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.     

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.     

Photosynthesis, reserve mobilization and enzymes of sucrose metabolism in soybean (Glycine max) cotyledons

Soybean (Glycine max L. [Merr] cv. Ransom II) seedlings were grown under a light/ dark regime or in continuous darkness. Cotyledons were harvested daily for measurements of reserve mobilization, net carbon exchange rate, chlorophyll content and activities of certain enzymes involved in sucrose metabolism. Seedlings lost dry weight for the first 3 to 4 days after planting, then maintained a constant dry weight in the etiolated seedlings, and gained dry weight (via net fixation of CO₂) in the light-grown seedlings. In general, the patterns of reserve mobilization were as expected based on the collective work of other investigators. Soluble sugars were mobilized first, followed by protein and lipid. Galactinol, previously uncharacterized in soybean cotyledons, was present at low concentrations and was rapidly depleted within 2 days after planting. Mobilization of reserves was most important during the first 8 days after planting, whereas net cotyledonary photosynthesis began at 6 days after planting and was the primary source of assimilates after 8 days. Maximum rates of cotyledon photosynthesis were higher [up to 18 mg CO₂ (g dry weight)^?1 h^?1] than previously reported and accounted for about 75% of the assimilates transported from the cotyledons to the growing seedling during the functional life of the cotyledon. Enzyme activities in light-grown cotyledons peaked 7 to 10 days after planting and then declined. Sucrose phosphate synthase (EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) activities were similar in etiolated and light-grown seedlings, whereas uridine-5′-di-phosphatase (EC 3.6.1.6) activity was substantially higher in light-grown seedlings. During the period of reserve mobilization, the maximum sucrose phosphate synthase activity in cotyledonary extracts was in excess of the calculated rate of sucrose formation. However, when the cotyledons had highest net photosynthetic rates (14 days after planting), sucrose phosphate synthase activity was similar to the rate of carbon assimilation. It appears that soybean cotyledons are adapted for high rates of sucrose formation (from reserve mobilization and/or photosynthesis) for export to the rapidly growing tissues of the seedling.     

Indole acetic acid mimics the effect of salt stress in relation to enzymes of carbohydrate metabolism in chickpea seedlings

The effect of addition of indole acetic acid (3 M) andNaCl (75 mM) on growth and enzymes of carbohydrate metabolism inchickpea seedlings was compared. In comparison with control seedlings, theseedlings growing in the presence of indole acetic acid (IAA) had reducedamylase activity in cotyledons and enhanced sucrose synthase (SS) and sucrosephosphate synthase (SPS) activities in cotyledons and shoots at all days ofseedling growth. Compared with control seedlings, sucrose content was higher incotyledons, shoots and roots and reducing sugar content was lower in shoots ofIAA treated seedlings. A low invertase (acid and alkaline) activity in shoots ofIAA treated seedlings could lead to reduced sink strength and hence decreasedgrowth of seedlings. Effects of NaCl stress on growth and activities of amylase,SS and SPS in cotyledons and invertase, SS and SPS in shoots were similar tothose observed with addition of IAA.     

Salinity Induces Accumulation of Soluble Sugars and Alters the Activity of Sugar Metabolising Enzymes in Rice Plants

Changes in the starch and sucrose contents, and the sucrose phosphate synthase, acid invertase, and starch phosphorylase activities were studied in the seedlings of salt sensitive and salt tolerant rice cultivars growing under two NaCl concentrations (7 and 14 dS m^-1) for 20 d. Under salinity, the starch content in roots declined more in salt sensitive cvs. Ratna and Jaya than in salt tolerant cvs. CSR-1 and CSR-3 and was unchanged in shoots. The contents of reducing and non-reducing sugars, and the activity of sucrose phosphate synthase was increased more in the sensitive than in the tolerant cultivars. Acid invertase activity decreased in shoots of the salt tolerant cultivars, whereas increased in salt sensitive cultivars. Starch phosphorylase activity decreased in all cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in soluble carbohydrates and related enzymes induced by low temperature during early developmental stages of quinoa (Chenopodium quinoa) seedlings

Low temperature represents one of the principal limitations in species distribution and crop productivity. Responses to chilling include the accumulation of simple carbohydrates and changes in enzymes involved in their metabolism. Soluble carbohydrate levels and invertase, sucrose synthase (SS), sucrose-6-phosphate synthase (SPS) and alpha-amylase activities were analysed in cotyledons and embryonic axes of quinoa seedlings grown at 5 degrees C and 25 degrees C in the dark. Significant differences in enzyme activities and carbohydrate levels were observed. Sucrose content in cotyledons was found to be similar in both treatments, while in embryonic axes there were differences. Invertase activity was the most sensitive to temperature in both organs; however, SS and SPS activities appear to be less stress-sensitive. Results suggest that 1) metabolism in germinating perispermic seeds would be different from endospermic seeds, 2) sucrose futile cycles would be operating in cotyledons, but not in embryonic axes of quinoa seedlings under our experimental conditions, 3) low temperature might induce different regulatory mechanisms on invertase, SS and SPS enzymes in both cotyledons and embryonic axes of quinoa seedlings, and 4) low temperature rather than water uptake would be mainly responsible for the changes observed in carbohydrate and related enzyme activities.     

Measurement of enzymes related to sucrose metabolism in permeabilized Chlorella vulgaris cells

Sucrose phosphate synthase (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14), sucrose synthase (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were measured in toluene permeabilized cells of Chlorella vulgaris Beijerinck. All three activities were detected at all stages of the growth curve; sucrose synthase and sucrose phosphate synthase showed a zone of maximum activity, while invertase increased with time of growth. Sucrose phosphate synthase and sucrose synthase (sucrose synthesis direction) were stimulated by divalent cations and inhibited by UDP. This inhibition could be reversed by Mg²⁺ or Mn²⁺. Sucrose phosphate synthase activity was inhibited by inorganic phosphate and was enhanced by glucose-6-phosphate, but was insensitive to sucrose. Arbutine decreased sucrose synthase activity in both directions. Sucrose cleavage was inhibited by divalent cations and by pyrophosphate. The effects on the enzyme activities of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid, abscisic acid and kinetin in the growth medium were investigated. Sucrose synthase activity was practically unaffected by all plant hormones tested, except for the presence of kinetin which stimulated the activity. Sucrose phosphate synthase activity was increased by both kinetin and abscisic acid. The effect of the latter was partially reversed by the presence of gibberellic acid. 2,4-D and kinetin were potent stimulators of invertase activity.     

Sucrose Metabolism in Lupinus albus L. Under Salt Stress

Salt stress (50 and 150 mM NaCl) effects on sucrose metabolism was determined in Lupinus albus L. Sucrose synthase (SS) activity increased under salt stress and sucrose phosphate synthase activity decreased. Acid invertase activity was higher at 50 mM NaCl and decreased to control levels at 150 mM NaCl. Alkaline invertase activity increased with the salt stress. Glucose content decreased with salt stress, sucrose content was almost three times higher in plants treated with 150 mM NaCl and fructose content did not change significantly. The most significant response of lupin plants to NaCl excess is the increase of sucrose content in leaves, which is partially due to SS activity increase under salinity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of osmo- and hydropriming of chickpea seeds on seedling growth and carbohydrate metabolism under water deficit stress

Seven-day-old seedlings obtained from seeds primed with mannitol (4%)and water showed three to four fold more growth with respect to root and shootlength in comparison with seedlings obtained from non-primed seeds. Seedlingswere grown under water deficit stress conditions created by 15% polyethyleneglycol (PEG) 6000 in the medium. Priming of chickpea seeds with NaCl and PEGwasnot effective in increasing seedling growth under these water deficit stressconditions. The activities of amylase, invertases (acid and alkaline), sucrosesynthase (SS) and sucrose phosphate synthase (SPS) were higher in shoots ofprimed seedlings. An increase in the activities of SS, and both the acid andalkaline invertases was also observed in roots of primed seedlings. The twofoldincrease in specific activity of sucrose phosphate synthase was observed incotyledons of primed seedlings. The higher amylase activity in shoots of primedseedlings enhanced the rapid hydrolysis of transitory starch of the shootleading to more availability of glucose for shoot growth and this was confirmedby the low level of starch in shoots of primed seedlings.     

Effects of nitrogen and phosphorus deficiencies on levels of carbohydrates, respiratory enzymes and metabolites in seedlings of tobacco and their response to exogenous sucrose

A simple method of growing plants in agar was exploited to investigate the effect of long-term nitrogen (N) and phosphorus (P) deficiencies on respiratory metabolism and growth in shoots and roots of Nicotiana tabacum seedlings, and their interaction with exogenously supplied sucrose. Levels of hexose phosphates and 3-phosphoglyceric acid (3-PGA) were low in P-deficient shoots and roots and high in N-deficient shoots and roots. The ratio of hexose phosphates to 3-PGA and levels of fructose-2,6-bisphosphate were high in P-deficient plants and low in N-deficient plants. These data reflect differences in the way metabolism was perturbed, yet both deficiencies were associated with increased root growth relative to shoot growth, starch accumulation in the shoots, and soluble carbohydrate accumulation, especially hexoses, in the roots. Enzymes for sucrose degradation (sucrose synthase, acid and alkaline invertase) and glycolysis (phosphofructokinase, pyrophosphate-dependent phospho-fructokinase and pyruvate kinase) remained unaltered or declined in the shoots and roots. The accumulation of hexoses in roots of N- and P-deficient plants may result from maintenance of high invertase activities relative to sucrose synthase and glycolytic enzymes in the roots. The possibility that hexose accumulation may drive preferential root growth osmotically in N and P deficiencies is discussed. The addition of sucrose to roots to further investigate the interaction of carbohydrates with growth and allocation in low N and low P produced clear effects even though endogenous levels of soluble carbohydrate were already high in the nutrient-deficient plants. In complete nutrition, growth was stimulated, protein content particularly of the roots was increased and there was a preferential increase in activity of sucrose synthase in roots. At low P, enzyme activities in roots were increased, including sucrose synthase, and protein content increased, particularly in the roots, but there was no increase in growth. In N-deficient plants, exogenous sucrose led to decreased protein, Rubisco and chlorophyll content in shoots, in contrast to the other conditions, and a higher protein content and a general increase of catabolic enzyme activities and growth in the roots.     

Sucrose phosphate synthase and other sucrose metabolizing enzymes in fruits of various species

Recent reports have suggested that sucrose phosphate synthase (EC 2.4.1.14), a key enzyme in sucrose biosynthesis in photosynthetic “source” tissues, may also be important in some sucrose accumulating “sink” tissues. These experiments were conducted to determine if sucrose phosphate synthase is involved in sucrose accumulation in fruits of several species. Peach (Prunus persica NCT 516) and strawberry (Fragaria x ananassa cv. Chandler) fruits were harvested directly from the plant at various stages of fruit development. Kiwi (Actinidia chinensis), papaya (Carica papaya), pineapple (Ananas comosus) and mango (Mangifera indica) were sampled in postharvest storage over a period of several days. Carbohydrate concentrations and activities of sucrose phosphate synthase, sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. All fruits contained significant activities of sucrose phosphate synthase. Moreover, in fruits from all species except pineapple and papaya, there was an increase in sucrose phosphate synthase activity associated with the accumulation of sucrose in situ. The increase in sucrose concentration in peaches was also associated with an increase in sucrose synthase activity and, in strawberries, with increased activity of both sucrose synthase and neutral invertase. The hexose pools in all fruits were comprised of equimolar concentrations of fructose and glucose, except in the mango. In mango, the fructose to glucose ratio increased from 2 to 41 during ripening as sucrose concentration more than doubled. The results of this study indicate that activities of the sucrose metabolizing enzymes, including sucrose phosphate synthase, within the fruit itself, are important in determining the soluble sugar content of fruits of many species. This appears to be true for fruits which sweeten from a starch reserve and in fruits from sorbitol translocating species, raffinose saccharide translocating species, and sucrose translocating species.     

Increase of sucrose synthase activity in wheat plants after a chilling shock

Plants of wheat (Triticum aestivum) were grown at 23°C. After 17 days they were suddenly transferred to 4°C under the same light conditions. The change in temperature produced an increase in the level of sucrose and fructans. Following the chilling shock, enzymes related to sucrose metabolism were measured. The activities of fructose 1,6-biphosphatase, UDPGlc pyrophosphorylase, sucrose phosphate synthase (SPS), UDPase and invertase were not modified even after 8 days at 4°C. On the contrary, the activity of sucrose synthase (SS) (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) rose continuously, immediately after the chilling shock.     

Effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism in seedlings of wheat cultivars

The effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism (alpha and beta amylases, sucrose phosphate synthase, sucrose synthase, acid and alkaline invertases) in wheat (Triticum aestivum L.) was investigated in the seedlings of drought-sensitive (PBW 343) and drought-tolerant (C 306) cultivars. The water deficit was induced by adding 6% mannitol (water potential -0.815 Mpa) in the growth medium. The water deficit reduced starch content in the shoots of tolerant seedlings as compared to the sensitive ones, but increased sucrose content in the shoots and roots of tolerant seedlings, indicating their protective role during stress conditions. It also decreased the alpha-amylase activity in the endosperm of seedlings of both the cultivars, but increased alpha and beta amylase activities in the shoots of tolerant ones. Sucrose phosphate synthase (SPS) activity showed a significant increase at 6 days of seedling growth (DSG) in the shoots of stressed seedlings of tolerant cultivar. However, SPS activity in the roots of stressed seedlings of sensitive cultivar was very low at 4 DSG and appeared significantly only at day 6. Sucrose synthase (SS) activity was lower in the shoots and roots of stressed seedlings of tolerant cultivar than sensitive ones at early stage of seedling growth. Higher acid invertase activity in the shoots of seedlings of tolerant cultivar appeared to be a unique characteristic of this cultivar for stress tolerance. Alkaline invertase activity, although affected under water deficit conditions, but was too low as compared to acid invertase activity to cause any significant affect on sucrose hydrolysis. In conclusion, higher sucrose content with high SPS and low acid invertase and SS activities in the roots under water deficit conditions could be responsible for drought tolerance of C 306.     

Light Induced Enhancement in Proline Levels Under Stress is Regulated by Non-Photosynthetic Events

Vigna radiata (L.) seedlings (5-d-old) were exposed to different concentrations of NaCl in light and in dark. The content of proline in the shoots increased with an increase in NaCl concentration, in light as well as in dark. But, irrespective of the concentration of NaCl, proline accumulation in the shoots was higher in light than in dark. Pretreatment of seedlings with dichlorophenyl dimethyl urea (DCMU) did not make any significant difference in light promoted stress induced proline accumulation. As DCMU is a potent inhibitor of photosynthetic electron transport, the light reaction of photosynthesis was not responsible for the observed light promotion of stress induced proline accumulation. In another set of experiments, 5-d-old green as well as etiolated seedlings were exposed to NaCl stress in the presence of different concentrations of sucrose. Irrespective of the concentration of sucrose used, proline content in shoots of stressed seedlings was higher in light than in dark. Although, sucrose enhanced NaCl stress induced increase in proline content in dark by about 32 %, this enhancement was not comparable to the 286 % increase in proline content brought about by light. These results showed that certain factors other than photosynthesis play a role in light promotion of stress induced proline accumulation.     

Sucrose Metabolism at Three Leaf Development Stages in Bean Plants

Sucrose metabolism was studied at three leaf development stages in two Phaseolus vulgaris L. cultivars, Tacarigua and Montalban. The changes of enzyme activities involved in sucrose metabolism at the leaf development stages were: (1) Sink (9-11 % full leaf expansion, FLE): low total sucrose phosphate synthase (SPS) activity, and higher acid invertase (AI) activity accompanied by low sucrose synthase (SuSy) synthetic and sucrolytic activities. (2) Sink to source transition (40-47 % FLE): increase in total SPS and SuSy activities, decrease in AI activity. (3) Source (96-97 % FLE): high total SPS activity, increased SuSy activities, decreased AI activity. The hexose/sucrose ratio decreased from sink to source leaves in both bean cultivars. The neutral invertase activity was lower than that of AI; it showed an insignificant decrease during the sink-source transition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cloning, characterization and tissue specific expression of a sucrose synthase gene from tropical epiphytic CAM orchid Mokara Yellow

A full-length cDNA encoding sucrose synthase was isolated from the tropical epiphytic CAM orchid Mokara Yellow. The cDNA is 2748bp in length containing an open reading frame of 2447bp encoding 816 amino acids with a predicted molecular mass of 93.1 kDa. The deduced amino acid sequence of M. Yellow sucrose synthase (Msus1) shares more than 80% identity with those from other monocotyledonous plants. The sucrose synthase gene was demonstrated to encode a functional sucrose synthase protein by expression as recombinant protein in Escherichia coli. Northern blot analysis showed that the expression pattern of Msus1 mRNA is tissue specific with highest levels in strong sinks such as expanding leaves and root tips, but not detectable in mature leaves and flowers. Incubation with sugars resulted in a significant increase in the steady-state Msus1 mRNA levels in shoots of seedlings.     

Partial purification and characterization of the soluble DNA polymerase (polymerase-α) from seedlings of Pisum sativum L.

Radish seedlings (Raphanus sativus L. Saxa Treib) were grown in the dark with or without added kinetin (2 mg/l=9.29 M). Low-temperature (77°K) fluorescence emission and absorption spectra of etiolated cotyledons were registered at increasing seedling age before and immediately, 30 s and 30 min after one 1-ms flash. Kinetin was found to induce a higher accumulation of the phototransformable protochlorophyll(ide) P_657–650 in the etiolated cotyledons, especially from day 6 to day 10 after germination. The amount of the P_657–650 protochlorophyll(ide) resynthesized during a 30-min dark period after a 1-ms flash decreased with seedling age. It was smaller in cotyledons from kinetin-treated seedlings at day 6 after germination and at that age only. The ability to perform the Shibata shift decreased with increasing seedling age. In cotyledons from 10- and 13-day-old seedlings, the shift was accomplished to a greater extent when the plants were grown in the presence of kinetin.     

Effect of cytokinins on <Emphasis Type="BoldItalic">in vitro</Emphasis> development of autotrophism and acclimatization of <Emphasis Type="Italic">Annona glabra</Emphasis> L.

The role of cytokinins in the differentiation of the photosynthetic apparatus in micropropagated plants and their effect on the plant’s ability to transition from a heterotrophic to an autotrophic condition during acclimatization was investigated. Annona glabra L. shoots were cultured on woody plant medium supplemented with sucrose and different cytokinins to evaluate leaf tissue for chloroplast development, chloroplast numbers, photosynthetic pigmentation, total photosynthetic potential, and soluble sugar content. Plants were transferred to the rooting medium in the presence or absence of sucrose and then acclimatized. Kinetin and benzyladenine (BAP) stimulated chloroplast differentiation. Inclusion of zeatin in the medium induced the formation of greater numbers of chloroplasts in the leaves, while plants cultivated in the presence of only kinetin and BAP demonstrated greater chlorophyll a and carotenoid content. The use of kinetin and BAP during in vitro culture promoted accumulation of dry matter during the acclimatization phase, especially in plants rooted under autotrophic conditions (without sucrose). Kinetin and BAP promoted development of more leaf area and greater plant survival rates in plant acclimatization on both autotrophic and heterotrophic media. The inhibitory effects of thidiazuron on the differentiation of chloroplasts, accumulation of chlorophyll a, and photosynthetic potential were examined.     

Activities of Sucrose Synthase and Acid Invertase in Pea Seedling Organs

The organ topography of sucrose synthase and soluble acid invertase in pea seedlings at heterotrophic stage (3–14 days) was studied. Sucrose synthase was most active in the roots, with the highest activity on the 6–8th days. In the leaves, its activity decreased from day 3 to day 14. In the stems, sucrose synthase activity was at an invariantly low level. The patterns of sucrose synthase activity in etiolated and green plants were similar. As distinct from sucrose synthase, invertase activity was the highest in the stem, especially in etiolated plants. The peak of its activity was observed on the 6-8th days. In the leaves, invertase activity was lower but its pattern was the same. In the roots, acid invertase activity decreased from the 3rd day and did not depend on illumination. The conclusion is that differences in sucrose synthase and acid invertase activities in roots, leaves, and stem are determined by differences in the import of hydrolytic products of stored compound from the cotyledons as well as by different demands of these organs for these products for the processes of organ expansion and for the maintenance of organ metabolism.     

Sucrose accumulation and enzyme activities in callus culture of sugarcane

The activities of sucrose phosphate synthase (SPS), sucrose synthase (SUSY), neutral invertase (NI) and soluble acid invertase (SAI) were measured in callus cultures of four Mexican sugarcane cultivars (Saccharum spp.) with a different capacity to accumulate sucrose in stem parenchyma cells. The results indicated that sucrose accumulation in callus was positively correlated to the activity of SPS and SUSY and negatively to the activity of SAI and NI while SPS explained most of the variation found for sucrose accumulation and NI least.The research was funded by the department of Biotechnology and Bioengineering CINVESTAV Mexico City, and F. G.-M. received grant-aided support from CONACyT, Mexico.