Effect of Kinetin on Starch and Sucrose Metabolising Enzymes in Salt Stressed Chickpea Seedlings

Higher amylase activity in cotyledons of kinetin treated salt stressed (75 mM NaCl) chickpea (Cicer arietinum L. cv. PBG-1) seedlings, as compared to salt stressed seedlings was observed during a growth period of 7 d. The activities of acid and alkaline invertases were maximum in shoots and minimum in cotyledons under all conditions. The reduced shoot invertase activities under salt stress were enhanced by kinetin with a simultaneous increase in reducing sugar content. Kinetin increased the activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in both the cotyledons and shoots of stressed seedlings. Kinetin appears to increase the turnover of sucrose in the shoots of stressed seedlings.     

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.     

Gibberellic acid and kinetin partially reverse the effect of water stress on germination and seedling growth in chickpea

The percent germination and seedling growth of chickpea (Cicer arietinum L. cv. PBG-1) decreased with increasing concentrations of exogenous polyethylene glycol 6000 (PEG). With 15% PEG in the growth medium germination was only 33% while with 10% PEG it was 58% as compared to 93% in control. Addition of gibberellic acid (GA₃) and kinetin to medium containing 10% PEG increased the germination and seedling growth and the effect was maximum with 6 µM GA₃ which was a better inducer of growth and germination under reduced water potential than kinetin. However, indole acetic acid (IAA) inhibited germination and growth of stressed seedlings. The activity of amylase in cotyledons under stress was significantly increased with GA₃ while kinetin and IAA were less effective. Gibberellic acid also enhanced the mobilization of starch from cotyledons of stressed seedlings resulting in low starch levels in cotyledons compared with stressed seedlings.     

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.     

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.     

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.     

Sucrose metabolism and fruit growth in parthenocarpic vs seeded blueberry (Vaccinium ashei) fruits

Although fruit set and development are induced by applications of gibberellins, final fruit weight of gibberellin-induced parthenocarpic fruit is often less than that of pollinated fruit. We examined changes in the activities of sucrose-metabolizing enzymes and sugar accumulation in developing fruits of cultivated blueberry (Vaccinium ashei Reade) and their correlation with fruit growth upon pollination or exogenous applications of gibberellic acid (GA₃). The objective was to determine if differences in fruit growth could be attributed to differences in enzyme activities and subsequent sugar accumulation in fruits. The fruit development period of GA₃-treated fruits was 15 days longer than that of pollinated fruits. At maturity, GA₃-treated fruit accumulated an average of 180 mg dry weight while pollinated fruit accumulated 390 mg dry weight. Dry weight accumulation in nonpollinated fruits was negligible and these fruits abscised by 45 days after bloom (DAB). The total carbon (C) cost (dry weight C + respiratory C) for fruit development was 109 and 244 mg C fruit^-1 for GA₃-treated and pollinated fruits, respectively. Hexose concentration increased to 100 mg (g fresh weight)^-1 at ripening in both GA₃-treated and pollinated fruits. Nonpollinated fruits reached a maximum hexose concentration at 45 DAB. Sucrose phosphate synthase (EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) activities reached a maximum of ≤5.0 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits. Soluble acid invertase (EC 3.2.1.26) activity increased to about 60 μmol (g fresh weight)^-1 h^-1 in both GA₃-treated and pollinated fruits at ripening, while in nonpollinated fruits, a maximum soluble acid invertase activity of 0.12 μmol (g fresh weight)^-1 h^-1 was measured at 24 DAB. Insoluble acid invertase activity declined during the early stages of fruit growth and remained relatively low throughout fruit development. Neutral invertase activity was low throughout development, increasing to 5 μmol (g fresh weight)^-1 h^-1 at ripening in GA₃-treated and pollinated fruits. Our studies demonstrate that blueberry fruit development does not appear to be limited by sucrose metabolizing enzyme activity and/or the ability to accumulate sugars in either GA₃-treated or pollinated fruits.     

Effects of gibberellic Acid and gold light on germination, enzyme activities, and amino Acid pool size in a dwarf strain of watermelon

Gibberellic acid (GA₃) promotes and continuous gold light inhibits germination of seeds of a dwarf strain (WB-2) of watermelon [Citrullus lanatus (Thunb.) Matsu. and Nakai]. Osmotic inhibition of germination with mannitol in light-grown seeds of WB-2 was only slightly reversed by GA₃ at the concentrations used, whereas, GA₃ substantially relieved osmotic inhibition in dark-grown seeds.

The effects of GA₃ and gold light on development of catalase and invertase activities and on levels of free amino acids in germinating seeds of WB-2 were examined. Light depressed development of catalase and invertase activity. Levels of free amino acids increased more slowly in embryonic axes of light- than dark-incubated seeds, but in cotyledons higher levels of amino acids were maintained in light-grown seeds. GA₃ accelerated the development of catalase activity in whole embryos and invertase activity in embryonic axes, but did not significantly affect invertase activity in cotyledons during germination. GA₃ had little effect on amino acid pools in cotyledons and embryonic axes.

     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside     

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.     

Evaluation of the effect of three growth regulators in the germination of <Emphasis Type="Italic">comparettia falcata</Emphasis> seeds under <Emphasis Type="Italic">in vitro</Emphasis> conditions

Summary The effect of 3-indoleacetic acid (IAA), 6-furfurylaminopurine (kinetin), and gibberellic acid (GA₃) on germination of the orchid Comparettia falcata was evaluated in a factorial experiment (4×4×4) with Murashige and Skoog (1962) basal medium. It was established that seeds of this orchid could be maintained under aseptic conditions as long as the necessary nutrients and appropriate concentrations of growth regulators were provided. Of the three growth regulators used, IAA significantly decreased seed germination of Comparettia falcata. There was a synergistic effect in the kinetin:GA₃ combination that produced a positive response in both percentage seed germination and development of seedlings. This study describes a single medium-based protocol able to achieve more than 160000 seedlings within 21 wk, starting from a single capsule, sufficient for both large-scale propagation and in vitro conservation of this threatened orchid.     

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.     

Der Einfluß von Wachstumsregulatoren auf Protein- und Enzym-Spektren kultivierter Explantate aus Rüben von Cichorium intybus L.

Summary On a simple nutrient medium in explants from roots of Cichorium intybus form shoots visible after about 14 days. Gibberellic acid (GA₃) does not influence the spontaneous development of the chicory explants. GA₃ in combination with kinetin inhibits shoot formation whereas kinetin alone promotes the process. On the other hand high concentrations of IAA inhibit the regeneration of shoots.The soluble proteins of chicory cultures treated with growth regulators were examined by disc-electrophoresis. It was shown that the proteins detected by staining with Amido black, phosphatases, esterases and glutamate dehydrogenase (GDH) present in the original root tissue remained constant under the different culture conditions during a period of 12 days. The quantitative changes of some of the proteins, phosphatases and esterases observed during the culture period were identical for all the different cultures in spite of the different morphogenetic behaviour. Only the activities of GDH and peroxidase changed after treatment with different growth regulators; however, in these cases, there was also no direct connection with the morphogenetic responses of the cultures.The specific activity of the GDH-band was promoted by IAA and at the same time the formation of peroxidases was inhibited. Kinetin delayed the formation of peroxidases during the first days of the culture period but promoted it later on. There was a repression by IAA of a specific kationic peroxidase. In the tissues treated with GA₃ the activity of peroxidases was always higher than in the control tissue. This effect of GA₃ can be partly explained by the fact that GA₃ inhibits the release of peroxidases of the explants into the nutrient medium.     

Effect of growth regulators onVicia faba plants irrigated by sea water Leaf area,pigment content and photosynthetic activity

The antagonistic effects of some growth regulators [i.e. indol-3-yl-acetic acid (IAA), gibberellic acid (GA₃) or kinetin] on stress imposed by sea water on leaf area, pigment and photosynthetic activity in leaves of broad bean plants at different stages of development were investigated. Seed priming with GA₃ alleviated either partially or completely the effects induced by the two levels of sea water (10 and 25 %) used on leaf area at all experimental stages. However, IAA, GA₃ and kinetin inhibited leaf growth by themselves in almost all measurements. Seed pretreatment with kinetin alleviated the inhibition of pigment production in sea water-irrigated plants. Furthermore, GA₃ or kinetin nullified the deleterious effects imposed by irrigation with sea water particularly the high level (25 %) on photosynthetic¹⁴CO₂ fixation.     

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     

Effects of prohexadione on cambial and longitudinal growth and the levels of endogenous gibberellins A1, A3, A4, and A9 and indole-3-acetic acid in Pinus sylvestris shoots

Prohexadione, a gibberellin (GA) biosynthesis inhibitor, was applied in ethanol around the circumference at the midpoint of the previous year terminal shoot of dormant Pinus sylvestris seedlings. After cultivating the seedlings under environmental conditions favorable for growth for 10 weeks, longitudinal and cambial growth were measured, and the endogenous levels of GA₁, GA₃, GA₄, GA₉, and indole-3-acetic acid (IAA) were determined by combined gas chromatography-mass spectrometry, using deuterated GAs and [¹³C₆]IAA as internal standards. Prohexadione application inhibited elongation and xylem and phloem production in the current year terminal shoot and xylem production in the previous year terminal shoots. Concomitantly, in both ages of shoots the cambial region contents of GA₁; GA₃, and GA₄ were decreased, whereas the level of GA₉ was increased. However, the IAA content was not altered in the terminal bud on the current year terminal shoot or in the cambial region of the current year or previous year terminal shoots. The results provide additional evidence that: (1) GAs are involved in the regulation of cambial growth, as well as longitudinal growth, in Pinus sylvestris shoots; (2) they act directly, rather than indirectly, by altering the IAA level; and (3) the GA₉ GA₄ GA₁ pathway is a major route of GA biosynthesis in conifer species.Abbreviations GA gibberellin - IAA indole-3-acetic acid - HPLC high performance liquid chromatography - GC gas chromatography - SIM selected ion monitoring - MS mass spectrometry     

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.     

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.     

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.     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid