Phosphite-induced changes of the transcriptome and secretome in Solanum tuberosum leading to resistance against Phytophthora infestans.

BACKGROUND: Potato late blight caused by the oomycete pathogen Phytophthora infestans can lead to immense yield loss. We investigated the transcriptome of Solanum tubersoum (cv. Desiree) and characterized the secretome by quantitative proteomics after foliar application of the protective agent phosphite. We also studied the distribution of phosphite in planta after application and tested transgenic potato lines with impaired in salicylic and jasmonic acid signaling. RESULTS: Phosphite had a rapid and transient effect on the transcriptome, with a clear response 3 h after treatment. Strikingly this effect lasted less than 24 h, whereas protection was observed throughout all time points tested. In contrast, 67 secretome proteins predominantly associated with cell-wall processes and defense changed in abundance at 48 h after treatment. Transcripts associated with defense, wounding, and oxidative stress constituted the core of the phosphite response. We also observed changes in primary metabolism and cell wall-related processes. These changes were shown not to be due to phosphate depletion or acidification caused by phosphite treatment. Of the phosphite-regulated transcripts 40% also changed with ß-aminobutyric acid (BABA) as an elicitor, while the defence gene PR1 was only up-regulated by BABA. Although phosphite was shown to be distributed in planta to parts not directly exposed to phosphite, no protection in leaves without direct foliar application was observed. Furthermore, the analysis of transgenic potato lines indicated that the phosphite-mediated resistance was independent of the plant hormones salicylic and jasmonic acid. CONCLUSIONS: Our study suggests that a rapid phosphite-triggered response is important to confer long-lasting resistance against P. infestans and gives molecular understanding of its successful field applications.

Redox chemistry in the phosphorus biogeochemical cycle.

The element phosphorus (P) controls growth in many ecosystems as the limiting nutrient, where it is broadly considered to reside as pentavalent P in phosphate minerals and organic esters. Exceptions to pentavalent P include phosphine--PH3--a trace atmospheric gas, and phosphite and hypophosphite, P anions that have been detected recently in lightning strikes, eutrophic lakes, geothermal springs, and termite hindguts. Reduced oxidation state P compounds include the phosphonates, characterized by C-P bonds, which bear up to 25% of total organic dissolved phosphorus. Reduced P compounds have been considered to be rare; however, the microbial ability to use reduced P compounds as sole P sources is ubiquitous. Here we show that between 10% and 20% of dissolved P bears a redox state of less than +5 in water samples from central Florida, on average, with some samples bearing almost as much reduced P as phosphate. If the quantity of reduced P observed in the water samples from Florida studied here is broadly characteristic of similar environments on the global scale, it accounts well for the concentration of atmospheric phosphine and provides a rationale for the ubiquity of phosphite utilization genes in nature. Phosphine is generated at a quantity consistent with thermodynamic equilibrium established by the disproportionation reaction of reduced P species. Comprising 10-20% of the total dissolved P inventory in Florida environments, reduced P compounds could hence be a critical part of the phosphorus biogeochemical cycle, and in turn may impact global carbon cycling and methanogenesis.

Monitoring of phosphorus oxide ion for analytical speciation of phosphite and phosphate in transgenic plants by high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

Large amounts of phosphate fertilizers utilized in agriculture and their relatively poor efficiency are of high ecological and economic concern. Therefore, transgenic plants capable of metabolizing phosphite are being engineered. In support of this biotechnological task, analytical speciation of phosphorus in biological tissues is required. In this study, plant extracts were analyzed by liquid chromatography-inductively coupled plasma mass spectrometry at m/z of elemental phosphorus and phosphorus oxide ions. Using polymeric-based anion exchange column and millimolar concentration of nitric acid in potassium phthalate mobile phase (pH 2.5), phosphite and phosphate ions were baseline resolved with retention times 6.95 ± 0.03 and 7.90 ± 0.03 min and with a total chromatographic run time 10 min. The detection limits were 1.58 and 1.74 µg P L(-1) at m/z 47, as compared to 2.18 and 2.04 µg P L(-1) at m/z 31, respectively. The results obtained in real world samples for the two detection modes were in good agreement, yet signal acquisition at m/z 47 enabled better precision without collision/reaction cell (RSD below 2%) as compared to RSD around 4% obtained at m/z 31 using He-pressurized cell (3.5 mL min(-1)).

Phosphite in sedimentary interstitial water of Lake Taihu, a large eutrophic shallow lake in China.

The seasonal occurrence and distribution of phosphite (HPO3(2-), P) in sedimentary interstitial water from Lake Taihu was monitored from 2011 to 2012 to better understand its possible link to P cycle in the eutrophic shallow lake. Phosphite concentrations ranged from < MDL to 14.32 ± 0.19 µg P/kg with a mean concentration of 1.58 ± 0.33 µg P/kg, which accounts for 5.51% total soluble P (TSP(s)) in surficial sediments (0-20 cm). Spatially, the concentrations of sedimentary phosphite in the lake's northern areas were relatively higher than those in the southern areas. Higher phosphite concentrations were always observed in seriously polluted sites. Generally, phosphite in the deeper layers (20-40 cm and 40-60 cm) showed minor fluctuations compared to that in the surficial sediments, which may be associated with the frequent exchange at the sediment-water interface. Phosphite concentrations in surficial or core sediments decreased as spring > autumn > summer > winter. Higher phosphite levels occurred in the areas with lower redox (Eh), higher P contents, and particularly when metal bonded with P to form Al-P(s) and Ca-P(s). Phosphite may be an important media in the P biogeochemical cycle in Lake Taihu and contribute to its internal P transportation.

Speciation of phosphorus oxoacids in natural and waste water samples.

Phosphorus is a key nutrient and in natural environments regulates trophic status and consequently water quality. Therefore monitoring of phosphorus content in natural and wastewater is essential. Although several phosphorus species can be found in the environment, the majority of the methods developed are for orthophosphate determination. High performance liquid chromatography (HPLC) coupled with inductively coupled plasma with atomic emission spectroscopy (ICP-AES) has been first used in this study for the speciation of the most common phosphorus oxoanions in aquatic environments: orthophosphate, phosphite, hypophosphite, pyrophosphate and tripolyphosphate. The chromatograms have been obtained by registering the phosphorous 213.618 nm emission intensity variation with time. The pH and the ionic strength of the mobile phase have been the most critical variables of the chromatographic separation. Moreover, methanol addition promotes the elution of the most retained species. Finally, by using ammonium nitrate and a gradient elution, increasing ionic strength and decreasing the pH, the separation has been achieved in 12 min. Limits of detection have been included within the 1-5 mg L(-1) range. The developed methodology has been tested with spiked tap water and effluent water of a wastewater treatment plant (WWTP) obtaining recoveries in the range of 91.5-114.1% for a 20 mg P L(-1) spike concentration.

Elucidating the redox cycle of environmental phosphorus using ion chromatography.

Historically, it was assumed that reactive, inorganic phosphorus present in pristine environments was solely in the form of orthophosphate. However, this assumption contradicts theories of biogenesis and the observed metabolic behavior of select microorganisms. This paper discusses the role of ion chromatography (IC) in elucidating the oxidation-reduction cycle of environmental phosphorus. These methods employ suppressed-IC, coupled with tandem conductivity and electrospray mass spectrometry detectors to identify and quantify phosphorus oxyanions in natural water, synthetic cosmochemical, and biological samples. These techniques have been used to detect phosphite and orthophosphate in geothermal hot springs. Hypophosphite, phosphite, and orthophosphate have been detected in synthetic schreibersite corrosion samples, and termite extract supernatant. Synthetic schreibersite corrosion samples were also analyzed for two poly-phosphorus compounds, hypophosphate and pyrophosphate, and results show these samples did not contain concentrations above the 1.3 and 2.0 µM respective 3σ limit of detection. These methods are readily adaptable to a variety of matrices, and contribute to the elucidation of the oxidation-reduction cycle of phosphorus oxyanions in the environment. In contrast to most studies, these techniques have been used to show that phosphorus actively participates in redox processes in both the biological and geological world.

Ion chromatographic determination of phosphorus soluble in different extracting media in fertilizers.

A new method based on ion chromatography (IC) was developed for the determination of phosphorus in fertilizers. Fertilizers were extracted with water, mineral acids, and 2% formic acid, 2% citric acid, and neutral ammonium citrate solutions according to European Regulation No. 2003/2003 of the European Parliament and the Council of October 13, 2003, or the Decree of the Italian Agriculture Minister of June 17, 2002; the extracts were analyzed by direct injection, after simple filtration, by IC on an IonPac AS19 (250 x 4 mm id) column, using a KOH (21-50-21 mM) gradient and suppressed conductivity detection. The calibration plot was linear over the range of 5-50 mg/L (r(2) of >0.999). The method was evaluated by comparison with a gravimetric method according to established norms. Associated uncertainty at the 95% confidence level was established as 0.47% for the determination of 3-46% P2O5 by IC. A good chromatographic separation of phosphorus forms such as phosphates and phosphites, and some other important anions like nitrates, chlorides, and sulfates present in many commercial fertilizers was also possible, with a linear response over the range of 5-50 mg/L. After a more complete validation, this IC determination of phosphorus could replace more tedious methods such as those using gravimetric determinations.

Phosphates, phosphites, and phosphides in environmental samples.

The common assumption that phosphorus occurs exclusively as phosphate in the environment is deserving of increased scrutiny. If a sample contained reduced phosphorus compounds (P in an oxidation state of less than +5), standard methods of phosphorus determination would incorrectly classify the compounds mostly as organic P, although significant fractions were sometimes misclassified as orthophosphates and condensed P. The disappearance of gaseous hydrogen phosphide (PH3) from samples was a function of solution composition, in that certain acids and metals enhanced removal whereas other constituents increased PH3 stability. No previously used extraction method could detect a significant portion of reduced phosphorus in representative samples by measuring PH3 evolution, particularly for highly recalcitrant iron phosphides. Despite analytical limitations, clear evidence was gathered that reduced phosphorus compounds can be leached from cast iron to water and that reduced phosphorus is also present in the scale (rust) that forms on the metal.