磷酸饥饿时番茄幼苗酸性磷酸酶活性的变化与Pi吸收的关系

磷酸饥饿时,番茄幼苗根部及地上部酸性磷酸酶活性均显著增强,根部细胞表面酸性磷酸酶及根部外泌的酸性磷酸酶活性亦明显提高。动力学分析表明,磷酸饥饿提高了番茄幼苗根部的酸性磷酸酶对其底物的亲和力。另外,磷酸饥饿对番茄幼苗根部酸性磷酸酶活性的最适ｐＨ值没有影响。钼酸对番茄幼苗根部酸性磷酸酶活性有强烈的抑制作用,对番茄幼苗Ｐｉ吸收速率也有十分明显的抑制效果。以上结果表明,磷酸饥饿时,番茄幼苗Ｐｉ吸收的适应性变化可能与根部酸性磷酸酶特别是根部细胞表面酸性磷酸酶及其外泌酸性磷酸酶的参与密切关联。     

Relationship between the Changes of Acid Phosphatase Activities and Pi -uptake of Tomato Seedlings during Phosphate Starvation

The acid phosphatase activities from roots and both stems and leaves of tomato seedlings all in-creased markedly under phosphate starvation. Phosphate starvation also increased the activities of acid phos-phatase from cell surface of, and released by roots of tomato seedlings. The kinetic analysis of acid phos-phatase of roots of tomato seedlings revealed that phosphate starvation increased the affinity of the enzyme to its substrate. The results also revealed that phosphate starvation had no effect on the optimum pH (pH 4.93) of the acid phosphatase of roots of tomato seedlings. It was also found that molybdate strongly inhibited not only the activities of acid phosphatase but also Pi- uptake rates of tomato seedlings.     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: I. Excretion of Acid Phosphatase by Tomato Plants and Suspension-Cultured Cells

Both tomato (Lycopersicon esculentum cv VF 36) plants and suspension cultured cells show phosphate starvation inducible (psi) excretion of acid phosphatase (Apase). Apase excretion in vitro was proportional to the level of exogenous orthophosphate (Pi). Intracellular Apase activity remained the same in both Pi-starved and sufficient cells, while Apase excreted by the starved cells increased by as much as six times over unstressed control cells on a dry weight basis. At peak induction, 50% of total Apase was excreted. Ten day old tomato seedlings grown without Pi showed slight growth reduction versus unstressed control plants. The Pi-depleted roots showed psi enhancement of Apase activity. Severely starved seedlings (17 days) reached only one-third of the biomass of unstressed control plants but, because of a combination of psi Apase excretion by roots and a shift in biomass to this organ, they excreted 5.5 times the Apase activity of the unstressed control. Observed psi Apase excretion may be part of a phosphate starvation rescue system in plants. The utility of the visible indicator dye 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine as a phenotypic marker for plant Apase excretion is demonstrated.     

Purification and characterization of secreted acid phosphatase in phosphorus-deficient Arabidopsis thaliana

Arabidopsis roots responded to the absence of an exogenous phosphate source with an increase in the specific activities of secreted acid phosphatases. Increases in enzyme activity were revealed beginning 2 days after P-withdrawal, reaching a maximum at 6 days. We characterized the secreted acid phosphatase. Two proteins, migrating at 52 and 63 kDa in SDS-PAGE, co-purified with the activity. Purified enzyme had a pH optimum of 5 and a pI of 5.9. In addition to the general phosphatase substrate, p -nitrophenyl-phosphate, the enzyme readily hydrolysed pyrophosphate, polyphosphate, ATP and PEP. Low or negligible activity was observed with glucose-1P, fructose-1P and phytic acid. The activity of the purified secreted acid phosphatase was stimulated by calcium and inhibited by molybdate, phosphate, fluoride, vanadate and nitrate. Activity was not inhibited by tartrate. The substrate profile and the biochemical properties suggest that Arabidopsis secreted acid phosphatase may have a role in mobilizing organic phosphate in the soil.     

Quantitative Aspects of Leaf Acid Phosphatase Activity and the Phosphorus Status of Tomato Plants

The relation between tomato leaf acid phosphatase activity andleaf tissue P content has been examined, and a study made ofthe effects of leaf development, variation in nitrogen supply,and variation in the growing medium on this relationship. Tomatoplants were grown in sand and given various concentrations ofphosphate. Plants were also grown for an initial period in peatcontaining an adequate level of phosphate, then transferredto peat to which was added 0 or 2.3 kg superphosphate m^–3and supplied with either 50 of 300 µg N ml^–1. Expressed on a unit tissue f. wt basis, acid phosphatase activityin the control plants in sand (given 41 µg P ml^minus;1)was highest in extracts from the expanding leaves and decreasedwith leaf maturity. However, when given a reduced supply ofphosphate, the enzyme activity in the more mature leaves wasequal to, or greater than, that in the expanding leaves. Thephosphatase activity increased first in the young, fully-expandedleaves and in the mature leaves (with 4.1 µg P ml^–1),but did not increase in the expanding leaves until the supplywas restricted to 2.1 µg P ml^–1. On closer examination,the increase in enzyme activity appeared to be associated withthe P level in the leaf tissues, the activity increasing whenthe level fell below about 0.25 per cent (g P per 100 g drywt tissue). The same relation was found with the plants grownin peat, and was independent of the concentration of nitrogensupplied to the plants. The fully expanded leaves showed the best enzyme response whenthe phosphate supply was restricted and the activity reflectedclosely the local levels of tissue P. The assay of the enzymein unpurified leaf extracts is simple and rapid, and could beused in a test to detect P-deficiency in tomato plants. Lycopersicon esculentum L, tomato, acid phosphatase activity, phosphorus status     

Phosphatase induction in roots of winter wheat during adaptation to phosphorus deficiency

The effect of phosphate supply during the first 4 weeks of the life cycle of wheat ( Triticum aestivum L. cv. Martonvásári-8) was investigated by following growth of seedlings, P levels in roots and shoots, changes of soluble phosphatases in roots and alteration of Ca²⁺ - and Mg²⁺-ATPase activity in the microsomal fraction. Plants were grown in complete nutrient solution supplemented with different levels of phosphate. Maximal growth rate was attained at 0.2 m M phosphate. The total P level in plants increased with increasing phosphate concentration in the growth solution, however, it decreased with age. Microsomal ATPase activity in 14-day-old plants increased with phosphorus deficiency. Using phosphocellulose column chromatography, a phosphatase (EC 3.1.3.2) induced by phosphorus deficiency was purified and partially characterized from the 30 000 g supernatant from roots of 14- to 30-day-old wheat plants. Na-pyrophosphate, p -nitrophenylphosphate, ATP, ADP, AMP, O-phosphoryl- l -serine and glucose-6-phosphate were all substrates for the enzyme. Its native molecular weight was 42 kDa as determined by Sephadex G-200 column chromatography. Readdition of phosphate to the growth solution resulted in a gradual decrease of the phosphatase activity, probably due to repression of its synthesis. We hypothesize that the extra phosphatase may participate in the adaptation mechanism under phosphorus-deficient conditions.     

γ-氨基丁酸对低氧胁迫下甜瓜幼苗多胺代谢的影响

以‘西域一号’甜瓜为试验材料,采用营养液水培法,研究了低氧胁迫下外源添加γ-氨基丁酸(GABA)对甜瓜幼苗多胺代谢的影响.结果表明:与通气对照相比,低氧胁迫处理的甜瓜幼苗谷氨酸脱羧酶(GAD)活性和GABA含量显著提高,同时多胺合成酶活性提高诱导多胺含量显著增加,但二胺氧化酶(DAO)和多胺氧化酶(PAO)活性也显著提高;根系精氨酸脱羧酶(ADC)活性提高幅度较大,导致根系游离态腐胺含量较高,而叶片乌氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(SAMDC)活性提高幅度较大,导致叶片游离态亚精胺(Spd)含量较高;根系游离态DAO和PAO活性显著低于叶片,其细胞壁结合态PAO活性显著高于叶片.与低氧胁迫处理相比,低氧胁迫下外源添加GABA处理的甜瓜幼苗叶片和根系中GABA和谷氨酸含量均显著提高,而GAD活性显著降低;精氨酸、鸟氨酸、甲硫氨酸含量的提高促使多胺合成酶活性显著提高,从而诱导多胺含量显著增加,DAO和PAO活性显著降低.     

磷饥饿时番茄幼苗根系质膜H+-ATP酶活性

磷饥饿提高了番茄幼苗质膜H+-ATP酶活性并促进了番茄幼苗根部的H+分泌。动力学分析表明,磷饥饿使番茄幼苗根部质膜H+-ATP酶的Km值明显降低,亦即提高了该酶对其底物的亲和力,但对该酶的Vmax影响不大。另外,磷饥饿并不改变ATP酶的最适pH值(最适pH值为6.5)。钒酸盐显著抑制番茄幼苗根部质膜ATP酶的活性以及H+分泌,也显著抑制番茄幼苗的Pi吸收。与对照相比,上述抑制作用在饥饿处理的植物中表现得更强。以上结果表明,磷饥饿时高亲和性Pi传递系统的诱导很可能包含质膜H+-ATP酶的参与。     

Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana

Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants. Received: 23 September 1999 / Accepted: 10 November 1999     

外源GSH对盐胁迫下番茄幼苗生长及抗逆生理指标的影响

采用营养液栽培法,研究外源谷胱甘肽(GSH)对NaCl胁迫下番茄幼苗生长、根系活力、电解质渗透率和丙二醛(MDA)、脯氨酸(Pro)、可溶性糖含量以及超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性的影响,为利用外源物质减轻盐胁迫伤害提供理论依据。结果显示:(1)NaCl胁迫显著抑制了番茄幼苗的生长、根系活力和SOD、POD、CAT活性,提高了电解质渗透率及MDA、Pro、可溶性糖含量;(2)外源喷施GSH能够诱导NaCl胁迫下番茄幼苗叶片抗氧化酶SOD、POD、CAT活性上调,电解质渗透率及MDA含量下降,Pro和可溶性糖含量恢复至对照水平;(3)外源喷施还原型谷胱甘肽抑制剂(BSO)使NaCl胁迫下番茄幼苗的根系活力以及抗氧化酶SOD、POD、CAT活性下降,脯氨酸含量提高;(4)喷施GSH可诱导BSO和NaCl共处理番茄植株的根系活力、SOD、POD、CAT活性提高,MDA和Pro含量降低。研究表明,外源GSH可通过提高促进盐胁迫下番茄幼苗植株渗透调节能力及清除活性氧的酶促系统的防御能力、降低细胞膜脂过氧化程度、保护膜结构的完整性,从而有效缓解NaCl胁迫对番茄幼苗生长的抑制,提高其耐盐性。     

γ-氨基丁酸对低氧胁迫下甜瓜幼苗多胺代谢的影响

以‘西域一号’甜瓜为试验材料,采用营养液水培法,研究了低氧胁迫下外源添加γ-氨基丁酸(GABA)对甜瓜幼苗多胺代谢的影响.结果表明：与通气对照相比,低氧胁迫处理的甜瓜幼苗谷氨酸脱羧酶(GAD)活性和GABA含量显著提高,同时多胺合成酶活性提高诱导多胺含量显著增加,但二胺氧化酶(DAO)和多胺氧化酶(PAO)活性也显著提高;根系精氨酸脱羧酶(ADC)活性提高幅度较大,导致根系游离态腐胺含量较高,而叶片鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(SAMDC)活性提高幅度较大,导致叶片游离态亚精胺(Spd)含量较高;根系游离态DAO和PAO活性显著低于叶片,其细胞壁结合态PAO活性显著高于叶片.与低氧胁迫处理相比,低氧胁迫下外源添加GABA处理的甜瓜幼苗叶片和根系中GABA和谷氨酸含量均显著提高,而GAD活性显著降低;精氨酸、鸟氨酸、甲硫氨酸含量的提高促使多胺合成酶活性显著提高,从而诱导多胺含量显著增加,DAO和PAO活性显著降低.     

Lower temperature and orthophosphate control of alkaline phosphatase in a cold-requiring plant

The elevated activity of alkaline phosphatase observed in roots and shoots of Verbascum rosettes grown in vitro at 15 °C may be the result of a reduced supply of orthophosphate. The rosette reveals greater activities of this enzyme under conditions of lower external phosphate or chilling temperature. Either of these treatments gives lower amounts of endogenous phosphate. The stimulation of phosphatase in tissues having lower levels of phosphate is thus a possible feature of other plant species which also display freezing tolerance.     

Inhibition of ribonuclease and protease activities in arsenic exposed rice seedlings: role of proline as enzyme protectant

When seedlings of two rice (Oryza sativa L.) cvs. Malviya-36 and Pant-12 were raised under 25 and 50 microM As2O3 in the medium an increase in the level of RNA, proteins and proline accompanied with a decline in the level of free amino acid pool was observed under arsenic supplementation compared to controls. In situ As3+ treatment caused a marked inhibition in activities of ribonuclease (RNase, EC 3.1.27.1), protease and leucine aminopeptidase (LAP, EC 3.4.11.1) whereas the activity level of carboxypeptidase (EC 3.4.16.5) was enhanced. In vitro supply of As2O3 in the enzyme assay medium beyond 400 microM resulted in gradual inhibition of RNase and beyond 5 microM inhibition of LAP activities. Addition of 1M proline in the assay medium significantly restored the loss in RNase activity due to in vitro arsenic treatment or due to osmotic stress created by incorporation of polyethylene glycol (PEG). Isoform pattern of RNase extracted from As3+ -exposed seedlings showed a significant alteration compared to its pattern in unexposed seedlings. Results suggest that arsenic exposure impairs hydrolysis of RNA and proteins in rice seedlings due to inhibition of RNase and proteases activities and that proline accumulating under As3+ toxicity appears to serve as enzyme protectant.     

Ribonuclease activity in roots of soybean seedlings subjected to chilling stress and recovery

Many developmental and physiological changes, including alterations of enzyme activities, occur in plants under low temperature stress. In this study the total ribonuclease activity was determined in crude extracts from root tips of soybean seedlings germinated at 25 °C, subjected to chilling conditions (10°C) and recovered at optimal temperature (25°C). Measurements of RNase activity were performed every 24 hours starting from the third to the 10-th day of growth. We found that chilling caused a considerable increase in ribonuclease activity (in comparison with the control), with an activity peak on the fourth day of the cold treatment. The enzyme activity in root extracts of the plants recovered after cold stress decreased along with the time of recovery. No differences were found in approximate molecular weight (35 kDa) and pH optimum (6.0) for ribonucleases extracted from control and chilled soybean roots.     

Acid phosphatase activities during the germination of Glycine max seeds.

In this paper, we describe a study concerning the determination of some characteristics of soybean seedlings and the detection of acid phosphatase activities towards different substrates during the germination. Enzyme activities with p-nitrophenylphosphate (pNPP) and inorganic pyrophosphate (PPi) as substrates were detected from the 5th and 7th days after germination, respectively. Acid phosphatase activities with tyrosine phosphate (TyrP), glucose-6-phosphate (G6P) and phosphoenol pyruvate (PEP) were also observed but to a lesser extent. Under the same conditions, no enzyme activity was detected with phytic acid (PhyAc) as substrate. The appearance of phosphatase activity was coincident with the decrease of inorganic phosphate content during germination; over the same period, the protein content increased up to the 5th day, decreased until the 8th day, and remained constant after this period. Relative to phosphatase activity in the cotyledons, the activities detected in the hypocotyl and roots were 82% and 38%, respectively. During storage the enzyme maintained about 63% of its activity for 3 months at 5 degrees C. The specificity constant (Vmax/Km) values for pNPP and PPi were 212 and 64 mu kat mM-1 mg-1, respectively. Amongst the substrates tested, PPi could be a potential physiological substrate for acid phosphatase during the germination of soybean seeds.     

Acid phosphatase changes associated with development of male sterile and fertile maize (Zea mays L.)

Free and bound acid phosphatase were investigated biochemically and electrophoretically in male sterile and fertile plants. Scutella at the 72-hr seedling stage and anthers with enclosed reproductive tissue at the premeiosis, meiosis, and mature developmental stages were tested. Biochemical data show that while the total amount of free acid phosphatase behaved similarly in fertile and sterile plants, specific activities decreased in fertile plants and remained unchanged or increased in sterile plants. Total amounts of bound acid phosphatase increased significantly in fertile plants while specific activities decreased. In sterile anthers both specific activity and amount of bound acid phosphatase decreased significantly (except cms-S). Electrophoretic results indicated that the basic form of the enzyme was very similar in each location.Published with the approval of the Director of the West Virginia Agricultural Experiment Station as Scientific Paper No. 1573. Based in part on a dissertation by C. V. W. submitted to the Division of Plant Science, West Virginia University, Morgantown, West Virginia.     

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.     

Interrelationship between metabolic and genetic regulation of alkaline and acid phosphatases in E. coli cells]

The effect of exogenous orthophosphate and mutations in regulatory genes of alkaline phosphatase on the level of nonspecific acid phosphatase was studied. The level of this enzyme as well as the level of alkaline phosphatase were shown to be regulated by exogenous orthophosphate being derepressed under phosphate starvation. The derepression of acid phosphatase is accompanied by more rapid secretion of enzyme from membranes to soluble fraction. Mutations in all the four regulatory genes decrease the level of enzyme in cells. Genes phoR and phoS, participating in regulation of alkaline phosphatase, are required for the derepression of acid phosphatase under the conditions of phosphate starvation.