Aleurone layer enzyme synthesis

Chrispeels, M. J. Varner, J. E. Hormonal control of enzyme synthesis On the mode of action of gibberellic acid and abscisin in aleurone layers of barley. Plant Physiol., 1967, 42(7), 1008-1016. 

When isolated aleurone layers of barley are incubated in a solution containing GA, they produce and secrete several hydrolytic enzymes. GA-dependent de novo synthesis has been demonstrated for a-amylase (13), protease (14), and 6-1,3-glucanase and ribonuclease (15). In addition, a GA-dependent release of ribonuclease and 6-1,3-glucanase has been demonstrated. The increase in activity of at least one hydrolase, 6-amylase, in the presence of GA is due to release of preformed enzyme and not to de novo synthesis. 

In germinating seeds (such as those of clover, fenugreek, and lucerne129), the cells of the aleurone layer have been shown to be responsible for the in vivo synthesis, and secretion into the endosperm storage-tissue, of the extracellular D-mannanases and related enzymes that are responsible for the metabolism of the reserve galac-tomannan. 

Morre et al. (1970) detected the synthesis of PC from CDP-choline in extracts from onion stem. The activity was associated with a particulate fraction. Devor and Mudd (1971b) found that the 100,000-g pellet from spinach leaves had the highest total amount and specific activity of this enzyme. The metal ion requirement could be satisfied by Mn + (saturating at 0.3 mM) or Mg + (saturating at 13 mM). The for CDP-choline was 10 pM, and the pH optimum was 7.5-8.0. Only modest stimulation could be achieved by the addition of DG. Johnson and Kende (1971) measured the enzyme in particulate fractions (44,000-g pellet) from barley (Hordeum vulgare L.) aleurone layers and found the activity greatly stimulated when the aleurone layers were exposed to gibberellin before extraction and assay of the enzyme. 

Synthesis of RNA during early imbibition necessitates the presence of an active RNA polymerase. Both dry wheat [64] and rye [46] embryos are rich sources of DNA-dependent RNA polymerases, and these enzymes appear to be available in sufficient quantities to catalyse RNA synthesis immediately upon imbibition. In fact, RNA polymerase activity in imbibing, isolated wheat embryos even declines after the second hour from the start of imbibition [84]. This might not be a characteristic of the intact embryo, however, for in intact wheat grains RNA polymerase activity increases after germination [85]. Whether this increase is in the growing embryo (seedling) or in the aleurone layer has not been determined. 

A system to hydrolyse aleurone-layer proteins to provide amino acids for the synthesis of hydrolytic enzymes this system is probably under the control of GA from the embryo. 

They argued that if the extracted a-amylase contained (and was consequently heavier) it must have been synthesized from the amino acids. This would be proof of de novo synthesis of the enzyme which could be separated from light ( O) enzyme on the basis of its density. In order to obtain a convenient label for the light enzyme, Filner and Varner first incubated aleurone layers in GA3, plus H-lysine. The H-labelled a-amylase-produced was collected and placed on an equilibrium density gradient (cesium chloride). An initial experiment showed a coincidence between the radioactive ( H) peak and that of assayable a-amylase activity (Fig. 7.2A). The fact that a-amylase could incorporate H-lysine was, of course, good evidence for its de novo synthesis, but the final proof came when it was shown that assayed a-amylase synthesized in GA3-treated aleurone layers incubated in H2 0 was heavier than the radioactive marker ( H) peak (Fig. 7.2B). 

Fig. 7.3A and B. The increase with time in (A) the level of translatable messenger RNA for a-amylase, and (B) the increase in the rate of synthesis of the enzyme in vivo in response to GA treatment. U units of a-amylase activity. For (A) poly-(A) RNA was extracted from aleurone layers treated with GA for different time periods, and used to support a-amylase synthesis in vitro. After Higgins etal., 1976 [50]... 

Finally, a brief word about cyclic AMP. Activation of the enzyme adenyl cyclase which catalyses cyclic 3, 5 -adenosine monophophate synthesis constitutes the initial molecular event in the target cells of several mammalian hormones. The nucleotide then initiates a series of events leading to a final response characteristic of the hormone. Despite numerous studies on the barley aleurone layer there is no convincing evidence that GA action is mediated via cyclic AMP [73] in fact the weight of evidence is against this nucleotide playing an important role in any plant tissue [7]. 

Most of the evidence suggests that the enzymes are newly synthesized, though in the majority of cases rigorous proof is unavailable. Enzyme development in a number of seeds is prevented by inhibitors of protein and/or RNA synthesis. For example, dipeptidase and isocitrate lyase development in Cucurbita maxima [92, 105] are suppressed by protein synthesis inhibitors, while actinomycin D, which inhibits some DNA-dependent RNA synthesis, prevents the increase in lipase and isocitrate lyase of castor beans [14, 77]. Studies with inhibitors can be criticized on several grounds especially since these chemicals may have previously unsuspected side effects. But more satisfactory evidence of the kind known for barley aleurone layers has been found in some seeds. De novo synthesis of isocitrate lyase in cotyledons of Citrullus vulgaris (watermelon) [52] and of endopeptidase in mung bean [21 a] have been shown to occur by means of density-labelling experiments with D2O. 

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