Effect of Salinity on the Activities of RNase, DNase and Protease during Germination and Early Seedling Growth of Mung Bean

Seeds and seedlings of mung bean (Phaseolus aureus Roxb.) were treated separately with NaCl, KCl, Na₂SO₄ and K₂SO₄ solutions of 5 and 10 S/cm conducitivity. The activity of RNase, DNase and protease were estimated in cotyledons, embryo axis, leaves, and roots. Salinity caused inhibition of RNase activity in the cotyledons and roots, but increase in embryo axis and leaves. Activity of DNase was also increased; sodium salt was more effective than potassium irrespective of associated anion. Salinity in general either reduced or had no effect on protease activity in all organs, with the exception of NaCl which doubled it in leaves.     

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.     

Gibberellin A3 reverses the effect of salt stress in chickpea (Cicer arietinum L.) seedlings by enhancing amylase activity and mobilization of starch in cotyledons

The percentage germination of chickpea seeds (Cicer arietinum L.cv. PBG-1) gradually decreased with increasing concentration of NaCl in the growth medium and was completely inhibited with 200 mM NaCl. In the presence of 75 mM NaCl, only 51% of the seeds germinated. Gibberellic acid (GA3) and kinetin at 6 µM concentration induced the maximum increase in % germination and seedling growth under salt stress. However, IAA further inhibited both the germination and growth of stressed seedlings. The reduction in amylase activity in cotyledons of stressed seedlings was partially reversed with GA3 and kinetin whereas IAA did not show any positive effect. GA3 was more effective than kinetin in enhancing the reduced germination and seedling growth of chickpea seeds along with amylase activity in cotyledons under NaCl induced saline conditions. The reduced uptake of radiolabelled 14C sucrose by cotyledons and its reduced distribution in the shoots and roots of stressed seedlings was increased with addition of GA3 in the medium. Cotyledonary amylase was separated into amylase 1 and amylase 2 by sephadex G 150 column chromatography. The reduced activities of both amylase 1 and amylase 2 in cotyledons under salt stress was returned to near normal levels with GA3 and there was also an increase in starch utilization, resulting in its lower concentration in cotyledons of GA3-supplemented stressed cotyledons.     

A Rapid Method for Evaluation of Autoxidation and Antioxidants

To examine the mechanism of starch degradation in legume cotyledons and the physiological role of α-glucosidase, mung bean seeds were germinated in the presence of Bay m 1099, an α-glucosidase inhibitor. Bay m 1099 (10 μg/ml medium), which minimized the growth deterioration of the mung bean seedlings, caused no changes in the overall rate of starch degradation and of soluble carbohydrate production in the cotyledons, although α-glucosidase activity had been completely suppressed. Total amylase and phosphorylase activities were not influenced by Bay m 1099. These results suggest that the mung bean α-glucosidase is less responsible for starch degradation, unlike wheat α-glucosidase [Konishi et al., Biosci. Biotech. Biochem., 58, 135-139 (1994)].     

Quantitative and Qualitative Changes in Peroxidase during Germination of Mung Bean under Salt Stress

Seeds of mung bean (Phaseolus aureus Roxb.) cv. Pusa Baisakhi were surface sterilized with sodium hypochlorite solution and sown both in Petri dishes and in sand culture containing aqueous solutions of four different salts, viz. NaCI, KCI, Na₂SO₄ and K₂SO₄, each at 5 and 10 S/cm. Peroxidase activity and its isoenzymes were studied in different plant parts at suitable time intervals during germination. Activity of peroxidase increased in embryo axis and leaves but decreased in cotyledons and roots with different salt treatments to varying degrees. A highly significant inverse correlation (r= -0.931 was found between the peroxidase level and the growth of embryo axis under saline conditions. The number of isoenzymes of peroxidase increased with increase in the time of germination. Salinity treatments resulted in the appearance of new isoenzymes in all the plant organs except roots where the isoenzymic pattern remained unchanged. Different types of salinity resulted in the appearance or/and disappearance of different isoenzymes.     

Changes in nitrogen content and protein profiles following <Emphasis Type="Italic">in vitro</Emphasis> selection of NaCl resistant mung bean and tomato

Mung bean and tomato were in vitro selected on media containing 0, 25, 50, 100 and 150 mM NaCl. Two types of media (hormone supplemented media, CB and hormone free media, MS) were used for mung bean using cotyledon explants whereas two types of explants (cotyledons and shoot apices) were used for tomato on MS media. Total-N, protein content, nitrite reductase (NiR) activity and protein protein profiles were checked in selected plants and compared to original non selected ones. NaCl at low concentrations slightly increased total-N in shoots and roots of in vitro selected mung bean and tomato whereas higher concentrations induced significant reductions. Similar increases in protein content were detected at lower concentrations with no significant effects thereover. On the contrary, NaCl gradually inhibited NiR activity. Similar responses of total-N, protein and NiR activity, but with greater magnitudes, were detected in original plants. In addition, NaCl significantly reduced dry weights of shoots and roots of either in vitro selected or, in particular, original intact plants. Moreover, electrophoresis (SDS-PAGE) of protein from shoots of either in vitro selected or intact plants showed that NaCl induced new protein bands while some others were concomitantly disappeared. The induction of one or more of the 86.4, 79, 77.6, 77 and 71.5 kDa bands following in vitro selection and/or the disappearance of the 86 kDa band from intact plants seemed necessary for mung bean resistance. Also, the presence of 86.2 kDa band and/or the loss of the 85.8 and 57.5 kDa bands might be included in tomato resistance. Of these induced bands in mung bean selected on CB media, only two bands were detected in plants selected on MS media. In tomato, two bands lost following selection from cotyledons but only one band lost following selection from shoot apices. These changes in protein pattern therefore might serve as adaptive regulators for resistance to NaCl.     

Effect of NaCl on Biomass and Contents of Sugars, Proline and Proteins in Seedlings and Leaf Explants of Nicotiana tabacum Grown in vitro

Effects of NaCl on growth in vitro and contents of sugars, free proline and proteins in the seedlings and leaf explants of Nicotiana tabacum cv. Virginia were investigated. The fresh and dry mass of the seedlings decreased under salinity. These growth parameters in leaf explants decreased at 50 mM NaCl and increased up to 150 mM NaCl and then decreased at higher level of salinity. Free proline content in both seedlings and leaf explants increased and polysaccharide content decreased continuously with increasing of NaCl concentration. Reducing sugars, oligosaccharides, soluble sugars and total sugars contents in both seedlings and leaf explants decreased up to 150 mM NaCl and then increased at higher concentrations of NaCl.     

Changes in isoenzymes of soluble malate dehydrogenase during germination of mung bean (Phaseolus aureus Roxb.) under salt stress

Seeds of mung bean (Phaseolus aureus Roxb.) cv. Pusa Baisakhi were surface sterilized and sown both in Petri dishes and sand culture containing aqueous solutions of four different saltsviz. NaCl, KC1, Na₂SO₄ and K₂SO₄ each at 5 and 10 m ?^-1 cm^-1. Malate dehydrogenase (MDH) isoenzymes were studied in different plant parts of mung bean at suitable intervals during germination under four different salts. In cotyledons, 96 h after sowing only one isoenzyme was left in control as compared to three under salt treatment. In the embryo axis, 96 h after sowing, sulphate salts resulted in the disappearance of isoenzymes with R₁ 0.43 and 0.62, whereas isoenzyme with R₁ 0.62 was missing only at a higher concentration of chloride salts. Chloride salts also resulted in the disappearance of band with R₁ 0.15, both in the embryo axis and leaves. However, in the roots the isoenzymic pattern remains the same with all the salt treatments.     

Does osmotic regulation of amylase activity in bean cotyledons exist?

The hypothesis that the promotive effect of the embryo axis of the germinating bean seed on amylase activity in the cotyledons is mediated by an osmoregulative mechanism was examined. After 2 days of germination the action of the axis on amylolytic activity was already clearly revealed, whereas at the same time it did not have any influence on osmotic pressure in the cotyledons. When the axis was attached to one cotyledon during 4 days of incubation, osmotic pressure in the cotyledon was lower than its value in the cotyledons of the intact seedling, whereas amylolytic activity was similar in both treatments. It was concluded that the tested hypothesis is not valid in the case of the bean seedling. External osmotic agents brought about a decrease in the level of amylase in the cotyledons, but this does not prove that osmotic changes which are brought about by production of internal metabolites are involved in the regulation of amylase synthesis.     

Effect of Phytohormone Pretreatment on Nitrogen Metabolism in Vigna radiata Under Salt Stress

Application of NaCl (electrical conductivity 4.0 mS cm^–1) resulted in about 52, 50 and 55 % reduction in total nitrogen contents in mung bean [Vigna radiata (L.) Wilczek] leaf, root and nodule, respectively. In nodule, nitrogenase activity was reduced by about 84 % under stress as compared with the control set. Glutamine synthetase activity was reduced by about 31, 16 and 23 %, glutamate oxoglutarate aminotransferase activity was reduced by 78, 57 and 42 % and glutamate dehydrogenase activity was reduced by 9, 8 and 42 % in leaf, root and nodule, respectively, under salt stress. The pretreatment with indole-3-acetic acid, gibberellic acid and kinetin, each ranging from 0.1 to 10 µM, in restoring the metabolic alterations imposed by NaCl salinity was investigated in mung bean. The three phytohormones used were able to overcome to variable extents the adverse effects of stress imposed by NaCl solution.     

Combined effect of salicylic acid and salinity on some antioxidant activities,oxidative stress and metabolite accumulation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>

It has been shown that salicylic acid (SA) acts as an endogenous signal molecule responsible for inducing abiotic stress tolerance in plants. The effect of SA and sodium chloride (NaCl) on growth, metabolite accumulation, oxidative stress and enzymatic and non-enzymatic antioxidant responses on common bean plants (Phaseolus vulgaris, cv. F-15) was studied. Results revealed that either SA or NaCl decrease, shoot, root and total plant dry weights. SA treatments decreased the contents of proline, and reduced forms of ascorbate and glutathione, however, the content of soluble sugars (TSS), thiobarbituric acid-reactive substances (TBARs) and oxidized ascorbate remained unaffected. On the other hand, salinity significantly reduced the levels of endogenous SA but increased the content of proline, soluble sugars, TBARs, ascorbate and glutathione, as well as all increasing the levels of antioxidant enzyme activities assayed, except CAT. The application of SA improved the response of common bean plants to salinity by increasing plant dry weight and decreasing the content of organic solutes (proline and TSS) and damage to the membrane (TBARs). Moreover, SA application under saline conditions decreased the levels of antioxidant enzyme activities POX, APX and MDHAR which could indicate successful acclimatization of these plants to saline conditions.     

Effects of NaCl Salinity on Nitrogenous Compounds and Proteases during Germination of Vigna sinensis Seeds

Serido bean (Vigna sinensis (L.) Savi) seeds were sown in water and in NaCl solution of —4.3 bars water potential. Total, insoluble and soluble proteins as well as soluble amino nitrogen and proteolytic activity of cotyledons were studied after 0, 1, 3, 5, 7, and 9 days of germination. Protein breakdown and turnover was delayed by the NaCl treatment as compared to the control. This was not due to the total amount of proteolytic activity, which was unchanged by salinity; even though the specific activity decreased due to the delayed breakdown of proteins under salt stress. The inhibitory effects of salinity on seed protein reserve mobilization may be due more to inhibition of translocation of hydrolysis products than to inhibition of protease activity.     

Effect of iso-osmotic levels of salts and PEG-6000 on enzymes in germinating pea seeds

Effects of iso-osmotic levels of salts (NaCl, CaCl₂, Na₂SO₄) and PEG-6000 on the activity of hydrolytic and nitrogen assimilatory enzymes in pea embryo axis and coty ledon were studied. The activity of nitrate reductase and nitrite reductase in embryo axis and cotyledon and the activity of protease and α-amylase in cotyledon decreased with decreasing medium osmotic potential as compared to control at all the stages of seedling growth. The activity of protease and amylase increases with increasing levels of stress in embryo axis. Sodium chloride induced, stress had more deleterious effects on the activity of nitrate reductase, nitrite reductase and αamylase followed by other salts and PEG-6000. On the other hand, CaCl₂ induced salt stress was more depressive for protease activity. The maximum increase in the activity of protease and amylase was observed in embryo axis at higher concentration of salts and PEG-6000.     

Substrate-dependent auxin production by Rhizobium phaseoli improves the growth and yield of Vigna radiata L. under salt stress conditions

Rhizobium phaseoli strains were isolated from the mung bean nodules, and, the most salt tolerant and high auxin producing rhizobial isolate N20 was evaluated in the presence and absence of L-tryptophan (L-TRP) for improving growth and yield of mung bean under saline conditions in a pot experiment. Mung bean seeds were inoculated with peat-based inoculum and NP fertilizers were applied at 30-60 kg ha-1, respectively. Results revealed that imposition of salinity reduced the growth and yield of mung bean. On the contrary, separate application of L-TRP and rhizobium appeared to mitigate the adverse effects of salt stress. However, their combined application produced more pronounced effects and increased the plant height (28.2%), number of nodules plant-1 (71.4%), plant biomass (61.2%), grain yield (65.3%) and grain nitrogen concentration (22.4%) compared with untreated control. The growth promotion effect might be due to higher auxin production in the rhizosphere and improved mineral uptake that reduced adverse effects of salinity. The results imply that supplementing rhizobium inoculation with L-TRP could be a useful approach for improving growth and yield of mung bean under salt stressed conditions.     

The role of cotyledon metabolism in the establishment of quinoa (Chenopodium quinoa) seedlings growing under salinity

A growth chamber experiment was conducted to assess the effect of salinity on emergence, growth, water status, photosynthetic pigments, osmolyte accumulation, and ionic content of quinoa seedlings (Chenopodium quinoa). The aim was to test the hypothesis that quinoa seedlings are well adapted to grow under salinity due to their ability to adjust the metabolic functionality of their cotyledons. Seedlings were grown for 21 days at 250 mM NaCl from the start of the germination. Germination percentage and cotyledon area were not affected by salt whereas seedling height decreased 15%. FW increased in both control and salt-treated cotyledons, but the increase was higher under salinity. DW only increased in salt-treated cotyledons. The DW/FW ratio did not show significant differences between treatments. Relative water content, chlorophyll, carotenoids, lipids, and proteins were significantly lower under salinity. Total soluble sugars, sucrose and glucose concentrations were higher in salt-treated than in control cotyledons. Ion concentration showed a different distribution pattern. Na⁺ and Cl^? concentrations were higher under salinity, while an inverse result was observed for K⁺ concentration. Proline and glycinebetaine concentrations increased under salinity, but the increase was higher in the former than the latter. The osmoprotective role of proline, glycinebetaine, and soluble sugars is discussed.     

Germination and Photosynthetic Responses to Salinity and Alkalinity in <i>Avicenna marina</i> Propagules

Avicenna marina (Forssk.) Vierh is a halophytic mangrove. The reproductive unit is green and has photosynthetic propagules. Mangroves are naturally exposed to fluctuations in some abiotic factors at the soil surface, including salinity and alkalinity. The objective of this study was to determine the effects of two salts including NaCl and NaHCO₃ on germination processes and discuss the relationships between cotyledon photosynthesis and embryo axis growth in A. marina propagules. These propagules came from Al Birk, located on the shoreline of the Saudi Red Sea. The results showed that the studied salts did not affect neither the final germination percentage nor the embryo axis growth. However, rooting and root growth were delayed by both salts at 300 mM and were strongly inhibited by 600 mM NaHCO₃. Both NaCl and NaHCO₃ reduced the photosynthetic activity. These two salts did not affect the other photosynthetic parameters, including stomatal conductance, net transpiration, and intercellular CO₂. Thereafter, the reduction in net photosynthesis was not related to any limitation of stomatal conductance. The early germination phase was independent of cotyledon photosynthesis, whereas rooting and root growth may be limited by reduced photosynthesis under NaCl and NaHCO₃.     

Amylase Activities in Attached and Excised Cotyledons and in Embryonic Axes of Pisum sativum L.

Amylase activity increased in attached cotyledons of peas, Pisumsativum L. var. Bördi, only during imbibition and remainedalmost constant up to 96 h after germination, but in excisedcotyledons the activity increased slightly at first then markedly.In contrast, the content of the reducing sugars was higher inattached cotyledons than in excised ones. A similar inverserelationship has been found between the concentration of reducingsugars in axes (both attached and excised) and amylase activity. The leakage from intact seeds contained more reducing sugarsthan the leakage from excised cotyledons, whereas the amountof proteins released from the cotyledons was four times greaterduring imbibition. This increase in amylase activity in excisedcotyledons is not thought to be the result of axis excision,but to be the result of the leakage of sugars from the cotyledonsduring incubation. These results suggest that the concentration of reducing sugarsmay be a factor that regulates amylase activity in vivo in boththe cotyledons and axis during the germination of pea seeds. (Received August 4, 1982; Accepted December 14, 1982)     

Influence of the axis on the enzymes of protein and amide metabolism in the cotyledons of mung bean seedlings

The growth of the mung bean (Vigna radiata) seedling is accompanied by the biosynthesis and accumulation of the endopeptidase vicilin peptidohydrolase and the catabolism of the reserve proteins in the cotyledons. If the axis is removed from the dry seeds and the cotyledons incubated on moist sand the accumulation of vicilin peptidohydrolase is reduced by 77% and the catabolism of reserve proteins slowed to 25% of the rate in intact seedlings. The cotyledons and the cotyledon exudate are rich in asparagine and this amino acid accounts for more than half of the reduced nitrogen exported from the cotyledons. Glutamine synthetase and asparagine synthetase, two key enzymes in the pathway of asparagine synthesis, are under temporal control in the cotyledons. Their activities increase 3.5- and 10-fold, respectively, then decline again. These increases in enzyme activity occur to the same extent in excised cotyledons and are prevented when the cotyledons are incubated in 5 micromolar cycloheximide. The results indicate that the axis may control certain key metabolic events in the cotyledons, such as the synthesis of vicilin peptidohydrolase, while many other anabolic activities may not depend on a growing axis.     

Studies on energy status and mitochondrial respiration during growth and senescence of mung bean cotyledons

Several characteristics of mitochondrial respiration and energy status have been studied during growth and senescence of mung bean ( Phaseolus radiatus L.) cotyledons. The results showed that mitochondrial oxygen consumption, respiratory control, ADP:O ratios, and energy charge changed in the cotyledons during germination and growth of the seedlings. The respiration rate of intact cotyledons approximately reflected the trend of the oxidative activities of the isolated mitochondria. An increase was observed in both whole cotyledon respiration and mitochondrial oxygen uptake at the onset of senescence of mung bean cotyledons (day 3 after germination), which thereafter declined gradually. The capacity and activity of the alternative pathway increased markedly in mitochondria isolated from senescent cotyledons. After the onset of senescence, the mung bean cotyledon mitochondria exhibited a decrease both in the respiratory control ratios and ADP:O ratios, and the cotyledons exhibited a gradual decline in energy charge. All these results showed an irreversible deterioration of energy conservation in mung bean cotyledons. The role(s) of the alternative pathway in senescent mung bean cotyledons is discussed.     

Inhibitory effect of polyamines on the activity of endopeptidase in mung bean cotyledons

The activity of cysteine endopeptidase (EP) in the cotyledons of mung bean seeds increased with time after germination. When cotyledons were excised from the embryonic axis in the course of seedling growth, the activity of EP in the excised cotyledon markedly dropped during the following incubation of 1 d. However, the level of EP protein in excised cotyledons, as examined by immunoblotting, was similar to that in axis-attached cotyledons at the corresponding stage. Thus, it seems that the low activity of EP in excised cotyledons is not due to a decrease in the content of EP protein, but due to a loss of the activity of existing EP. Treatment of attached cotyledons with polyamines (PAs; putrescine and spermidine [Spd]) resulted in a decrease in EP activity, while the same PA-treatment brought about little alteration in the level of EP protein. This indicates that PAs somehow produce an inhibitory effect on the activity of EP. Axis-removal resulted in an accumulation of Spd in the cotyledon. The possibility is suggested that PA, especially Spd, is involved in the inhibition of EP activity in excised mung bean cotyledons.