Excess nickel modulates activities of carbohydrate metabolizing enzymes and induces accumulation of sugars by upregulating acid invertase and sucrose synthase in rice seedlings

The effects of increasing concentrations of nickel sulfate, NiSO₄ (200 and 400 μM) in the growth medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism were examined in seedlings of the two Indica rice cvs. Malviya-36 and Pant-12. During a 5–20 day growth period of seedlings in sand cultures, with Ni treatment, no definite pattern of alteration in starch level could be observed in the seedlings. In both roots and shoots of the seedlings Ni treatment led to a significant decrease in activities of starch degrading enzymes α-amylase, β-amylase, whereas starch phosphorylase activity increased. The contents of reducing, non-reducing, and total sugars increased in Ni-treated rice seedlings with a concomitant increase in the activities of sucrose degrading enzymes acid invertase and sucrose synthase. However, the activity of sucrose synthesizing enzyme sucrose phosphate synthase declined. These results suggest that Ni toxicity in rice seedlings causes marked perturbation in metabolism of carbohydrates leading to increased accumulation of soluble sugars. Such perturbation could serve as a limiting factor for growth of rice seedlings in Ni polluted environments and accumulating soluble sugars could serve as compatible solutes in the cells under Ni toxicity conditions.     

Effect of aluminium on metabolism of starch and sugars in growing rice seedlings

The effect of increasing concentrations of Al₂(SO₄)₃ in situ on the content of starch, sugars and activity behaviour of enzymes related to their metabolism were studied in growing seedlings of two rice cvs. Malviya-36 and Pant-12 in sand cultures. Al₂(SO₄)₃ levels of 80 and 160 μM in the growth medium caused an increase in the contents of starch, total sugars as well as reducing sugars in roots as well as shoots of the rice seedlings during a 5–20 days growth period. The activities of the enzymes of starch hydrolysis α-amylase, β-amylase and starch phosphorylase declined in Al-exposed seedlings, whereas the activities of sucrose hydrolyzing enzymes sucrose synthase and acid invertase increased in the seedlings due to Al³⁺ treatment. The enzyme of sucrose synthesis, sucrose phosphate synthase showed decreased activity in Al³⁺ treated seedlings compared to controls. Results suggest that Al³⁺ toxicity in rice seedlings impairs the metabolism of starch and sugars and favours the accumulation of hexoses by enhancing the activities of sucrose hydrolyzing enzymes.     

Biochemical Changes in the Rice Grain during Germination

Changes in the content of starch, protein, and RNA and in the activity of their hydrolases in the rice endosperm (Oryza sativa L., variety IR8) were determined during the first week of germination without added nutrient both in the dark and in the light. Changes were generally more rapid in the dark than in the light. Oxygen uptake and RNase activity started to increase and the root protruded on the second day, followed by the coleoptile on the third day, and the primary leaf on the fourth day. ATP level was at a maximum on the fourth day. The activity of amylases and R enzyme increased progressively, but that of phosphorylase tended to decrease during starch degradation. A new α amylase isozyme band appeared during germination. Glucose was the major product of starch degradation. Sucrose, maltose, maltotriose, raffinose, and fructose were also detected. Protease activity reached a maximum on the fifth or sixth day and closely paralleled the increase in soluble amino N and soluble protein.     

外源调节剂对淹涝水稻幼苗株高及碳水化合物消耗的影响

为研究淹涝条件下水稻幼苗株高及碳水化合物消耗对不同外源生长调节剂的响应,本试验选用籼型常规稻IR64和导入耐淹涝基因Sub1的IR64-Sub1为试验材料,秧龄20 d时喷施1-氨基环丙烷-1-羧酸(ACC)、多效唑(PB)、赤霉素(GA)3种外源生长调节剂,以喷施清水为对照(CK).喷施处理2 d后进行0、4、8、12、16 d没顶淹涝胁迫,淹涝胁迫结束后常温恢复7 d,取样分析不同外源生长调节剂对水稻成活率、株高、叶绿素降解及恢复、地上部非结构性碳水化合物(NSC)消耗的影响.结果表明:淹涝导致水稻幼苗株高显著增长,叶片SPAD值快速下降,叶片可溶性糖迅速消耗,但耐淹涝品种IR64-Sub1淹水前茎鞘NSC含量明显高于IR64,淹涝中NSC消耗速率低于IR64,淹水结束后地上部淀粉含量高于IR64.外源PB处理显著抑制水稻幼苗株高增长、叶绿素降解及NSC消耗,提高存活率,且对IR64-Sub1效果更为显著.外源GA处理水稻幼苗叶绿素降解、株高增长和NSC消耗最快,植株恢复能力最低,耐淹涝能力最差,但与IR64相比,GA对IR64-Sub1淹涝耐性的抑制明显减弱.外源ACC促进伸长效果明显低于外源GA处理.淹水前喷施PB可有效抑制植株水下伸长,延缓叶绿素降解,减缓NSC消耗,保留更多NSC,为淹水胁迫解除后水稻快速恢复提供有利条件,这对于易涝地区减轻涝渍危害具有重要意义.     

Effect of arsenic on behaviour of enzymes of sugar metabolism in germinating rice seeds

Arsenic (As) is a potential contaminant of groundwater as well as soil in many parts of the world. The effects of increasing concentration of As (25 μm and 50 μm As₂O₃) in the medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism i.e. α-amylase, β-amylase, starch phosphorylase and acid invertase were studied in germinating seeds of two rice cvs. Malviya-36 and Pant-12 during 0–120 h period. As toxicity in situ led to a marked decline in the activities of α-amylase, β-amylase in endosperms as well as embryoaxes of germinating rice seeds. The activity of acid invertase increased in endosperms as well as embryoaxes whereas starch phosphorylase activity declined in endosperms but increased in embryoaxes under As treatment. In endosperms a decline in starch mobilization was observed under As toxicity, however under similar conditions the content of total soluble sugars increased in embryoaxes. The observed inhibition in activities of amylolytic enzymes might contribute to delayed mobilization of endospermic starch which could affect germination of seeds in As polluted environment, while the induced acid invertase activity and increased sugar accumulation in embryoaxes could serve as a possible component for adaptation mechanism of rice seedlings grown under As containing medium.     

Regulation of sugar metabolism in rice (Oryza sativa L.) seedlings under arsenate toxicity and its improvement by phosphate

The effect of arsenate with or without phosphate on the growth and sugar metabolism in rice seedlings cv. MTU 1010 was studied. Arsenate was found to be more toxic for root growth than shoot growth and water content of the seedlings gradually decreased with increasing concentrations. Arsenate exposure at 20 μM and 100 μM resulted in an increase in reducing sugar content and decrease in non-reducing sugar content. There was a small increase in starch content, the activity of starch phosphorylase was increased but α-amylase activity was found to be decreased. Arsenate toxicity also affected the activities of different carbohydrate metabolizing enzymes. The activities of sucrose degrading enzymes viz., acid invertase and sucrose synthase were increased whereas, the activity of sucrose synthesizing enzyme, viz. sucrose phosphate synthase declined. The combined application of arsenate with phosphate exhibited significant alterations of all the parameters tested under the purview of arsenate treatment alone which was congenial to better growth and efficient sugar metabolism in rice seedlings. Thus, the use of phosphorus enriched fertilizers may serve to ensure the production of healthy rice plants in arsenic contaminated soils.     

Carbohydrate metabolism in growing rice seedlings under arsenic toxicity

We studied in the seedlings of two rice cultivars (Malviya-36 and Pant-12) the effect of increasing levels of arsenic in situ on the content of sugars and the activity of several enzymes of starch and sucrose metabolism: alpha-amylase (EC 3.2.1.1), beta-amylase (EC 3.2.1.2), starch phosphorylase (EC 2.4.1.1), acid invertase (EC 3.2.1.26), sucrose synthase (EC 2.4.1.13) and sucrose phosphate synthase (EC 2.4.1.14). During a growth period of 10-20 d As2O3 at 25 and 50 microM in the growth medium caused an increase in reducing, non-reducing and total soluble sugars. An increased conversion of non-reducing to reducing sugars was observed concomitant with As toxicity. The activities of alpha-amylase, beta-amylase and sucrose phosphate synthase declined, whereas starch phosphorylase, acid invertase and sucrose synthase were found to be elevated. Results indicate that in rice seedlings arsenic toxicity causes perturbations in carbohydrate metabolism leading to the accumulation of soluble sugars by altering enzyme activity. Sucrose synthase possibly plays a positive role in synthesis of sucrose under As-toxicity.     

Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

Amylolytic activity and carbohydrate levels in relation to coleoptile anoxic elongation in Oryza sativa genotypes

Among starchy seeds, rice has the unique capacity to germinate successfully under complete anaerobiosis. In this conditions, starch degradation is supported by a complete set of starch-degrading enzymes that are absent or inactive in cereals except rice. A characterization of carbohydrate metabolism and starch-degrading enzyme activity across twenty-nine genotypes of Oryza sativa L. is presented here. The zymogram of amylolytic activities present in rice embryos and endosperms under anaerobic conditions seven days after sowing (DAS) revealed marked differences among cultivars. Coleoptile elongation was positively correlated with total amylolytic activities and α-amylase activity in embryos, and negatively correlated with α-amylase activity in endosperm. Moreover, carbohydrate content in embryos was found to be positively correlated with total amylolytic activities under anaerobic conditions, while a negative relationship was recorded in the endosperm. Carbohydrate status in rice seedlings has a primary importance in sustaining coleoptile elongation towards the surface. The relationship between carbohydrate level in embryo and anoxic germination, as well as with total amylolytic activities present in rice embryo under anaerobic condition 7 DAS, is consistent with the role of sugar metabolism to support rice germination under oxygen-deprived environment.     

淹水对三峡库区消落带香附子生理特性的影响

为了阐明香附子对三峡库区消落带水淹的耐淹机理,该研究模拟三峡库区消落带夏季汛期水淹环境,设置常规水分管理(CK)、根部水淹(T1)、半淹(T2)以及全淹(T3)4个不同处理组,研究香附子对不同淹水环境的生理响应机制。结果显示:(1)经过45d的水淹处理,所有水淹处理香附子叶片叶绿素a、叶绿素b、类胡萝卜素以及总叶绿素的含量均显著下降,但叶绿素a/b值仍维持在CK水平;(2)淹水并没有增加T1和T3处理植株可溶性蛋白、脯氨酸和可溶性糖含量,但显著增加了T2植株叶片脯氨酸和可溶性糖的含量;(3)随着水淹时间的延长,各水淹处理植株叶片丙二醛含量逐渐升高,且自水淹15d后均与CK植株差异显著;(4)所有水淹处理香附子植株SOD、CAT和APX等抗氧化酶活性均有所增加,但随着水淹时间的继续延长,T3植株叶片SOD和CAT活性逐渐下降。(5)经过45d的水淹处理,各水淹处理植株淀粉含量在叶和根中均显著增加。研究表明,虽然淹水对香附子产生了一定的伤害,但香附子能够通过调节体内各种保护酶活性和渗透调节物质含量来增加对水淹的抵御能力,而且根部高浓度淀粉含量也为香附子耐受水淹胁迫提供了稳定的能量供应。     

Metabolic changes in rice seedlings with different submergence tolerance after desubmergence

When flash flood intolerant rice cultivars are submerged, they show greater morphological changes such as elongation and chlorosis than tolerant cultivars. These morphological responses are caused by ethylene produced during submergence, however, a visible damage of intolerant cultivars is markedly developing after desubmergence rather than during submergence, which is probably due to oxidative damage. We studied the effect of ethylene produced during submergence on antioxidant content and oxidative damage after desubmergence. When rice (Oryza sativa) was submerged for 8 days, both tolerant cultivar (BKNFR) and intolerant cultivars (Mashuri and IR42) showed a decrease in ascorbate concentration during submergence. After 3 days of desubmergence, the tolerant cultivar showed a rapid recovery of total ascorbate and ascorbic acid, whereas intolerant cultivars showed a slow recovery of them, an increase in malondialdehyde formation, and low survival rate (about 30%). However, applying 200 mg l⁻¹ of AgNO₃ as an ethylene antagonist to intolerant cultivars suppressed the decrease in ascorbate and the increase in malondialdehyde formation after desubmergence, and improved survival rate to about 60%. Ascorbic acid supply to leaf discs from submerged IR42 suppressed increase in malondialdehyde formation by incubation under the light for 24 h. In addition, strong negative correlations were observed between malondialdehyde formation with ascorbate concentration (r=−0.93) and with percentage of survival (r=−0.98). Our results indicate that the accumulated ethylene during submergence adversely affected antioxidant mechanism in intolerant rice cultivars after desubmergence, and ascorbic acid was an important antioxidant in vivo for the recovery of submerged rice seedlings.     

Preferential Induction of Alcohol Dehydrogenase in Coleoptiles of Rice Seedlings Germinated in Submergence Condition

Difference in the growth response to submergence between coleoptiles and roots of rice (Oryza sativa L.) was investigated in 9-d-old rice seedlings. The coleoptile length in the submergence condition was much greater than that in aerobic condition, whereas the root length in the submergence condition was less than that in the aerobic condition. Alcohol dehydrogenase (ADH) activity in the coleoptiles in the submergence condition was much greater than that in the aerobic condition, but ADH activity in the roots in the submergence condition increased slightly. These results suggest that the preferential ADH induction in rice seedlings may contribute to the difference in the growth response between the coleoptiles and roots under low oxygen conditions.     

In-vitro degradation of starch granules isolated from spinach chloroplasts

The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated using an in-vitro system composed of native chloroplast starch granules, purified chloroplast and non-chloroplast forms of phosphorylase (EC 2.4.1.1) from spinach leaves, and -amylase (EC 3.2.1.1) isolated from Bacillus subtilis. Starch degradation was followed by measuring the release of soluble glucans, by determining phosphorylase activity, and by an electron-microscopic evaluation following deep-etching of the starch granules. Starch granules were readily degraded by -amylase but were not a substrate for the chloroplast phosphorylase. Phosphorolysis and glucan synthesis by this enzyme form were strictly dependent upon a preceding amylolytic attack on the starch granules. In contrast, the non-chloroplast phosphorylase was capable of using starch-granule preparations as substrate. Hydrolytic degradation of the starch granules was initiated at the entire particle surface, independently of its size. As a result of amylolysis, soluble glucans were released with a low degree of polymerization. When assayed with these glucans as substrate, the chloroplast phosphorylase form exhibited a higher apparent affinity and a higher reaction velocity compared with the non-chloroplast phosphorylase form. It is proposed that transitory starch degradation in vivo is initiated by hydrolysis; phosphorolysis is most likely restricted to a pool of soluble glucan intermediates.Abbreviations Glc1P Glucose 1-phosphate - Mes 2(N-morpholino)ethanesulfonic acid - Pi Orthophosphate     

Starch synthases and starch branching enzymes from Pisum sativum

Concentrations of ADPglucose:α-1,4-glucan-4-glucosyltransferase (starch synthase) and α-1,4 glucan: α-1,4-glucan-6-glycosyltransferase (branching enzyme) from developing seeds of Pisum sativum were measured. Primed starch synthase activity increased from 8 to 14 days after anthesis and decreased by 50 % at 26 days. Citrate-stimulated starch synthase activity was highest at 10 days after anthesis decreasing to low levels by 22 days. Branching enzyme activity increased from 8 to 18 days after anthesis and decreased little by 26 days. Two fractions of starch synthase were recovered by gradient elution from DEAE-cellulose of extracts from 12- and 18-day-old seeds. The two fractions differed in primer specificity, K_m for ADPG and relative amounts of citrate-stimulated activity. A major and minor fraction of branching enzyme were observed in extracts from both 12- and 18-day-old seeds. Marked differences in the relative abilities ofthe two branching enzyme fractions to stimulate phosphorylase and to branch amylose as well as pH optima were found. Although the content of the starch synthase and branching enzyme fractions varied with seed age, little difference was seen in the properties of chromatographically similar fractions. Therefore, the changes in starch synthase and branching enzyme activity during pea seed development resulted from changes in the concentrations of a few enzyme forms, but not the appearance of different enzyme forms.     

Effect of submergence on translocation,starch content and amylolytic activity in deep-water rice

Submergence induces rapid internodal elongation in deep-water rice (Oryza sativa L. cv. Habiganj Aman II). We investigated the metabolic activities which help to support such fast growth. Three days of submergence in water under continuous light led to the mobilization of 65% of the starch from those regions of rice internodes which had been formed prior to submergence. Disappearance of starch was accompanied by a 70-fold enhancement of amylolytic activity. Similar increases in amylolytic activity were detected in response to ethylene and gibberellic acid. Submergence also caused a 26-fold increase in the translocation of newly synthesized photosynthetic assimilates from the leaves to the internodes and younger regions of the culms. These physiological processes are likely to provide the metabolic energy required for internodal elongation in response to submergence.Abbreviation GA₃ gibberellic acid     

Mobilization of Starch after Submergence of Air-Grown Rice Coleoptiles. Implications for Growth and Gravitropism

Submergence of air-grown rice seedlings (Oryza sativa L. var. Sasanishiki) induces elongation of the coleoptile. We investigated whether rapid underwater extension is associated with a loss of starch. After 1 d of submergence the starch content was reduced by 70%. This loss of reserve carbohydrate was accompanied by a 38% increase in the concentration of glucose in the cell sap of the coleoptiles. The submerged (starch-depleted) coleoptiles had a slower negative gravitropism than the air-grown controls, although the rate of elongation in the horizontal position was not impaired. We conclude that the submergence-induced mobilization of starch provides substrates and osmotica for the rapidly growing cells. In addition, our results indicate that a full complement of starch is necessary for normal gravitropism in the rice coleoptile.     

Temperature induced changes in the starch components and biosynthetic enzymes of two rice varieties

The effects of temperature on starch and amylose accumulation, fine structure of amylopectin and activities of some enzymes related to starch synthesis in developing rice endosperms was examined. Two early indica rice varieties were used, differing in amylose concentration (AC, %), namely Jia 935 (low AC) and Jia 353 (high AC). The results showed that the effects of high temperature on AC and amylopectin fine structure were variety-dependent. High temperature caused a reduction in amylose concentration and an increase in the short chain (CL<22) proportion of amylopectin for Jia 935; while opposite was true for Jia 353. High temperature also reduced and increased the activity of granule-bound starch synthase (GBSS) in Jia 935 and in Jia 353, respectively. This suggests that a change in the ratio of amylose/starch due to temperature was attributable to a change in GBSS activity. Moreover, obvious differences between the two rice varieties were detected in the activities of sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (ADPG-Ppase), soluble starch synthase (SSS), starch branching enzyme (SBE), starch de-branching enzyme (SDBE) and starch phosphorylase (SPase) to high temperature. Accumulation rate of amylose was significantly and positively correlated with GBSS for Jia 935, but not for Jia 353. Amylose accumulation was also significantly and positively correlated with the activities of SDBE, SBE, ADPG-Ppase and SuSy for both varieties. The results suggest that the ratio of amylose to starch in rice endosperm is not only related to GBSS, but also affected by the activities of SDBE, SBE, ADPG-Ppase and SuSy.     

Physiological and metabolic enzymes activity changes in transgenic rice plants with increased phosphoenolpyruvate carboxylase activity during the flowering stage

To compare the differences in physiology and metabolism between phosphoenolpyruvate carboxylase (PEPC) transgenic rice and its control, untransformed wild rice, dry matter accumulation, soluble sugar, starch and protein contents and enzyme activities were determined in different plant parts during flowering. Results revealed that PEPC transgenic rice had higher dry weights for leaf, stem and sheath as well as panicle than the untransformed wild rice did, with the largest increase in the panicle. Soluble sugar and protein content in the grains of PEPC transgenic rice were significantly enhanced while starch content changed less. PEPC transgenic rice exhibited high levels of PEPC activity, manifesting in high net photosynthetic rates during flowering. Moreover, transgenic rice with high PEPC expression levels also had elevated levels of the enzymes such as sucrose-p-synthase and sucrose synthase, which may confer a higher capacity to assimilate CO₂ into sucrose. Little increase in grain starch content was observed in transgenic plants due to the stable activities of starch synthase and Q enzyme. However, the PEPC transgenic rice plant induced the activities of nitrate reductase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, glutamine synthetase, and asparagine synthase to high levels, as compared with the untransformed rice plant. PEPC activity was correlated with protein content in grains and the enzymes of nitrogen metabolism, suggesting that high PEPC activity in transgenic rice might be able to redirect carbon and nitrogen flow by regulating some enzymes related to carbon or nitrogen metabolisms. These results may help to understand how the C₃ plants possessing a C₄-like photosynthesis pathway worked by expression of PEPC.     

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.