K. N. Chandra Sekhar

Identification and immunocytochemical localization of α-galactosidase in resting and germinated date palm (Phoenix dactylifera L.) seeds

Abstractα-Galactosidases (EC 3.2.1.22) from resting and germinated date (Phoenix dactylifera L.) seeds were compared and localized using immunocytochemical methods. The enzyme was present in both the endosperm and embryo of resting seeds, in the endosperm undergoing digestion where the greatest specific activity was present, and in the haustorium of seedlings. The enzyme had a molecular mass of 140000 as determined by gel filtration and a pH optimum of 4.5. At least seven forms of the enzyme with isoelectric points ranging from 3.85 to 5.2 were detected in the haustorium whereas only four of these forms were present in the endosperm. The relative activity levels of the various forms also differed between the two tissues. On Western blots all enzyme forms were recognized by antibodies raised against mung-bean (Vigna radiata) α-galactosidase. Using immunogold techniques, label was shown to be present in the protein bodies of the resting embryo cells but to decrease in this organelle as the reserve protein was mobilized and to appear diffusely in the cytoplasm in subsequent stages. In resting endosperm cells, label occurred in the protein bodies and in a thin region of inner wall. In endosperm undergoing digestion, where different stages of protoplast and wall breakdown occurred, immunogold staining was localized in the flocculent contents of vacuoles which resulted from storageprotein breakdown, then dense staining occurred in the inner wall of cell cavities formed by the complete dissolution of the cytoplasm, and finally, staining was uniformly diffuse throughout the remaining endosperm wall adjacent to the haustorium surface. These observations indicate that the α-galactosidase present in cell walls of the date palm endosperm during mannan mobilization is not secreted by the haustorium but instead is probably a pregermination product stored mainly in the protein bodies of resting endosperm and is released to the wall following loss of membrane integrity.

Electrophoretic Characterization and Immunological Localization of Coconut (Cocos nucifera L.) Endosperm Storage Proteins

Proteins extracted from coconut (Cocos nucifera L.) endosperm were analyzed using sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, tube gel isoelectric focusing, and two-dimensional electrophoresis. Antisera to soybean 11S and 7S globulins were used with Western blotting techniques to test for immunological similarities. Endosperm was fixed and processed for light microscopy and for immunocytochemistry. The nonreduced proteins fractionated into four major bands, whereas the reduced proteins fractionated into seven major bands ranging from 55 to 17 kilodaltons (kD). All major bands were glycosylated and each consisted of at least two polypeptides with different isoelectric points. A minor band at 67 kD and two minor bands at 22 kD were recognized by antibodies to 7S soybean globulin and a major and minor band at 35 and 32 kD, respectively, were recognized by antibodies to 11S soybean globulins. An antibody raised against the 55 kD protein showed no cross reactivity with any other bands. Cellular structure differs depending on location within the endosperm, especially with respect to cell size and characteristics of the protein bodies. The cells are relatively thin-walled and contain abundant lipid bodies and protein bodies with included protein crystalloids. Distinct localization patterns are apparent for legumin, vicilin, and the 55 kD protein. Legumin (11S) and vicilin (7S) are present in the crystalloids, whereas the 55 kD protein is present only in the protein body matrix.

Role of α-galactosidase in cell wall metabolism of date palm (Phoenix dactylifera) endosperm

SummaryThe endosperm of developing date palm (Phoenix dactylifera) seeds was sampled at regular intervals from pollination to mature fruit. The galactose content of the cell wall mannans was assessed. Accumulation of α-galactosidase, a cell wall hydrolase, during endosperm development was analyzed by isoelectric focusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis in combination with Western blotting and immunolocalization on tissue sections. N-terminal amino acid sequence of the first 15 amino acids showed homology with amino acids 71 to 85 of the sequence reported for the mature guar protein. Four forms of the enzyme with isoelectric points ranging from 4.4 to 5.2 appeared by 11 weeks after pollination, and all forms remained until maturity. A major band of 41 kDa and several lower Mr, lightly staining bands cross reacted with the anti-α-galactosidase antiserum. The major band remained until maturity while the lightly staining bands gradually disappeared. In the mobilizing endosperm of germinated seeds, two darkly staining bands were observed at 41 and 40 kDa. At 9 weeks after pollination, the endosperm was cellular and the silver enhanced gold label localizing α-galactosidase occurred predominantly in the cell periphery. By 11 weeks, the label was present in the cytoplasm, but lacking on the thickening cell wall. α-Galactosidase accumulated in the protein bodies along with the storage protein. At 13 to 17 weeks, the label accumulated and then was lost in a centrifugal pattern (from the middle lamella inward) from the cell walls as they matured and was lost in the cytoplasm. The mature endosperm cells had intense label present only over the protein bodies and over the inner cell wall. These observations suggest that α-galactosidase is synthesized during endosperm development and unique forms of the enzyme are associated with cell wall maturation and cell wall mobilization in this species.