METABOLIC RESPONSES OF THE DIATOM ACHNANTHES BREVIPES (BACILLARIOPHYCEAE) TO NUTRIENT LIMITATION

The diatom Achnanthes brevipes C.A. Ag. was cultured in the presence of limiting concentrations of nitrogen (N) or inorganic phosphate (P_i). Growth, in terms of final yield, was more affected by N limitation than P_i limitation; N limitation had a greater effect also on protein and chlorophyll content. Carbohydrate concentrations increased under both nutrient starvation treatments, but N or P_i limitation had different effects. Total (intracellular plus extracellular) sugar content increased when cells were exposed to both types of nutrient limitation, but the extracellular polysaccharide fraction increased only in the presence of P_i starvation. Analyses were performed to identify the metabolic changes occurring in cells exposed to low phosphate because this was the main condition that affected carbohydrate extrusion. Activities of several enzymes involved in carbohydrate metabolism showed that under P_i limitation there was no activation of alternative reactions that were found to result in P_i liberation, instead of its consumption, in some higher plants and in the green alga Selenastrum minutum Naeg. Collins. Results showed that activities of pyruvate kinase, phosphorylating NAD-dependent 3-phosphate-glyceraldehyde dehydrogenase, and 3-phospho-glycerate kinase were inhibited under P_i-limited conditions compared with control cells, indicating limited glucose catabolism. Activity of uridine diphosphate glucose pyrophosphorylase, a key enzyme for the biosynthesis of the storage compound crysolaminarin, was also partly inhibited in P_i-stressed cells. Our findings suggest that carbohydrate catabolism in A. brevipes is limited under P_i deficiency, whereas extracellular extrusion of carbohydrate is favored.     

Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.)

We investigated (1) the effect of constant and altered inorganic phosphate (P_i) supply (1–100 mmol m^–3) on proteoid root production by white lupin ( Lupinus albus L.); and (2) the variation in citrate efflux, enzyme activity and phosphate uptake along the proteoid root axis in solution culture. Proteoid root formation was greatest at P_i solution concentrations of 1–10 mmol m^–3 and was suppressed at 25 mmol m^–3 P_i and higher. Except at 1 mmol m^–3 P_i, the formation of proteoid roots did not affect plant dry matter yields or shoot to root dry matter ratios, indicating that proteoid roots can form under conditions of adequate P supply and not at the expense of dry matter production. Plants with over 50% of the root system as proteoid roots had tissue P concentrations considered adequate for maximum growth, providing additional evidence that proteoid roots can form on P-sufficient plants. There was an inverse relationship between the P_i concentration in the youngest mature leaf and proteoid root formation. Citrate efflux and the activities of enzymes associated with citric acid synthesis (phosphoenolpyruvate carboxylase and malate dehydrogenase) varied along the proteoid root axis, being greatest in young proteoid rootlets of the 1–3 cm region from the root tip. Citrate release from the 0–1 and 5–9 cm regions of the proteoid root was only 7% (per unit root length) of that from the 1–3 cm segment. Electrical potential and ³²P_i uptake measurements showed that P_i uptake was more uniform along the proteoid root than citrate efflux.     

Carbohydrate partitioning, photosynthesis and growth in Lemna gibba G3. II. Effects of phosphorus limitation

The relationship between CO₂ assimilation rate, growth and partitioning of carbon among starch, sucrose, glucose and fructose were studied in phosphorus (P_i)-limited Lemna gibba L. G3. Two experimental models were used: 1) Cultures were grown at various stable, suboptimal rates regulated by the supply of P_i; 2) cultures growing at optimal rates were transferred to P_i-free medium. The response to a P_i deficiency can be divided into two phases. Phase I is characterized by hyperactivity of the sucrose synthesis pathway, leading to high levels of glucose and fructose. Phase II is characterized by starch accumulation associated with a decrease in the cytoplasmic pools of soluble sugars owing to inhibition of carbon export from the chloroplast. A strong negative correlation was found between the CO₂ assimilation rate and starch levels. No significant correlation was found between assimilation and ATP levels and decrease in relative growth rate did not significantly affect the adenylate energy charge (EC). The regulatory aspects of the partitioning of carbon among soluble sugars and starch as well as the negative correlation between carbohydrate levels and CO₂ assimilation at P_i-limited growth are discussed.     

Involvement of the ethylene-signalling pathway in sugar-induced tolerance to the herbicide atrazine in Arabidopsis thaliana seedlings

Soluble sugars can induce tolerance to otherwise lethal concentrations of the herbicide atrazine in Arabidopsis thaliana seedlings. This sugar-induced tolerance involves modifications of gene expression which are likely to be related to sugar and xenobiotic signal transduction. Since it has been suggested that ethylene- and sugar-signalling pathways may interact, the effects of glucose (Glc) and sucrose (Suc) on seedling growth and tolerance to atrazine were analysed in etr1-1, ein2-1, ein4, and sis1/ctr1-12 ethylene-signalling mutant backgrounds, where key steps of ethylene signal transduction are affected. Both ethylene-insensitive and ethylene-constitutive types of mutants were found to be affected in sugar-induced chlorophyll accumulation and root growth and in sugar-induced tolerance to atrazine. Interactions between ethylene and sugars were thus shown to take place during enhancement of seedling growth by low-to-moderate (up to 80 mM) sugar concentrations. The strong impairment of sugar-induced atrazine tolerance in etr1-1, ein2-1, and ein4 mutants demonstrated that this tolerance required active signalling pathways and could not be ascribed to mere metabolic effects nor to mere growth enhancement. Sugar-induced atrazine tolerance thus seemed to involve activation by sugar and atrazine of hexokinase-independent sugar signalling pathways and of ethylene signalling pathways, resulting in derepression of hexokinase-mediated Glc repression and in induction of protection mechanisms against atrazine injury.     

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.     

A transgenic dTph1 insertional mutagenesis system for forward genetics in mycorrhizal phosphate transport of Petunia

The active endogenous dTph1 system of the Petunia hybrida mutator line W138 has been used in several forward-genetic mutant screens that were based on visible phenotypes such as flower morphology and color. In contrast, defective symbiotic phosphate (P_i) transport in mycorrhizal roots of Petunia is a hidden molecular phenotype as the symbiosis between plant roots and fungi takes place below ground, and, while fungal colonization can be visualized histochemically, P_i transport and the activity of P_i transporter proteins cannot be assessed visually. Here, we report on a molecular approach in which expression of a mycorrhiza-inducible bi-functional reporter transgene and insertional mutagenesis in Petunia are combined. Bi-directionalization of a mycorrhizal P_i transporter promoter controlling the expression of two reporter genes encoding firefly luciferase and GUS allows visualization of mycorrhiza-specific P_i transporter expression. A population of selectable transposon insertion mutants was established by crossing the transgenic reporter line with the mutator W138, from which the P _i transporter downregulated ( ptd1 ) mutant was identified, which exhibits strongly reduced expression of mycorrhiza-inducible P_i transporters in mycorrhizal roots.     

The relationship between phosphate status and cyanide-resistant respiration in bean roots

Bean ( Phaseolus vulgaris L.) seedlings were cultured on complete or phosphate-deficient nutrient medium. After 14 days of culture on phosphate-deficient medium the visible symptoms of P_i deficiency were observed only in the shoot, the fresh and dry weights of the roots were slightly higher than in control plants. The decreased P_i content in the roots had little effect on total respiration rate but had an effect on the level of inhibition of respiration by cyanide. The high resistance of respiration to cyanide observed in P_i-deficient roots was the result of the suppression of cytochrome path activity and an increased participation of the alternative, cyanide-resistant pathway. The cytochrome pathway activity increased when inorganic phosphate was supplied to P_i-deficient roots for 1 or 3.5 h. It is speculated that the suppression of cytochrome pathway in P_i-deficient roots may result from restriction of the phosphorylating capacity or a partial inhibition of cytochrome oxidase activity.     

Chemical nature of P efflux from P-adequate Spirodela and Lemna plants

The chemical nature of P released (efflux) from healthy (P-adequate) Spirodela oligorrhiza (Kurz) Hegelm, and Lemna major L. plants growing in sterile solution culture was investigated using thin-layer chromatography and autoradiography. Ester patterns of P efflux material from [ 32 P_i] labelled plants was compared with P ester patterns in the tissue of the same plants.
Nearly all of the released P was P_i (99.8% in Spirodela , 99.6% in Lemna ) there being little consistent evidence for the release of significant amounts of organic P (0.2% and 0.4% of released P – Spirodela and Lemna respectively). Efflux of P predominantly as organic P (hexose or nucleotide P) would have resulted in an important loss of energy for the plant. The corresponding proportion of organic P in the tissue of the labelled plants from which the P efflux material arose was 15% ( Spirodela ) and 8% ( Lemna ). Consequently there was no evidence for the preferential release of phosphorylcholine, a compound implicated in P efflux because of its high membrane permeability. Autoradiographic resolution of the chromatogram was improved by labelling plants at high ³²P specific activity [185 GBq (mol P_i)⁻¹] compared with levels considered safe [37 GBq (mol P_i)_-1] without any detrimental effects.
Spirodela and Lemna plants appear to exert a close control over both the quantity (rates of P efflux) and quality (proportion of P_i to organic P) of their P efflux. The possible role of externally located phosphatases in minimising the release of organic P is also discussed.     

Alkaline phosphatase activity in two planktonic desmid species and the possible role of an extracellular envelope

1. Cosmarium abbreviatum var. planctonicum , a desmid from oligo‐mesotrophic lakes, had a higher maximum alkaline phosphatase activity (APA) and affinity constant under both continuous and pulsed inorganic phosphorus (P_i) limitation than Staurastrum chaetoceras , a desmid predominantly encountered in eutrophic lakes.
2. APA of both species increased when measured in starved cells subjected to pulsed P_i conditions when compared to continuous P_i limitation.
3. The portion of extracellular relative to cellular APA was higher in S. chaetoceras than in C. abbreviatum , indicating that S. chaetoceras secreted the enzymes more readily into its environment.
4. The difference in APA could explain the dominance of C. abbreviatum during competition between these two species under conditions of continuous organic P (P_o) limitation, but not the outcome under a pulsed P_o shortage. The dominance of S. chaetoceras in the latter experiment can, however, be explained by species‐specific P_i‐uptake characteristics.
5. After a saturating pulse of P_i, no increase in P_i in the extracellular mucus layer of C. abbreviatum was found and it was therefore concluded that the mucilage sheath does not store P. However, the sheath could have a main function as an accumulation site of cellular APA, providing the cell with P_i.     

Photosynthetic response of P-deficient Gracilaria tenuistipitata under two different phosphate treatments

Phosphorus-deficient Gracilaria tenuistipitata Zhang et Xia was cultured for 15 days at two different inorganic phosphate (P_i) concentrations: 3 μM (low P_i treatmenl) or 30 μM phosphate (high P_i treatment). The amount of ribulose-l,5-bisphosphate carboxy-lase/oxygenase (Rubisco), phycobiliproteins, Chl a and total soluble proteins were higher in the high P_i than in the low P_i treatment. The total N content of the low P_i plants was lower than in plants grown at high P_i concentrations whereas the amount of total C was highest in low P_i plants. The increase of Rubisco content in the high P_i treatment (3-fold) was parallel to the enhancement of the maximum photosynthetic rate which increased 5-fold. This correspondence was also found in the low P_i treatment in which Rubisco content and maximum photosynthesis did not change significantly from the initial values. The photosynthetic efficiency was also higher at high than at low P_i. The high P_i plants also showed higher dark respiration and growth rates. The data presented here suggest that marine macroalgae submitted to P_i deficiency exhibit a decrease in growth caused not only by P_i implication on energy transfer in photosynthesis and respiration, but also by the diminution of the amount of photosynthetic pigments and Rubisco.     

Phosphate uptake by the yeast, Rhodotorula rubra, and the green alga, Selenastrum capricornutum Printz, after phosphate additions to steady-state continuous cultures

Abstract We examined phosphate (P_i) uptake by two well-characterized microorganisms: a green alga ( Selenastrum capricornutum ) and a heterotrophic yeast ( Rhodotorula rubra ). Phosphate uptake was measured in dual- and single-species continuous cultures after perturbation of a phosphorus (P)-limited steady-state culture by additions of varying concentrations of P_i. We found that, under these conditions, both organisms had very high transport rates for P_i. The yeast was able to attain higher internal P concentrations than predicted from either steady-state or from P-starved batch culture data. Because the yeast was able to sequester and store P_i more efficiently than the alga under dilute P_i continuous culture conditions, co-existence of the two organisms was ultimately controlled by the concentration of carbon available for growth of the yeast.     

Accumulation and conversion of sugars by developing wheat grains. 4. Effects of phosphate and potassium ions in endosperm slices

Abstract. Transverse slices through developing grains of Triticum aestivum cv. SUN 9E 16 d after anthesis were incubated in simple defined media with various radioactive labels. In some enzymic assays slices were pretreated with 2.5% Triton X-100 or with 5% butanol to remove cellular membranes and endogenous substrates.
Endogenous potassium leaked from endosperm slices into 30mol m⁻³ sucrose while sucrose was converted partly into starch. Exogenous alkali-ions, except Li⁺, stimulated conversion of sucrose to insoluble matter, specifically to starch with K⁺. Starch synthetase activity of Triton-pretreated slices was stimulated by K⁺ at both high and low substrate ADPG concentration, but was not affected by phosphate (25 mol m⁻³).
Phosphate in the medium had no effect on incorporation of sucrose or glucose into alcohol-insoluble material or starch in fresh slices (internal inorganic phosphate (P,) concentration was about 11 mol m⁻³). Three- to four-fold contrasts in internal P_i level, achieved by prolonged preincubations in different media, did not show an inhibition of starch synthesis by P_i. However, phosphate (25mol m⁻³) inhibited starch synthesis, that was mediated by ADPG pyrophosphorylase in butanol-pretreated endosperm slices by 15–18%.
It is concluded that starch synthesis in wheat endosperm is not regulated directly by apoplastic P_i; level.     

Effects of salinity and phosphate on ion distribution in lupin leaflets

Lupin ( Lupinus luteus L. cv. Weiko III) were grown in nutrient solution over a range of inorganic phosphate (P_i) concentrations, with or without 50 m M NaCl. Plants with high P_i (2 m M ) and salt showed progressive leaf necrosis and had higher concentrations of total phosphate than plants grown with high P_i alone. Most of the extra total phosphate in salt treated plants was in the P_i form. P_i supply did not influence Na⁺, K⁺ or Cl⁻ concentrations in epidermal vacuoles or mesophyll cells. However, epidermal vacuoles accumulated more monovalent cations (Na⁺ and K⁺) than Cl⁻, and in vacuoles of plants grown with 0.1 m M P_i additional P_i was accumulated, possibly to maintain charge balance. Plants grown with 2 m M P_i did not accumulate additional P_i in epidermal vacuoles, but showed higher phosphorus levels in cell walls. It is suggested that at moderate phosphorus concentrations P_i plays a role in epidermal osmotic adjustment, possibly explaining the beneficial role of additional phosphorus on salt stressed plants. At high P_i supply with salt, P_i does not contribute to osmotic adjustment and instead accumulates in cell walls. However, the cause of leaf damage under conditions of high phosphorus supply and salinity is still not entirely clear.     

Phosphate starvation responses are mediated by sugar signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phosphate (Pi) is one of the least available plant nutrients in soils. It is associated with dynamic changes in carbon fluxes and several crucial processes that regulate plant growth and development. Pi levels regulate the expression of large number of genes including those involved in photosynthesis and carbon metabolism. Herein we show that sugar is required for Pi starvation responses including changes in root architecture and expression of phosphate starvation induced (PSI) genes in Arabidopsis. Active photosynthesis or the supplementation of sugar in the medium was essential for the expression of PSI genes under Pi limiting conditions. Expression of these genes was not only induced by sucrose but also detected, albeit at reduced levels, with other metabolizable sugars. Non-metabolizable sugar analogs did not induce the expression of PSI genes. Although sugar input appears to be downstream of initial Pi sensing, it is absolutely required for the completion of the PSI signaling pathway. Altered expression of PSI genes in the hexokinase signaling mutant gin2 indicates that hexokinase-dependent signaling is involved in this process. The study provides evidence for requirement of sugars in PSI signaling and evokes a role for hexokinase in some components of Pi response mechanism.     

Inorganic Pi Increases Neuronal Survival in the Acute Early Phase Following Excitotoxic/Oxidative Insults

Abstract: Inorganic phosphate (P_i) plays a vital role in intracellular energy metabolism. Its many effects include stimulation of glucose use, enhancement of high-energy phosphate concentrations, and modulation of cytosolic free [Ca²⁺]. Cultured fetal rat cortical neurons constitutively import P_i, and cytosolic levels positively correlate with [ATP], [NADPH], and energy charge. In the present study, we demonstrate that the concentration of intracellular P_i is an important determinant of acute neuronal survival after an excitotoxic or oxidative insult to cultured fetal rat cortical neurons. Extracellular P_i dose-dependently enhanced survival of cortical neurons after exposure to NMDA at early (≤6 h) time points after termination of the insult. P_i similarly increased neuronal survival after exposure to kainic acid or H₂O₂. P_i-exposed neurons had higher basal intracellular [P_i], [ATP], and [GSH], and slightly lower cytosolic free [Ca²⁺], compared with P_i-deprived neurons. P_i-exposed neurons maintained increased [ATP] after exposure to NMDA and displayed reduced formation of reactive oxygen species after exposure to kainic acid or H₂O₂, compared with P_i-deprived neurons. These findings demonstrate that changes in extracellular and intracellular P_i can affect neuronal survival after excitotoxic or oxidative insults.     

The dormancy-breaking stimuli “chilling,hypoxia and cyanamide exposure” up-regulate the expression of α-amylase genes in grapevine buds

It has been suggested that respiratory stress is involved in the mechanism underlying the dormancy-breaking effect of hydrogen cyanamide (H₂CN₂) and sodium azide in grapevine buds; indeed, reductions in oxygen levels (hypoxia) and inhibitors of respiration promote bud-break in grapevines. In this study, we showed that, hypoxia increased starch hydrolysis soluble sugar consumption and up-regulated the expression of α-amylase genes (Vvα-AMYs) in grapevine buds, suggesting that these biochemical changes induced by hypoxia, may play a relevant role in the release of buds from endodormancy (ED). Three of the four Vvα-AMY genes that are expressed in grapevine buds were up-regulated by hypoxia and a correlation between changes in sugar content and level of Vvα-AMY gene expression during the hypoxia treatment was found, suggesting that soluble sugars mediate the effect of hypoxia on Vvα-AMY gene expression. Exogenous applications of soluble sugars and sugar analogs confirmed this finding and revealed that osmotic stress induces the expression of Vvα-AMY1 and Vvα-AMY3 and that soluble sugars induces Vvα-AMY2 and Vvα-AMY4 gene expression. Interestingly, the plant hormone gibberellic acid (GA₃) induced the expression of Vvα-AMY3 and Vvα-AMY4 genes, while dormancy breaking stimuli, chilling and cyanamide exposure, mainly induced the expression of Vvα-AMY1 and Vvα-AMY2 genes, suggesting that these two α-amylase genes might be involved in the release of grapevine buds from the ED.