The secretion of a-amylase from single isolated (Hordeum vulgare L. cv Himalaya) aleurone layers was studied in an automated flow-through apparatus. The apparatus, consisting of a modified sample analyzer linked to a chart recorder, automatically samples the flow-through medium at 1 minute intervals and assays for the presence of a-amylase. The release of a-amylase from aleurone layers begins after 5 to 6 hours of exposure to gibberellic acid and reaches a maximum rate after 10 to 12 hours. The release of a-amylase shows a marked dependence on Ca2", and in the absence of Ca2" it is only 20% of that in the presence of 10 millimolar Ca2". Withdrawal of Ca22 from the flow-through medium results in the immediate cessation of enzyme release and addition of Ca" causes immediate resumption of the release process. The effect of Ca2" is concentration-dependent, being half-maximal at 1 millimolar Ca2+ and saturated at 10 mnllimolar Ca'+. Ruthenium red, which blocks Ca2" but not Mg2+ efflux from barley aleurone layers, renders a-amylase release insensitive to Ca2`withdrawal. Inhibitors of respiratory metabolism cause a burst of a-amylase release which lasts for 0.5 to 5 hours. Following this phase of enhanced a-amylase release, the rate of release declines to zero. Pretreatment of aleurone layers with HCI prior to incubation in HCN also causes a burst of a-amylase release, indicating that the inhibitor is affecting the secretion of a-amylase and not its movement through the cell wall. The rapid inhibition of a-amylase release upon incubation of aleurone layers at low temperature (5°C) or in 0.5 molar mannitol also indicates that enzyme release is dependent on a metabolically linked process and is not diffusionlimited. This conclusion is supported by cytochemical observations which show that, although the cell wall matrix of aleurone layers undergoes extensive digestion after gibberellin treatment, the innermost part of the cell wall is not degraded and could influence enzyme release.A characteristic of the aleurone layer of cereal seeds is its capacity to secrete a wide spectrum of hydrolytic enzymes, which play a role in endosperm degradation during germination. In barley aleurone, the synthesis of many of these enzymes is controlled by GA3 produced by the embryo (17). Little is known, however, about the control of enzyme release or the cellular mechanism ofprotein transport. The release ofhydrolytic enzymes from aleurone cells requires transport of the enzyme across the plasmalemma and movement through the matrix of the highly thickened cell wall.Varner and Mense (18) examined a-amylase release from single aleurone layers of barley in a flow-through device and concluded that enzyme release into the incubation medium could be resolved 'Supported by National Science Foundation Grant PCM 13286 to R.L. J.'To whom reprint requests should be addressed.3Abbreviations: GA, gibberellin(s); DNP, 2,4-dinitrophenol; CCCP, carbonyl cyanide-m-chlorophenyl-hydrazone.into two parts, secretion and release. The process of enzym...
The effects of exposure to low temperature on photosynthesis and protein phosphorylation in chilling-sensitive and cold-tolerant plant species were compared. Chilling temperatures resulted in light-dependent loss of photosynthetic electron transport in chilling-sensitive rice (Oryza sativa L.) but not in cold-tolerant barley (Hordeum vulgare L. LHCII (9). Phosphorylation is achieved through the lightstimulated activity of one or more thylakoid membrane-bound kinases (7) and can be reversed via a membrane-bound phosphoprotein phosphatase (8). Light stimulation of protein kinase activity is thought to be the result of kinase activation via reduction of the plastoquinone pool (1). The relative degree of LHCII phosphorylation is thought to physically mediate the interaction between PSII and LHCII (5) and thus regulate the balance of excitation energy arriving at the two reaction centers, providing a mechanism that controls the relative turnover of the two photosystems. In this study we have investigated the possibility that the effects of chilling on regulatory phenomena, and on protein phosphorylation in particular, are responsible for the sensitivity of rice to chilling temperatures.
The effect of temperature on a-amylase synthesis and secretion from barley (c.v. Himalaya) half-seeds and aleurone layers is reported. Barley half-seeds incubated at 15 C in gibberellic acid (GA) concentrations of 0.5 and 5 micromolar for 16 hours do not release a-amylase. Similarly, isolated aleurone layers of barley do not release a-amylase when incubated for 2 or 4 hours at temperatures of 15 C or below following 12 hours incubation at 25 C at GA concentrations from 50 nanomolar to 50 micromolar. There is an interaction between temperature and GA concentration for the process of a-amylase release from aleurone layers; thus, with increasing GA concentration, there is an increase in the Qio of this process. A thermal gradient bar was used to resolve the temperature at which the rate of aamylase release changes; thermal discontinuity was observed between 19 and 21 C. The time course of the response of aleurone tissue to temperature was determined using a continuous monitoring apparatus. Results show that the effect of low temperature is detectable within minutes, whereas recovery from exposure to low temperature is also rapid. Although temperature has a marked effect on the amount of a-amylase released from isolated aleurone layers, it does not significantly affect the accumulation of a-amylase within the tissue. At all GA concentrations above 0.5 nanomolar, the level of extractable a-amylase is unaffected by temperatures between 10 and 28 C. It is concluded that the effect of temperature on aamylase production from barley aleurone layers is primarily on the process of enzyme secretion.Temperature extremes often exert a dramatic effect on plant growth. In chilling-sensitive species, temperatures below 15 C cause a marked reduction in metabolic activity and even death (6, 7). High temperatures, like those of desert regions, also impair physiological functions in plants not adapted to such temperature extremes. Physiological dysfunctions at temperature extremes have been attributed to changes in the physical nature of membrane lipids. In chilling-sensitive plants, electron spin resonance has established that changes in membrane fluidity occur upon exposure to cold (6), and a similar conclusion is inferred for the effects of high temperatures on photosynthesis of non-heat-resistant plants (15).Although plants from temperate zones do not generally show chilling injury at temperatures below 15 C and there is no evidence that their membranes undergo phase changes, certain aspects of the physiology of these plants show discontinuities with reduced temperature (2,8,9,12,13,16 temperate species is sensitive to temperature extremes, and cereal seeds are examples of temperate species whose germination is sensitive to low temperatures (2, 12). Both wheat and rice germinate poorly at temperatures below 18 C, and Arrhenius plots of germination of several rice varieties show thermal discontinuities in both germination and seedling growth between 17 and 18 C (12). Shoot elongation of temperate species in response to GA ...
The phosphorylation of thylakoid proteins of rice (Oryza sativa L.) was studied in vitro using 'y-32PIATP. Several thylakoid proteins are labeled, including the light-harvesting complex of photosystem II. Protein phosphorylation is sensitive to temperature, pH, and ADP, ATP, and divalent cation concentrations. In the range pH 7 to 8.2, phosphorylation of the light-harvesting polypeptides declines above pH 7.5, whereas labeling of several other thylakoid polypeptides increases. Increasing divalent cation concentration from 3 to 20 millimolar results in a decrease in phosphorylation of the 26 kilodalton light-harvesting complex polypeptide and increased phosphorylation of several other polypeptides. ADP has an inhibitory effect on the phosphorylation of the light-harvesting complex polypeptides. Phosphorylation of the 26 kilodalton lightharvesting polypeptide requires 0.45 millimolar ATP for half-maximal phosphorylation, compared to 0.3 millimolar for the 32 kilodalton phosphoprotein. Low temperature inhibits the phosphorylation of thylakoid proteins in chilling-sensitive rice. However, phosphorylation of histones by thylakoid-bound kinase(s) is independent of temperature in the range of 25 to 5C, suggesting that the effect of low temperature is on accessibility of the substrate, rather than on the activity of the kinase. (8,20).The inhibition of thylakoid protein phosphorylation by low temperatures in chilling-sensitive rice also blocks the normal occurrence of state I-state II transitions both in vivo and in vitro (21). In the absence of this regulatory process the plastoquinone pool may become highly reduced. Photoinhibition resulting from turnover of the 32-kD herbicide binding protein has been attributed to reaction center binding of reduced plastoquinone (13).The effect of chilling on thylakoid protein phosphorylation could be due entirely to the effect of low temperatures on the phosphorylation reaction, or it may have a component that is an indirect effect mediated through a change in, for example, pH of the stroma. In this study, the phosphorylation of thylakoid proteins in rice was examined to determine whether variation in pH or cation, ATP or ADP concentrations have large enough effects that they should be considered as possible mediators of a component of the chilling-induced inhibition ofphosphorylation seen in vivo.To investigate possible causes of the low temperature effect of in vitro phosphorylation, the effects of low temperature on kinetics of thylakoid protein phosphorylation and on phosphorylation of histones, an exogenous substrate for thylakoid kinases (1,19)
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