Effects of plant growth regulators on secondary wall thickening of cotton fibres

The effect of plant growth regulators on the secondary wall thickeningof cotton fibre was studied. The results indicated that the GA_S andiP+iPA levels in the fibre of field-grown cotton plantsremained almost constant but the IAA and ABA levels changed considerably duringfibre development. Although the change in both IAA and ABA levels seemed not tobe closely related with the rate of cellulose accumulation, there was still arelationship between the ratio of ABA to IAA and secondary wall thickening. Inin vitro studies, ABA (50mol·L^–1) markedly enhanced theaccumulation of dry matter and cellulose in the fibre cell wall duringsecondarywall thickening, but no similar effect was observed with NAA, GA₃ orkinetin treatments. The role of ABA in secondary wall thickening of cottonfibreis discussed.     

Changes in secondary metabolites and fiber quality of cotton (Gossypium hirsutum) seed under consecutive water stress and in silico analysis of cellulose synthase and xyloglucan endotransglucosylase

Global warming has led to severe drought conditions. The selection of plant varieties that can withstand drought and produce increased yields are of utmost importance. In the current study, secondary metabolites, seed trait and fiber characteristic of cottonseeds (Gossypium hirsutum) exposed to double and third water stress exposure was investigated. Total phenol and tannin content in W₁S₃₃ increased significantly after third water stress exposure. Accumulation of wax was enhanced in seeds of W₃S₃₃ and W₃S₃₄ that were subjected to third water stress. Fiber quality parameters decreased when cottonseeds were rainfed. High irrigation resulted in fragile and delicate fiber. Seeds grown under 66% FC irrigation saved water and produced seeds that had the potential of producing high quality fibers. In silico analysis was performed on cellulose synthase A (CesA) and xyloglucan endotransglycosylase (XET) enzymes present in Gossypium hirsutum. The intracellular locations of the CesA and XET1 enzymes are the plasma membrane and cell wall, respectively. Proline is conserved in the C-terminal of the CesA enzyme and plays an important role in enzyme functionality. This study provides a better understanding as to the mechanisms by which the plant can tolerate and combat water stress conditions as well as reduce water consumption. In order to grow cotton seeds with desirable morphometric characteristics and optimal fibers under water stress exposure and in dry areas, it is better to use seeds that are irrigated under optimal irrigation conditions, ie 66% FC.     

Atomic force microscopy of cotton fiber cell wall surfaces in air and water: quantitative and qualitative aspects

Cotton (Gossypium hirsutum cv. MD51) fiber cell walls were analyzed with an atomic force microscope to determine the effect of chemical treatments on cell wall organization and topography. Analysis of fibers in either air or water and without any staining or coating produced high-resolution images of cell wall microstructure which could be used for detailed quantitative analysis. Treatment of fibers with 1% H₂O₂ had little effect on surface morphology. Alkali removed much of the cuticle, some primary wall components, and revealed mostly thin-diameter microfibrils. Acidic Updegraff reagent fragmented the fibers, removed much of the cuticle, and revealed mostly thick microfibrils. The surface roughness of fibers treated sequentially with alkali and acid was quantitatively distinguishable from all other fiber types based on the standard deviation of the height data, amplification of surface area, and integration of the scan line data. Analysis of the fractal dimension enabled untreated and peroxide-treated fibers to be clearly distinguished from the other fiber types. Segmentation of the fractal data revealed specific portions of the fractal dimension which were especially useful for defining the size of structures that differentiated fiber types. Areas containing microfibrils could be quantitatively differentiated from non-microfibrillar areas. In water, some alkali-treated fibers had microfibrils that were relatively small in diameter while others appeared to consist of crystalline arrays of smaller fibrils. Received: 10 December 1996 / Accepted: 29 January 1997     

A cell wall protein down-regulated by auxin suppresses cell expansion in Daucus carota (L.)

We investigated the function of the auxin-regulated cell wall gene DC 2.15, a member of a small gene family, present in Daucus carota (L.) and other plants. Cultured cells derived from carrot hypocotyls transformed by the DC 2.15 cDNA in antisense direction were ten-fold longer than wild-type cells, indicating a function of the corresponding protein in suppression of cell expansion. The analysis of carrot plants expressing the DC 2.15 gene in antisense direction showed that the corresponding protein and/or related proteins probably are involved in leaf and vascular bundle development. The antisense plants generally displayed a retarded growth phenotype and delayed greening in comparison to wild-type plants. The asymmetric architecture of the wild-type leaves was degenerated in the DC 2.15 antisense plants and the leaves showed a torsion within and along their major vein. The vascular bundles showed a lowered ratio of the phloem/xylem area in cross sections of the leaf middle vein whereas the bundle sheath and the cambium showed no obvious phenotype. Expression of a promoter-GUS construct was found primarily in vascular bundles of stems, leaves and in the nectar-producing flower discs. The observed pleiotropic antisense phenotype indicates, by loss of function, that one or several related cell wall proteins of this gene family are necessary to realize several complex developmental processes.     

Polysaccharides, tightly bound to cellulose in cell wall of flax bast fibre: Isolation and identification

Part of matrix polymers of flax bast fibre cell wall is tightly bound to cellulose and can not be extracted by conventional methods. To analyze these polymers, the residue, remaining after cell wall treatment with chelators and alkali, was dissolved in solution of lithium chloride in N,N-dimethylacetamide. Cellulose was precipitated by water and completely degraded by cellulase, giving the possibility to separate matrix polysaccharides, which remained in polymeric form. The obtained polymers were fractionated by gel permeation chromatography and characterized by monosaccharide analysis, staining with LM5 antibody and Yariv reagent, ¹H and ¹³C NMR. The total yield of the polysaccharides that are tightly bound to cellulose in flax fibre, was 4.6%. The major fractions (molecular mass 100–400 kDa) were composed of galactose, accompanied by two other significant monomers, GalA and Rha, with the ratio 1.1–1.4. Composition and structure of the cellulose bound galactan permit to consider it as fragment of the high-molecular mass (2000 kDa) galactan, synthesized by the developing fibres, while forming the secondary cell wall of gelatinous type.     

Influence of external factors on callose and cellulose synthesis during incubation in vitro of intact cotton fibres with [14C]sucrose

Seed clusters, with adhering fibres, from individual locules of 36-d-old fruit capsules of Gossypium arboreum L. were fed with [¹⁴C]sucrose in vitro. The fibres synthesised, under standard conditions, (13)--D-glucan (callose) and (14)--D-glucan (cellulose) in the ratio of approx. 2:1. Under a great variety of different conditions this product ratio remained more or less constant, even when total glucan synthesis was strongly inhibited with 2,4-dinitrophenol or phloretin, or when stimulated with abscisic acid. In attempts to favour cellulose synthesis, no conditions were found where the ratio was substantially reduced. On the other hand, the ratio could be appreciably increased by inhibiting cellulose synthesis, e.g. with 2,6-dichlorobenzonitrile or coumarin, by anionic detergents such as sodium dodecyl sulphate, by low temperatures, or by increasing the osmotic strength of the incubation medium up to conditions causing plasmolysis. Specific degradation of callose, during incubation of the seed clusters, by exogenous exo-(13)--D-glucanase significantly diminished incorporation of radioactivity into cellulose.Abbreviations DMSO dimethyl sulphoxide - EDTA ethylenediaminetetraacetic acid     

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.     

Development of cell wall appendages inAcanthosphaera zachariasi (Chlorococcales): Kinetics,site of cellulose synthesis and of microfibril assembly,and barb formation

Summary The investigation of the formation of cell wall appendages inAcanthosphaera by means of light and electron microscopy and by the use of dyes which interfere with microfibril assembly resulted in several observations which are helpful to an understanding of the formation of normal cell walls. The barbs are built up in the ER, pass through the Golgi apparatus, and are extruded exocytotically after cytokinesis, a remarkable example of the secretion of a structured product. Each cellulose microfibril in a spike develops in a distinct pit of the plasmalemma. The pits are aggregated in a pit field, generating one spike, and are closely adjacent to a basal vesicle which might have morphogenetic and/or regulatory functions. The pits are the site of cellulose synthesis; here the plasmalemma is conspicuously thickened. As shown directly and by the application of Calcofluor white and Congo red, the microfibrils assemble at a certain distance from the plasma membrane,i.e. cellulose synthesis and microfibril assembly are separated by a gap. It is discussed whether single glucan chains or small bundles of them are released from the plasmalemma. The elongation rate of the spikes indicates that about 1000 glycosidic linkages per glucan chain per minute are formed.     

The effects of human leukemia inhibitory factor (hLIF) and culture medium on in vitro differentiation of cultured porcine inner cell mass (pICM)

Summary Isolation and maintenance of porcine embryonic stem (pES) cells have been hindered by the inability to inhibit differentiation of the porcine inner cell mass (pICM) in vitro. Culture conditions currently in use have been developed from mouse ES cell culture and are not effective for maintaining the pICM. Optimizing culture conditions for the pICM is essential. We have developed a grading system to detect changes in the differentiation status of in vitro cultured pICM. Porcine ICMs (Day 7) were isolated by immunosurgery and cultured for 4 d in either Dulbecco’s modified Eagle’s medium (DMEM)-based medium (D medium) or DMEM/Ham’s F-10 (1:1)-based medium (D/H medium) with or without human Leukemia Inhibitory Factor (hLIF, 1000 iu/ml). Colonies were photographed daily for morphological analysis. pICMs were categorized into one of two types based on their morphological profile: type A, nonepithelial or type B, epithelial-like. Eight investigators evaluated pICM differentiation using standardized differentiation profiles. Each pICM series was graded on a scale of 1 (fully undifferentiated) to 5 (fully differentiated) for each time point. Differentiation was verified by alkaline phosphatase activity, cytokeratin staining, and scanning electron microscopy. Neither hLIF nor culture medium delayed differentiation of pICMs (P=0.08 and P=0.25, respectively). The grading system employed was an effective tool for detecting treatment effects on differentiation of the developing pICM. These results demonstrate that hLIF cannot significantly inhibit differentiation of the pICM, and is unlikely to assist in porcine ES cell isolation. Future experiments utilizing homologous cytokines may prove more beneficial.     

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into -1,3- and -1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of ¹⁴C incorporated from [¹⁴C] glucose was present in the hot-water-soluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as -1,3-linked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as -1,4-linked glucose indicative of cellulose.     

Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579-containing proteins that are essential for normal xylan deposition in the secondary cell wall

There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co‐expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T‐DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 (IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of these irx15 irx15l double mutants are disorganized, compared with the wild type or other previously described xylan mutants, and exhibit dramatic increases in the quantity of sugar released in cell wall digestibility assays. Furthermore, localization studies using fluorescent fusion proteins label both the Golgi and also an unknown intracellular compartment. These data are consistent with irx15 and irx15l defining a new class of genes involved in xylan biosynthesis. How these genes function during xylan biosynthesis and deposition is discussed.     

Over‐expression of specific HvCslF cellulose synthase‐like genes in transgenic barley increases the levels of cell wall (1,3;1,4)‐β‐d‐glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)‐β‐d ‐glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)‐β‐d ‐glucans in the Poaceae is mediated, in part at least, by the cellulose synthase‐like CslF family of genes. Over‐expression of the barley CslF6 gene under the control of an endosperm‐specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)‐β‐d ‐glucan content in grain of transgenic barley. Analyses of (1,3;1,4)‐β‐d ‐glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)‐β‐d ‐glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)‐β‐d ‐glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)‐β‐d ‐glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)‐β‐d ‐glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)‐β‐d ‐glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)‐β‐d ‐glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.