Gibberellic Acid Controls the Secretion of Acid Phosphatase in Aleurone Layers and Isolated Aleurone Protoplasts of Avena fatua

The role of gibberellic acid (GA₃) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2 [EC] .) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA₃control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion     

Influence of Gibberellic Acid and the Rht3 Gene on Choline and Phospholipid Metabolism in Wheat Aleurone Tissue

The effect of gibberellic acid (GA3) on phospholipid metabolismand -amylase production was studied in aleurone tissue of twonear-isogenic lines of wheat (Triticum aesuvum L.). Incubationof embryoectomized seeds from a GA-responsive line (rht3, tall)with GA₃ caused the induction of -amylase activity after a lagphase of 30 h. In the case of embryoectomized seeds from a ‘GA-insensitive’line (Rh13, dwarf), however, the lag phase was extended up to50 h. During the first 14 h following imbibition, GA₃ inhibitedcholine uptake and its subsequent incorporation into phosphatidylcholine in the Rhr3 line but not in the rht3 line. GA₃ promotedphospholipid breakdown in both the lines during this period,however. GA₃ also terminated independent turnover of the cholineN-methyl groups in phosphatidyl choline and promoted turnoverof the whole choline headgroup. These results are discussedin relation to the possibility that phosphatidyl choline turnoveris an integral part of the GA₃ signal-transduction mechanismin aleurone tissue. Key words: GA3, Rht3 gene, choline, phospholipid     

The Pattern and Control of Phospholipid Metabolism in Wheat Aleurone Tissue

Phospholipid synthesis in wheat aleurone tissue was found tobe induced by imbibition. Radiotracer experiments using [Me-¹⁴C]cholineprecursor showed that the synthesized phospholipid was locatedin the microsomal fraction of the tissue. The most active periodof phospholipid synthesis was during the first day of germinationbefore gibberellic acid (GA₃) responses were evident. GA₃ was,in fact, without effect upon the rate of phospholipid synthesis,but evidence is presented which indicates that the hormone increasedthe rate of phospholipid breakdown. Abscisic acid (ABA) alsoinduced phospholipid breakdown, and ABA did not inhibit theGA₃-induced breakdown. The relationship of phospholipid synthesisand membrane formation to the induction of hydrolase productionby GA₃ is discussed.     

拟南芥磷酸酶基因亚细胞定位与组织表达

通过克隆拟南芥磷酸酶PP2C家族基因At3g51370,构建了绿色荧光蛋白融合表达载体,用基因枪将构建好的载体轰击洋葱表皮细胞进行瞬时表达分析,发现该At3g51370基因表达蛋白定位在细胞核中;用实时定量PCR方法分析At3g51370基因的组织表达特性,发现该基因在花器官中的表达量明显高于其它组织.进一步构建了含At3g51370基因的启动子和GUS报告基因的植物表达载体,经农杆菌介导转化拟南芥,对转基因拟南芥进行GUS组织化学染色,分析该启动子在不同生长时期与不同组织中的转录活性,结果发现,在幼苗期At3g51370基因主要集中在根尖分生组织和顶端分生组织表达,在成年植株中则集中在生殖器官如花和果荚柄等部位表达,在光照和黑暗条件下,At3g51370基因的表达特性没有明显差异.研究表明,At3g51370可能与其它核定位的PP2C磷酸酶一样参与了基因表达的调控,可能在拟南芥早期发育阶段的细胞增值分裂相关信号转导途径中发挥功能,并在花器官的发育过程中行使功能,且不参与光信号转导.     

拟南芥3个蛋白磷酸酶2C基因编码蛋白的亚细胞定位及组织表达分析

利用gateway技术从拟南芥中克隆了3个蛋白磷酸酶2C基因At5G66080、At1G68410和At5G06750,3个基因的ORF全长分别为1 158 bp、1 311 bp和1 182 bp,分别编码一条385、376和393个氨基酸残基的多肽.构建了3个基因的植物表达载体35S:GFP:At5G66080、35S:GFP:At1G68410和35S:GFP:At5G06750,采用基因枪法进行的洋葱表皮细胞GFP瞬时表达实验表明,荧光信号主要分布在细胞核上,显示这3个基因的产物可能在细胞核上发挥作用.利用实时荧光定量PCR研究At5G66080、At1G68410和At5G06750基因在不同组织中的表达特性,结果表明:3个基因在各个器官均有表达,但表达量不同;At5G66080、At1G68410和At5G06750基因在花中表达量最大;At5G66080和At5G06750基因在根、叶和叶柄中的表达量次之,在茎中的表达量最低;At1G68410基因在根中的表达量次之,在茎、叶和叶柄中的表达量较低.     

Biochemical Characterization and Subcellular Localization of the Red Kidney Bean Purple Acid Phosphatase

Phosphatases are known to play a crucial role in phosphate turnover in plants. However, the exact role of acid phosphatases in plants has been elusive because of insufficient knowledge of their in vivo substrate and subcellular localization. We investigated the biochemical properties of a purple acid phosphatase isolated from red kidney bean (Phaseolus vulgaris) (KBPAP) with respect to its substrate and inhibitor profiles. The kinetic parameters were estimated for five substrates. We used 31P nuclear magnetic resonance to investigate the in vivo substrate of KBPAP. Chemical and enzymological estimation of polyphosphates and ATP, respectively, indicated the absence of polyphosphates and the presence of ATP in trace amounts in the seed extracts. Immunolocalization using antibodies raised against KBPAP was unsuccessful because of the non-specificity of the antiserum toward glycoproteins. Using histoenzymological methods with ATP as a substrate, we could localize KBPAP exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. KBPAP activity was not detected in the embryo. In vitro experiments indicated that pectin, a major component of the cell wall, significantly altered the kinetic properties of KBPAP. The substrate profile and localization suggest that KBPAP may have a role in mobilizing organic phosphates in the soil during germination.     

The Role of Membrane-Bound Enzymes in an Early Response of Aleurone Tissue to Gibberellic Acid

Treatment of aleurone layers of barley seed with gibberellicacid increases the observable phosphorylcholine glyceride transferaseactivity in a membrane fraction prepared from extracts of thealeurone cells. This gibberellic acid-dependent increase inglyceride transferase activity requires neither RNA synthesisnor protein synthesis. Membrane fractions prepared from mixturesof extracts of gibberellic acid-treated layers and control layershave a specific activity of glyceride transferase higher thanexpected on the basis of simple addition of the activities fromthe two sources. Therefore, some kind of activation is occurring.     

The Ultracytochemical Localzation of Acid Phosphatase Activity in Wheat During Fertilization

No acid phosphatase activity was observed in the mature embryo sac of wheat (Triticum aestivum) except the chalazal cytoplasm Of the central cell before fertilization. During fertilization, acid phosphataseactivity was observed in the following loci: part of chromatin of the egg nucleus and most of the mitochondria in the egg cytoplasm; the perinuclear spaces of the egg and sperm nuclei at the fusion of the egg and sperm nuclei; the chalazal cytoplasm and some vacuoles of the degenerated synergid; two sperm nuclei within the cytoplasm of female cells; the cell wall of each cell of the embryo sac and that of the nucellar cells surrounding the embryo sac. No acid phosphatase was observed in the two-celled proembryo. Dense enzyme reaction product was localized in the chromatin of the free nuclei at early stage of the endosperm. The characteristic of acid phosphatase distribution during fertilization may be associated with the physiological change of the egg Cell, the reorganization of mitochondria in the egg cell cytoplasm, the degeneration of one of the two synergids, the physiological state of the sperm nuclei and the nuclear membrane fusion of the egg and sperm nuclei.     

Gibberellic Acid and Ion Release from Barley Aleurone Tissue: Evidence for Hormone-dependent Ion Transport Capacity

The release of potassium, magnesium, and phosphate ions from aleurone cells of barley (Hordeum vulgare L. cv. Himalaya) is a gibberellic acid-dependent process. The release of these ions is preceded by a lag period of 6 to 8 hours after gibberellic acid addition. The effect of gibberellic acid on the release of ions is not mediated through an effect on ion solubilization. Thus, gibberellic acid does not apreciably affect the sum of extracted and released ions relative to controls. Rather, the effect of the hormone is on the release process itself. Inhibitors of oxidative phosphorylation when added with gibberellic acid or at times up to 6 hours after gibberellic acid inhibition release. When these inhibitors are added after ion release has begun, however, rapid efflux of ions occurs. These results suggest a strong correlation between energy levels and ion transport capacity. Inhibitors of RNA and protein synthesis also inhibit gibberellic acid-stimulated ion release. Evidence suggests that RNA and protein synthesis are required to establish and maintain ion release capacity of aleurone cells.     

Gibberellic Acid-induced Increase in Activity of a Particular Isozyme of Acid Phosphatase in Wheat Half-seeds

Embryoless half-seeds of Triticum aestivum L. contain at leastnine acid phosphatase isozymes of isoelectric pH ranging from4.0 to 7.2. Treatment with GA₃ resulted in activation of a particularisozyme of pI 4.0. Three major isozymes (pi 4.0, 4.9 and 6.2)differed in their relative specificities. A similar increaseof the pI 4 isozyme was also observed in the endosperm of germinatingwheat seeds. (Received April 7, 1981; Accepted July 1, 1981)     

Ultracytochemical Localization of Acid Phosphatase in Nucellar Cells of Wheat During Degeneration

The distribution of acid phosphatase activity in nucellar ceils of wheat ( Triticum aestivum L. ) during degeneration has been studied using the lead precipitation method at the electron microscopic level. Acid phosphatase was localized in the slightly condensed nuclear chromatin in nucellar cells without any sign of ultrastructural degeneration. As the nuceilar cells started degenerating, the enzyme activity in the cell was observed, in the order from small vacuoles to cell walls, mitochondria, plastids and endoplasmic reticulum. Enzyme activity was the highest in most components of the nuceilar ceils adjacent to the embryo sac where the degeneration of nucellar cells was the strongest, but it was not observed in the nuclei of the degenerated nucel]ar cells. The results indicated that the degeneration of nucellar cells was a progressive and orderly process and supported that the degeneration of nuceilar cells was a programmed cell death.     

Gibberellic Acid-stimulated de Novo Synthesis of a Particular Isozyme of Acid Phosphatase in Wheat Half-seeds

GA₃ stimulated incorporation of radioactive methionine intoan acid phosphatase isozyme possessing an isoelectric pH at4.0 (pI 4 isozyme). This effect of GA₃ was completely inhibitedby cycloheximide, but not by cordycepin. The results suggestthe existence of preformed mRNA coding for pI 4 isozyme in thewheat aleurone layer and the stimulation of its translationby GA₃. (Received April 7, 1981; Accepted July 1, 1981)     

Enzymes of Intermediary Metabolism in the Aleurone Cells of Germinating Wheat

Several enzymes of the glycolysis-gluconeogenesis sequence,the pentose cycle, the tricarboxylic acid cycle, and the electrontransport chain, were studied in the aleurone tissue of germinatingTriticum vulgare. Since all the enzymes are located either inthe cytosol or in mitochondria, they acted as parameters ofthe development of metabolic capacity in these compartmentsduring germination. Each of the enzymes studied was presentin the aleurone tissue of the ungerminated grain. After 12 –18 h germination, the level of each enzyme began to increase,following a sigmoidal course which levelled off by the fourthday. This pattern of enzyme development in the aleurone tissuewas independent of the presence of the embryo, and, therefore,of hormonal control from that tissue (in contrast to the glyoxysomalenzymes). Studies using metabolic inhibitors indicated thatthe developments of the different cytosolic enzyme activitieswere dependent to different degrees upon RNA and protein synthesis.     

The Formation of Endoplasmic Reticulum in the Aleurone Cells of Germinating Wheat: an Ultrastructural Study

In the aleurone cells of the quiescent wheat grain endoplasmicreticulum was sparse and present as short profiles. During germinationlong profiles of endoplasmic reticulum developed near to thenuclear membrane and in close association with plastids. Muchof this development occurred during the first 2 d of germination,but further development and stacking of the membranes appearedto take place after this time. The development of the long profileswas independent of the presence of the embryo and thereforeof control by gibberellic acid. On the contrary, after the secondday of germination gibberellic acid produced a decline in theamount of endoplasmic reticulum associated with vesiculationof the reticulum profiles. The results of this ultrastructuralstudy are discussed in the context of available informationon the biosynthesis of phospholipids in aleurone tissue. Someaspects of our results are at variance with those of otherswho have studied the aleurone tissue of barley. These differencesare discussed and suggestions for their resolution are made.     

Quantification of Tartrate Resistant Acid Phosphatase Activity Using a Computerized Image Analysis System

Tartrate resistant acid phosphatase (TRAP) has been accepted as a marker for identification of osteoclasts. A method is reported here for quantitating TRAP using an image analysis system. The amount of the enzyme specific to osteoclasts can be used to differentiate osteoclasts from other cells capable of TRAP expression. TRAP expression characteristic of the osteoclast was compared with that of multinucleated giant cells (MNGC)s recruited to the site of subcutaneously implanted mineralized bone matrix. Two weeks post-implantation, the pellets were removed and processed for the demonstration of TRAP along with rat proximal tibiae. A large amount of TRAP was consistently expressed by the in situ osteoclasts. The MNGCs associated with the mineralized bone implants expressed little if any TRAP reaction product. Using this system, the amount of TRAP reaction product or any other enzyme reaction product expressed can be objectively and reproducibly quantitated.     

Quantification of Tartrate Resistant Acid Phosphatase Activity Using a Computerized Image Analysis System

Tartrate resistant acid phosphatase (TRAP) has been accepted as a marker for identification of osteoclasts. A method is reported here for quantitating TRAP using an image analysis system. The amount of the enzyme specific to osteoclasts can be used to differentiate osteoclasts from other cells capable of TRAP expression. TRAP expression characteristic of the osteoclast was compared with that of multinucleated giant cells (MNGC)s recruited to the site of subcutaneously implanted mineralized bone matrix. Two weeks post-implantation, the pellets were removed and processed for the demonstration of TRAP along with rat proximal tibiae. A large amount of TRAP was consistently expressed by the in situ osteoclasts. The MNGCs associated with the mineralized bone implants expressed little if any TRAP reaction product. Using this system, the amount of TRAP reaction product or any other enzyme reaction product expressed can be objectively and reproducibly quantitated.     

Production of Gibberellic Acid Using a Dairy Waste as the Basal Medium

Filtered whey has been shown to be a feasible basal medium for the production of gibberellic acid. A maximum yield of 750 mg/liter has been obtained, and the only supplement required was Mg²⁺ ion at a final concentration of 10 mg/liter.     

The Hazard of Acid Differentiation in Gomori's Method for Acid Phosphatase

In his original method for the histochemical demonstration of acid phosphatase, Gomori prescribed differentiation of incubated sections by rinsing them in 14% aqueous acetic acid, to remove the nonspecifically precipitated lead deposits. According to him, the enzymatically produced lead phosphate is not washed out by this procedure. As a result of recent improvements in tissue preparation and shorter incubation time, this staining reaction as it is used now is quite sensitive to an acetic acid wash. If this wash is used as recommended originally, it may completely abolish a truly positive reaction. To avoid falsely negative results, and to compare sections of normal and pathological tissue, omission of this differentiation by acetic acid is essential. The risk of mistaking nonspecific lead precipitates in the interpretation of a positive reaction is very small, and can be avoided by running a negative control slide in which no lead phosphate can be produced enzymatically.     

The Induction of Glyoxysomal Enzyme Activities in the Aleurone Cells of Germinating Wheat

The aleurone cells of quiescent Triticum vulgare grain wereobserved to contain glyoxysomes, but enzymes known to be locatedin this organelle were not detected. During germination thenumber of glyoxysomes increased, and their associated enzymeactivities appeared, increasing up to the fifth or sixth day.The appearance of ß-oxidation, isocitratase, and malatesynthetase activities were largely dependent upon the presenceof the embryo. Gibberellic acid (GA₂) was effective in replacingthe embryo in this role. It is proposed, therefore, that thedevelopment of glyoxysomal enzyme activities and probably ofthe glyoxysomes themselves, is a gibberellic acid-dependentprocess. The developments of citrate synthetase and malate dehydrogenaseactivities were only partly dependent upon gibberellic acid.Since it is known that these enzymes are located in other compartmentsbesides the glyoxysomes, it is proposed that their gibberellicacid-dependent activities are located in glyoxysomes while theirgibberellic acidindependent activities are located in the cytosoland/or the mitochondria. The developmental courses of the gibberellicacid-independent activities and the results of studies usinginhibitors of protein synthesis support this hypothesis     

A Chemical Relaxation Study of Human Prostatic Acid Phosphatase

Chemical relaxation methods and a dilution technique were applied to the study of the hydrolysis of p-nitrophenyl phosphate by human prostatic acid phosphatase. Although the reaction mechanism was not elucidated, rate constants and equilibrium constants were obtained for the reaction of enzyme and p-nitrophenol to form a complex. A slow, 2-sec relaxation effect which showed no concentration dependence was observed in various reaction mixtures, including some lacking the substrate and products of the hydrolytic reaction. The conclusion drawn is that there are two forms of the prostatic enzyme, which are normally in equilibrium with each other, but which undergo a relatively slow interconversion when this equilibrium is perturbed. A preliminary calculation indicates that these forms are present in the equilibrium ratio of 2:1.