Roles of hormones in the responses of excised tomato cotyledons to mannitol induced water stress

Excised tomato cotyledons were aseptically cultured in mannitol induced water stress solutions, and a number of changes were determined. Stressed cotyledons had less fresh weights than normal cotyledons throughout the experiment. Stressed cotyledons initially showed reduced proteins, RNA and chlorophyll contents as compared to normal cotyledons, but eventually showed increases which often surpassed values in normal cotyledons. Endogenous auxin and cytokinin activities were initially lower in stressed cotyledons than in normal cotyledons, but were later higher in stressed cotyledons than in normal cotyledons. Gibberellin changes were slight. Applications of exogenous kinetin were able to reverse the effects of stress in reducing fresh weights, proteins, RNA and chlorophyll contents. Applications of GA₃ only partially reverse stress Effects while IAA had no effects. Stressed cotyledons also senesced more slowly than normal cotyledons. The conclusion was reached that the cotyledons were initially injured by the stress but were able to adapt to the stress and that cytokinin activities were fundamental to the responses.     

Photosynthesis and photorespiration in presenescent, senescent, and rejuvenated soybean cotyledons

Various growth and physiological parameters were measured in germinating, presenescent, and senescing soybean (Glycine max [L.] Merr.) cotyledons and in cotyledons rejuvenated by epicotyl removal 18 days after planting. The maximal measured carbon dioxide exchange rates (CER) in the cotyledons were in the range of those reported for field-grown soybean leaves. Rejuvenated cotyledons accumulated total chlorophyll in excess of the maximum observed in presenescent cotyledons. When photosynthetic rates were expressed per cotyledon, the CER in rejuvenated tissue recovered to the maximal rates observed in presenescent cotyledons. Ribulose-1,5-bisphosphate carboxylase/oxygenase in rejuvenated cotyledons also recovered to the maximal amount seen in presenescent cotyledons so that CER appeared to be a function of ribulose-1,5-bisphosphate carboxylase/oxygenase content during most of the period studied. Observations of the postillumination outburst of CO₂ and ¹⁴C label in glycine indicated that photorespiration was occurring in the cotyledons and that photorespiration relative to photosynthesis was different in rejuvenated compared with presenescent cotyledons.     

Inhibition by calcium of senescence of detached cucumber cotyledons: effect on ethylene and hydroperoxide production

The effect of Ca on senescence was followed in detached cucumber (Cucumis sativus L.) cotyledons floating on various solutions in the dark. Compared with those in water, cotyledons in 10⁻⁴ molar CaCl₂ exhibited reduced chlorophyll loss and H₂O₂ production, reduced and delayed ethylene production, and did not undergo a burst in CO₂ production. In contrast, Mg had little effect on cotyledon senescence, whereas K stimulated chlorophyll loss but did not increase H₂O₂ accumulation of ethylene and CO₂ production. This reduction in the rate of senescence by Ca could also be achieved by increasing the endogenous levels of Ca in the cotyledons before excision, although the reduction was less than that with Ca in the external solution. The addition of H₂O₂ to the solutions on which cotyledons were floated stimulated chlorophyll breakdown, but effects on ethylene and CO₂ were not consistent.     

Relationship of IAA-oxidase Activity to Gibberellinand IAA-induced Elongation of Light-grown Cucumber Seedlings

The growth and IAA-oxidase activity of light-grown cucumber seedlings (cv. Aonagajibae) were investigated in response to GA₃ and IAA. Both GA₃ and IAA induced significant elongation of the hypocotyl. The fresh and dry weights of the hypocotyl increased due to GA₃ or IAA treatment, whereas no significant change was observed in the cotyledons of GA₃-treated seedlings as compared with the controls. The fresh and dry weights of IAA-treated cotyledons were both lower than those of controls. Treatment with GA₃ or IAA resulted in retardation of IAA-oxidase activity in the hypocotyl and cotyledons. The degree of retardation was less in the cotyledons than in the hypocotyl. An inverse relationship was recognized between GA₃- or IAA-induced elongation and IAA-oxidase activity in the hypocotyl. The auxin-mediated mechanism for gibberellin action was discussed.     

Developmental formation of glutamine synthetase in greening pumpkin cotyledons and its subcellular localization

During the germination of pumpkin (Cucurbita sp. Amakuri Nankin) seeds in dark, the activity of glutamine synthetase in cotyledons gradually increased, reaching a maximum at 5 to 6 days. A measurable enhancement (about 4-fold) of the enzyme activity occurred when the seedlings were exposed to continuous illumination from day 4 up to day 8. Glutamine synthetase activity was detectable only in the cytosolic fraction in the etiolated cotyledons, whereas it was found both in the cytosolic and chloroplast fractions in the green cotyledons. The two isoenzymes of glutamine synthetase have been separated by DEAE-cellulose column chromatography of extracts from the green cotyledons. These data indicate that during the greening process the chloroplastic glutamine synthetase is newly synthesized. The roles of cytosolic and chloroplastic glutamine synthetase in germinating pumpkin cotyledons concerning assimilation of NH₃ are discussed.     

Origin and evolution of C4 photosynthesis in the tribe Salsoleae (Chenopodiaceae) based on anatomical and biochemical types in leaves and cotyledons

 The Chenopodiaceae genus Salsola contains a large number of species with C₄ photosynthesis. Along with derivative genera they have a prominent position among the desert vegetation of Asia and Africa. About 130 species from Asia and Africa were investigated to determine the occurrence of C₃ versus C₄ syndrome in leaves and cotyledons, and to study specific anatomical and biochemical features of photosynthesis in both photosynthetic organs. The species studied belong to all six previously identified sections of the tribe Salsoleae based on morphological characters. Types of photosynthesis were identified using carbon ¹³C/¹²C isotope fractionation. The representatives of all systematic groups were investigated for mesophyll anatomy and biochemical subtypes by determination of enzyme activity (RUBPC, PEPC, NAD- and NADP-ME and AAT) and primary photosynthetic products. Two photosynthetic types (C₃ and C₄) and two biochemical subtypes (NAD- and NADP-ME) were identified in both leaves and cotyledons. Both Kranz and non-Kranz type anatomy were found in leaves and cotyledons, but cotyledons had more diversity in anatomical structure. Strong relationships between anatomical types and biochemical subtypes in leaves and cotyledons were shown. We found convincing evidence for a similar pattern of structural and biochemical features of photosynthesis in leaves and cotyledons within systematic groups, and evaluated their relevance at the evolutionary level. We identified six groups in tribe Salsoleae with respect to photosynthetic types and mesophyll structure in leaves and cotyledons. Two separate lineages of biochemical and anatomical evolution within Salsoleae were demonstrated based on studies of leaves and cotyledons. The sections Caroxylon, Malpighipila, Cardiandra and Belanthera have no C₃ species and only the NAD-ME C₄ subtype has been found in leaves. We suggest the C₄ species in the NADP-ME lineage evolved in Coccosalsola and Salsola sections, and originated in the subsection Arbuscula. Coccosalsola contains many species with C₃ and/or C₃-C₄ intermediate photosynthesis. Within these main evolutionary lineages, species of different taxonomic groups (sections and subsections) had differences in anatomical or/and biochemical features in leaves and cotyledons. We conclude that structural and biochemical changes in the photosynthetic apparatus in species of the tribe Salsoleae were a key factor in their evolution and broad distribution in extreme desert environments. Received January 25, 2001 Accepted July 17, 2001     

Regulation of ascorbate peroxidase activity in dark-grown radish cotyledons by a catalase inhibitor, 3-Amino-1,2,4-Triazole

The present study was performed to see the physiological role of cytosolic ascorbate peroxidase (APX) and its relationship to other enzymes involved in the H₂O₂ scavenging metabolism, and also to elucidate the regulation of APX expression in dark-grown radish (Raphanus sativus L. cv Taiwang) cotyledons. To do so, 3-amino-l,2,4-triazole (aminotriazole), a known specific inhibitor of catalase, was used to simulate a catalase-deficient phenomenon in cotyledons. Aminotriazole, in very low concetration (10^-4 M), inhibited remarkably the development of catalase activity in cotyledons during dark germination. This inhibition of catalase by aminotriazole, however, did not result in any significant changes in the growth response and the H₂O₂ level of developing cotyledons. In addition, the development of guaiacol peroxidase (GPX) activity was also not significantly affected. Unlike GPX, cytosolic APX activity was induced rapidly and reached a 1.7-fold increase in aminotriazole treated cotyledons at day 7 after germination. However,in vitro incubation of cytosolic APX preparation from cotyledons with aminotriazole did not result in any significant change in activity. One cytosolic APX isozyme (APXa) band involved in this APX activation was predominantly intensified in a native polyacrylamide gel by activity staining assay. This means that this APXa isozyme seems to play a key role in the expression of cytosolic APX activity. On the other hand, 2-day-old control seedlings treated with exogenous 1 mM H₂O₂ for 1 h showed a significant increase of cytosolic APX acitivity even in the absence of aminotriazole. Also, 2 μM cycloheximide treatment substantially inhibited the increase of APX activity due to aminotriazole. Based on these results, we suggest that a radish cytosolic APX could probably be substituted for catalase in H₂O₂ removal and that the expression of APX seems to be regulated by a change of endogenous H₂O₂ level which couples to APX protein synthesis in a translation stage in cotyledons.     

Changes in photosynthetic capacity and polypeptide patterns during natural senescence and rejuvenation of <Emphasis Type="Italic">Cucurbita pepo</Emphasis> L. (zucchini) cotyledons

Natural senescence of Cucurbita pepo (zucchini) cotyledons was accompanied by a gradual degradation of reserve proteins (globulins) and an intensive decrease in the content of both large subunit (LSU) and small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The net photosynthetic rate, the primary photochemical activity of PSII, estimated by the variable fluorescence (F_v)/maximal fluorescence (F_m) ratio (F_v/F_m) and the actual quantum yield of PSII electron transport in the light-adapted state (ΦPSII) also progressively decreased during natural senescence. In contrast, the fraction of the absorbed light energy, which is not used for photochemistry (LNU) increased with progression of senescence. The decline in the photosynthetic rate started earlier in ontogenesis compared with the down-regulation of the functional activity of PSII, thus suggesting the existence of protective mechanisms which maintain higher efficiency of the photochemical electron transport reactions of photosynthesis compared with the dark reactions of the Calvin cycle during earlier stages of natural senescence. Decapitation of the epicotyl above the senescing cotyledons resulted in full recovery of the polypeptide profile in the rejuvenated cotyledons. In addition, the photosynthetic rate increased reaching values that exceeded those measured in juvenile cotyledons. The photochemical efficiency of PSII also gradually recovered, although it did not reach the maximum values measured in the presenescent cotyledons.     

Calcium-induced formation of chlorophyll b and light-harvesting chlorophyll complex in cucumber cotyledons in the dark

Dark-grown cucumber seedlings were exposed to intermittent light (2 min light and 98 min dark) and then cotyledons were incubated with 50 mM CaCl₂ in the dark. Chlorophyll (Chl) a was selectively accumulated under intermittent light and Chl b was accumulated during the subsequent dark incubation with CaCl₂. The change in chlorophyll-protein complexes during Chl b accumulation induced by CaCl₂ in the dark was investigated by SDS-polyacrylamide gel electrophoresis. Chlorophyll-protein complex I and free chlorophyll were major chlorophyll-containing bands of the cotyledons intermittently illuminated 10 times. When these cotyledons were incubated with CaCl₂ in the dark, the light-harvesting Chl complex was formed. When the number of intermittent illumination periods was extended to 55, small amounts of Chl b and light-harvesting Chl complex were recognized at the end of intermittent light treatment, and these two pigments were further increased during the subsequent incubation of the cotyledons with CaCl₂ in the dark compared to water controls.     

Effect of the Hypocotyl Hook on Chlorophyll Accumulation in Excised Cotyledons of Cucumis sativus L

Hypocotyl hooks have been shown to influence greening in excised cucumber (Cucumis sativus) cotyledons. The properties of the lag phase are greatly affected by the presence or absence of the hook tissue. A 45-second light pretreatment followed by 4 hours of darkness is sufficient to remove the lag phase from cotyledons with hooks, while hookless cotyledons require 2 hours of continuous illumination followed by 1 hour of dark incubation to break the lag phase. The effect of hooks on cotyledon greening is enhanced if the hooks are shielded from light. Cutting off the hooks after lag phase removal caused a marked decrease in chlorophyll accumulation in the cotyledons. These observations may indicate that the hypocotyl hooks produce a substance or substances needed in the greening process, which are translocated to the cotyledons. Indoleacetic acid, abscisic acid, gibberellin A₃, 6-benzylamino purine and δ-aminolevulinic acid do not show any activity; on the other hand, ethylene appears to replace partially the hypocotyl hooks.     

Activities of Ribulose Bisphosphate Carboxylase and Phosphoenolpyruvate Carboxylase and C-Bicarbonate Fixation during in Vitro Culture of Pinus radiata Cotyledons

The activities of ribulose bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC), as indicators of autotrophic and nonautotrophic CO₂ fixation, were measured in excised cotyledons of Pinus radiata D. Don cultured for 21 days under shoot-forming (SF) and nonshoot-forming (NSF) conditions. The activity of RuBPC was found to increase in both SF and NSF cultures during the initial 5 days of culture. However, it leveled off from day 5 to day 10 and subsequently began to decrease until the end of the culture period under the SF conditions. In contrast, in the NSF cultures, RuBPC activity increased until day 15, when it was twofold higher than the maximum activity found in the SF cultures. An increase in PEPC activity of about 2.5 times the level of activity in freshly excised cotyledons was observed during the initial 5 days of culture under the SF conditions. PEPC activity began to decline after day 5 until it reached the level of activity seen in NSF cotyledons by day 15. In contrast, the activity of PEPC did not show any significant increase during the initial 10 days of culture under the NSF conditions. The K_m (phosphoenolpyruvate) of PEPC from SF cotyledons was about 35% higher than that of NSF cotyledons. Cotyledons from two culture periods (days 5 and 15) were incubated for 15 seconds with NaH¹⁴CO₃. The label in the malate and asparatate fractions as a percentage of total ¹⁴C incorporation was 3 times higher in the SF cotyledons than in the NSF cotyledons. A higher incorporation of ¹⁴C into products of photosynthesis under the NSF conditions was also observed.     

Effects of Ammonium and Potassium Ions on Some Physiological and Biochemical Processes of Excised Cucumber Cotyledons

Incubation of excised cucumber cotyledons (Cucumis sativus L.) with NH₄Cl solutions exceeding 0.001 M inhibited their greening, fresh weight increases, and incorporation of ¹⁴C-leucine into insoluble N compounds. The absorption of ¹⁴C-leucine during incubation and retention of moisture by the excised cotyledons after incubation were greatly diminished by the NH₄Cl treatments. Treatment with KCl solutions of the same concentrations as the NH₄Cl stimulated the greening, fresh weight increases, and the absorption and incorporation of ¹⁴C-leucine. Desiccation of cotyledons stored at 5°C for 10 days was inhibited by dilute KCl solutions. The toxicity of NH₄Cl was alleviated by KCl solutions at low concentrations.     

Gibberellin induces endopeptidase activity in detached cotyledons of Pisum sativum

Endopeptidase activity in cotyledons of 5-day seedlings of Pisum sativum increased rapidly during germination. However, the increase of the activity in detached cotyledons was depressed. We examined whether a growth regulator can be substituted for the embryonic axis on the development of endopeptidase activity. As monitored by an assay with azoalbumin, the development of endopeptidase activity from crude extracts of detached cotyledons appeared to be slightly accelerated by incubation with 10^–5 M GA₃. However, the pattern after gelatin-polyacrylamide gel suggested that the activity induced in detached cotyledons during a 5-d incubation at 10^–7 M GA₃ was the same as that in attached ones during germination for 5 days and an even greater increase in activity was obtained with 10^–5 M GA₃. These results suggest that GA₃ from the embryonic axis induces endopeptidase activity in attached cotyledons at the first stage of germination.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - GA gibberellic acid     

Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons

The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5- to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO₃. Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO₃, subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO₃-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active.     

Effects of calcium on chlorophyll synthesis and stability in the early phase of greening in cucumber cotyledons

The effects of calcium on chlorophyll accumulation and its stability in the early phase of greening in cucumber cotyledons were investigated. Chlorophyll accumulation was hardly affected by dark preincubation of cotyledons with 10 millimolar calcium solution, but was inhibited almost completely when 50 or 100 millimolar solution was used. On the other hand, 50 millimolar calcium inhibited δ-aminolevulinic acid formation in the light by only 75%. Calcium had little effect on the lag for initiation of protochlorophyllide₆₅₀ regeneration, but slowed down the rate of accumulation of protochlorophyllide₆₅₀. In calcium-treated cotyledons, the chlorophyll formed by primary photoconversion was quickly decomposed in the dark. The present results show that calcium inhibited chlorophyll accumulation by inhibiting δ-aminolevulinic acid formation in the light and by stimulating the decomposition of newly formed chlorophyll, both effects being completely prevented by potassium.     

Comparison of gibberellin-like substances from cotyledons and phloem exudate collected from cotyledons of Pharbitis nil in relation to photoperiodic flowering

Gibberellin (GA)-like substances were analyzed in extracts from cotyledons and phloem exudate collected from cotyledons in photoinduced and vegetative seedlings of the short-day plant Pharbitis nil Chois. var. Violet, using high performance liquid chromatography (HPLC) and the dwarf rice bioassay, to see whether any specific GA-like substances were transported from the photoinduced cotyledons via phloem. Cotyledon extracts exhibited five peaks of free GA-like activity in HPLC, whereas only one or two active peaks were detected in phloem exudate extracts. The level of free GA-like activity was considerably lower in phloem exudate than in the cotyledons. In five out of six analyses of cotyledons and phloem exudate, there were substantially higher levels of free GA-like substances in photoinduced plants. Conjugated GA-like substances were present in much higher levels than free GA-like substances in the cotyledon extracts but the levels were not influenced by daylength. In phloem exudate extracts there was no conjugated GA-like substances. The free GA-like substances that are transported via phloem cochromatographed with GA_5/20 and GA₁₉ on HPLC. These were significantly higher in photoinduced plants and thus could have some influence on the photoperiodically-induced flowering in P. nil.     

A Total Synthesis of (±)-Pachyrrhizone

We examined whether developing cotyledons of soybean seed had photosynthetic activities. The cotyledons evolved oxygen under illumination and the activity was inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The rate of oxygen evolution decreased during the development of seeds; about 30μ miol O₂ · mg chlorophyll^-1 · hr^-1 at the early developing stage and about 10μ miol O₂-mg chlorophyll^-1 · hr^-1 at the late developing stage. The rate of oxygen uptake remained at an almost constant level of 40^mol 0₂-mg chlorophyll^-1 · hr^-1 throughout the development. Photosynthetic ¹⁴CO₂-fixation by the cotyledon was observed. Ribulose bisphosphate carboxylase was immunochemically detected in the developing cotyledons. These results show that functional photosynthetic apparatus is present in the developing cotyledons of soybean seeds.     

Biochemical characteristics of thylakoid membranes in chloroplasts of dark-grown pine cotyledons

Japanese black pine (Pinus thunbergii) cotyledons were found to synthesize chlorophylls in complete darkness during germination, although the synthesis was not as great as that in the light. The compositions of thylakoid components in plastids of cotyledons grown in the dark and light were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of polypeptides and spectroscopic determination of membrane redox components. All thylakoid membrane proteins found in preparations from light-grown cotyledons were also present in preparations from dark-grown cotyledons. However, levels of photosystem I, photosystem II, cytochrome b_[ill]/f, and light-harvesting chlorophyll-protein complexes in dark-grown cotyledons were only one-fourth of those in light-grown cotyledons, on a fresh weight basis. These results suggest that the low abundance of thylakoid components in dark-grown cotyledons is associated with the limited supply of chlorophyll needed to assemble the two photosystem complexes and the light-harvesting chlorophyll-protein complex.     

Aminopeptidase activity associated with alpha1-conarachin (peanut protein)

Aminopeptidases were investigated in protein extracts of dormant and germinated peanut cotyledons by electrophoretic immunochemical techniques. Considerable activity was observed in protein zones that migrated toward the anode after simple electrophoresis. Of the immunogenic proteins in dormant seed, aminopeptidase activity was associated only with the immunocomplex of α₁-conarachin, a globular protein in peanuts. The specific aminopeptidase activity of total extracts was marginally higher than that of purified α-conarachin. Specific iso-enzyme(s) adsorption on the antigen-antibody complex of α₁-conarachin might be attributed in part, to these phenomena. Reactions of protein extracts from germinated cotyledons with immune sera made against protein in germinated tissues—roots and cotyledons—showed the identical associative interaction. Some of the determinant groups on α₁-conarachin in the germ and cotyledon were apparently maintained during early phases of germination.