Changing the Dimensions of Suberin Lamellae of Green Cotton Fibers with a Specific Inhibitor of the Endoplasmic Reticulum-Associated Fatty Acid Elongases.

The fibers of the green lint mutant of cotton (Gossypium hirsutum L.) contain large amounts of wax and are suberized. More than 96% of the bifunctional aliphatic suberin monomers ([alpha],[omega]-alkanedioic acids and [omega]-hydroxyalkanoic acids) have chain lengths of C22 and C24 in green cotton fiber suberin. In fibers grown in the presence of S-ethyl-N,N-dipropylthiocarbamate (EPTC), a specific inhibitor of the endoplasmic reticulum-associated fatty acid elongases, the aliphatic suberin monomers were shortened to chain lengths of C16 and C18. Whereas the amounts of most suberin monomers were not negatively affected by the inhibitor treatment, the amounts of [alpha],[omega]-alkanedioic acids and of glycerol were reduced by more than 80%. Analysis in the transmission electron microscope showed a reduction in suberin content after EPTC treatment. The suberin layers were discontinuous and consisted of fewer lamellae than in the controls. A small proportion (up to 22%) of the electron-translucent suberin lamellae were thinner after EPTC treatment, probably because of the shortening of the aliphatic suberin monomers. A larger proportion of the electron-translucent lamellae were thicker than the lamellae in the controls. Possible explanations for this observation are discussed.

Polysaccharides in the culture medium of cotton (Gossypium hirsutum L.) ovules cultured in vitro.

Polysaccharides secreted into the culture medium (PCM) from cotton (Gossypium hirsutum L.) ovules, culturedin vitro, have been examined. The amount of the polysaccharides increases with the duration of the culture, but except for the galactose content, does not vary markedly with the culture age. Analysis shows that the polysaccharide mixture contains mainly pectins, but there is also a significant amount of xyloglucan. The source of the polysaccharides is most likely the callus tissue which develops on the ovules.

ß-Glucanases in developing cotton (Gossypium hirsutum L.) fibres.

Cotton fibres possess several ß-glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(1→3)-ß-D-glucanase (EC 3.2.1.58) but which also had the characteristics of a ß-glucosidase (EC 3.2.1.21). Endo-(1→3)-ß-D-glucanase activity (EC 3.2.1.39) and much lower levels of (1→4)-ß-D-glucanase activity were also detected. The exo-(1→3)-ß-glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(1→3)-ß-glucanase activity remained constant throughout fibre development. The ß-glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (1→6)-ß-glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O-ß-gentiobiosylglucose.

Changes in the non-structural carbohydrate content of cotton (Gossypium spp.) fibres at different stages of development.

The neutral sugars (glucose, fructose, and sucrose) and the sugar phosphates (glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate) soluble in hot aqueous 80% methanol from the fibres of cotton - Gossypium arboreum L., G. barbadense L., and G. hirsutum L. - were determined at various stages of fibre development. In addition, the (1→3)-ß-D-glucan content was measured and in the case of G. arboreum the rate of (1→3)-ß-D-glucan and cellulose synthesis was determined with [(14)C]sucrose as the precursor. For each of the species a similar chronology was obtained for the changes in content of the various non-structural carbohydrates. At the early stages of secondary wall formation, glucose and fructose exhibited a maximum which was closely followed by a maximum in the (1→3)-ß-D-glucan content and in the sugar phosphates. On the other hand, the sucrose content increased regularly until fibre maturity. The rates of synthesis of (1→3)-ß-D-glucan and of cellulose were highest following the maximum in the (1→3)-ß-D-glucan content, when the latter was being depleted.

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 µM were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo-ß-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 ß-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 ß-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.

Studies on an immunologic method for the diagnosis of varicose veins.

A glycoprotein fraction was isolated from varicose veins by a successive extraction procedure. Antibodies to this fraction were shown to exist in the serum of persons suffering from varicose veins. The pathology of this illness is discussed in relation to the autoimmune reaction, and the basis for a method for the early diagnosis of varicose veins is proposed.

ß-Glucanases and non-cellulosic glucans in the developing oat plant.

The changes in the levels of various ß-glucan hydrolase activities in the second internode of the stem of the developing oat plant have been examined. Concurrent changes in the non-cellulosic ß-glucans contained in the corresponding total hemicelluloses were also studied. Possible relationships between the observed changes and the growth and development of the plant tissue are discussed.