Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin

Clones encoding a thiolprotease (tpp) have been isolated from a cDNA library of unpollinated, senescent pea ovaries and its pattern of expression during both ovary senescence and parthenocarpic development have been studied. The sequence of the tpp cDNA displays a high similarity with other plant and animal thiolproteases of the papain group. The homology is highest around the Cys-His of the active centre; a 109 amino acid sequence at the carboxy terminus was found to be homologous only to thiolproteases of plant origin; this part of the mRNA is also present in another pea mRNA that exhibits similar patterns of induction. tpp mRNA shows a temporal pattern of accumulation that precedes that observed for proteolytic activity. Such accumulation did not occur when ovaries were induced to grow parthenocarpically by gibberellic acid (GA) treatment; furthermore the initial low level of expression present in ovaries decreased after GA treatment, indicating that the gene is down-regulated by gibberellins. Spatially, tpp mRNA is localized mainly within the ovule and ovary vascular elements, and transiently within the endocarp of senescent ovaries. This pattern of expression precedes the development of the cytopathogenic effects observed as unpollinated ovaries undergo senescence.     

Correlation of spermine levels with ovary senescence and with fruit set and development inPisum sativum L.

Separation and quantitation of polyamines from unpollinated pea (Pisum sativum L.) ovaries and young fruits induced by application of gibberellic acid to unpollinated ovaries showed, in both cases, a decrease in putrescine and spermidine levels between anthesis and 4 d later. By contrast, spermine levels increased prior to the onset of senescence of the unpollinated ovaries (3 d post anthesis) and decreased during fruit development. Low levels of putrescine, spermidine and spermine were also observed in young fruits obtained by self-pollination and by treatment of unpollinated ovaries with 2,4-dichlorophenoxyacetic acid. In-vitro culture of ovary explants in a medium containing spermine showed that a reduction of the growth of gibberellic acid-treated unpollinated ovaries was associated with a rise in the level of spermine in the fruits. The results obtained indicate that changes in spermine levels are involved in the control of ovary senescence and of fruit set and development.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophen-oxyacetic acid - GA₃ gibberellic acid - HPLC high-performance liquid chromatography     

Biochemical and histochemical detection of endoproteolytic activities involved in ovary senescence or fruit development in Pisum sativum

The appearance of endoproteolytic activities related to the senescence of unpollinated pea ( Pisum sativum L. cv. Alaska) ovaries, or with fruit development induced by gibberellic acid (GA₃), was examined simultaneously by biochemical and histochemical techniques using gelatin as substrate. Biochemical detection was carried out by gelatin-containing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Histochemical detection was performed using a gelatin film technique. No differences in endopeptidase activity were found in extracts from non-treated or developing ovaries during the two first days post-anthesis. After day 3 non-treated ovaries showed a marked increase in activity as well as two new bands with proteolytic activity, associated with the beginning of the senescence. At the same time a new activity was also located at the endocarp. In developing ovaries activity was only observed around vascular cells of the mesocarp at the end of the period studied (4–5 days post-anthesis). Activity detected in the ovules was essentially the same in both GA₃-treated and non-treated ovaries.     

Carbamoyl phosphate synthetase, ornithine transcarbamylase, and aspartate transcarbamylase activities in the pea ovary : changes with senescence of the unpollinated ovary or with fruit set induced by gibberellic Acid

Carbamoyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), and aspartate transcarbamylase (ATC) were assayed in extracts from unpollinated ovaries of Pisum sativum L. CPS and OTC activities were, per milligram protein, the highest reported in a plant tissue, representing an estimated 0.1% of the protein in the ovary. The OTC/CPS and ATC/CPS ratios were about 100 and 0.5, respectively, indicating that most of the carbamoyl phosphate is used for arginine synthesis. The weight, protein content, and CPS, OTC, and ATC activities per ovary were determined during the senescence of the ovary and also during fruit set induced by treatment with gibberellic acid (GA₃). In the nontreated ovary the weight and the protein first increased and then decreased dramatically, but the decrease in protein took place much earlier. In the GA₃-treated ovaries the increase in weight was considerably greater than the increase in the protein. Whether or not the ovaries were treated with GA₃, CPS, OTC, and ATC activities closely followed the changes in protein, and thus their ratios and specific activities remained essentially constant. It appears that treatment with GA₃ increases the amount of protein and enzymic activities by preventing a large increase in the rate of protein degradation. In addition, the effects of acetylglutamate, ornithine, and UMP on CPS activity were studied. The pea enzyme exhibits regulatory properties intermediate between those of Escherichia coli and the ureotelic liver enzymes.     

Gene expression during two alternative pathways of ovary development in Pisum sativum: fruit development and ovary senescence

Pea ovaries are induced to enter a fruit development pathway involving physiological and morphological changes by pollination or application of plant growth regulators. In the absence of these stimuli, overies stop growing and enter an alternative pathway of senesecence that leads to their degeneration. We have used two dimensional polyacrylamide gel electrophoresis in search of molecular changes underlying fruit development and ovary senescence at the level of total accumulated proteins, newly synthesized proteins, and translatable, RNA populations. We have found changes in gene expression during the processes of ovary formation and ovary senescence. Stimuli that induce fruit set do not appreciably alter the overall patterns of synthesized proteins or translatable RNAs, indicating that fruit development is apparently a natural continuation of ovary formation. However, ovary senescence is an alternative pathway that involves the presence of new RNA messengers and proteins as well as the disappearance of others. These changes were detected earlier than any morphological or structural changes could be observed in the ovary.     

The Control of Leaf Senescence in Kleinia articulata by Photoperiod

Experiments with attached and detached leaves of K. articulatahave shown that senescence rates are determined by the daylengthprevailing during early growth (leaf-expansion stage) and thatthis effect is lasting, long days leading to early leaf death.Daylength also affects longevity after full expansion has occurred,long days hastening senescence. Tests with numerous plant-growthregulators have revealed beneficial effects of gibberellic acid,while kinetin is detrimental to survival at 50 ppm. Entire detachedleaves and isolated petioles behave similarly to leaves on theplant, but leaf discs do not behave in the same way. Other parametersaffected by daylength include: leaf shape, the capacity of leavesto form roots, and enlargement of mesophyll cells normal tothe leaf surface.     

Arginine Decarboxylase and Putrescine Oxidase in Ovaries of Pisum sativum L. (Changes during Ovary Senescence and Early Stages of Fruit Development)

Enzymatic activities involved in putrescine metabolism in ovaries of Pisum sativum L. during ovary senescence and fruit set were investigated. Accumulation of putrescine was observed during incubation of extracts from gibberellic acid-treated unpollinated ovaries (young developing fruits) but not in extracts from untreated ovaries (senescent ovaries). Extracts from pea ovaries showed arginine decarboxylase (ADC) activity, but ornithine decarboxylase and arginase activity were not detected. ADC activity decreased in presenescent ovaries and increased markedly after induction of fruit set with gibberellic acid. Increases in ADC activity were also observed with application of other plant growth substances (benzy-ladenine and 2,4-dichlorophenoxyacetic acid), after pollination, and in the slender (la crys) pea mutant. By contrast, putrescine oxidase activity increased in presenescent ovaries but did not increase during early fruit development. All of these results suggest that ADC and putrescine oxidase are involved in the control of putrescine metabolism. Ovary senescence is characterized by the absence of putrescine biosynthesis enzymes and increased levels of putrescine oxidase and fruit development by an increase in ADC and a constant level of putrescine oxidase.     

Changes in the Level of Peptidase Activities in Pea Ovaries during Senescence and Fruit Set Induced by Gibberellic Acid

The activities and changes in the levels of exopeptidase and endopeptidase activities were characterized in unpollinated ovaries of Pisum sativum L. cv Alaska during senescence and early fruit development induced by gibberellic acid (GA₃). Two aminopeptidases and one iminopeptidase were electrophoretically separated. These peptidases were sensitive to inhibitors of sulfhydryl proteases. Carboxypeptidase activity was inhibited by phenylmethyl sulfonyl fluoride. An azocasein-degrading endopeptidase, sensitive to thiol protease inhibitors, was also found. An increase in the specific activity of aminopeptidase during both fruit development and ovary senescence was observed. In contrast, the specific activity of carboxypeptidase and endopeptidase increased only during senescence of the ovary. Changes in exopeptidase activity in senescing ovaries could be mainly the consequence of a greater stability to proteolysis while the rise in endopeptidase activity appeared to be due to new or increased synthesis of the enzyme. These results suggest that endopeptidase, and not amino or carboxypeptidase, plays a key role in the senescence of pea ovaries and that the changes in unpollinated ovaries leading to ovary senescence or fruit development can be controlled by gibberellins.     

The effect of plant hormones on the components of the secretory pathway in human normal and tumor cells

Plant hormones play a key role in plant growth and differentiation. Certain plant hormones are known to be potential antitumor agents, affect the secretory activity of animal cells, and are produced by mammalian cells as proinflammatory cytokines. The goal of this research was to study the effect of abscisic and gibberellic acids on the secretory system of human epidermoid A431 carcinoma cells and HaCaT keratinocytes. Immunocytochemical and morphometric analysis showed that a subtoxic concentration of abscisic and gibberellic acids induced extension of the ER network and increased the size of the Golgi complex. Electron-microscope studies confirmed the hypertrophic changes of the Golgi complex: swelling of cisternae in the trans-Golgi compartment after exposure to abscisic acid and swelling of cis- and trans-compartments after exposure to gibberellic acid. The Click-iT technique revealed elevation of total protein synthesis only in A431 cells exposed to abscisic acid. Our data suggest that the hypertrophy of Golgi may reflect enhanced secretory activity in A431 cells exposed to abscisic acid. In other experiments, Golgi hypertrophy was not accompanied with increased protein synthesis that suggested the stress-related changes of ER and Golgi complex. Our results demonstrate that morphologically similar reaction manifested in hypertrophy of Golgi complex, in response to plant hormones, is the result of different functional activities, and that molecular mechanisms underlying induced changes need further investigations.     

Biochemical changes in testis and ovary of Chrysocoris stolli Wolf. after the application of juvenoid and ecdysterone

The changes in the metabolic status of both testis and ovary of Chrysocoris stolli following the treatment with juvenile hormone analogue (JHa) and ecdysterone were studied. After the exogenous application of JHa in selective dose, total carbohydrate, glycogen, trehalose, cholesterol, ascorbic acid and inorganic phosphorus increased significantly whereas free fatty acid (FFA), phospholipid, total protein, RNA and DNA decreased significantly in comparison to control of both testis and ovary. Total lipid significantly decreased in testis and significantly increased in ovary after JHa injection. The activities of cellular enzymes like alkaline phosphatase, 5' nucleotidase, catalase and peroxidase significantly decreased while acid phosphatase and GPT significantly increased after the JHa application in comparison to control both in testis and ovary. Activities of GOT and general esterase significantly decreased in testis and increased in ovary after JHa application. The exogenous application of ecdysterone also brought about the similar kind of responses as was noticed in case of JHa treatment but these two treatments differed in some cases such as ecdysteroid that produced some results which were just the reverse of what was produced by JHa treatment. The results obtained here were explained in terms of mode of action of these two hormones.     

Ribulose-1,5-bisphosphate carboxylase and fruit set or degeneration of unpollinated ovaries of Pisum sativum L.

The polypeptide patterns obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis of undigested and autodigested extracts from pea (Pisum sativum L.) ovaries at the early stages of development or degeneration have been studied. Development of unpollinated ovaries was stimulated by application of different plant growth regulators (gibberellic acid, 2,4-dichlorophenoxyacetic acid, and N⁶-benzyladenine) or by plant topping. Polypeptide bands of similar mobility to ribulose-1,5-bisphosphate carboxylase (RuBPCase) subunits (16 and 55 kDa) could be detected in all types of autodigested extracts from stimulated ovaries. However these bands were absent in electrophoretic patterns of autodigested extracts from unstimulated ovaries after 3 d post anthesis and in patterns of autodigested mixtures of these extracts with either those from stimulated ovaries or those from unstimulated ovaries before day 3. These observations indicate that a proteolytic activity which promotes the hydrolysis of RuBPCase appears in unstimulated ovaries about 3 d after anthesis. This event coincides with the loss of the capacity of unpollinated ovaries to develop in response to gibberellic acid and with the degeneration of the ovary wall.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS-PAGE sodium dodecylsulphate-polyacrylamide gel electrophoresis     

Changes in invertase activities precede ovary growth induced by gibberellic acid in

Developing pods of pea ( Pisum sativum L. cv. Alaska no 7) were used to study the enzymes of sucrose metabolism. Acid and neutral invertase (EC 3.2.1.26). sucrose synthase (SS, EC 2.4.1.13) and sucrose phosphate synthase (SPS, EC 2.4.1.14) have been localized in the soluble fraction. Acid invertase activity was also present in the insoluble fraction and in pea ovary apoplast. In pea pods, sucrose breakdown was dominated by the invertase pathway. SS specific activity only increased at late stages of parthenocarpic pod development, while SPS did so in pods obtained by pollination. Changes in time course of invertase activities have been correlated with the growth rate of fruits induced to develop either by fertilization or by exogenous application of giberellic acid (GA), 2,4-dichloro-phenoxy acetic acid (2,4-D) or 6-benzylaminopurine (6-BAP). The soluble neutral activities might be associated with pod elongation, while the acid ones were rather related to assimilate import by the induced fruits. Application of gibberellic acid to non-pollinated ovaries significantly enhanced the soluble neutral invertase activity before any ovary outgrowth was detected (up to 2 h after treatment). Within the same period of time. GA-treated ovaries showed a decrease in the acid invertase activity of the soluble fraction and an increase of the acid invertase activity in the apopiast. preceding in time the increment of the acid invertase activity associated with the insoluble fraction. Our results suggest that the early GA response may be mediated through a promotion of processes of protein secretion.     

Characterization of the Arrest in Anther Development Associated with Gibberellin Deficiency of the gib-1 Mutant of Tomato

The role of gibberellins in flower bud development was investigated by studying the gib-1 mutant of tomato, Lycopersicon esculentum. This gibberellin-deficient mutant initiates flower buds, but floral development is not completed unless the mutant is treated with gibberellin. Treatment with other plant growth regulators does not induce normal flower development. Development of gib-1 flower buds, as measured by progress toward anthesis, ceases at a bud length of 2.5 millimeters; however, increase in size of the bud continues. Buds between 2.5 and 3.7 millimeters are developmentally arrested but still are capable of developing normally after treatment with gibberellic acid. Anthers of these developmentally arrested buds contain pollen mother cells that are in the G1 phase of premeiotic interphase. Following treatment of developmentally arrested buds with gibberellic acid, premeiotic DNA synthesis and callose accumulation in pollen mother cells are evident by 48 hours posttreatment, and within 66 hours, prophase I of meiosis- and meiosis-related changes in tapetum development are observable.     

Immunohistochemical localization of ornithine decarboxylase in the rat ovary

Summary The present report describes the immunocytochemical localization of ornithine decarboxylase in the prepubertal rat ovary after administration of human chorionic gonadotropin (HCG). Numerous ornithine decarboxylase immunoreactive cells appeared in the thecal layer as well as in the interstitial gland tissue after treatment with HCG. The granulosa cells, the ovum and the ovarian stroma were devoid of immunoreactive ornithine decarboxylase. In contrast to the ovary of HCG-treated rats, the ovary of prepubertal rats given the vehicle alone contained only a few weakly immunoreactive cells.     

Separation of Photolabile-Phytochrome and Photostable-Phytochrome Actions on Growth and Microtubule Orientation in Maize Coleoptiles (A Physiological Approach)

The effect of isoprenoid growth regulators on avocado (Persea americana Mill. cv Hass) fruit growth and mesocarp 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was investigated during the course of fruit ontogeny. Both normal and small-fruit phenotypes were used to probe the interaction between the end products of isoprenoid biosynthesis and the activity of HMGR in the metabolic control of avocado fruit growth. Kinetic analysis of the changes in both cell number and size revealed that growth was limited by cell number in phenotypically small fruit. In small fruit a 70% reduction in microsomal HMGR activity was associated with an increased mesocarp abscisic acid (ABA) concentration. Application of mevastatin, a competitive inhibitor of HMGR, reduced the growth of normal fruit and increased mesocarp ABA concentration. These effects were reversed by co-treatment of fruit with mevalonic acid lactone, isopentenyladenine, or N-(2-chloro-4-pyridyl)-N-phenylurea, but were not significantly affected by either gibberellic acid or stigmasterol. However, stigmasterol appeared to partially restore fruit growth when co-injected with mevastatin in either phase II or III of fruit growth. In vivo application of ABA reduced fruit growth and mesocarp HMGR activity and accelerated fruit abscission, effects that were reversed by co-treatment with isopentenyladenine. Together, these observations indicate that ABA accumulation down-regulates mesocarp HMGR activity and fruit growth, and that in situ cytokinin biosynthesis modulates these effects during phase I of fruit ontogeny, whereas both cytokinins and sterols seem to perform this function during the later phases.     

Gibberellic acid effects on the ultrastructure of endocarp cells of unpollinated ovaries of Pisum sativum

Ultrastructural changes of endocarp cells of unpollinated pea ( Pisum sativum L. cv. Alaska) ovaries treated and non-treated with gibberellic acid (GA₃) were studied. Following treatment with GA₃, the differentiation of cells of the middle zone of the endocarp into sclerenchyma occurred in 3 phases: initial, elongation and maturation. In the initial phase a proliferation of endocarp cells was produced in both GA₃treated and non-treated ovaries. Changes in the cell membrane system (Golgi apparatus, endoplasmic reticulum, plasmalemma) and the formation of primary and secondary cell wall were observed in the 2nd and 3rd phases of treated ovaries. In nontreated ovaries senescence followed the initial phase. These results indicate that GA₃ induced the processes involved in the elongation and maturation phases.     

On the nature of the physiological responses of Avena stem segments to gibberellic Acid treatment

Gibberellic acid was found to cause elongation in Avena sativa (oat) stem segments whether it was applied continuously or as a short pulse. The shorter the pulse time became, the higher was the gibberellic acid concentration needed to cause elongation; the segmental growth apparently depends upon the amount of gibberellic acid taken up by the segments. Avena segments showed a decreased growth response to gibberellic acid if the treatments were initiated at increasingly later times after excision from the plant. This decreased responsiveness to gibberellic acid was inhibited by low temperature (0-4 C), but accelerated by anaerobiosis. On the other hand, growth stimulation by a gibberellic acid pulse at the start of incubation was not altered by cold treatment but was nullified by a nitrogen atmosphere. Both the readiness of the segments for growth stimulation by gibberellic acid and its action in promoting growth clearly involve temperature-dependent, aerobic metabolism.