Protease activity increased in attached cotyledons of germinated peas (Pisum sativum L. cv. Alaska) as the stored proteins declined but did not increase in excised cotyledons incubated for the same length of time. Cotyledons of seeds germinated in the presence of a casein hydrolysate solution developed less protease activity than did those germinated on water. These results suggest that accumulation of amino acids regulates the protease level in the cotyledons of germinating peas.In contrast to protease, a-and 8-amylase increased during incubation of excised pea cotyledons. Their increase was inhibited by abscisic acid. Abscisic acid did not inhibit "4C-leucine incorporation into protein or reduce the respiratory rate in the cotyledons; hence, its effect on amylase formation was not the result of a general inhibition of metabolism. An ether-soluble acid fraction, which would contain any abscisic acid present in the material, inhibited amylase formation more when it was obtained from imbibed seeds than when it was obtained from cotyledons of seeds germinated for 10 days. These and other results suggest that amylase formation in germinating peas is regulated by abscisic acid.It is well known that gibberellin produced in the embryos of germinating seeds of barley and other cereals moves to the aleurone layers and promotes the formation of amylases, proteases, and other hydrolases which degrade the reserve materials in the seeds, making them available for the growing embryo.In contrast, available knowledge of the control of enzyme formation in the cotyledons of germinating peas and beans is limited. It has been suggested that a factor, perhaps a gibberellin, was involv%ed in amylase formation in germinating peas (15) and beans (5), and it was also reported that kinetin is involved in this process in germinating beans (6). Beevers et al.(1. 2) concluded that the degradation of protein at the early germination stage in peas could not be explained simply by the amount of protease present (measured using casein as a substrate) and suggested the existence of another mechanism for controlling the rate of reserve protein degradation. Penner 1 This research was supported by the United States Atomic Energy Commission, Contract AT (1 1-1 (12) reported that the presence of the embryonic axis in squash during the first 32 hr of germination was prerequisite for the development of maximum proteolytic activity, and that presence of a cytokinin in the culture solution could reproduce the effect of the embryonic axis. Ihle and Dure (7) found that protease formation during precocious germination (that is, germination of excised, immature embryos) in cotton was inhibited by abscisic acid, if the embryos were excised during the middle stage of embryogenesis, but not if they were excised at a later stage.We report further observations on the regulation of protease and amylase formation in peas. MATERIALS AND METHODSPea seeds (Pisum sativum L. cv. Alaska) were sterilized for 15 min with 1% (w/v) sodium hypochlorite solution (Fish...

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In contrast to earlier reported results of similar experiments in peas, in which almost no increase in protease activity occurred in incubated detached cotyledons, we report here an increase in protease activity in both attached and detached bean cotyledons. Detached bean cotyledons showed continually increasing protease activity up to the 12th day, while that in attached cotyledons declined after 6 days. The Rapid liberation of low molecular weight nitrogenous compounds at the expense of stored proteins at the early stage of bean germination was reported by Pusztai and Duncan (7). They also reported in that paper a time course of protease activity which reached maximum on the 8th day of germination. Seeschaaf and Pirson (8) reported that in half cotyledons of blue lupine, protein breakdown proceeded, and the amounts of protease and several other enzymes, including 6-phosphogluconic acid dehydrogenase, increased during incubation of detached cotyledons more than those in attached cotyledons did during germination. They found that the increase occurred only in the absence of the buds; removal of roots or hypocotyl had no effect. Addition of IAA to incubated cotyledons inhibited the increase of 6-phosphogluconic acid dehydrogenase activity. They concluded that auxin from the bud controls enzyme levels in the cotyledons. However, there are no data to show whether auxin has a similar effect on digestive enzymes. a-Amylase activity was also reported to increase dur1Brooklyn Botanic Garden Contribution No. 203. This work was supported by Grant GB 32196 from the National Science Foundation.ing both germination and incubation of axis-free half cotyledons of beans by Dale (1) and by Gepstain and Ilan (2). Dale suggested that gibbereilin is involved in a-amylase formation, while Gepstain and Ilan observed an enhancing effect of kinetin. Yomo has reported that formation of a-amylase in detached bean cotyledons was inhibited by ABA during incubation (10). Yomo and Varner (11) found that in detached cotyledons (a) a-amylase activity was also inhibited by ABA, and (b) protein breakdown and protease formation were very slight compared with attached cotyledons. We report here on the protein breakdown and the formation of protease in comparison with that of a-amylase, during incubation of excised bean cotyledons, in relation to effects of cycloheximide and ABA. MATERIALS AND METHODSGeneral Procedure. Bean (Phaseolus vulgaris L. cv. Kentucky Wonder) seeds were sterilized in 1 % aqueous NaOCl (Allied Chemical) solution for 10 min and imbibed at 22 C for 16 hr. Imbibed seeds were germinated on moist vermiculite in the dark. Cotyledons from germinated seedlings are referred to as attached cotyledons. For incubation experiments, the imbibed seeds were cut transversely in half and the axisfree half cotyledons (detached cotyledons) were sterilized for 1 min in 0.2% NaOCl solution which had been neutralized with 2 N HCI just before use. They were then put in a 125-ml Erlenmeyer flask containing 10 ml of 2% agar solution whic...

Embryos were excised from barley seeds, their homogenates were incubated with ficin, and their content in gibberellin-like substance was assayed by means of α-amylase-producing activity, but no gibberellin-like substance could be detected.Embryos free from endosperm which were cultured for five days produced measurable amounts of gibberellin-like substance. This substance was not produced when a carbon source was absent from the medium, or when air was removed after 2 days of aerobic culture. CCC and Phosfon D (at the concentration of 2×10(-4) M and 0.8×10(-4) M, respectively) inhibited the formation of the gibberellin-like substance in cultured embryos without affecting their growth.Mevalonic acid could be used as a carbon source in the culture of the embryos. The formation of the gibberellin-like substance was in this case inhibited by 0.8×10(-4) M Phosfon D, but was not inhibited by 2×10(-3) M CCC.

Protease formation in Phaseolus vulgaris L. cotyledons during seed germination was studied histochemically using a gelatin-film-substrate method. Protease activity can be detected by this method on the 5th day of germination, at approximately the same time that a rapid increase of activity was observed by a test-tube assay with casein as a substrate. At the early stage of germination, protease activity was observed throughout the cotyledon except in two or three cell layers below the cotyledon surface and in several cell layers around the vascular bundles. A highly active cell layer surrounding the protease-inactive cells near the vascular bundles is suggested to be a source of the protease.