Characterization of cell-wall polymers from cotton ovule culture fiber cells by gel permeation chromatography

Summary Cotton (Gossypium hirsutum, Texas Marker-1) fiber cells originating from ovule culture have been analyzed by gel permeation chromatography of dimethyl acetamide/lithium chloride-soluble components and compared within planta-grown fibers. The profile of cell-wall polymer molecular weights indicated that fibers grown for 21 d in culture more closely resembled fibers growingin planta for 30 d post-anthesis than fully mature fibers. The weight average molecular weight was 3 400 000 and number average molecular weight of polymers from ovule culture fibers was 109 000. Analysis of the polymer weight fraction distribution revealed that ovule culture fibers were similar to 30 d post-anthesis immature fibers but lacked a low molecular weight (log M 3–4) polymer fraction. Assessment of the polymer branching frequency showed that ovule culture fibers were intermediate in branching between 30 d post-anthesis fiber and maturein planta fiber. In summary, polymers deposited in cell walls of ovule culture fibers appear to grossly mimic the polymers accumulated during normal fiber biogenesisin planta, yet subtle differences may explain why ovule culture fibers rarely reach their full genetic potential in length.     

A novel cottong ovule culture: Induction, growth, and characterization of submerged cotton fibers (Gossypium hirsutum L.)

Summary The growth of submerged cotton (Gossypium hirsutum L.) fibers from cultured ovules has been investigated. The results indicate that exogenous plant hormone levels regulate the induction of submerged fiber growth. The age of ovules at induction is also important. Cell diameter, wall thickness, and cell length of submerged fibers were measured and compared with air-grown fibers and fibers grown in vivo (produced by cotton plants grown in the greenhouse). Various cellwall thickening patterns were observed among submerged fibers, while only one predominant cell-wall deposition pattern was produced in air-grown fibers and in fibers produced in vivo. The diameter of submerged fibers was about the same as that of air-grown fibers but about 22% less than that of fibers grown, in vivo. It appears that the secondary cell wall thickenings are initiated earlier in submerged fibers. The cell-wall thickness of submerged fibers, at 41 d post anthesis (DPA), was 51% greater than that of fibers grown in vivo, whereas the cell-wall thickness of air-grown fibers was 42% less than that of fibers produced in vivo. The cell length of submerged fibers was approximately half that of fibers grown in vivo. and the air-grown fiber length was about two-thirds of fibers grown in vivo. The age of ovules at induction affects the outcome of the air-grown fiber-cell length, but does not appear to affect the length of submerged fiber cells. To produce submerged fiber growth, we found that the optimal age of ovules at induction was 0 DPA, and the optimal medium (with a GA₃ of 0.5 μM and an IAA range of 5-20 μM) depends on the time of ovule induction (−2 to+2DPA). We conclude that conditions leading to submerged cotton fiber growth have great potential for (a) direct monitoring of growth and making precise, detailed measurements during fiber growth and development; (b) producing cellulose and fibers in vitro more efficiently than earlier ovule-culture methods; and (c) using these unique cultures to obtain a better understanding of signal transduction and gene expression leading to growth, development, and programmed cell death in the life history of the cotton fiber.     

Cotton-fiber germin-like protein. II: Immunolocalization,purification, and functional analysis

Cotton (Gossypium hirsutum L.) contains a germin-like protein (GLP), GhGLP1, that shows tissue-specific accumulation in fiber. The fiber GLP is an oligomeric, glycosylated protein with a subunit size of approximately 25.5 kDa. Accumulation of GhGLP1 occurs during the period of fiber elongation [4–14 days post-anthesis (DPA)]. During early phases of fiber development (2–4 DPA), GhGLP1 localizes to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7–10 DPA), GhGLP1 localizes to the apoplast. In other plants, germins and GLPs have been reported to have enzymatic activities including oxalate oxidase (OxO), superoxide dismutase, and ADP-glucose pyrophosphatase. Cotton fiber extracts did not contain OxO activity, nor did intact fibers stain for OxO activity. A four-step purification protocol involving ammonium sulfate precipitation of a 1.0 M NaCl extract, ion-exchange chromatography on DEAE-Trisacryl M, lectin-affinity chromatography, and gel filtration chromatography resulted in electrophoretically pure GhGLP1. While 1.0 M NaCl extracts from 10–14 DPA fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a GLP accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.Abbreviations ABP Auxin binding protein - AGPPase ADP-Glucose pyrophosphatase/phosphodiesterase - bis-PNPP Bis-p-nitrophenol phosphate - ConA Concanavalin A - DOA Day of anthesis - DPA Days post-anthesis - GLP Germin-like protein - Mn-SOD Manganese superoxide dismutase - OxO Oxalate oxidase - PBS Phosphate-buffered saline     

Characterization of inhibitors of cellulose synthesis in cotton fibers

Several compounds were tested for their ability to inhibit the in-vivo synthesis of cellulose and other cell-wall polysaccharides in fibers of cotton (Gossypium hirsutum L.) developing on in-vitro cultured ovules. Inhibitory effects were measured by the ability of the compounds to inhibit the incorporation of radioactivity from [U-¹⁴C]glucose into these cell-wall polymers. Of the compounds surveyed, 2,6-dichlorobenzonitrile (DCB) was the most effective and specific one for its effects on cellulose synthesis when compared to its effect on the synthesis of other cell-wall components. At 10 M DCB caused 80% inhibition of cellulose synthesis, and the effect was reversed upon removal of the DCB, with recovery to 90% of the control rate. Two analogs of DCB, 2-chloro-6-fluorobenzonitrile and 2,6-dichlorobenzene carbothiamide, were as specific and nearly as effective as DCB with respect to their effects on cellulose synthesis. Coumarin, generally regarded as an inhibitor of cellulose synthesis in other plant systems, was effective in cotton fibers in millimolar concentrations and, like DCB, was relatively specific with regard to its effect on cellulose synthesis. DCB and coumarin inhibited the synthesis of both primary and secondary wall cellulose. Bacitracin, an inhibitor of the cycling of phosphorylated polyprenols involved in cell-wall synthesis in bacteria, and ethylenediaminetetracetic acid (EDTA) and ethyleneglycol-bis-(-amino-ethylether)-N,N-tetracetic acid (EGTA), chelators of civalent cations, were also effective, although only at relatively high concentrations, in inhibiting incorporation of radioactivity into cellulose.Abbreviations DCB 2,6-dichlorobenzonitrite - CFB 2-chloro-6-fluorobenzonitrile - EDTA ethylenediaminetetracetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)-N,N-tetracetic acid     

Changes in the sugar composition and molecular mass distribution of matrix polysaccharides during cotton fiber development

Cotton (Gossypium herbaceum L.) fiber development consists of a fiber elongation stage (up to 20 d post-anthesis) and a subsequent cell wall thickening stage. Cell wall analysis revealed that the extractable matrix (pectic and hemicellulosic) polysaccharides accounted for 30-50% of total sugar content in the fiber elongation stage but less than 3% in the cell wall thickening stage. By contrast, cellulose increased dramatically after the fiber elongation ceased. The amounts of extractable xyloglucans and arabinose- and galactose-containing polymers per seed increased in the early fiber elongation stage and decreased thereafter. The amounts of extractable acidic polymers and non-cellulosic beta-glucans (mainly composed of beta-1,3-glucans) increased in parallel with fiber elongation and then decreased. The molecular masses of extractable non-cellulosic beta-glucans, and arabinose- and galactose-containing polymers decreased during both fiber elongation and cell wall thickening stages. The molecular mass of extractable xyloglucans also decreased during the fiber elongation stage, but this decrease ceased during the cell wall thickening stage. Conversely, the molecular size of acidic polymers in the extractable pectic fraction increased during both stages. Thus, not only the amounts but also the molecular size of the extractable matrix polysaccharides showed substantial changes during cotton fiber development.     

Glucan synthesis by intact cotton fibres fed with different precursors at the stages of primary and secondary wall formation

Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 M were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo--1,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight material was found in cellulose and at least 2/3 in -1,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation -1,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.Abbreviations UDPG uridinediphosphoglucose - GDPG guanosinediphosphoglucose - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid - DMSO dimethyl-sulfoxide - DNP 2,4-dinitrophenol - DPA days post anthesis     

Regulation of cotton fiber elongation by xyloglucan endotransglycosylase/hydrolase genes

Ligon lintless mutant (li1li1) with super-short fibers (5-8 mm in length) and its wild type (Li1Li1) with normal fibers (30 mm in length) were used to study the function of xyloglucan endotransglycosylase/hydrolase (XTH) genes during fiber elongation in cotton. Wild-type cotton attained the fiber elongation stage earlier (5 days post-anthesis, DPA), than the Ligon lintless mutant (12 DPA) with a higher fiber elongation velocity of about 1.76 mm/day. Xyloglucan contents in Ligon lintless mutant fibers were 5-fold higher than the wild type during 9-15 DPA. It was also observed that the activity of XTH in wild-type cotton fibers was about 2-fold higher than that of the Ligon lintless mutant with a peak at 12 DPA. DNA blot analysis indicated that the XTH gene in the Ligon lintless mutant and its wild type belonged to a multiple allelic series. However, RNA blot analysis and quantitative real-time PCR exhibited an earlier expression (10 DPA) of XTH in wild type as compared to delayed (15 DPA) expression in the Ligon lintless mutant. The study also revealed that 9-15 DPA might be a key phase for upregulation of fiber elongation via increasing XTH activity. Higher XTH activity can cleave down the xyloglucan-cellulose chains thus loosening fiber cell wall and promoting fiber cell elongation in wild type as compared to its mutant.     

Specific and direct measurement of theβ-1,3-glucan in developing cotton fiber

The reaction of N,N-diethylaziridinium chloride with raw cotton (Gossypium hirsutum L.) seed fibers to introduce N,N-diethylaminoethyl (DEAE) substituents at a low degree of substitution was used for demonstrating the presence of O(4)H, characteristic of a -1,3-glucan. The derivatized 1,3-glucan/cellulose was hydrolyzed to DEAE-glucoses that were analyzed by gas-liquid chromatography. Capillary columns proved effective for measuring the small amounts of 4-O-DEAE-glucose in the presence of major amounts of 2-O- and 6-O-DEAE-glucoses. Analyses of raw cotton fibers were carried out through fiber development (20, 27, 34, 41 and 48 d post anthesis, DPA) and field exposure (62, 83 and 104 DPA) periods. The yields of 4-O- and other individual DEAE-glucoses and the yield of 4-O-DEAE-glucose in relation to 2-O-DEAE-glucose were particularly informative concenring the role of the -1,3-glucan in cellulose. The results confirmed the early production and almost immediate decrease of the -1,3-glucan and demonstrated continued production of accessible cellulose followed by a sharp decrease in accessibility after boll opening. The -1,3-glucan content of the raw cotton fiber, estimated from the yield of 4-O-DEAE-glucose (representing 1,3-glucan) and the yield of 2-O-DEAE-glucose (approximating 1,3-glucan plus cellulose) was 10%, 4%, 1% and 0.6% at, in the order given, 20, 27, 48, and 104 DPA. These results are in general agreement with other conventional analyses.Abbreviations DPA days post-anthesis - DEAE diethylaminoethyl     

In-situ Raman microprobe studies of plant cell walls: Macromolecular organization and compositional variability in the secondary wall of Picea mariana (Mill.) B.S.P.

Native-state organization and distribution of cell-wall components in the secondary wall of woody tissue from P. mariana (Black Spruce) have been investigated using polarized Raman microspectroscopy. Evidence for orientation is detected through Raman intensity variations resulting from rotations of the exciting electric vector with respect to cell-wall geometry. Spectral features associated with cellulose and lignin were studied. The changes in cellulose bands indicate that the pyranose rings of the anhydroglucose repeat units are in planes perpendicular to the cross section, while methine C–H bonds are in planes parallel to the cross section. Changes in bands associated with lignin indicate that the aromatic rings of the phenyl-propane units are most often in the plane of the cell-wall surface. However, regions where lignin orientation departs from this pattern also occur. These results represent direct evidence of molecular organization with respect to cellular morphological features in woody tissue, and indicate that cell-wall components are more highly organized than had been recognized. Studies carried out in order to establish the usefulness and sensitivity of the Raman technique to differences of composition within the cell walls provide evidence of variations in the distribution of cellulose and lignin. Such compositional differences were more prominent between the walls of different cells than within a particular cell wall.     

Xyloglucan breakdown during cotton fiber development

Cotton (Gossypium herbaceum L.) fibers elongated almost linearly up to about 20 days post anthesis. The molecular mass of xyloglucans in fiber cell walls decreased gradually during the elongation stage. When enzymatically active (native) cell wall preparations of fibers were autolyzed, the molecular mass of xyloglucans decreased. The decrease was most prominent in wall preparations obtained from the rapidly elongating fibers. The xyloglucan-degrading activity was recovered from the fiber cell walls with 3 mol/L NaCl, and the activity was high at the stages in which fibers elongated vigorously. These results suggest the possible involvement of xyloglucan metabolism in the regulation of cotton fiber elongation.     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

Changes in biochemical composition of the cell wall of the cotton fiber during development

The composition of the cell wall of the cotton fiber (Gossypium hirsutum L. Acala SJ-1) has been studied from the early stages of elongation (5 days postanthesis) through the period of secondary wall formation, using cell walls derived both from fibers developing on the plant and from fibers obtained from excised, cultured ovules. The cell wall of the elongating cotton fiber was shown to be a dynamic structure. Expressed as a weight per cent of the total cell wall, cellulose, neutral sugars (rhamnose, fucose, arabinose, mannose, galactose, and noncellulosic glucose), uronic acids, and total protein undergo marked changes in content during the elongation period. As a way of analyzing absolute changes in the walls with time, data have also been expressed as grams component per millimeter of fiber length. Expressed in this way for plant-grown fibers, the data show that the thickness of the cell wall is relatively constant until about 12 days postanthesis; after this time it markedly increases until secondary wall cellulose deposition is completed. Between 12 and 16 days postanthesis increases in all components contribute to total wall increase per millimeter fiber length. The deposition of secondary wall cellulose begins at about 16 days postanthesis (at least 5 days prior to the cessation of elongation) and continues until about 32 days postanthesis. At the time of the onset of secondary wall cellulose deposition, a sharp decline in protein and uronic acid content occurs. The content of some of the individual neutral sugars changes during development, the most prominent change being a large increase in noncellulosic glucose which occurs just prior to the onset of secondary wall cellulose deposition. Methylation analyses indicate that this glucose, at least in part, is 3-linked. In contrast to the neutral sugars, no significant changes in cell wall amino acid composition are observed during fiber development.     

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.