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.     

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.     

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.     

Immunolocalization of a thiol-protease induced during the senescence of unpollinated pea ovaries

A thiol-endoprotease induced during the senescence of unpollinated ovaries of Pisum sativum L. cv. Alaska has been localized at both cellular and subcellular levels using purified antibodies. Immunoblot analysis showed a single band of 30 kDa in extracts from senescent ovaries 3 and 4 days post-anthesis. Immunolocalization showed the accumulation of the protease within the exocarp and in the outer cell layers of the mesocarp of the senescent ovaries, although with an asymmetric distribution as illustrated in transverse sections. Ultrastructural localization indicates that the protease is associated with the tonoplast and with electron dense materials within the vacuole, where lysis of cell components occurs in senescent ovaries.     

Purification and characterization of a thiol-protease induced during senescence of unpollinated ovaries of Pisum sativum

A senescence-specific protease has been purified from senescent unpollinated ovaries of Pisum sativum L. cv. Alaska by acidic extraction. (NH₄)₂SO₄ fractionation, ion exchange chromatography on CM-Sephadex, and affinity chromatography on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-Sepharose. Characterization of the purified protease indicated that it is a thiol-endoprotease (EC 3. 4. 22 class) active over a wide pH range. Purified antibodies against this protease inhibit the degradation of Rubisco in autodigested extracts of senescent ovaries, suggesting that Rubisco might be a substrate for the protease in senescent pea ovaries. The relative levels of the protease were determined by an enzyme-linked immunosorbent assay (ELISA) along the processes of ovary senescence and gibberellic acid (GA)-induced fruit development, indicating its induction at the beginning of senescence and the suppression of its synthesis by GA treatment.     

Interaction of 4-chloroindole-3-acetic acid and gibberellins in early pea fruit development

In pea, normal pod (pericarp) growth requires the presence of seeds; and in the absence of seeds, gibberellins (GAs) and/or auxins can stimulate pericarp growth. To further characterize the function of naturally occurring pea GAs and the auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), on pea fruit development, profiles of the biological activities of GA3, GA1, and 4-Cl-IAA on pericarp growth were determined separately and in combination on pollinated deseeded ovaries (split-pericarp assay) and nonpollinated ovaries. Nonpollinated ovaries (pericarps) responded differently to exogenous GAs and 4-Cl-IAA than pollinated deseeded pericarps. In nonpollinated pericarps, both GA3 and 4-Cl-IAA stimulated pericarp growth, but GA3 was significantly more active in stimulating all measured parameters of pericarp growth than 4-Cl-IAA. 4-Cl-IAA, GA1, and GA3 were observed to stimulate pericarp growth similarly in pollinated deseeded pericarps. In addition, the synergistic effect of simultaneous application of 4-Cl-IAA and GAs on pollinated deseeded pericarp growth supports the hypothesis that GAs and 4-Cl-IAA are involved in the growth and development of pollinated ovaries.     

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     

The place of brassinolide in the sequential response to plant growth regulators in elongating tissue

In the sequential response to plant growth regulators in young elongating tissue from peas and wheat the peak of sensitivity to 24-epi-brussinolide (1 μM) occurs after those of gibberellin and cytokinin and begins before that of auxin in isolated wheat ( Triticum vulgare L. ev. Egret) coleoptiles aged from 21-96 h. In dwarf pea ( Pisum sativum L. cv. Greenfeast) segments, the peak of sensitivity also lies between those of gibberellin and auxin, and it also occurs before sensitivity to auxin in sections from first leaves of wheat. All the leaf sections and all but the most mature coleoptiles and pea segments were sensitive to fusicocein (1 μM).     

Cell division and cell enlargement in fruit of Lagenaria leucantha as influenced by pollination and plant growth substances

The effects of NAA (naphthaleneacetic acid), GA₃ (gibberellic acid), CPPU (N-(2-chloro-4-pyridyl)-N'-phenylurea) and pollination on fruit set, cell division and enlargement were studied in Lagenaria leucantha, an important vegetable. NAA and GA₃ were ineffective in inducing parthenocarpy, whereas CPPU induced parthenocarpic fruit significantly larger than fruit that resulted from pollination. Cell division, which occurred during the first 4 days after pollination was not reactivated by NAA or GA₃, but was effectively reactivated by CPPU. The cell number of the total cross-section of CPPU-treated fruit was 117.4% of that of pollinated fruit and 154.4% of that of unpollinated at 12 DAA (days after anthesis) respectively. The CPPU-induced parthenocarpic fruit had the largest cell cross-sectional area followed, successively, by pollinated fruit, NAA-treated fruit, GA₃-treated fruit and unpollinated fruit. These results indicate that CPPU induced parthenocarpic fruit growth by directly reactivating cell division and expansion.     

Expression of thiol proteases decreases in tomato ovaries after fruit set induced by pollination or gibberellic acid

The modulation of the expression of thiol proteases during both senescence and development was investigated Proteolytic activity and some thiol proteases were analyzed in unpollinated tomato (Lycopersicon esculentum Mill. cv. Rutgers) ovaries during presenescence and during early fruit development induced by treatment with gibberellic acid (GA) or by natural pollination. Proteolytic activity in extracts was tested on azocasein and by observing degradation of the ribulose-1,5-bisphosphate carboxylase large subunit in western blots. There was no correlation between total activity and protein content. Thiol proteases were analyzed by western blot with antibodies raised against papain and a recombinant tomato C14 thiol protease. A 58-kDa polypeptide was recognized by both antibodies and two more polypeptides of 47 and 36 kDa were detected with the second one. All these polypeptide levels increased in untreated unpollinated ovaries at the presenescent stage. Natural pollination or GA treatment of unpollinated ovaries resulted in decreases of these polypeptides at an early developmental stage. The same pattern was observed for the levels of C14 mRNA. Our results suggest that the expression of C14 thiol protease occurs in unpollinated ovaries at the presenescent stage and that it can be suppressed by factors that induce fruit set and development.     

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.     

Stimulation of Pisum sativum epicotyl elongation by gibberellin and auxin. – Different effects of two hormones on osmoregulation and cell walls

The possible involvement of auxin in the action of gibberellin in stimulating cell elongation was examined by comparing the effects of gibberellic acid (GA) and IAA on the growth, osmoregulation and cell wall properties of the Alaska pea ( Pisum sativum L. cv. Alaska) subhook. Both GA and IAA stimulated cell elongation in the subhook region of derooted cuttings. Cotyledon excision decreased the stimulating effect of GA on the growth of the subhook region, but did not affect that of IAA. As the subhook region elongated, the osmotic potential of the cell sap and the total amount of osmotic solutes increased. Cotyledon excision accelerated the increase in the osmotic potential and suppressed the accumulation of osmotic solutes. In cuttings with cotyledons. GA partly counteracted the increase in the osmotic potential and substantially promoted the accumulation of osmotic solutes. On the other hand, in cuttings without cotyledons. GA did not affect the change in the osmotic potential although it slightly promoted the accumulation of osmotic solutes. IAA accelerated the increase in the osmotic potential, but did not affect the accumulation of osmotic solutes. IAA enhanced the extensibility of the cell wall, while GA did not affect it. These results suggest that at least in the Alaksa pea subhook region. GA does not stimulate cell elongation by affecting the level of auxin.     

Programme of senescence in petals and carpels of Pisum sativum L. flowers and its control by ethylene

The role of ethylene in the control of senescence of both petals and unpollinated carpels of pea was investigated. An increase in ethylene production accompanied senescence, and the inhibitors of ethylene action were effective in delaying senescence symptoms in different flower verticils. Pollination did not seem to trigger the senescence syndrome in the corolla as deduced from the observation that petals from pollinated and unpollinated flowers and from flowers whose carpels had been removed senesced at the same time. A cDNA clone encoding a putative ethylene-response sensor (psERS) was isolated from pea flowers, and the pattern of expression of its mRNA was studied during development and senescence of different flower tissues. The levels of psERS mRNA paralleled ethylene production (and also levels of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) mRNA) in both petals and styles. Silver thiosulfate treatments were efficient at preventing ACO and psERS mRNA induction in petals. However, the same inhibitor showed no ability to modify expression patterns in pea carpels around the anthesis stage, suggesting different controls for ethylene synthesis and sensitivity in different flower organs. Received: 18 June 1998 / Accepted: 22 December 1998     

Involvement of a neutral proteolytic activity in the senescence of unpollinated ovaries of Pisum sativum>

Carrasco, P. and Carbonell, J. 1988. Involvement of a neutral proteolytic activity in the senescence of unpollinated ovaries of Pisum sativum . - Physiol. Plant. 72: 610–616.
The study of proteolysis by autodigestion in extracts from developing (gibberellic acid-treated) or senescing (non-treated) unpollinated ovaries of Pisum sativum L. cv. Alaska indicated the presence of two main proteolytic activities sensitive to inhibitors of sulfhydryl proteases. One of them was active at acidic pH and was present both in developing and senescent ovaries. The other one, active at neutral pH, was only detected in non-treated ovaries and was identified by the hydrolysis of the large subunit of ribulose-l,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39). Senescence of non-treated ovaries was also associated with an increase in proteolytic activity measured with a Coomassie Blue reagent and by determination with ninhydrin of a-NH₂ groups released after autodigestion of the extracts. The presence of the neutral proteolytic activity only in senescing ovaries, but not in developing ones, suggests that it plays a key role in the senescence of pea ovaries and that its appearance is prevented by gibberellic acid.     

Effect of number and configuration of fruits, photon flux and age on the growth and dry matter distribution of fruits of Pisum sativum L.

Abstract. In experiments with Pisum sativum cv. Sleaford Orbiter in a controlled environment, the effect of fruit number and position, photon flux density and developmental stage on fruit growth was studied. During early development (up to 22 d from anthesis) growth of the first fruit was unaffected by the presence of one or two additional fruits irrespective of their position. When grown to maturity in competition with fruits at the same node a small decrease in weight of this fruit was observed. Where plants retained a full complement (20-30) of fruits the growth of the first fruit was markedly decreased at all stages of development (6-40 d). In all instances where competition was observed, the pericarp was more affected than the seeds. This was particularly so when photon flux was decreased 18-22 d from anthesis compared with a decrease at an earlier stage. Partition of dry matter between fruits showed a progressively increasing allocation to the later-formed fruits with time for all treatments. The actual proportions allocated to different fruits were not changed by the number of competing fruits. Decreasing photon flux by more than two-thirds decreased fruit growth rates but had little effect on dry matter partitioning in most cases, although where all fruits were retained, there was a tendency for fruits at the lower reproductive nodes to be less affected. These findings are discussed in relation to known sources of assimilate for fruits, assimilate transport and sink demand. It is suggested that partition of dry matter between fruits can be estimated on the basis of fruit size and the developmental trend in relative growth rate of fruits grown in the absence of competition for assimilate from other fruits.