An alkaline α-galactosidase transcript is present in maize seeds and cultured embryo cells, and accumulates during stress

Raffinose family oligosaccharides (RFO) accumulate in many developing seeds and are degraded during seed germination. However, acidic α-galactosidase (AGAL) activity and subcellular location do not correlate with raffinose depletion; alkaline α-galactosidases (AGA) may be responsible for RFO hydrolysis in germinating seeds. Three cDNA clones for AGA/SEED IMBIBITION PROTEIN were obtained from the Pioneer Hi-Bred maize expressed sequence database. Two of the clones were expressed in Escherichia coli, and the recombinant proteins, when incubated with naturally occurring galactosides or p-nitrophenyl α-d-galactose, exhibited AGA activity with maximum catalysis at pH 7.5 (ZmAGA1) or pH 8.5 (ZmAGA3). No raffinose biosynthetic capacity was observed with either enzyme. Maximal α-galactosidase activity in mature dehydrated, germinating and germinated maize (Zea mays) seeds occurred at pH 7.5. ZmAGA1 was the sole family member detected in seeds and maize Hi-II, embryo-derived, callus cells. Its transcript accumulated when seed germination was interrupted by heat, cold or dehydration stress, but not in response to NaCl. Tissue prints localized transcripts to the scutellum or the embryo axis, depending on the stress applied. In maize Hi-II callus cells, transcripts accumulated when callus was subjected to heat stress (42 °C), during which ZmAGA1 transcript accumulation was further induced by sucrose. Galactosides in a variety of forms, including raffinose, partially repressed the sucrose-induced accumulation of transcript in heat-stressed callus.

Pawpaw [Asimina triloba (L.) Dunal] Fruit Ripening. II. Activity of Selected Cell-wall Degrading Enzymes

Pawpaw fruit were harvested at the advent of the ripening process and were ripened at room temperature. Based on fruit firmness and respiration and ethylene production rates at harvest and during ripening, fruit were classified into one of four categories: preripening (no to very slight loss of firmness; preclimacteric), early ripening (some softening; increasing rates of ethylene and CO2 production), mid-ripening (soft; at or just past climacteric), and late ripening (very soft; postclimacteric). The activities of the cell-wall degrading enzymes polygalacturonase (PG), endo-(1→4)ß-D-glucanase (EGase), and endo-ß-1,4-mannanase (MAN) were low in the preripening and early ripening stages, increased dramatically by mid-ripening coincident with the respiratory and ethylene climacterics, and decreased at late ripening. However, pectin methylesterase (PME) activity per milligram protein was highest at the green stage when the fruit firmness was high and decreased as ripening progressed. Tissue prints indicated both EGase and MAN increased as ripening proceeded. The EGase activity was evident near the seeds and the surface of the fruit at preripening and eventually spread throughout, while MAN activity was evident near the fruit surface at preripening and was progressively expressed throughout the flesh as fruit ripened. The greatest decline in fruit firmness occurred between pre- and early ripening, before the peak activities of PG, EGase, and MAN, although MAN exhibited the greatest relative increase of the three enzymes in this period. The data suggest that PME may act first to demethylate polygalacturonate and may be followed by the action of the other enzymes resulting in cell wall disassembly and fruit softening in pawpaw.