Changes in the Pattern of Some Isoenzymes of the Corolla after Pollination in Phalaenopsis amabilis Blume

A well known correlative effect is that of senescence of the corolla after fertilization, but the mechanism as well as the biochemical changes which accompany it have not been elucidated completely (2-4). In the present work, the changes in the pattern of isozymes of glucose-6-P, malate and glutamate dehydrogenases, and peroxidases contained in the corolla of Phalaenopsis have been studied 12, 24, 48, and 120 hr after pollination. An identical study was carried out on an unpollinated control of fully expanded flowers during 16 days. The changes from unopened flower buds to fully expanded flowers were also observed (Fig. 1). The extract of fresh petals homogenized in a Potter-Elvehjem homogenizer and centrifuged at 1OO,OOOg for 15 min was separated by vertical electrophoresis on starch gel (11). The peroxidases were detected by the benzidine reaction (13). The NAD-dependent dehydrogenases were observed by the method of Market and Ursprung (5) and the glucose-6-P dehydrogenase was observed with a 20 mm glucose-6-P solution and NADP as coenzyme. The comparison of the pattern of isozymes was made on a fresh weight basis. Dehydrogenases. A decrease of dehydrogenases was found during aging of the corolla. Glucose-6-P dehydrogenase had only one band which decreased from unopened to fully expanded flower. Malate dehydrogenase as well as glutamate dehydrogenase showed two isozymic forms; band 2 decreased for both enzymes from unopened flower to fully expanded flower. The glucose-6-P dehydrogenase band disappears 24 to 48 hr after pollination. In contrast, the two malate dehydrogenase bands remain up to the 5th day after pollination. A change of pattern was evident, band 2 decreasing more rapidly than band 1. Glutamate dehydrogenase also showed two bands at the 5th day after pollination; band 1 decreased more rapidly than band 2. For the unpollinated flowers the pattern of enzymes did not change during aging of the fully expanded flowers up to 16 days. Alcohol, isocitrate, and formate dehydrogenases could not be detected by histochemical methods (5). The glucose-6-P dehydrogenase activity suggests that carbohydrate metabolism in the corolWl takes place via the pentose cycle; the Krebs cycle seems active and so is the capacity for metabolizing ammonia via glutamate dehydrogenase. Pollination would induce metabolic changes by affecting the dehydrogenase activities according to the following order: glucose6-P, malate and glutamate dehydrogenases. Peroxidases. The most complete pattern of peroxidases showed 7 bands, 3 anodic and 4 cathodic. The results showed that in the interval between unopened and fully expanded flower, bands 1, 2, 3, 4, and 5 became visible. Five days after pollination a major increase in the middle anodic band 2 and the fast moving cathodic band 7 could be observed, as well as the appearance of the cathodic band 6. The semiquantitative estimation showed an increase of peroxidase activity during aging of the corolla. In orchids, pollination is known to increase the auxin content and development of the ovary (14), and Gessner (2) and Hsiang Tsung Hsun (3, 4) have shown that pollination induces accumulation of substances in the ovary and column as a result of translocation from the perianth; this accumulation could modify the cytoplasm and the synthesis or activity of enzymes in the corolla. It is known that senescence induces a decrease of protein and nucleic acid content (1, 6-10). The low protein level would be the result not only of degradative processes and size of the protein precursor amino acid pool (12) but also of a diminution of the de novo synthesis mechanism as suggested by the final loss of all synthetic capacities. Considering that changes in the enzyme pattern would be due to activation and repression of the genes, the aging of the corolla seems related to a genic activation in the case of peroxidases and to a genic repression in the case of glucose-6-P dehydrogenase, malate dehydrogenase, and glutamate dehydrogenase. The deterioration of the processes involved with the synthesis of protein and RNA due to the increase of catabolic enzymes and the appearance of enzyme activators and inhibitors cannot be discarded. Since changes in the pattern of isoenzymes are also observed during opening of the flower, pollination only accelerates the senescence of the corolla in the fully expanded flower. Regulation of the enzyme pattern after pollination would be caused by the presence of an active growth center in the ovary which induces translocation of water and solutes from the corolla. Thus senescence of the corolla can be viewed as originating from endogenous correlative effects inducing cytoplasmic alterations which act as gene regulators.