Differential responses of oat cultivars to phosphate deprivation: plant growth and acid phosphatase activities

The effect of phosphate starvation on growth and acid phosphatases (APases) localization and activity in oat tissues was investigated. Oat cultivars (Avena sativa L.??Arab, Polar, Szakal) were grown for 1?C3?weeks in complete nutrient medium (+P) and without phosphate (?P). Pi concentration in plant tissues decreased strongly after culturing on ?P medium. Pi deficit reduced shoot growth, stimulated root elongation and increased ratio of root/shoot in all oat cultivars. Pi deficit had a greater impact on growth of oat cv. Polar than other varieties. A decrease in the internal Pi status led to an increase of acid phosphatase activities in extracts from shoots and roots, and in root exudates. The highest activity of secreted APases was observed for oat cv. Arab, during the third week of growth under Pi-deficient conditions. The activity of extracellular APase was high in young, growing zones of roots of ?P plants. Histochemical visualization indicated high activity of APases in the epidermis and vascular tissues of ?P plants. Pi deficiency increased intracellular APase activity in shoot mainly in oat cv. Polar, whereas APase activity in roots was the highest in oat cv. Szakal. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoform(s) may be affected by Pi deficiency. Three major APase isoforms were detected in all oat plants; one was strongly induced by Pi deficit. The studied oat cultivars differed in terms of acclimation to deficiency of phosphate??used various pools of APases to acquire Pi from external or internal sources.     

Acid phosphatases and growth of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars under diverse phosphorus nutrition

The morphological and physiological responses of barley to moderate Pi deficiency and the ability of barley to grow on phytate were investigated. Barley cultivars (Hordeum vulgare L., Promyk, Skald and Stratus) were grown for 1–3 weeks on different nutrient media with contrasting phosphorus source: KH₂PO₄ (control), phytic acid (PA) and without phosphate (−P). The growth on −P medium strongly decreased Pi concentration in the tissues; culture on PA medium generally had no effect on Pi level. Decreased content of Pi reduced shoot and root mass but root elongation was not affected; Pi deficit had slightly greater impact on growth of barley cv. Promyk than other varieties. Barley varieties cultured on PA medium showed similar growth to control. Extracellular acid phosphatase activities (APases) in −P roots were similar to control, but in PA plants were lower. Histochemical visualization indicated for high APases activity mainly in the vascular tissues of roots and in rhizodermis. Pi deficiency increased internal APase activities mainly in shoot of barley cv. Stratus and roots of cv Promyk; growth on PA medium had no effect or decreased APase activity. Protein extracts from roots and shoots were run on native discontinuous PAGE to determine which isoforms may be affected by Pi deficiency or growth on PA medium; two of four isoforms in roots were strongly induced by conditions of Pi deficit, especially in barley cv. Promyk. In conclusion, barley cultivars grew equally well both on medium with Pi and where the Pi was replaced with phytate and only slightly differed in terms of acclimation to moderate deficiency of phosphate; they generally used similar pools of acid phosphatases to acquire Pi from external or internal sources.     

磷胁迫诱导大豆叶片酸性磷酸酶同工酶的表达

通过田间试验对两种磷处理的274个大豆基因型叶片酸性磷酸酶活性进行筛选,并将其中8个进行营养液栽培试验以研究磷胁迫对其叶片酸性磷酸酶同工酶表达的影响.结果表明,大豆叶片酸性磷酸酶活性存在着明显的基因型差异,不施磷处理提高了大部分(约60%)供试基因型叶片酸性磷酸酶的活性.营养液栽培试验表明,低磷处理普遍提高了所有8个供试大豆基因型叶片酸性磷酸酶的活性.等电聚焦电泳结果表明,供试大豆基因型的老叶和新叶中均有6条酸性磷酸酶的同工酶带.低磷处理显著增加了叶片酸性磷酸酶酶带的活性,但是没有诱导新的酸性磷酸酶酶带产生.研究发现叶片酸性磷酸酶活性可作为反映大豆磷胁迫的酶学指标;磷胁迫诱导大豆叶片酸性磷酸酶活性的增加是由于已有同工酶活性的提高而不是由于特异性酶带的产生.     

磷胁迫诱导大豆叶片酸性磷酸酶同工酶的表达

通过田间试验对两种磷处理的274个大豆基因型叶片酸性磷酸酶活性进行筛选,行将其中8个进行营养液栽培试验以研究磷胁迫对其叶片酸性磷酸酶同工酶表达的影响。结果表明,大豆叶片酸性磷酸酶活性存在着明显的基因型差异,不施磷处理提高了大部分(约60％)供试基因型叶片酸性磷酸酶的活性。营养液栽培试验表明,低磷处理普遍提高了所有8个供试大豆基因型叶片酸性磷酸酶的活性。等电聚焦电泳结果表明,供试大豆基因型的老叶和新叶中均有6条酸性磷酸酶的同工酶带。低磷处理显著增加了叶片酸性磷酸酶酶带的活性,但是没有诱导新的酸性磷酸酶酶带产生。研究发现叶片酸性磷酸酶活性可作为反映大豆磷肋迫的酶学指标;磷胁迫诱导大豆叶片酸性磷酸酶活性的增加是由于已有同工酶活性的提高而不是由于特异性酶带的产生。     

Induction of maize acid phosphatase activities under phosphorus starvation

Large variation in phosphorus-(P) acquisition efficiency exists among maize inbred and hybrid genotypes. Acid phosphatases are a type of enzyme that affects P acquisition and P-use efficiency in plants. The objectives of this research were (1) to characterize acid phosphatase activity in maize grown hydroponically under P starvation, and (2) to determine if there is differential induction of acid phosphatases in two maize genotypes previously characterized as P efficient (Mo17) and P inefficient (B73). B73 and Mo17 seedlings were grown hydroponically and both intracellular and secreted acid phosphatase activities were characterized. Fresh seedling weight of both genotypes decreased under P starvation, but percent fresh weight allocated to roots increased 14 days after P starvation in B73. Soluble protein concentration in shoots and roots was affected little, but secreted protein decreased by 40 and 20% in B73 and Mo17 seedlings grown without P for 14 days. Intracellular and secreted acid phosphate activity increased substantially in leaves and roots in B73 and Mo17 in response to P starvation. Secreted APase activity per unit protein increased 310 and 300% in B73 and Mo17, respectively, 7 days after P withdrawal. One of the minor isozymes identified on non-denaturing PAGE, was increased specifically in response to P starvation in both maize genotypes. The patterns and levels of change in APase activities in B73 and Mo17 were not sufficiently different to account for the diverse growth response of these genotypes in low-P conditions. The results suggest that APases may not be a major mechanism for scavenging or acquiring P and changes in APases may reflect a state of P stress in both varieties. Other factors such as root architecture, secretion of low-molecular weight carboxylates and microbial interactions might explain the difference between these two genotypes.     

Molecular control of acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus-stressed white lupin

White lupin (Lupinus albus) grown under P deficiency displays a suite of highly coordinated adaptive responses. Included among these is secretion of copious amounts of acid phosphatase (APase). Although numerous reports document that plants secrete APases in response to P deficiency, little is known of the biochemical and molecular events involved in this process. Here we characterize the secreted APase protein, cDNA, and gene from white lupin. The secreted APase enzyme is a glycoprotein with broad substrate specificity. It is synthesized as a preprotein with a deduced M(r) of 52,000 containing a 31-amino acid presequence. Analysis of the presequence predicts that the protein is targeted to outside the cell. The processed protein has a predicted M(r) of 49,000 but migrates as a protein with M(r) of 70,000 on sodium dodecyl sulfate gels. This is likely due to glycosylation. Enhanced expression is fairly specific to proteoid roots of P-stressed plants and involves enhanced synthesis of both enzyme protein and mRNA. Secreted APase appears to be encoded by a single gene containing seven exons interrupted by six introns. The 5'-upstream putative promoter of the white lupin-secreted APase contains a 50-base pair region having 72% identity to an Arabidopsis APase promoter that is responsive to P deficiency. The white lupin-secreted APase promoter and targeting sequence may be useful tools for genetically engineering important proteins from plant roots.     

Stimulation of root acid phosphatase by phosphorus deficiency is regulated by ethylene in Medicago falcata

Plants have developed numerous strategies to cope with phosphorus (P) deficiency resulting from low availability in soils. Evolution of ethylene and up-regulation of root secreted acid phosphatase activity are common for plants in response to P deficiency. To determine the role of ethylene in response of plants to P deficiency, we investigated the effects of ethylene precursor (1-amino cyclopropane-1-carboxylic acid, ACC) and ethylene synthesis antagonists (aminoethoxyvinylglycine AVG, cobalt, Co²⁺) on P concentrations in roots and shoots of Medicago falcata seedlings grown in P-sufficient (500 μM H₂PO₄⁻) and P-deficient (5 μM H₂PO₄⁻) solution. After transferring M. falcata seedlings from P-sufficient to P-deficient solution for 2 days, root P concentration was significantly reduced. The reduction in root P concentration was reversed by AVG and Co²⁺, and a similar reduction in root P concentration of seedlings exposed to P-sufficient solution was observed by ACC. Expression of high-affinity phosphate transporters (MfPT1, MfPT5) was enhanced by P-deficiency and this process was reversed by AVG and Co²⁺. There was a marked increase in activity of root acid phosphatase (APase) and expression of gene encoding APase (MfPAP1) under P-deficient conditions, and the increase in APAse activity and expression of MfPAP1 was inhibited by AVG and Co²⁺. APase activity and expression of MfPAP1 expression in seedlings grown in P-sufficient solution were enhanced by ACC. Root and shoot P concentrations were increased when organic phosphorus was added to the P-deficient solution, and the increase in P concentration was significantly inhibited by AVG and Co²⁺. These results indicate that ethylene plays an important role in modulation of P acquisition by possibly mobilizing organic P via up-regulating root APase activity and high-affinity phosphate transporters.     

pho3: a phosphorus-deficient mutant of Arabidopsis thaliana (L.) Heynh

A novel P-deficient mutant of Arabidopsis thaliana, pho3, was isolated by screening for root acid phosphatase (APase) activity in plants grown under low-P conditions. pho3 had 30% less APase activity in roots than the wild type and, in contrast to wild-type plants, root APase activity did not increase in response to growth in low P. However, shoot APase activity was higher in pho3 than in the wild-type plants. In addition, the pho3 mutant had a P-deficient phenotype, even when grown in P-sufficient conditions. The total P content of 11-d-old pho3 plants, grown in agar media with a plentiful supply of P, was about 25% lower than the wild-type level in the shoot, and about 65% lower in the roots. In the rosette leaves of mature soil-grown pho3 plants the total P content was again reduced, to about 50% of wild-type levels. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased anthocyanin content (at least 100-fold greater than the wild type in soil-grown plants) and starch accumulation. The results suggest that the mutant is unable to respond to low internal P levels, and may lack a transporter or a signalling component involved in regulating P nutrition. Received: 21 March 2000 / Accepted: 15 August 2000     

Variation in phosphorus efficiency among 73 bread and durum wheat genotypes grown in a phosphorus-deficient calcareous soil

A greenhouse experiment was carried out to study the severity of phosphorus (P) deficiency symptoms on leaves, shoot dry matter production, and shoot concentration and content (the total amount per shoot) of P in 39 bread wheat (Triticum aestivum L.) and 34 durum wheat (Triticum durum L.) genotypes grown in a severely P-deficient calcareous soil with low (20mgPkg⁻¹ soil) and adequate (80mgPkg⁻¹ soil) P supply for 39 days. As the seed P concentration or content can affect plant performance under P-deficient conditions, the seeds of the genotypes used in the present study were also analyzed for P concentration. Phosphorus efficiency (relative shoot growth) of genotypes, calculated by the ratio of shoot dry matter production under low P to that under adequate P supply, significantly differed among the genotypes, and varied between 46.7% and 78.6%. Phosphorus efficiency ranged from 51% to 71% with an average of 61% for bread and from 47% to 79% with an average of 66% for durum wheat genotypes. There was no correlation between P efficiency ratio and P concentration of plants (R ²=0.0001), but P efficiency of all bread and durum wheat genotypes showed a very significant correlation with the P content (the total amount of P per shoot) (R ²=0.333^***). The relationship between the P efficiency and total amount of P per shoot was much more significant in bread (R ²=0.341^***) than in durum wheat (R ²=0.135^*). Like shoot P concentrations, also severity of visible leaf symptoms of P deficiency on older leaves, including leaf chlorosis and necrosis, did not correlate with P efficiency. In most cases, genotypes showing higher P efficiency had higher absolute shoot dry weight under P deficient conditions. Under P deficient conditions, the absolute shoot dry weight very significantly correlated with shoot P content (R ²=0.665^***), but the correlation between the absolute shoot dry weight and shoot P concentration tended to be negative. There was also variation in native seed P reserve of the genotypes, but this variation had no influence on the P efficiency. The results indicate that the total amount of P per shoot and shoot dry matter production at low P supply are most reliable parameters in ranking genotypes for P efficiency at early growth stage. In wheat germplasm tested in the present study, several wheat genotypes are available showing both very high P efficiency and very high shoot content and concentration of P suggesting that P acquisition ability should be most important mechanism for high P efficiency in such genotypes. On the other hand, there are also genotypes in the germplasm having more or less same P concentration or P content in shoot but differing substantially in P efficiency, indicating importance of P utilization at cellular level in P efficiency. All these results suggest that P efficiency mechanisms can be different from one genotype to other within a given plant species.     

The role of acid phosphatases in plant phosphorus metabolism

Hydrolysis of phosphate esters is a critical process in the energy metabolism and metabolic regulation of plant cells. This review summarizes the characteristics and putative roles of plant acid phosphatase (APase). Although immunologically closely related, plant APases display remarkable heterogeneity with regards to their kinetic and molecular properties, and subcellular location. The secreted APases of roots and cell cultures are relatively non-specific enzymes that appear to be important in the hydrolysis and mobilization of P_i from extracellular phosphomonoesters for plant nutrition. Intracellular APases are undoubtedly involved in the routine utilization of P_i reserves or other P_i-containing compounds. A special class of intracellular APase exists that demonstrate a clear-cut (but generally nonabsolute) substrate selectivity. These APases are hypothesized to have distinct metabolic functions and include: phytase, phosphoglycolate phosphatase, 3-phosphoglycerate phosphatase, phosphoenolpyruvate phosphatase, and phosphotyrosyl-protein phosphatase. APase expression is regulated by a variety of developmental and environmental factors. P_i starvation induces de novo synthesis of extra- and intracellular APases in cell cultures as well as in whole plants. Recommendations are made to achieve uniformity in the analyses of the different APase isoforms normally encountered within and between different plant tissues.     

Effect of aluminum on the in vitro activity of acid phosphatases of four potato clones grown in three growth systems

The aim of this study was to assess the effect of aluminum on the in vitro activity of acid phosphatases (APases) of four potato clones, Macaca and Dakota Rose (Al-sensitive), and SMIC148-A and Solanum microdontum (Al-tolerant), grown in vitro, in hydroponics or in a greenhouse. The enzyme was assayed in vitro in the presence of 0, 1.85, 3.70, 5.55 and 7.40 mM Al. In plantlets grown in vitro, root APases were inhibited by Al in all clones, while shoot APases were inhibited by Al in S. microdontum and Dakota Rose and increased in Macaca at all Al concentrations. In plantlets grown in hydroponics, root APases increased in Macaca at 1.85 mM Al, whereas decreased at all Al levels in S. microdontum. In greenhouse plantlets, root APases decreased at 7.40 mM Al in S. microdontum and SMIC148-A, and at 3.70, 5.55 and 7.40 mM Al in Dakota Rose. Shoot APases decreased in Macaca and SMIC148-A. Conversely, in Dakota Rose, APases increased at 1.85 and 3.70 mM Al. These results show that the effect of Al toxicity on in vitro APase activity depends not only on Al availability but also on the plant organ, genetic background, and the growth conditions. Therefore, it suggests that acid phosphatases activity assessed in vitro might not be a good parameter to validate the screening for adaptation of potato clones to Al toxicity.     

酸性磷酸酶参与大豆子叶磷转运和利用

本文以磷效率不同的两个大豆品种为材料,研究大豆幼苗期子叶酸性磷酸酶活性和同工酶谱对外源磷有效性的响应,及其参与子叶磷高效转运和利用的过程。结果表明：在幼苗生长前期,子叶酸性磷酸酶活性及其同工酶谱组成变化明显,而且不受外源磷有效性的调控;在幼苗生长的前8天,子叶全磷含量随着酸性磷酸酶的活性增加而显著降低,而且磷高效大豆品种比磷低效大豆品种具有较高的酸性磷酸酶活性和植株全磷含量。以上结果说明在大豆幼苗生长前期,由于大粒种子不仅具有较高的磷含量,而且具有较高子叶酸性磷酸酶活性,促进子叶有机磷的水解和转运是磷高效大豆品种适应低磷胁迫的生理机制之一。     

The Arabidopsis gene hypersensitive to phosphate starvation 3 encodes ethylene overproduction 1

When plants are subjected to a deficiency in inorganic phosphate (Pi), they exhibit an array of responses to cope with this nutritional stress. In this work, we have characterized two Arabidopsis mutants, hps3-1 and hps3-2 (hypersensitive to Pi starvation 3), that have altered expression of Pi starvation-induced (PSI) genes and enhanced production of acid phosphatase (APase) when grown under either Pi sufficiency or deficiency conditions. hps3-1 and hps3-2, however, accumulate less anthocyanin than the wild type when grown on a Pi-deficient medium. Molecular cloning indicated that the phenotypes of hps3 mutants were caused by mutations within the ETO1 (ETHYLENE OVERPRODUCTION 1) gene. In Arabidopsis, ETO1 encodes a negative regulator of ethylene biosynthesis, and mutation of ETO1 causes Arabidopsis seedlings to overproduce ethylene. The ethylene biosynthesis inhibitor aminoethoxyvinyl glycine or the ethylene perception inhibitor Ag(+) suppressed all the mutant phenotypes of hps3. Taken together, these results provide further genetic evidence that ethylene is an important regulator of multiple plant responses to Pi starvation. Furthermore, we found that a change in ethylene level has differential effects on the expression of PSI genes, maintenance of Pi homeostasis, production of APase and accumulation of anthocyanin. We also demonstrated that ethylene signaling mainly regulates the activity of root surface-associated APases rather than total APase activity.     

Energy status and functioning of phosphorus-deficient soybean nodules

Characterization of the effects of long-term P deficiency and of onset and recovery from P deficiency on bacteroid mass and number per unit nodule mass and energy status of soybean (Glycine max L. Merr.) nodules was used to investigate the mechanisms by which P deficiency decreases symbiotic N₂ fixation. The continuous P deficiency treatment (0.05 millimolar P) significantly decreased the whole plant dry mass, P, and N by 62, 90, and 78%, respectively, relative to the P-sufficient control (1.0 millimolar) at 44 days after transplanting. Specific nitrogenase activity was decreased an average of 28% over a 16-day experimental period by P deficiency. Whole nodules of P-deficient controls contained 70 to 75% lower ATP concentrations than nodules of P-sufficient controls. Energy charge and ATP concentrations in the bacteroid fraction of nodules were not significantly affected by P treatment. However, ATP and total adenylate concentrations and energy charge in the plant cell fraction of nodules were significantly decreased 91, 62, and 50%, respectively, by the P deficiency treatment. Specific nitrogenase activity, energy charge, and ATP concentration in the plant cell fraction increased to the levels of nonstressed controls within 2, 2, and 4 days, respectively, after alleviation of external P limitation, whereas bacteroid mass per unit nodule mass and bacteroid N concentration did not increase to the level of nonstressed controls until 7 days after alleviation of external P limitation. All of these parameters except bacteroid mass per unit nodule mass decreased to the levels of the P-deficient controls by 11 days after onset of external P limitation. Concentration of ATP in the bacteroid fraction was not significantly affected by alteration in the external P supply. Energy charge in the bacteroid fraction from plants recovering from P deficiency was decreased to a small (10%) but significant extent (P < 0.05) at two sampling dates relative to P-sufficient controls. These ATP concentration and energy charge measurements indicate that P deficiency impaired oxidative phosphorylation in the plant cell fraction of nodules to a much greater extent than in the bacteroids. The concurrence of significant changes in specific nitrogenase activity (2 days) and in the energy charge (2 days) and ATP concentration (4 days) in the plant cell fraction during recovery from external P limitation is consistent with the conclusion that P deficiency decreases the specific nitrogenase activity by inhibiting an energy-dependent reaction(s) in the plant cell fraction of the nodules.     

Acid phosphatase isozymes secreted under phosphate-deficient conditions inPholiota nameko

We previously reported the purification of an acid phosphatase (APase52) secreted from the mycelia ofPholiota nameko under phosphate-deficient conditions. In the present study, two other isozymes (APase47 and APase48) were found and their structures were compared with that of APase52. Thirteen amino acid residues at theN-terminus of APase47 were completely identical with those of APase48 and had partial homology with those of APase52. The deglycosylation of proteins indicated that three APase isozymes differ in theN-linked oligosaccharide content. The protease-generated peptide maps of the APases differed from one another in the band pattern. These results suggest that the APases are the products of different genes.     

Aluminum resistance of cowpea as affected by phosphorus-deficiency stress

Plants growing in acid soils suffer both phosphorus (P) deficiency and aluminum (Al) toxicity stresses. Selection of genotypes for adaptation to either P deficiency or Al toxicity has sometimes been unsuccessful because these two soil factors often interact. Two experiments were conducted to evaluate eight cowpea genotypes for Al resistance and to study the combined effect of P deficiency and Al toxicity stress on growth, P uptake, and organic acid anion exudation of two genotypes of contrasting Al resistance selected from the first experiment. Relative root inhibition by 30 μM Al ranged from 14% to 60% and differed significantly among the genotypes. Al significantly induced callose formation, particularly in Al-sensitive genotypes. P accumulation was significantly reduced (28% and 95%) by Al application for both the Al-resistant and the Al-sensitive genotypes. Al supply significantly enhanced malate release of root apices of both genotypes. However, the exudation rate was significantly higher in the Al-resistant genotype. P deprivation induced an enhanced malate exudation in the presence of Al only in the Al-resistant genotype IT89KD-391. Citrate exudation rate of the root apices was lower than malate exudation by a factor of about 10, and was primarily enhanced by P deficiency in both genotypes. Al treatment further enhanced citrate exudation in P-sufficient, but not in P-deficient plants. The level of citrate exudation was consistently higher in the Al-resistant genotype IT89KD-391 particularly in presence of Al.It is concluded that the Al-resistant genotype is better adapted to acid Al-toxic and P-deficient soils than the Al-sensitive genotype since both malate and citrate exudation were more enhanced by combined Al and P-deficiency stresses.