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.     

Water stress enhances beta-amylase activity in cucumber cotyledons

Cotyledons detached from 4-d-old cucumber (Cucumis sativus L.) seedlings were subjected to water stress (air-drying or PEG-treatment) to examine the effects of the stress on carbohydrate metabolism. Amylolytic activity in the cotyledon was increased about 6-fold by water stress within 1 d. The substrate specificity and the action pattern indicated that beta-amylase is responsible for the activity. Activities of azocaseinase, malate dehydrogenase and triose-phosphate isomerase were not affected by water stress, indicating that the effect of the stress on beta-amylase is rather specific. Cycloheximide-treatment strongly reduced the enhancement of beta-amylase activity. The hypocotyl of cucumber seedlings also exhibited an increase in the enzyme activity when subjected to water stress. The major free sugars in cucumber cotyledons were glucose, fructose, maltose, and sucrose; sucrose being the most abundant. Sucrose content in excised, unstressed cotyledons increased markedly during the incubation. Changes in other free sugars were small compared with that of sucrose. Starch also accumulated in unstressed cotyledons. In stressed cotyledons more sucrose and less starch accumulated than in unstressed ones. Such results were discussed in relation to the enhancement of beta-amylase activity.     

Control of galactosyl-sugar metabolism in relation to rate of germination

The storage sugars stachyose and raffinose (galactosyl derivatives of sucrose) are metabolized early during germination of soybean [ Glycine max (L.) Merr.] seeds. The activities of four enzymes involved in the catabolism of these sugars were monitored in soybean cotyledons and embryonic axes during a 7-day germination period. An increase in enzyme activities correlated with a decline in galactosyl sugars. In embryonic axes, uridine diphosphate glucose (UDPglc)-hexose-l-P uridyltransferase (EC 2.7.7.12), an enzyme characteristic of the Leloir pathway, predominated over galactose-1-phosphate uridyltransferase (EC 2.7.7.10), an enzyme characteristic of the pyrophosphorylase pathway; whereas in cotyledons, the situation was reversed. There were differences between two cultivars. Ransom and Amsoy, in the levels of UDPglc-4-epimerase (EC 5.1.3.2); but not in glucose-1-phosphate uridyltransferase (EC 2 7.7.9). An accelerated aging treatment had a significant effect on the development of embryonic axes, as measured by dry weight. In vitro aging of seeds reduced the rate of growth and resulted in higher levels of galactose-containing sugars and significantly lower levels of UDPglc-hexose-l-P uridyltransferase. Thus, reduced development may be related to inability to mobilize or utilize stored carbon reserves. However, it has not been proved that the reduced enzyme activity is responsible for the effects of accelerated aging on growth and sugar metabolism.     

Biochemical Changes in Excised Leaves of Oryza sativa Subjected to Water Stress

Excised rice (Oryza sativa L. cv. Ratna) leaves were used to compare the changes in the levels of various biochemical intermediates and enzyme activities during senescence in turgid and water-stressed conditions. Chlorophyll, total protein and soluble protein content decreased but α-amino nitrogen content increased during the senescence of turgid leaves. In the leaves subjected to water stress, these changes were accelerated, the acceleration being greater with higher degree of water stress. Starch, soluble sugars, total carbohydrates and non-reducing sugar content decreased during senescence of turgid leaves. Water stress accelerated the changes in the levels of starch and non-reducing sugar, but the changes in the levels of soluble sugars and total carbohydrates were retarded. Reducing sugar content increased at first and then decreased in the turgid leaves, and water stress accelerated the change. The decline in the catalase activity and the increase in the peroxidase activity with time was faster in the water-stressed leaves than in the turgid leaves. Acid inorganic pyrophosphatase activity increased, but alkaline inorganic pyrophosphatase activity decreased during the senescence of turgid leaves, and such changes were accelerated by water stress. The results suggest that water stress does not accelerate all the processes connected with leaf senescence.     

Differential response of carbon and nitrogen metabolism to fluoride application in fruiting structures of chickpea (Cicer arietinum)

The effect of sodium fluoride (10 and 50 mol·m⁻³) on the activities of sucrose metabolizing enzymes, transaminases and glutamine synthetase in relation to the transformation of free sugars to starch and protein in the fruiting structures (pod wall, seed coat, cotyledons) of chickpea was studied by culturing detached reproductive shoots in a liquid medium. Addition of fluoride to the culture medium drastically reduced starch content of the cotyledons and caused a marked build-up of total free sugars comprised mainly of reducing sugars in the pod wall and seed coat, and sucrose in the cotyledons. Concomitantly, the activity of soluble invertase was stimulated in the pod wall but reduced in the cotyledons. However, soluble protein content of both the pod wall and the cotyledons increased in conjunction with an increase in the activities of glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase and glutamine synthetase. Disruption of starch biosynthesis under the influence of fluoride and the resulting accumulation of free sugars possibly resulted in their favoured utilization in nitrogen metabolism. Labelling studies with [U-¹⁴C]-sucrose showed that the ¹⁴C incorporation into total free sugars was enhanced by fluoride in the pod wall but reduced in the seed coat and cotyledons, possibly due to an inhibitory effect on their translocation to the developing seeds.     

Hydrolytic enzyme activities and degradation of storage components in cotyledons of germinatingPhaseolus mungo seeds

Phaseolus mungo seeds were allowed to germinate in the dark, and time-course changes in contents of protein fractions, starch, soluble α-amino nitrogen and reducing sugars and in hydrolytic enzyme activities in cotyledons were investigated. In cotyledons of germinated seeds, marked increases in proteolytic (caseolytic, globulytic and gelatin-hydrolyzing) activities and amylolytic activity occurred with concurrent mobilization of storage proteins and starch. Removal of axis organs from seeds at very early stages of germination caused the deteriorated breakdown of storage components and decreased development of proteolytic enzymes in the cotyledons, but this treatment did not significantly affect the appearance of amylolytic activity. The experimental results are discussed in comparison with the hydrolytic enzyme activities of germinating seeds of other leguminous species.     

Physiological responses of somaclonal variants of triploid bermudagrass (<Emphasis Type="Italic">Cynodon transvaalensis</Emphasis> × <Emphasis Type="Italic">Cynodon dactylon</Emphasis>) to drought stress

Eight somaclonal variants with enhanced drought tolerance were isolated from regenerated plants of triploid bermudagrass (Cynodon dactylon × Cynodon transvaalensis cv., TifEagle). Three of them (10-17, 89-02, 117-08) with strong drought tolerance were selected for investigations of physiological responses to drought stress. Compared to the parent control, TifEagle, the somaclonal variants had higher relative water contents and relative growth, and lower ion leakages in the greenhouse tests, while no difference in evapotranspirational water losses and soil water contents was observed between the variants and TifEagle. The variants also had less leaf firing in the field tests under drought stress. Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities decreased gradually in responses to drought stress in all plants and exhibited negative correlations with ion leakage, indicating that the declined activities of these antioxidant enzymes were associated with drought injury in the triploid bermudagrass. However, CAT activities were significantly higher in all three variants than in TifEagle during drought stress. Two variants, 10-17 and 89-02, also had significantly higher APX activities than TifEagle before and during the first 4 days of drought treatments. These two lines also showed higher SOD activities after prolonged drought stress. Proline, total soluble sugars and sucrose were accumulated under drought stress in all plants and exhibited positive correlations with ion leakage. More proline and sugars were accumulated in TifEagle than in the variants. The results indicated that higher activities of the antioxidant enzymes in the variants during drought stress are associated with their increased drought tolerance.     

Effect of GA3, kinetin and indole acetic acid on carbohydrate metabolism in chickpea seedlings germinating under water stress

Enhanced amylase activity was observed during a 7-day-growth period in the cotyledons of PEG imposed water stressed chickpea seedlings grown in the presence of GA₃ and kinetin, when compared with stressed seedlings. During the first 5 days of seedling growth, the seedlings growing under water deficit conditions as well as those growing in the presence of PGRs had a higher amylase activity in shoots than that of control seedlings. Neither GA₃ nor kinetin increased the amylase activity of roots whereas IAA reduced root amylase activity. Activity of acid and alkaline invertases was maximum in shoots and at a minimum in cotyledons. Compared with alkaline invertase, acid invertase activity was higher in all the tissues. The reduced acid and alkaline invertase activities in shoots of stressed seedlings were enhanced by GA₃ and kinetin. Roots of stressed seedlings had higher alkaline invertase activity and GA₃ and IAA helped in bringing the level near to those in the controls. GA₃ and kinetin increased the sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities in cotyledons of stressed seedlings, whereas they brought the elevated level of SPS of stressed roots to near normal level. The higher level of reducing sugars in the shoots of GA₃ and kinetin treated stressed seedlings could be due to the high acid invertase activity observed in the shoots, and the high level of bound fructose in the cotyledons of stressed seedlings could be due to the high activity of SPS in this tissue.     

Effetto della Presenza di Zuccheri Sull'attivita Esochinasica in Cotiledoni Germinanti di Ricino

Abstract

The effects of sugars on the development of hexokinase and fructokinase activities in isolated cotyledons from germinating castor bean seeds. — The possibility of an inductive effect of hexose concentration on the rate of synthesis of enzymes involved in the phosphorylation of sugars has been investigated. Cotyledons were removed from castor bean seeds germinated 48 h at 27 °C in the dark, and incubated 12 h in water or in 0,05–0,1 M glucose or fructose. The activities of hexokinase and of fructokinase (determined spectrophotometrically in the soluble fraction from cell free extracts) was found to increase, upon incubation, at a rate more than 100% higher for the cotyledons incubated in the presence of sugars than for those in water. The results suggest some specificity of the effect of fructose on fructokinase and of glucose on hexokinase. « Insoluble » hexokinase was not affected by the sugars. Protein synthesis inhibitors such as actinomycin D and puromycin inhibited any increase of kinase activities in the isolated cotyledons.     

Effects of chilling stress on the accumulation of soluble sugars and their key enzymes in <Emphasis Type="Italic">Jatropha curcas</Emphasis> seedlings

As osmolytes and signaling molecules, soluble sugars participate in the response and adaptation of plants to environmental stresses. In the present study, we measured the effect of chilling (12 °C) stress on the contents of eight soluble sugars in the leaves, cotyledons, stems, and roots of Jatropha curcas seedlings, as well as on the activities of eight rate-limiting enzymes that are critical to the metabolism of those soluble sugars. Chilling stress promoted both starch hydrolysis and soluble sugar accumulation. The soluble sugar contents of the leaves and cotyledons were affected more than that of the stems and roots. Meanwhile, the activities of the corresponding metabolic enzymes (e.g., β-amylase, uridine diphosphate glucose phosphorylase, and sucrose phosphate synthase) also increased in some organs. The gradual increase of soluble neutral alkaline invertase activity in the four studied organs suggested that sucrose catabolic production, such as glucose and fructose, was especially important in determining resistance to chilling stress and hexose signal transduction pathway. In addition, the substantial accumulation of raffinose family oligosaccharides and increase in corresponding metabolic enzyme activity suggested that galactinol and raffinose play an important role in determining the chilling resistance of J. curcas. Together, these findings establish a foundation for determining the relationship between the chilling resistance and soluble sugar accumulation of J. curcas and for investigating the mechanisms underlying sugar signaling transduction and stress responses.     

The Effects of Germination on the Subcellular Distribution of Cholinesterase in Cotyledons of Phaseolus vulgaris

Homogenates of Phaseolus vulgaris cotyledons have been found capable of hydrolyzing acetylthiocholine. The hydrolysis occurs optimally at pH 8.0, and is inhibited by neostigmine but not eserine. Total activity of the enzyme increases about three-fold between the second and third days of germination, and remains high until day 6 before dropping coincident with the appearance of visible morphological symptoms of senescence in the tissue. Fractionation studies have revealed that the enzyme is enriched in preparations of purified cell wall and plasma membrane and is also present in a soluble fraction. The soluble enzyme accounts for more than 70% of the total cholinesterase activity two days after planting but by the fourth day of germination only about 30% of the total activity in the tissue is soluble. During the same period there is a large increase in the specific activities of both the cell wall and plasma membrane enzymes. By the seventh day of germination the particulate and soluble forms of the enzyme both show much reduced activities, but the specific activities of the cell wall and plasma membrane enzymes subsequently increase again. This is thought to reflect breakdown of protein other than cholinesterase in these structures as they in turn become subject to the increasing pressures of senescence. Cholinesterase in plant tissue presumably serves to regulate the endogenous titre of acetylcholine. The behaviour of this enzyme in bean cotyledons has been interpreted in terms of patterns of physiological and ultrastructural change known to characterize this tissue during germination.     

Osmoregulation in hypocotyls of etiolated mung bean seedlings with or without cotyledons in response to water-deficient stress

Effects of water-deficient stress and cotyledon excision on osmoregulation in hypocotyls of dark-grown mung bean seedlings were studied, and following results were obtained. Water-deficient stress inhibited hypocotyl elongation either in intact or decotylized seedlings. The inhibition was more conspicuous in decotylized seedlings than in intact ones. Water-deficient stress decreased osmotic potential in hypocotyls, while cotyledon excision increased it. The concentrations of soluble sugars, free amino acids and potassium ions in hypocotyls of intact or decotylized seedlings increased in response to water-deficient stress. Cotyledon excision reduced the concentration of soluble sugars and free amino acids, but it did not change the concentration of potassium ions, suggesting that a part of soluble sugars and free amino acids is transported from cotyledons. Unlike cotyledon excision, excision of the apex or roots had no influence on osmoregulation in response to water-deficient stress. Segments excised from hypocotyls had the ability to osmoregulate in response to water-deficient stress. Based on these results, the role of cotyledons in osmoregulation in response to water-deficient stress and quantitative relationships between osmotic potential and hypocotyl elongation in etiolated mung bean seedlings are discussed.     

Effect of kinetin (6-furfuryl aminopurine) on changes in activities of some enzymes of lipid-carbohydrate metabolism in relation to growth of isolated cotyledons of vegetable marrow (Cucurbita pepo L.)

Of the 0–35 ppm concentration employed, kinetin (6-furfurylaminopurine) at 25 ppm resulted in the maximum increase in size and fresh weight of excised cotyledons of vegetable marrow (Cucurbita pepo L.). The lipolysis of total lipids and triglycerides increased on kinetin treatment. The content of reducing sugars rose markedly and that of sucrose reduced drastically with kinetin treatment. No lipase activity could be detected in the control. The activity of this enzyme was always higher with kinetin treatment alone as compared to the combined action of kinetin and detergent. The activities of isocitrate lyase and invertase were maximum at the 3rd day of incubation and even more pronounced with kinetin treatment. The activities of sucrose phosphate synthase paralleled with the amount of sucrose.     

S‐nitrosylation/denitrosylation as a regulatory mechanism of salt stress sensing in sunflower seedlings

Nitric oxide (NO) and various reactive nitrogen species produced in cells in normal growth conditions, and their enhanced production under stress conditions are responsible for a variety of biochemical aberrations. The present findings demonstrate that sunflower seedling roots exhibit high sensitivity to salt stress in terms of nitrite accumulation. A significant reduction in S‐nitrosoglutathione reductase (GSNOR) activity is evident in response to salt stress. Restoration of GSNOR activity with dithioerythritol shows that the enzyme is reversibly inhibited under conditions of 120 mM NaCl. Salt stress‐mediated S‐nitrosylation of cytosolic proteins was analyzed in roots and cotyledons using biotin‐switch assay. LC‐MS/MS analysis revealed opposite patterns of S‐nitrosylation in seedling cotyledons and roots. Salt stress enhances S‐nitrosylation of proteins in cotyledons, whereas roots exhibit denitrosylation of proteins. Highest number of proteins having undergone S‐nitrosylation belonged to the category of carbohydrate metabolism followed by other metabolic proteins. Of the total 61 proteins observed to be regulated by S‐nitrosylation, 17 are unique to cotyledons, 4 are unique to roots whereas 40 are common to both. Eighteen S‐nitrosylated proteins are being reported for the first time in plant systems, including pectinesterase, phospholipase d ‐alpha and calmodulin. Further physiological analysis of glyceraldehyde‐3‐phosphate dehydrogenase and monodehydroascorbate reductase showed that salt stress leads to a reversible inhibition of both these enzymes in cotyledons. However, seedling roots exhibit enhanced enzyme activity under salinity stress. These observations implicate the role of S‐nitrosylation and denitrosylation in NO signaling thereby regulating various enzyme activities under salinity stress in sunflower seedlings.     

Respiratory changes with chilling injury of soybeans

The leakage of solutes from cotyledons of soybeans (cv. Chippewa 64) was markedly stimulated by a chilling treatment (1 to 4 C) during the 1st minute of imbibition, but chilling after even 1 minute of water uptake resulted in little or no leakage increase. The respiratory rate of soybean particles was reduced more than 60% if a chilling treatment (15 minutes at 1 to 4 C) was given during the first minutes of imbibition, and little or no reduction was obtained if the chilling treatment was begun at 5 to 15 minutes after the start of imbibition. Using KCN as an inhibitor of cytochrome oxidase pathway of respiration and salicylhydroxamic acid as an inhibitor of the alternative pathway, it was found that the chilling injury involved a major reduction in the cytochrome pathway in whole axes and cotyledons and an engagement of the alternative pathway of respiration in cotyledon tissue. The suggestion is made that the chilling injury involves lesions resulting from temperature stress during the reorganization of membranes with water entry, and that both the leakage and the respiratory effects are consequences of these membrane lesions.     

Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars and leads to higher water and nitrogen uptake

We previously reported on Vicia narbonensis seeds with largely decreased alpha- D-glucose-1-phosphate adenyltransferase (AGP; EC 2.7.7.27) due to antisense inhibition [H. Weber et al. (2000) Plant J 24:33-43]. In an extended biochemical analysis we show here that in transgenic seeds both AGP activity and ADP-glucose levels were strongly decreased but starch was only moderately reduced and contained less amylose. The flux control coefficient of AGP to starch accumulation was as low as 0.08, i.e. AGP exerts low control on starch biosynthesis in Vicia seeds. Mature cotyledons of antisense seeds had increased contents of lipids, nitrogen and sulfur. The protein content was higher due, in particular, to increased sulfur-rich albumins. Globulin fractions of storage proteins had a lower ratio of legumin to vicilin. Isolated cotyledons partitioned less [14C]sucrose into starch and more into soluble sugars with no change in the protein fraction. Respiration of isolated cotyledons and activities of the major glycolytic and carbohydrate-metabolizing enzymes were not affected. Sucrose and the hexose-phosphate pool were increased but UDP-glucose, 3-phosphoglyceric acid, phospho enolpyruvate, pyruvate, ATP and ADP were unchanged or even lower, indicating that carbon partitioning changed from starch to sucrose without affecting the glycolytic and respiratory pathways. Soluble compounds were increased but osmolality remained unchanged, indicating compensatory water influx resulting in higher water contents. Developmental patterns of water and nitrogen accumulation suggest a coupled uptake of amino acids and water into cotyledons. We conclude that, due to higher water uptake, transgenic cotyledons take up more amino acids, which become available for protein biosynthesis leading to a higher protein content. Obviously, a substantial part of amino acid uptake into Vicia seeds occurs passively and is osmotically controlled and driven by water influx.     

Vigna Radiata Seed Germination under Salinity

Salinity reduced mung bean (Vigna radiata Wilczek) radicle and root elongation, delayed and inhibited hypocotyl elongation and mobilization of reserves from the cotyledons to the embryo axis. Fresh and dry masses and water content of the embryo axes were reduced. Under salinity, a net leakage of K to the media increased with time and increasing NaCl concentrations. Sugars present in the cotyledons of seeds were of primary importance for growth of the embryo axis upto 18 h after sowing whereas breakdown of starch by amylase contributed later, the contribution being delayed and reduced with increasing NaCl concentration. Even when amylase activity in the cotyledons was progressively reduced with increasing NaCl concentration, the increasing contents of soluble sugars in the cotyledons indicated that sugars were not limiting for mung bean seedling growth under salinity.     

Culture of isolated zygotic embryos of Pinus radiata D. Don. Part II: Biochemical changes associated with the conversion of isolated embryos

Summary Isolated zygotic embryos of Pinus radiata D. Don germinated and their cotyledons, hypocotyl and root grew and developed further on the optimized culture medium, named LPSH2 based on the accompanying paper. The resulting plantlets appeared normal, but were one-third the size of the natural seedlings grown on water-agar medium only. When the isolated embryos were cultured on water-agar medium, they grew little and more importantly root development did not occur. Studies on biochemical changes during germination and early seedling growth showed that the patterns of changes in soluble sugar and starch content were generally different between isolated embryos and seedlings. Early on during culture the cotyledons and hypocotyls of natural seedlings had higher levels of soluble sugars and starch than the counterparts of the isolated embryos grown on the LPSH2-medium. Conversely, the root of the isolated embryos contained more soluble sugars and starch than that of the seedlings throughout the 3 wk of culture. However, little difference was found between the isolated embryos and seedlings as far as total protein concentrations and their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles were concerned.     

Distribution and Isoenzymes of Aspartate Aminotrans-f erase in Cotyledons of Germinating Pumpkins

During germination a steady decline in the reserve protein occurred in dark grown pumpkin cotyledons. By 9 days, 80% of this nitrogen reserve was hydrolyzed but only 50 % was removed from the cotyledons. The remaining nitrogen (30 %) was incorporated into water soluble protein which reached a maximum 9 days after germination. The increase in water soluble protein in pumpkin cotyledons parallel the increase in soluble and particulate aspartate aminotransferase (E.C.2.6.1.1.), suggesting that this enzyme is involved in nitrogen metabolism during germination. Little enzyme activity was found in pumpkin tissues other than the cotyledons. Four anodally moving isoenzymes were found in the soluble aspartate aminotrans-ferase fraction and 3 anodally moving isoenzymes were found in the particulate fraction. The slowest moving isoenzymes disappeared first during germination.