10 days postanthesis.     

A kinesin-like protein is essential for oriented deposition of cellulose microfibrils and cell wall strength

Cortical microtubules have long been hypothesized to regulate the oriented deposition of cellulose microfibrils. However, the molecular mechanisms of how microtubules direct the orientation of cellulose microfibril deposition are not known. We have used fibers in the inflorescence stems of Arabidopsis to study secondary wall deposition and cell wall strength and found a fragile fiber (fra1) mutant with a dramatic reduction in the mechanical strength of fibers. The fra1 mutation did not cause any defects in cell wall composition, secondary wall thickening, or cortical microtubule organization in fiber cells. An apparent alteration was found in the orientation of cellulose microfibrils in fra1 fiber walls, indicating that the reduced mechanical strength of fra1 fibers probably was attributable to altered cellulose microfibril deposition. The FRA1 gene was cloned and found to encode a kinesin-like protein with an N-terminal microtubule binding motor domain. The FRA1 protein was shown to be concentrated around the periphery of the cytoplasm but absent in the nucleus. Based on these findings, we propose that the FRA1 kinesin-like protein is involved in the microtubule control of cellulose microfibril order.     

Phytosterol content and the campesterol:sitosterol ratio influence cotton fiber development: role of phytosterols in cell elongation

Phytosterols play an important role in plant growth and development, including cell division, cell elongation, embryogenesis, cellulose biosynthesis, and cell wall formation. Cotton fiber, which undergoes synchronous cell elongation and a large amount of cellulose synthesis, is an ideal model for the study of plant cell elongation and cell wall biogenesis. The role of phytosterols in fiber growth was investigated by treating the fibers with tridemorph, a sterol biosynthetic inhibitor. The inhibition of phytosterol biosynthesis resulted in an apparent suppression of fiber elongation in vitro or in planta. The determination of phytosterol quantity indicated that sitosterol and campesterol were the major phytosterols in cotton fibers; moreover, higher concentrations of these phytosterols were observed during the period of rapid elongation of fibers. Furthermore, the decrease and increase in campesterol:sitosterol ratio was associated with the increase and decease in speed of elongation, respectively, during the elongation stage. The increase in the ratio was associated with the transition from cell elongation to secondary cell wall synthesis. In addition, a number of phytosterol biosynthetic genes were down-regulated in the short fibers of ligon lintless-1 mutant, compared to its near-isogenic wild-type TM-1. These results demonstrated that phytosterols play a crucial role in cotton fiber development, and particularly in fiber elongation.     

THE COMPOSITION OF THE DEVELOPING PRIMARY WALL IN ONION ROOT TIP CELLS. II. CYTOCHEMICAL LOCALIZATION

Jensen , William A. (U. California, Berkeley.) The composition of the developing primary wall in onion root tip cells. II. Cytochemical localization. Amer. Jour. Bot. 47(4) : 287—295. Illus. 1960.–The composition of the developing cell wall in the first 2 mm. of the onion root tip was studied using a cytochemical technique that permitted the detection of hemicellulose and the noncellulosic polysaccharides as well as the pectic substances and cellulose. The technique is based on the combination of a differential extraction procedure with the periodic acid-Schiff reaction for carbohydrates. The data obtained indicate that the cells of the apical initials are low in all wall substances but that all of the wall materials are present to some extent. Early in cell development, differences appear in the composition of the walls of the various tissues. The cortical cells are relatively high in the noncellulosic polysaccharides and cellulose while relatively low in the pectic substances and hemicellulose. Very early in development the protoderm is similar to the cortex, but differences develop during the radial enlargement of the cells. During this stage the walls of the protodermal cells are low in the noncellulosic polysaccharides and cellulose and high in pectic substances and hemicellulose. As elongation progresses, these differences are lost and the 2 tissues become very similar. The vascular cell walls are low in the noncellulosic polysaccharides and cellulose and are high in pectic substances and hemicellulose early in development. Later, hemicellulose becomes relatively more important. When the cell wall materials are sequentially extracted, no change in the general morphology of the cell occurs until only the noncellulosic polysaccharides and the cellulose remained. When the noncellulosic polysaccharides are then removed, the cells remain intact but are 30% less in diameter. This suggests that while cellulose is of critical importance, the noncellulosic polysaccharides may play a major role in determining the physical characteristics of the wall.     

beta-1,3-Glucan in Developing Cotton Fibers: Structure, Localization, and Relationship of Synthesis to That of Secondary Wall Cellulose

Evidence is presented for the existence of a noncellulosic β-1,3-glucan in cotton fibers. The glucan can be isolated as distinct fractions of varying solubility. When fibers are homogenized rigorously in aqueous buffer, part of the total β-1,3-glucan is found as a soluble polymer in homogenates freed of cell walls. The proportion of total β-1,3-glucan which is found as the soluble polymer varies somewhat as a function of fiber age. The insoluble fraction of the β-1,3-glucan remains associated with the cell wall fraction. Of this cell wall β-1,3-glucan, a variable portion can be solubilized by treatment of walls with hot water, a further portion can be solubilized by alkaline extraction of the walls, and 17 to 29% of the glucan remains associated with cellulose even after alkaline extraction. A portion of this glucan can also be removed from the cell walls of intact cotton fibers by digestion with an endo-β-1,3-glucanase. The glucan fraction which can be isolated as a soluble polymer in homogenates freed of cell walls is not associated with membranous material, and we propose that it represents glucan which is also extracellular but not tightly associated with the cell wall. Enzyme digestion studies indicate that all of the cotton fiber glucan is β-linked, and methylation analyses and enzyme studies both show that the predominant linkage in the glucan is 1 → 3. The possibility of some minor branching at C-6 can also be deduced from the methylation analyses. The timing of deposition of the β-1,3-glucan during fiber development coincides closely with the onset of secondary wall cellulose synthesis. Kinetic studies performed with ovules and fibers cultured in vitro show that incorporation of radioactivity from [¹⁴C]glucose into β-1,3-glucan is linear with respect to time almost from the start of the labeling period; however, a lag is observed before incorporation into cellulose becomes linear with time, suggesting that these two different glucans are not polymerized directly from the same substrate pool. Pulse-chase experiments indicate that neither the β-1,3-glucan nor cellulose exhibits significant turnover after synthesis.     

Changes in cell wall polysaccharides during elongation in a 2, 4-D free medium in a carrot suspension culture

Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570-577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4-D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA-soluble pectic substance, 5 and 24%, KOH-soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH-soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH-soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non-elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non-elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.     

Analysis of cell-wall polymers during cotton fiber development

Although the fibers of cotton (Gossypium hirsutum L.) are single cells with a secondary wall composed primarily of cellulose, the cell-wall polymers of the fibers are technically difficult to characterize with respect to molecular weights. This limitation hinders understanding how the fiber wall composition changes during development, particularly with respect to genotypic variations, and how the molecular composition is related to physical properties. We analyzed cell-wall polymers from cotton fibers (cultivar, Texas Marker-1) at several developmental stages (8–60 days post-anthesis; DPA) by gel-permeation chromatography of components soluble in dimethyl acetamide and lithium chloride. This procedure solubilizes fiber cell-wall components directly without prior extraction or derivatization, processes that could lead to degradation of high-molecular-weight components. Cellwall polymers from fibers at primary cell-wall stages had lower molecular weights than the cellulose from fibers at the secondary wall stages; however, the high-molecularweight cellulose characteristic of mature cotton was detected as early as 8 DPA. High-molecular-weight material decreased during the period of 10–18 DPA with concomitant increase in lower-molecular-weight wall components, possibly indicating hydrolysis during the later stages of elongation.Abbreviations DMAC dimethyl acetamide - DP degree of polymerization - DPA days post anthesis - GPC gel-permeation chromatography - MW molecular weight - MWD molecular-weight distribution - TM-1 Texas Marker 1