A comparative study of effects of increasing concentrations of phosphate and phosphite on rice seedlings

While phosphate (Pi) serves as an essential and indispensible plant nutrient, phosphite (Phi) acts as a potent herbicide. Despite their differential influence on plants, both the ions can attenuate phosphate starvation responses (PSRs). We analyzed and compared Pi and Phi uptake and accumulation, attenuation of PSRs and the morphological and physiological responses of the rice seedlings in response to the increasing concentrations of Pi and Phi. Our study revealed that increasing levels of Phi led to pronounced reduction in shoot and root mass in rice seedlings in comparison to similar Pi treatments. Phi inhibited root hair and root formation at 5 and 30 mM Phi concentrations, respectively. Whereas, higher Pi concentrations (40 and 50 mM) affected only root hair elongation. Increasing Phi dose led to drastic reduction in chlorophyll content which was not so in case of Pi. There was inverse relationship between external Pi/Phi level and anthocyanin content of the leaves. In comparison to 20 mM Pi treatment, similar dose of Phi led to significant downregulation of Pi transporters in both leaves and roots. Rice seedlings were found to accumulate mmol and µmol levels of Pi and Phi, respectively. Comparison of various PSR parameters revealed that in comparison to Pi, Phi exhibited greater degree of attenuation of PSRs. Lesser Phi accumulation and greater attenuation of PSRs by Phi indicate plant’s adaption to restrict entry of this toxic ion inside cells.     

THE EFFECTS OF PHOSPHITE ON PHOSPHATE STARVATION RESPONSES OF ULVA LACTUCA (ULVALES,CHLOROPHYTA)1

Effects of phosphite (Phi) on phosphate (Pi) starvation responses were determined in Ulva lactuca L. by incubation in Pi‐limited (1 μM NaH₂PO₄) or Pi‐sufficient (100 μM NaH₂PO₄) seawater containing 0–3 mM Phi. Exposure to 1 μM NaH₂PO₄ decreased the growth rate and the content of free Pi and esterified‐P but increased the activities of extracellular alkaline phosphatase (EC 3.1.2.1) and intracellular acid phosphatase (ACP; EC 3.1.2.2); two ACP isozymes observed by activity staining on isoelectric focussing (IEF) gel were induced. The K_m value of Pi uptake rate was decreased by incubation with 1 μM NaH₂PO₄ and the decrease in K_m value was inhibited by 2 mM Phi, reflecting the operation of a high‐affinity Pi uptake system at low Pi concentrations. In the presence of Phi, the growth rate of Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. As Phi concentrations were increased from 0 to 2 mM, the free Pi contents in both Pi‐sufficient and Pi‐starved thalli decreased, but the esterified‐P contents in Pi‐starved thalli increased, whereas those in Pi‐sufficient thalli increased at 1 mM Phi and decreased at 2 mM Phi. Cell wall localized AP activity in both Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. Intracellular ACP activity in Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM but was not affected in Pi‐sufficient thalli. The induction of ACP isozyme activity and high‐affinity Pi uptake system in Pi‐starved thalli was inhibited by Phi. The present investigation shows that Phi interrupts the sensing mechanisms of U. lactuca to Pi‐limiting conditions.     

Arabidopsis thaliana root and root exudate metabolism is altered by the growth-promoting bacterium Kosakonia radicincitans DSM 16656T

Plant and Soil - Although phosphite (PO3, Phi) cannot replace phosphate (PO4, Pi) as a nutritional source of phosphorus (P) for plants, decisions about using foliar Phi application in citrus groves...     

A novel dominant selectable system for the selection of transgenic plants under in vitro and greenhouse conditions based on phosphite metabolism

Antibiotic and herbicide resistance genes are currently the most frequently used selectable marker genes for plant research and crop development. However, the use of antibiotics and herbicides must be carefully controlled because the degree of susceptibility to these compounds varies widely among plant species and because they can also affect plant regeneration. Therefore, new selectable marker systems that are effective for a broad range of plant species are still needed. Here, we report a simple and inexpensive system based on providing transgenic plant cells the capacity to convert a nonmetabolizable compound (phosphite, Phi) into an essential nutrient for cell growth (phosphate) trough the expression of a bacterial gene encoding a phosphite oxidoreductase (PTXD). This system is effective for the selection of Arabidopsis transgenic plants by germinating T0 seeds directly on media supplemented with Phi and to select transgenic tobacco shoots from cocultivated leaf disc explants using nutrient media supplemented with Phi as both a source of phosphorus and selective agent. Because the ptxD/Phi system also allows the establishment of large‐scale screening systems under greenhouse conditions completely eliminating false transformation events, it should facilitate the development of novel plant transformation methods.     

Disruption of the phosphate-starvation response of oilseed rape suspension cells by the fungicide phosphonate

The influence of the anti-fungal agent phosphonate (Phi) on the response of oilseed rape (Brassica napus L. cv. Jet Neuf ) cell suspensions to inorganic phosphate (Pi) starvation was examined. Subculture of the cells for 7 d in the absence of Pi increased acid phosphatase (APase; EC 3.1.3.2) and pyrophosphate (PPi)-dependent phosphofructokinase (PFP; EC 2.7.1.90) activities by 4.5- and 2.8-fold, respectively, and led to a 19-fold increase in V _max and a 14-fold decrease in K _m (Pi) for Pi uptake. Addition of 2 mM Phi to the nutrient media caused dramatic reductions in the growth and Pi content of the Pi-starved, but not Pi-sufficient cells, and largely abolished the Pi-starvation-dependent induction of PFP, APase, and the high-affinity plasmalemma Pi translocator. Immunoblotting indicated the cells contain three APase isoforms that are synthesized de novo following Pi stress, and that Phi treatment represses this process. Phosphonate treatment of Pi-starved cells significantly altered the relative extent of in-vivo ³²P-labelling of polypeptides having M_rs of 66, 55, 45 and 40 kDa. However, Phi had no effect on the total adenylate pool of Pi-starved cells which was about 32% lower than that of Pi-sufficient cells by day 7. Soluble protein levels, and activities of pyruvate kinase (EC 2.7.1.40) and ATP-dependent phosphofructokinase (EC 2.7.1.11) were unaffected by Pi starvation and/or Phi treatment. The effects of Phi on the growth, and APase and PFP activities of Pi-starved B. napus seedlings were similar to those observed in the suspension cells. The results are consistent with the hypothesis that a primary site of Phi action in higher plants is at the level of the signal transduction chain by which plants perceive and respond to Pi stress at the molecular level. Received: 30 December 1996 / Accepted: 19 February 1997     

Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root development

Plants have evolved complex strategies to maintain phosphate (Pi) homeostasis and to maximize Pi acquisition when the macronutrient is limiting. Adjustment of root system architecture via changes in meristem initiation and activity is integral to the acclimation process. However, the mechanisms that monitor external Pi status and interpret the nutritional signal remain to be elucidated. Here, we present evidence that the Pi deficiency response, pdr2, mutation disrupts local Pi sensing. The sensitivity and amplitude of metabolic Pi-starvation responses, such as Pi-responsive gene expression or accumulation of anthocyanins and starch, are enhanced in pdr2 seedlings. However, the most conspicuous alteration of pdr2 is a conditional short-root phenotype that is specific for Pi deficiency and caused by selective inhibition of root cell division followed by cell death below a threshold concentration of about 0.1 mm external Pi. Measurements of general Pi uptake and of total phosphorus (P) in root tips exclude a defect in high-affinity Pi acquisition. Rescue of root meristem activity in Pi-starved pdr2 by phosphite (Phi), a non-metabolizable Pi analog, and divided-root experiments suggest that pdr2 disrupts sensing of low external Pi availability. Thus, PDR2 is proposed to function at a Pi-sensitive checkpoint in root development, which monitors environmental Pi status, maintains and fine-tunes meristematic activity, and finally adjusts root system architecture to maximize Pi acquisition.     

Growth response of komatsuna (Brassica rapa var. peruviridis) to root and foliar applications of phosphite

Soil and hydroponic culture experiments were conducted to investigate the effects of phosphite (Phi) as phosphorus (P) fertilizer via root and foliar applications on the growth and P supply of komatsuna. In both experiments, root P treatments were combinations of Phi and phosphate (Pi) at different Pi:Phi ratios, for a total of high P level (92 mg P pot^?1; the soil experiment) or low P level (0.05 mM P; the hydroponic experiment). Foliar P treatments were deionized water (control), a Pi solution and a Phi solution at low concentration of 0.05% P₂O₅. In both experiments, shoot dry weight of plants significantly decreased as Pi:Phi ratio decreased. In the soil experiment, plants grew abnormally at a Pi:Phi ratio of 25:75 and died when P was applied to soil entirely as Phi form (0:100 treatment). In the hydroponic experiment, no visible damage was found in shoot but root growth was strongly inhibited with severe damage symptoms at low Pi:Phi ratios. Total P concentration in plant decreased significantly with decreasing Pi:Phi ratio, especially in the hydroponic experiment. Foliar application of Phi although greatly increased total P of plants compared to that of Pi in both experiments, it did not improve but further decreased plant growth at low Pi:Phi ratios in the soil experiment and at all Pi:Phi ratios in the hydroponic experiment. The results of this study clearly indicated that Phi could not be used as P fertilizer by komatsuna plants via both application methods and could not substitute P at any rate at either low or high level. No beneficial effect of Phi was detected even when it was applied at low rate or applied in combination with Pi at different ratios. The effects of Phi were strongly dependent on the P nutrition status of plants; and plants that were not sufficiently fertilized with Pi may become vulnerable to Phi even at low levels.     

一种基于亚磷酸盐及其脱氢酶的植物磷利用和杂草控制系统的建立

磷是植物生长发育所必需的大量营养元素之一。土壤中存在大量的正磷酸盐 (Pi),但由于土壤化学和微生物转化使得土壤可利用磷的浓度并不高。土壤缺磷以及杂草的抗除草剂能力已成为当前农业可持续发展的重要限制因素,所以提高植物对土壤磷的吸收利用能力或寻求可替代正磷酸盐的磷肥以及开发新型杂草控制系统已成为亟待解决的问题。自然界中亚磷酸盐 (Phi) 是含量仅次于正磷酸盐的磷源,但仅在某些细菌中能被专一性的亚磷酸盐脱氢酶 (PTDH) 氧化利用,对植物的生长发育则具有抑制作用。利用这一特性,将从土壤宏基因组中直接扩增到的假单胞菌PTDH基因PsPtx通过农杆菌侵染法转入烟草中,并通过RT-PCR、垂直板幼苗生长、显性标记和生长竞争实验分析PsPtx转基因烟草的基因表达以及在Phi胁迫条件下的特性。结果显示,PsPtx在其转基因植株的根茎叶组织中都有几乎相同水平的表达;PsPtx转基因烟草不但能解除Phi对植物的毒害作用,并将它氧化成可用的Pi作为生长发育所需的磷源,而且在Phi胁迫条件下较野生型烟草有相当明显的生长竞争优势;另外PsPtx还具备成为植物遗传转化显性选择标记的优良特质。因此,PsPtx基因编码的亚磷酸盐脱氢酶可用于开发一种基于亚磷酸盐为磷肥和除草剂的植物磷利用和杂草控制系统,为当前农作物转基因研究存在的一些重大问题提供一个有效解决方案。     

31P NMR Measurements of the Cytoplasmic and Vacuolar Pi Content of Mature Maize Roots: Relationships with Phosphorus Status and Phosphate Fluxes

The vacuolar and cytoplasmic inorganic phosphate (Pi) contentof the mature regions of maize roots was measured by a ³¹P NMRtechnique which used an external standard to avoid the needfor tissue extraction and which exploited the relatively rapidrelaxation of cytoplasmic Pi in order to improve the detectionof this pool in fully-vacuolated cells. In mature roots of maize growing with abundant external phosphate,the concentration of Pi in the cytoplasm was approximately 6.5mol m^–3. When these plants were deprived of external phosphate,the vacuolar Pi content of the roots decreased rapidly, butthe cytoplasmic Pi concentration initially remained constantand did not begin to decline until P-stress became severe. Calculationsshow that withdrawal of Pi from the vacuoles into the cytoplasmunder these conditions would be against an electrochemical gradient. During P-starvation, an increased capacity for Pi influx developed,preceding any detectable change in the cytoplasmic Pi contentof the roots. This response is considered in terms of paralleleffects on transport sites for phosphate at the plasmalemmaand at the tonoplast. Comparisons of simultaneous rates of influxand net uptake implied that phosphate efflux accounted for <10% of influx in plants of a steady or declining P-status. However,direct measurements of efflux suggested that this process maybe temporarily accelerated when plants are recovering from P-stress. Key words: P-nutrition, subcellular compartmentation     

Phosphite disrupts the acclimation of Saccharomyces cerevisiae to phosphate starvation.

The influence of phosphite (H2PO3-) on the response of Saccharomyces cerevisiae to orthophosphate (HPO4(2-); Pi) starvation was assessed. Phosphate-repressible acid phosphatase (rAPase) derepression and cell development were abolished when phosphate-sufficient (+Pi) yeast were subcultured into phosphate-deficient (-Pi) media containing 0.1 mM phosphite. By contrast, treatment with 0.1 mM phosphite exerted no influence on rAPase activity or growth of +Pi cells. 31P NMR spectroscopy revealed that phosphite is assimilated and concentrated by yeast cultured with 0.1 mM phosphite, and that the levels of sugar phosphates, pyrophosphate, and particularly polyphosphate were significantly reduced in the phosphite-treated -Pi cells. Examination of phosphite's effects on two PHO regulon mutants that constitutively express rAPase indicated that (i) a potential target for phosphite's action in -Pi yeast is Pho84 (plasmalemma high-affinity Pi transporter and component of a putative phosphate sensor-complex), and that (ii) an additional mechanism exists to control rAPase expression that is independent of Pho85 (cyclin-dependent protein kinase). Marked accumulation of polyphosphate in the delta pho85 mutant suggested that Pho85 contributes to the control of polyphosphate metabolism. Results are consistent with the hypothesis that phosphite obstructs the signaling pathway by which S. cerevisiae perceives and responds to phosphate deprivation at the molecular level.     

Phosphite: a novel P fertilizer for weed management and pathogen control

The availability of orthophosphate (Pi) is a key determinant of crop productivity because its accessibility to plants is poor due to its conversion to unavailable forms. Weed's competition for this essential macronutrient further reduces its bio‐availability. To compensate for the low Pi use efficiency and address the weed hazard, excess Pi fertilizers and herbicides are routinely applied, resulting in increased production costs, soil degradation and eutrophication. These outcomes necessitate the identification of a suitable alternate technology that can address the problems associated with the overuse of Pi‐based fertilizers and herbicides in agriculture. The present review focuses on phosphite (Phi) as a novel molecule for its utility as a fertilizer, herbicide, biostimulant and biocide in modern agriculture. The use of Phi‐based fertilization will help to reduce the consumption of Pi fertilizers and facilitate weed and pathogen control using the same molecule, thereby providing significant advantages over current orthophosphate‐based fertilization.     

Phosphate Uptake by Excised Maize Root Tips Studied by in VivoP Nuclear Magnetic Resonance Spectroscopy

The extent of phosphate uptake measured by the relative changes in cytoplasmic Pi, vacuolar Pi, ATP, glucose-6-phosphate, and UDPG was determined using in vivo³¹P nuclear magnetic resonance spectroscopy. Maize (Zea mays) root tips were perfused with a solution containing 0.5 or 1.0 millimolar phosphate at pH ~6.5 under different conditions. In the aerated state, phosphate uptake resulted in a significant increase (>80%) in vacuolar Pi, but cytoplasmic Pi only transiently increased by 10%. Under N₂, the cytoplasmic Pi increased ~150% which could be attributed to a large extent to the breakdown of ATP, sugar phosphates and UDPG. Vacuolar Pi increased but only to the extent of ~10% of that seen under aerobic conditions. 2-deoxyglucose pretreatment was utilized to decrease the level of cytoplasmic Pi. When pretreated with the 2-deoxyglucose, the excised maize roots absorbed phosphate from the perfusate with a significant increase in the cytoplasmic Pi. The increase could only be traced to external phosphate since the concentrations of other phosphorus containing species remained constant during the uptake period. With 2-deoxyglucose pretreatment, phosphate uptake under anaerobic conditions was substantially inhibited with only the vacuolar phosphate showing a slight increase. When roots were treated with carbonyl cyanide m-chlorophenyl hydrazone, no detectable Pi uptake was found. These results were used to propose a H⁺-ATPase related transport mechanism for phosphate uptake and compartmentation in corn root cells.     

Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux

Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.     

Phosphate or phosphite addition promotes the proteolytic turnover of phosphate-starvation inducible tomato purple acid phosphatase isozymes

Within 48 h of the addition of 2.5 mM phosphate (HPO42-, Pi) or phosphite (H2PO3-, Phi) to 8-day-old Pi-starved (-Pi) tomato suspension cells: (i) secreted and intracellular purple acid phosphatase (PAP) activities decreased by about 12- and 6-fold, respectively and (ii) immunoreactive PAP polypeptides either disappeared (secreted PAPs) or were substantially reduced (intracellular PAP). The degradation of both secreted PAP isozymes was correlated with the de novo synthesis of two extracellular serine proteases having M(r)s of 137 and 121 kDa. In vitro proteolysis of purified secreted tomato PAP isozymes occurred following their 24 h incubation with culture filtrate from Pi-resupplied cells. The results indicate that Pi or Phi addition to -Pi tomato cells induces serine proteases that degrade Pi-starvation inducible extracellular proteins.     

A phosphorus-31 nuclear magnetic resonance study of elicitor-mediated metabolic changes in Catharanthus roseus suspension cultures

Summary The induction of metabolic changes in suspension cultured cells of Catharanthus roseus upon elicitation has been investigated. Addition of a yeast glucan preparation to the growth medium resulted in induction of phenylalanine ammonia lyase. Phosphate uptake and metabolism of elicited cells was followed by ³¹P nuclear magnetic resonance. The uptake rate of Pi from the medium by oxygenated cells of C. roseus was reduced immediately after elicitation. Despite this reduced Pi uptake elicited cells had significantly increased amounts of ATP (twofold increase within 6 h). Cytoplasmic levels of Pi, phosphomonoesters, and Uridine Diphasphate glucose (UDP-Glc) were unaffected by eliciation. Furthermore, the cytoplasmic and vacuolar pH remained constant after addition of elicitor.     

The possible involvement of a phosphate-induced transcription factor encoded by phi-2 gene from tobacco in ABA-signaling pathways

A novel phosphate-induced gene, phi-2, has been identified by its induction on addition of phosphate to phosphate-starved tobacco BY-2 cells. The predicted gene product of phi-2 has significant homology to a group of bZIP proteins involved in ABA-signaling pathways, and phi-2 also responded to ABA treatment. A previously isolated phosphate-induced gene, phi-1, (Sano et al. (1999) Plant Cell Physiol. 40: 1) was also responsive to ABA. Although phosphate addition induced semi-synchronous cell division in phosphate-starved tobacco BY-2 cells, ABA adversely affected cell division. Detailed examination revealed that the high levels of phosphate required to induce semi-synchronous cell division seemed to be perceived as indicators of stress by the cells. One of the stress indicators perceived by the cells is a cytoplasmic pH change, to which phi-2 and phi-1 genes respond. The different components of the cell's response to phosphate induction are discussed.     

Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) suspension cell cultures

Phosphite (H₂PO₃^–, Phi) prevents the acclimation of plants and yeast to orthophosphate (Pi, HPO₄^2–) deprivation by specifically obstructing the derepression of genes encoding proteins characteristic of their Pi-starvation response. In this study, we report that prolonged (i.e., 3–4 weeks) culture of Brassica napus L. suspension cells in Pi-deficient (–Pi) media leads to programmed cell death (PCD). However, when the B. napus cells were subcultured into –Pi media containing 2 mM Phi, they initiated PCD within 5 days, with 95% cell death observed by day 9. Dying cells exhibited several morphological and biochemical features characteristic of PCD, including protoplast shrinkage, chromatin condensation, and fragmentation of nuclear DNA. Immunoblotting indicated that B. napus cells undergoing PCD upregulated a 30-kDa cysteine endoprotease that is induced during PCD in the inner integument cells of developing B. napus seeds. It is concluded that PCD in B. napus suspension cells is triggered by extended Pi starvation, and that Phi treatment greatly accelerates this process. Our results also infer that the adaptive value of acclimating at the molecular level to Pi-stress is to extend the viability of –Pi B. napus cell cultures by about 3 weeks.Abbreviations APase acid phosphatase (EC 3.1.3.2) - BnCysP B. napus cysteine proteinase - DAPI 4,6-diamidino-2-phenylindole - FDA fluorescein diacetate - PCD programmed cell death - Phi phosphite - +Pi and –Pi Pi-sufficient and -deficient, respectively - PI propidium iodide - PSI Pi-starvation inducible     

Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate

Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport.