Cytoplasmic pH-Stat during Phenanthrene Uptake by Wheat Roots: A

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Cytoplasmic pH-Stat during Phenanthrene Uptake by Wheat Roots: A Mechanistic Consideration Xinhua Zhan, Xiu Yi, Le Yue, Xiaorong Fan, Guohua Xu, and Baoshan Xing Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00697 • Publication Date (Web): 29 Apr 2015 Downloaded from http://pubs.acs.org on May 5, 2015

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Environmental Science & Technology

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Cytoplasmic pH-Stat during Phenanthrene Uptake by Wheat Roots: A Mechanistic Consideration

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Xinhua Zhan,*,† Xiu Yi,† Le Yue,†,‡ Xiaorong Fan,† Guohua Xu,† and Baoshan Xing‡

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†

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Jiangsu Province, 210095, P.R. China

College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing,

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‡

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01003, United States

Stockbridge School of Agriculture, University of Massachusetts, Amherst, Massachusetts

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KEYWORDS: polycyclic aromatic hydrocarbons, cytoplasmic acidification, pH regulation,

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phenanthrene uptake, wheat roots

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ACS Paragon Plus Environment


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ABSTRACT: Dietary intake of plant-based foods is a major contribution to the total exposure of

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polycyclic aromatic hydrocarbons (PAHs). However, the mechanisms underlying PAH uptake by

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roots remain poorly understood. This is the first report, to our knowledge, to reveal cytoplasmic

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pH change and regulation in response to PAH uptake by wheat roots. An initial drop of

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cytoplasmic pH, which is concentration-dependent upon exposure to phenanthrene (a model

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PAH), was followed by a slow recovery, indicating the operation of a powerful cytoplasmic pH

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regulating system. Intracellular buffers are prevalent and act in the first few minutes of

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acidification. Phenanthrene activates plasmalemma and tonoplast H+ pump. Cytolasmic

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acidification is also accompanied by vacuolar acidification. In addition phenanthrene decreases

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the activity of phosphoenolpyruvate carboxylase and malate concentration. Moreover,

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phenanthrene stimulates nitrate reductase. Therefore, it is concluded that phenanthrene uptake

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induces cytoplasmic acidification and cytoplasmic pH recovery is achieved via physicochemical

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buffering, proton transport outside cytoplasm into apoplast and vacuole, and malate

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decarboxylation along with nitrate reduction. Our results provide a novel insight into PAH

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uptake by wheat roots, which is relevant to strategies for reducing PAH accumulation in wheat

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for food safety, and improving phytoremediation of PAH-contaminated soils or water by

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agronomic practices.

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TOC/Abstract Art

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Polycyclic aromatic hydrocarbons (PAHs) are an important group of widespread environmental

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pollutants, stemming from incomplete combustion or pyrolysis of organic matters. These

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compounds have been of major health concern due to their well-documented carcinogenicity,

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mutagenicity and toxicity to both human and non-human organisms,1,2 and have been listed as

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priority pollutants by most countries.3 Surface soil as a reservoir holds more than 90% of PAHs

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in the environment.4 The contamination of soils by PAHs can cause the subsequent

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contamination of plants grown in these soils. It has been reported that, in China, over 20% of

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staple crops contain PAHs that exceed the allowable limits.5 This may pose human health

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hazards because dietary intake has been identified as the dominant route of exposure to PAHs for

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non-smoking people, with plant-based foods constituting a major contributor to the total PAH

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intake.6-8 It is important, therefore, to understand the exact mechanisms underlying PAH uptake

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by plant roots for assessment of crop contamination and development of safe cropping systems

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and for effective phytoremediation technologies.

INTRODUCTION

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Our previous results have demonstrated that PAHs can enter crop roots via both passive and

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active transports,9 and active transport is mediated by proton/PAH symporters.10 In theory, the

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symport of proton/PAHs will result in a decrease in cytoplasmic pH. However, to date little

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information is available regarding cytoplasmic pH change during PAH uptake by plant roots. The

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cytoplasmic pH-stat relating to PAH influx into plant roots is critical to clearly elucidate the

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process of PAH uptake.

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It is well known that all living plant cells maintain a cytoplasmic pH close to neutrality. 4


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Proton-coupled cotransport processes and H+-consuming or H+-generating metabolic reactions

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will produce excess H+ or OH- ions in the cytoplasm. If these H+ or OH- ions were not removed

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physically or chemically from the cytoplasm, they would lead to enormous pH changes in the

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cytoplasm: thus the pH would decrease to 0 or increase to 14 in less than a cell generation time.

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Plants need to keep cytoplasmic pH at a relative constant level to maintain the electrochemical

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potential gradient for ion uptake and to optimize enzyme activity.11,12 A small change in

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cytoplasmic pH can severely alter metabolisms and damage cells.13 Accordingly, the

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maintenance of H+-homeostasis and cytoplasmic pH is ultimately important.

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The mechanisms contributing to intracellular pH regulation in plant cells can be essentially

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classified into two types. A metabolic-based regulatory mechanism, i.e. the biochemical pH-stat,

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is a critical component in cytoplasmic pH regulation.14,15 It relies on metabolites acting as strong

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pH buffers and pH-dependent metabolic reactions such as the carboxylation and decarboxylation

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of organic acids like malate to generate or consume H+.16 The second regulatory mechanism,

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defined as the biophysical pH-stat, is the membrane transport of H+ between the cytoplasm and

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the two main acidic compartments, the apoplast and vacuole. This is primarily facilitated by

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directly energized H+ pumps, including the P-type H+-ATPase (P-ATPase) at the plasma

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membrane, which pumps H+ into the apoplast, and the V-type H+-ATPase (V-ATPase) at the

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tonoplast, which in tandem with a second vacuolar H+ pump, the H+-pyrophosphatase (H+-PPase),

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pumps H+ into the vacuole.17 Whether these mechanisms are involved in cytoplasmic pH

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regulation with respect to PAH uptake by plant roots has not been addressed and understood.

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In this paper, we hypothesize that the symport of proton/PAHs causes a decrease in 5



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cytoplasmic pH, which subsequently recovers due to the elicitation of the biophysical and

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biochemical pH-stats. The aims of this study were i) to characterize the change in cytoplasmic

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pH during plant root uptake of PAHs; ii) to identify the mechanisms involved in the regulation of

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cytoplasmic pH in plant root cells; and iii) to understand cytoplasmic pH homeostasis in relation

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to plant root uptake of PAHs. To our knowledge this is the first report of cytoplasmic pH change

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and regulation in plant roots with respect to PAH uptake.

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MATERIALS AND METHODS

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Chemicals. Phenanthrene, a model compound of PAHs,18,19 was purchased from Fluka

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Chemical Corporation (phenanthrene purity >97%). Its molecular weight is 178.2 g mol-1, and

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water solubility is 7.3 µmol L-1 at 25 °C. All organic solvents used were of HPLC grade.

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Plant Preparation and Growth Conditions. Wheat (Triticum aestivum L.) seeds were

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surface sterilized in 10% H2O2 for 10 min. After thorough rinse with Millipore (Milli-Q, Billerica,

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MA, USA) water, they were then germinated on moistened filter paper for 4 d at 25 °C in the

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dark. The wheat seedlings were transplanted to black plastic pot containing 2500 mL

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half-strength aerated Hoagland nutrient solution for 5 d and then transferred to the full-strength

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Hoagland solution for 5 d. The nutrient solution prepared with Millipore water and the initial pH

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of the solution was adjusted to 5.5. Wheat seedlings were grown in a climate chamber under

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controlled conditions (photoperiod 16 h light/8 h dark; light intensity 400 µmol m-2 s-1; day/night 6


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temperature of 25/20 °C; relative humidity 60%). After a 10-d growth in Hoagland nutrient

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solution, the wheat seedlings were immersed in Millipore water for 24 h and then employed in

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the subsequent experiments for electrophysiology, enzyme activity and analysis of organic acids.

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Measurement of Cytoplasmic and Vacuolar pH. Double-barreled proton-selective

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microelectrodes were employed to measure pH in cytoplasm and vacuole. The microelectrodes

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were prepared as described by Walker et al.20 and Schlue et al.21 Calibration of the

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proton-selective electrodes was performed before and after each measurement. All pH calibration

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solutions contained 120 mM KCl, 10 mM NaH2PO4, and 20 mM buffer: either 5 mM

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2-(N-morpholino) ethanesulfonic acid (Mes)-H2SO4 (pH 4.0), Mes-NaOH (pH 6.0),

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3-N-morpholino propansulfonic acid-NaOH (pH 7.0), or N-tris[hydroxymethyl]methyl-

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3-amino- propanesulfonic acid-NaOH (pH 8.5).

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Calibration and intracellular pH measurements were made using a high-input impedance

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differential electrometer (model FD223; World Precision Instruments, Sarasota, FL, USA). The

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electrometer output passed via an A/D converter (Labmaster DMA/PGH; Scientific Solutions,

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Solon, Ohio, USA), at a sampling frequency of 10 Hz, to an Opus PC V microcomputer. Em (i.e.

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the reference electrode potential), Em+pH (i.e. the measured output of the pH selective electrode)

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and pH were simultaneously recorded with a three-channel chart recorder. AxoScope 10.2

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software (Molecular Devices, Sunnyvale, CA) was used to analyze the data.

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15-d-old wheat root tip was excised, and mounted in a Plexiglas chamber attached to the

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stage of an Olympus compound microscope, which was fixed to the surface of a 7



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vibration-damped table (Kinetic Systems Inc.). The Plexiglas chamber was perfused with basal

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solution (5 mM MES, 0.5 mM CaCl2, 0.05 mM NaCl, 0.05% methanol, pH 5.5) at a flow rate of

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10 mL min-1 for 2 h before the measurements. Thereafter, the double-barreled microelectrodes

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were inserted into root mature epidermal cell (1 to 2 cm from the root apex) with a hydraulically

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driven Narashige micromanipulator mounted on the microscope stage. When the resting potential

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was constant, basal solution was replaced by test solution (i.e., basal solution containing 0, 0.1,

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0.6 or 1.2 mg L-1 phenanthrene). The measurements were conducted in the dark at 25 °C.

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Plasma Membrane H+-ATPase Activity Assay. Plasma membrane vesicles were isolated

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according to Yan et al.22 Plasma membrane H+-ATPase activity was determined as described by

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Zhan et al.10 Phenanthrene concentrations treated in reaction media were 0, 0.1, 0.6 and 1.2 mg

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L-1, respectively. Plasma membrane H+-ATPase activity was expressed as µmol inorganic

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phosphate (Pi) mg-1 protein h-1. Determination of plasma membrane H+-ATPase activity is shown

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in the Supporting Information (SI).

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Vacuolar H+-ATPase and H+-Pyrophosphatase (PPase) Activity Assay. Tonoplast

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vesicles were isolated from wheat roots using differential centrifugation as described by Giannini

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and Briskin23 and Valencia et al.24 Vacuolar H+-ATPase and PPase activity was determined

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colorimetrically by measuring the release of Pi as described by Façanha and De Méis.25 The

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reaction media contained phenanthrene concentrations of 0, 0.1, 0.6 and 1.2 mg L-1. Vacuolar

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PPase activity was calculated as half of the rate of Pi released from inorganic pyrophosphate

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(PPi). Vacuolar H+-ATPase and PPase activity were expressed as µmol Pi mg-1 protein h-1.

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Measurement of vacuolar H+-ATPase and PPase activity is displayed in the Supporting

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Information (SI).

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Nitrate Reductase Activity Assay. In vivo nitrate reductase (NR) activity was measured

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after a 4-h induction with100 mM KNO3 for wheat roots. 1 g of wheat roots induced (1 cm in

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length) was vacuum infiltrated in 10 mL of reaction solution containing 200 mM KNO3, 50 mM

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trishydroxymethyl aminomethane (Tris)-HCl (pH 7.5), with phenanthrene concentrations of 0,

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0.1, 0.6 and 1.2 mg L-1. Samples were incubated in a shaking water bath for 2 h at 32 °C in the

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dark. After the incubation, 2 mL of the incubation solution was diluted with water to 4 mL. 2 mL

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of 1% sulfanilic acid in 1.5 M HCl was added to the diluted incubation solution, followed by 2

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mL of N-(1-naphthyl) ethylenediamine-HCl (200 mg L-1). After 30 min, the absorbance was

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measured at 540 nm.26 NR activity was expressed as µmol NO2 – g-1 fresh weight (FW) h-1.

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Phosphoenolpyruvate Carboxylase (PEPC) Activity Assay. 5-10 g of roots was ground in

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a pre-chilled mortar with 30 mL of extraction solution (100 mM Tris-H2SO4, 1 mM EDTA, 10

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mM MgC12, 5% v/v glycerol, 7 mM β-mercaptoethanol, 1 mM phenylmethylsulphonyl fluoride

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and 10 µg mL-1 leupetin, pH 8.2). The extract was centrifuged for 20 min at 15000 g, and the

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supernatant was centrifuged again at 100,000 g for 30 min at 4 °C. Proteins extracted were 9



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dialyzed with the same homogenization buffer solution. The PEPC activity was

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spectrophotometrically measured by monitoring NADH oxidation at 340 nm for 3 min at 25°C.

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The assay medium (3 mL) contained 10 mM MgC12, 2.5 mM phosphoenolpyruvate (PEP), 0.2

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mM NADH, 10 mM NaHCO3, 15 µg mL-1 malic dehydrogenase (MDH), 100 mM Tris- H2SO4,

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pH 7.0, 200 µL of desalted enzyme solution. The phenanthrene concentrations were 0, 0.1, 0.6

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and 1.2 mg L-1, respectively. The reaction was initiated by adding 0.2 mL 40 mM PEP and the

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change in A340 monitored. Standard protein was used as γ-globulia. Enzyme activity was

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expressed as nmol CO2 mg-1 protein min-1.

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Organic Acid Analysis. Intact wheat seedlings were incubated in Hoagland nutrient

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solution (pH 5.5) containing 0, 0.1, 0.6 or 1.2 mg L-1 phenanthrene for 4 h at 25 °C. There were

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triplicates per treatment. After harvest, root tips (2 cm in length) were excised, rinsed with

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distilled water, blotted with paper towels, weighed and immediately frozen in liquid N2 and

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stored at −70 °C for organic acid determination. The frozen roots were ground in a cold mortar

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with 2mL of cold 80% (v/v) ethanol to form slurries and samples were sonicated for 2min in an

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ice bath. The mixture was centrifuged at 10,000 ×g for 20 min at 4 °C and the pellet was

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extracted twice with 2 mL ice-cold water. The supernatants from each of these extractions were

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pooled and concentrated in a Speed Vac SC110 rotoevapopator (Thermo-Savant, Holbrook, NY,

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USA). The dried residues were dissolved in deionized water and filtered through a membrane

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filter (0.45 µm, Fisher Scientific, USA). Concentrations of organic acids were measured by

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HPLC consisted of an automatic injector (Waters 717), a binary high-pressure pump (Waters

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1525), and a UV detector (Waters 2487). Separation was performed with a Shodex RSpak

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KC-811 (ø 8.0×300 mm, 6 µm particle) column. The temperature of the HPLC column was kept

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constant at 50 °C. The used mobile phase was 6 mM perchloric acid, with a flow rate of 0.7 mL

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min-1. The injection volume was 10 µL. Organic acids were detected at 218 nm. Root tissue

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organic acid concentrations were quantified as µmol g-1 fresh weight.

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Statistical Analyses. Statistical analyses were performed with SAS software version 9.1.3

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(SAS Institute Inc., Cary, NC, USA). Enzymatic activities and concentrations of organic acids

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were subjected to one-way analysis of variance (ANOVA) and compared using the Duncan’s test

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at the 0.05 probability level. The comparison of different enzymes was conducted with paired

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t-test at 95% confidence level.

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RESULTS

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Cytoplasmic and Vacuolar pH. To evaluate the change in cytoplasmic and vacuolar pH

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during phenanthrene uptake, double-barreled proton-selective microelectrodes were employed.

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The cytoplasmic pH of wheat root cells was 7.1 to 7.3 (Figure 1a). Addition of phenanthrene

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caused acidification of the cytoplasm followed by a gradual recovery. Upon stepwise increase of

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the external phenanthrene concentration, enhanced acidification of the cytoplasm was observed.

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The decrease in cytoplasmic pH was 1.10 ± 0.05, 2.14 ± 0.19, and 2.33 ± 0.15 units at 11



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phenanthrene concentrations of 0.1, 0.6 and 1.2 mg L-1, respectively. The cytoplasmic

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acidification triggered by phenanthrene uptake was markedly dependent on phenanthrene

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concentration (i.e., the higher the phenanthrene concentration, the stronger the cytoplasmic

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acidification).

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Figure 1b shows the evolution of the vacuolar pH of wheat root cells during phenanthrene

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uptake. The vacuolar pH was 5.3 to 5.5. It dramatically decreased after adding phenanthrene.

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The vacuolar acidification showed the similar pattern as the cytoplasm, but the amplitude of

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maximum acidification of vacuole was smaller than that of cytoplasm. The values of vacuolar

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acidification were 1.11 ± 0.07, 1.26 ± 0.13, and 1.50 ± 0.09 pH units at phenanthrene

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concentrations of 0.1, 0.6 and 1.2 mg L-1, respectively.

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Activity of Plasma Membrane H+-ATPase, Vacuolar H+-ATPase and Vacuolar

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H+-PPase. To check whether the biophysical process is involved in the regulation of cytoplasmic

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pH during phenanthrene uptake, the activity of plasma membrane H+-ATPase, vacuolar

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H+-ATPase and vacuolar H+-PPase was measured (Figures 2 and 3). Phenanthrene activated the

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plasma membrane H+-ATPase and the tonoplast H+-ATPase (Figure 2). Activation of plasma

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membrane and tonoplast H+-ATPase was obviously phenanthrene-dependent in a nonlinear

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manner within concentrations of 0-1.2 mg L-1 (P

Cytoplasmic pH-Stat during Phenanthrene Uptake by Wheat Roots: A

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