Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

Regulation of the synthesis of barley aleurone alpha-amylase by gibberellic Acid and calcium ions

The effects of gibberellic acid (GA₃) and calcium ions on the production of α-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA₃ or Ca²⁺ show qualitative and quantitative changes in hydrolase production following incubation in either GA₃ or Ca²⁺ or both. Incubation in H₂O or Ca²⁺ results in the production of low levels of α-amylase or acid phosphatase. The addition of GA₃ to the incubation medium causes a 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of Ca²⁺ at 10 millimolar causes a further 8- to 9-fold increase in α-amylase release and a 75% increase in phosphatase release. Production of α-amylase isoenzymes is also modified by the levels of GA₃ and Ca²⁺ in the incubation medium. α-Amylase 2 is produced under all conditions of incubation, while α-amylase 1 appears only when layers are incubated in GA₃ or GA₃ plus Ca²⁺. The synthesis of α-amylases 3 and 4 requires the presence of both GA₃ and Ca²⁺ in the incubation medium. Laurell rocket immuno-electrophoresis shows that two distinct groups of α-amylase antigens are present in incubation media of aleurone layers incubated with both GA₃ and Ca²⁺, while only one group of antigens is found in media of layers incubated in GA₃ alone. Strontium ions can be substituted for Ca²⁺ in increasing hydrolase production, although higher concentrations of Sr²⁺ are required for maximal response. We conclude that GA₃ is required for the production of α-amylase 1 and that both GA₃ and either Ca²⁺ or Sr²⁺ are required for the production of isoenzymes 3 and 4 of barley aleurone α-amylase.     

Calmodulin stimulation of unidirectional calcium uptake by the endoplasmic reticulum of barley aleurone

The role of calmodulin (CaM) in gibberellic acid (GA₃)-stimulated Ca²⁺ uptake was investigated in endomembranes isolated from aleurone cells of barley (Hordeum vulgare L.). Unidirectional Ca²⁺ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA₃ and Ca²⁺ compared with those isolated from layers incubated in Ca²⁺ alone. However, the level of uptake from Ca²⁺-treated tissue could be stimulated to that of the GA₃-treated cells by applying exogenous CaM which increased the V _max of the Ca²⁺ transporter approximately threefold. Calcium uptake in ER from GA₃-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA₃-treated tissue was unaffected. Treatment with GA₃ also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca²⁺ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca²⁺ uptake into the ER induced by GA₃ may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca²⁺-dependent α-amylase synthesis in the lumen of the ER.     

Ca-stimulated secretion of alpha-amylase during development in barley aleurone protoplasts

The effects of gibberellic acid (GA₃) and Ca²⁺ on the synthesis and secretion of α-amylase from protoplasts of barley (Hordeum vulgare L. cv Himalaya) aleurone were studied. Protoplasts undergo dramatic morphological changes whether or not the incubation medium contains GA₃, CaCl₂, or both. Incubation of protoplasts in medium containing both GA₃ and Ca²⁺, however, causes an increase in the α-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA₃ or Ca²⁺ alone. Isoelectric focusing shows that adding Ca²⁺ to incubation media containing GA₃ increases the levels of α-amylase isozymes having high isoelectric points (pI). In the presence of GA₃ alone, only isozymes with low pIs accumulate. The increase in α-amylase activity in the incubation medium begins after 36 hours of incubation, and secretion is complete after about 72 hours. Protoplasts require continuous exposure to Ca²⁺ to maintain elevated levels of α-amylase release. Immunoelectrophoresis shows that Ca²⁺ stimulates the release of low-pI α-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA₃ alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaCl₂. The response is not specific for Ca²⁺ since Sr²⁺ can substitute, although less effectively than Ca²⁺. Pulse-labeling experiments show that α-amylase isozymes produced by aleurone protoplasts in response to GA₃ and Ca²⁺ are newly synthesized. The effects of Ca²⁺ on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on α-amylase stability or on protoplast viability. We conclude that Ca²⁺ directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca²⁺-stimulated enzyme release.     

Tannins as gibberellin antagonists in the synthesis of alpha-amylase and Acid phosphatase by barley seeds

The tannins chebulinic acid or tara tannin were added to an incubation system in which GA₃ induces enzyme synthesis in endosperm half seeds of barley (Hordeum vulgare L.). The activity of amylase and acid phosphatase in the incubation medium was reduced compared to the activity in the medium after incubation with GA₃ alone. When embryo half seeds of barley were incubated with chebulinic acid or tara tannin in the absence of added GA₃, the enzyme activity of the incubation medium was also reduced. The activity of preformed enzymes obtained from endosperm half seeds previously induced with GA₃ was not reduced by the addition of tannin. Comparisons were made of the amount of enzyme activity from breis of aleurone layers incubated with GA₃ in the presence and absence of tannins. The amounts of activity were relatively small and approximately equal in both cases, indicating that secretion from the aleurone was not blocked by the tannins. The reduction of enzyme activity caused by tannins in both endosperm and embryo half seeds could be completely reversed by the addition of GA₃.     

Enhancement by gibberellic acid and asymmetric distribution of lysophospholipase in germinating barley

Germination of whole barley seeds for 4 and 6 days followed by measurement of lysophospholipase (lysolecithin acyl hydrolase, LAH) in the embryo-containing and embryo-free halves revealed a gradient of activity between the two halves of the seed. Most of the activity appeared in the embryo-containing half. This gradient decreased slightly in the aleurone and dramatically in the starchy endosperm during the 2 day germination interval. Embryo-containing and embryo-free half seeds of surface sterilized barley were placed separately on sterile agar plates. After 4 and 6 days LAH was observed in both the aleurone and starchy endosperm of the embryo-containing halves. In the embryo-free halves, LAH appeared at low levels in the aleurone and was virtually absent in the starchy endosperm. The scutellum of germinating seeds contains LAH activity. Exposure of embryo-free half seeds to GA₃ for 24 hr showed enhancement of acidic and alkaline LAH activities in the aleurone fraction and in the GA₃-medium in which the half seeds were treated. The LAH activity of the starchy endosperm of these half seeds was little changed by GA₃ treatment. Exposure of isolated aleurones to GA₃ for 24 hr resulted in substantial enhancement of acidic and alkaline LAH activities in the bathing medium and in fractions prepared from the aleurone. The physiological significance of the influence of GA₃ on LAH activity during barley germination is discussed.     

Calcium regulation of the secretion of α-amylase isoenzymes and other proteins from barley aleurone layers

The effect of calcium on the secretion of α-amylase (EC 3.2.1.1) and other hydrolases from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was studied. Withdrawal of Ca²⁺ from the incubation medium of aleurone layers preincubated in 5 μM gibberellic acid (GA₃) and 5 mM CaCl₂ results in a 70–80% reduction in the secretion of α-amylase activity to the incubation medium. Agar-gel electrophoresis shows that the reduction in α-amylase activity following Ca²⁺ withdrawal is correlated with the disappearance of group B isoenzymes from the incubation medium. The secretion of isoenzymes of group A is unaffected by Ca²⁺. The addition of Ca²⁺ stimulates the secretion of group-B isoenzymes but has no measurable effect on either the α-amylase activity or the isoenzyme pattern of aleurone-layer extracts. Pulse-labelling experiments with [³⁵S]methionine show that Ca²⁺ withdrawal results in a reduction in the secretion of labelled polypeptides into the incubation medium. Immunochemical studies also show that, in the absence of Ca²⁺, α-amylase isoenzymes of group B are not secreted into the incubation medium. In addition to its effect on α-amylase, Ca²⁺ influences the secretion of other proteins including several acid hydrolases. The secretion of these other proteins shows the same dependence on Ca²⁺ concentration as does that of α-amylase. Other cations can promote the secretion of α-amylase to less and varying extents. Strontium is 85% as effective as Ca²⁺ while Ba²⁺ is only 10% as effective. We conclude that Ca²⁺ regulates the secretion of enzymes and other proteins from the aleurone layer of barley.     

Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic Acid

Polyamine metabolism and its relation to the induction of α-amylase formation in the aleurone layers of barley seeds (Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA₃) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).

Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA₃ (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of α-amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA₃. Kinetic studies show that both applied [U-¹⁴C]ornithine and [U-¹⁴C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA₃. Additionally, added GA₃ does not affect the uptake and turnover of [1,4-¹⁴C]Put, nor does it affect the conversion of Put → Spd or Spd → Spm. Treatment of the aleurone layers with GA₃ for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase.

Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-¹⁴C] adenosine into ATP. The lower the level of Spd the less α-amylase formation is induced by added GA₃. The reduction of GA₃-induced α-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA₃, with respect to induction of α-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction.

     

A comparison of the effects of Ca2+ and gibberellic acid on enzyme synthesis and secretion in barley aleurone

The effects of the addition and withdrawal of gibberellic acid (GA₃) and Ca²⁺ on enzyme synthesis and secretion by barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. Incubation of layers in GA₃ plus Ca²⁺ affects the total amount of secreted α-amylase (EC 3.2.1.1) and acid phosphatase (EC 3.1.3.2) by promoting the appearance of different isoenzymic forms of these enzymes. The release of α-amylase isoenzymes 1–4 in response to GA₃ plus Ca²⁺ has a lag of 6 h. When layers are incubated in GA₃ alone for 6 h prior to the addition of Ca²⁺, isoenzymes 1–4 appear in the medium after only 30 min. When the addition of Ca²⁺ to layers pretreated in GA₃ is delayed beyond 12 h, its effectiveness in stimulating the synthesis and release of isoenzymes 3 and 4 is diminished. After 35 h of preincubation in GA₃, addition of Ca²⁺ will not stimulate synthesis of α-amylase isoenzymes 3 and 4. Aleurone layers preincubated for 6 h in GA₃ will respond to Ca²⁺ when the GA₃ is withdrawn from the incubation medium by producing α-amylase isoenzymes 1–4. The converse is not the case, however, since layers preincubated in Ca²⁺ for 6 h will not produce all isoenzymes of α-amylase when subsequently incubated in GA₃. The Ca²⁺-stimulated release of α-amylase from GA₃ pre-treated layers is dependent on the time of incubation in Ca²⁺ and the concentration of the ion. The response to Ca²⁺ is temperature-dependent, and other divalent cations such as Mg²⁺ cannot substitute for Ca²⁺. We conclude that Ca²⁺ influences α-amylase release by influencing events at the biochemical level.     

Hormonal Response and Seed Viabil ity of Parteresia coarctata,a Brackish Water Species

Stimulation of α-amylase activity was observed in Porteresia coarctata immature seeds (20-day-old) when de-embryonated prewashed half seeds were incubated in media containing gibberellic acid (GA₃, 10^?5M). No such activity was observed in mature seeds even when GA₃ concentration was increased up to five fold. ABA suppressed the GA₃ enhanced α-amylase synthesis up to nearly 70% in the immature seeds. Absence of this enzyme activity in mature seeds may be due to high levels of ABA. The immature aleurone showed a 23 kD polypeptide induced by ABA.     

Development of (1-->3,1-->4)-beta-d-Glucan Endohydrolase Isoenzymes in Isolated Scutella and Aleurone Layers of Barley (Hordeum vulgare)

An immunological assay has been used to investigate the synthesis of (1→3,1→4)-β-glucanase (EC 3.2.1.73) isoenzymes from isolated barley aleurone layers and scutella. Enzyme release from both tissues is enhanced by 1 micromolar gibberellic acid and 10 millimolar Ca²⁺, although increases induced by gibberellic acid are observed only in the presence of Ca²⁺. Isoenzyme I is synthesized predominantly in the scutellum, while isoenzyme II is synthesized exclusively in the aleurone. A third, putative isoenzyme III has been detected in significant proportions in scutellar secretions and may also be secreted from aleurone layers. Both gibberellic acid and Ca²⁺ appear to preferentially enhance isoenzyme II secretion from the aleurone and isoenzyme III secretion from scutella. The patterns of isoenzyme secretion are suggestive of tissue-specific differences in expression of the genes which code for (1→3,1→4)-β-glucanase isoenzymes. Qualitatively similar results were obtained with barley cultivars harvested in Australia and North America.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

Effects of gibberellic acid and abscisic acid on α-amylase activity and ultrastructure of aleurone cells ofAvena sativa L.

The effects of GA₃ and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied. α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA₃ solution or 100 μM GA₃+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA₃+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA₃+10 μM ABA.     

No Effect of 5-Fluorouracil on the Properties of Purified alpha-Amylase from Barley Half-seeds

α-Amylase has been purified from de-embryonated seeds of barley (Hordeum vulgare L. cv. Betzes) which have been incubated on 10⁻⁶ m gibberellic acid (GA₃) following 3 days of imbibition in buffer. Incubation of the half-seeds in up to 10⁻² m 5-fluorouracil (5-FU) during the entire incubation period, including imbibition, had no effect on any of the following characteristics of purified α-amylase: thermal stability in the absence of calcium, molecular weight of the enzyme, isozyme composition, specific activity, or the amount of α-amylase synthesized by the aleurone tissue. The synthesis of rRNA and tRNA was strongly inhibited by 5-FU, indicating that the analog had entered the aleurone cells. These results are not in agreement with those of Carlson (Nature New Biology 237: 39-41 [1972]) who found that treatment of barley aleurone with 10⁻⁴ m 5-FU prior to the addition of GA₃ resulted in decreased thermal stability of GA₃-induced α-amylase and who interpreted this as evidence that the mRNA for α-amylase was synthesized during the imbibition of the aleurone tissue and independently of gibberellin action. Results of the present experiments indicate that the thermal stability of highly purified α-amylase is not altered by treatment of barley half-seeds with 5-FU, and that 5-FU cannot be used as a probe to examine the timing of α-amylase mRNA synthesis.     

Alpha-amylase secretion by single barley aleurone layers

The secretion of α-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 α-amylase. The release of α-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 α-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 Ca²⁺ from the flow-through medium results in the immediate cessation of enzyme release and addition of Ca²⁺ 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 millimolar Ca²⁺. Ruthenium red, which blocks Ca²⁺ but not Mg²⁺ efflux from barley aleurone layers, renders α-amylase release insensitive to Ca²⁺ withdrawal. Inhibitors of respiratory metabolism cause a burst of α-amylase release which lasts for 0.5 to 5 hours. Following this phase of enhanced α-amylase release, the rate of release declines to zero. Pretreatment of aleurone layers with HCl prior to incubation in HCN also causes a burst of α-amylase release, indicating that the inhibitor is affecting the secretion of α-amylase and not its movement through the cell wall. The rapid inhibition of α-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 diffusion-limited. 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.     

Effects of gibberellic acid and environmental factors on cytosolic calcium in wheat aleurone cells

Gibberellins (GAs) control a wide range of physiological functions in plants from germination to flowering. The cellular mechanisms by which gibberellic acid (GA₃) acts have been most extensively studied in the cereal aleurone. In this tissue, alterations in cellular calcium are known to be important for the primary response to GA, which is the production and secretion of hydrolytic enzymes. The extent to which cytosolic Ca²⁺ mediates the early events in GA action, however, is not known. In order to address this question, changes in cytosolic Ca²⁺ in wheat (Triticum aestivum L. cv. Inia) aleurone cells that occur rapidly after treatment with GA were characterized. In addition, GA-induced changes were compared with changes induced by three environmental stimuli that are known to modify the GA response: osmotic stress, salt (NaCl), and hypoxia. The Ca²⁺-sensitive dye fluo-3 was used to photometrically measure cytosolic Ca²⁺. It was found that GA₃ induced a steady-state increase in cytosolic Ca²⁺ of 100–500 nM. This increase was initiated within a few minutes of treatment with GA and was fully developed after 30–90 min. The changes in cytosolic Ca²⁺ that were induced by GA were distinct from those induced by mannitol, NaCl, or hypoxia. Mannitol caused a steady-state decrease whereas NaCl and hypoxia both increased cytosolic Ca²⁺. In the case of NaCl this increase was transient but for hypoxia the increase was prolonged as long as hypoxic conditions were maintained. Gibberellin-induced changes in cytosolic Ca²⁺ were not induced by the inactive GA, GA₈, nor did the GA-insensitive wheat mutant, D6899, respond to active GA₃ with altered cytosolic Ca²⁺. It is concluded that changes in cytosolic Ca²⁺ are an early and integral part of the GA response in aleurone cells. The data also indicate, however, that changes in Ca²⁺ are not sufficient, by themselves, to induce the GA response of aleurone cells.Abbreviations AM acetoxymethyl ester - GA gibberellin - GA₃ gibberellic acid - Mes 2-[N-morpholino]ethanesulfonic acid - PM plasma membrane The author is very grateful to Dr. T-h. D. Ho for his gift of D6899 grain and to Dr. R. Hooley for supplying the inactive GA₈. This work was supported by National Science Foundation Grant DCB-9206692.     

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA₃) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA₃. In contrast, GA₃ is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA₃ is similar to that observed for α-amylase. Treating aleurone layers with both GA₃ and abscisic acid prevents all the GA₃ effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA₃ response, it is concluded that the proteases represent the GA₃-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.