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

Abstract

α-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.

Ultrastructural aspects of metabolic pathways and translocation routes during mobilization of seed reserves in Acrocomia aculeata (Arecaceae)

“…The evidence of apoplastic transport and the gradual changes in the matrix of the protein bodies indicate those substances could be products of hydrolysis and enzymes. The transport of enzymes from protein bodies to adjacent cells via the cell wall is congruent with previous observations of P. dactylifera, which, although possessing plasmodesmata in the thickened walls of the endosperm, also shows the dissemination of enzymes through the cell wall (Sekhar and DeMason 1990).…”

“…Endo-β-mannanase and α-galactosidase, enzymes involved in the hydrolysis of galactomannans, occur in the endosperm of dry seeds of A. aculeata and have high activity in the portion of endosperm adjacent to the haustorium (Bicalho et al 2016;Mazzottinidos-Santos et al 2017). Several enzymes were diagnosed in the endosperm of P. dactylifera, among which the site of α-galactosidase was identified in protein bodies by immunocytochemistry (DeMason et al 1985;Sekhar and DeMason 1990). In the endosperm of P. dactylifera and W. filifera, acid phosphatase, an enzyme involved in the mobilization of carbohydrates and phosphates, is also stored in protein bodies (DeMason et al 1989), reinforcing our interpretation.…”

“…On the other hand, the endosperm has been indicated to have a certain degree of autonomy in the digestion and mobilization of its reserves. Sekhar and DeMason (1990), Zienkiewicz et al (2014), and Mazzottini-dos-Santos et al 2017suggested that protein bodies of endosperm cells possess hydrolytic enzymes, similar to the aleurone layer of amylaceous seeds (Bewley et al 2013). These observations provide evidence that protein bodies are related to lipid and mannan mobilization.…”

Storage proteins and cell wall mobilisation in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

“…McCleary and Matheson (1974) also detected multiple isoforms of a-galactosidase in guar seeds, although only one of them (40 kDa) is associated with the endosperm (Overbeeke et al 1989). In seeds of alfalfa, bean, carob, coffee, palm, soybean, lupin and white clover, at least two isoforms of a-galactosidase are present, with molecular weights ranging between 17 and 57 kDa (Courtois and Petek 1966;Mccleary and Matheson 1974;Williams et al 1978;Itoh et al 1979;Plant and Moore 1982;Chandra Sekhar and DeMason 1990;Dhar et al 1994).…”

Genes with homologies to known α‐galactosidases are expressed during senescence of barley leaves

“…In plants h-galactosidases catalyze the hydrolysis of various storage substances like galactooligosaccharides, -lipids and -proteins. Since seeds contain large amounts of such storage compounds, which are utilized as an energy source during germination, work on plant h-galactosidases has mostly focused on seed tissues Wallenfels 1974, Chandra Sekhar andDe Mason 1990). h-Galactosidase activities, however, have been also measured in photosynthetic active tissues (Gatt and Becker 1970, Thomas and Webb 1977, Smart and Pharr 1980.…”