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Atmospheric Environment 169 (2017) 278e286

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Atmospheric Environment

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Effects of submicron ammonium sulfate particles on the growth andyield of komatsuna (Brassica rapa L. var. perviridis)

Akira Motai a, Satoshi Nakaba b, I. Wuled Lenggoro c, Makoto Watanabe b,Yoshiharu Wada d, Takeshi Izuta b, *

a The United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japanb Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japanc Department of Chemical Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japand Faculty of Agriculture, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan

h i g h l i g h t s

� Growth and yield of komatsuna were reduced by exposure to (NH4)2SO4 particles.� Exposure to fine particles did not affect leaf chlorophyll and nitrogen contents.� Exposure to fine particles reduced stomatal conductance during the day time.

a r t i c l e i n f o

Article history:Received 11 June 2017Received in revised form29 August 2017Accepted 19 September 2017Available online 21 September 2017

Keywords:Ammonium sulfate particleBrassica rapa L. var. perviridisGrowthPM2.5

Stomatal conductance

* Corresponding author.E-mail address: izuta@cc.tuat.ac.jp (T. Izuta).

https://doi.org/10.1016/j.atmosenv.2017.09.0271352-2310/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

The aim of this study was to determine the effects of submicron ammonium sulfate (AS) particles onkomatsuna (Brassica rapa L. cv. Hakkei) plants. First, we optimized a leaf-washing method to measure theamount of AS particles deposited on the leaf surface of the plants. Then, we used this method todetermine the retention time of particles deposited on the leaf surface of the plants. We also investigatedthe effects of AS particles on the growth and yield of the plants. Almost all the AS particles deposited onthe leaf surface were removed within 1 min washing time with ultrapure water, and ion leaching fromthe leaf was relatively slow but continuous during the leaf-washing procedure. On the basis of theseresults, we determined that 1 min was a suitable washing time to remove most of the AS particles whileminimizing the influence of ion leaching from the leaf. The amount of particulate SO4

2� deposited on theleaf surface decreased over time, probably because AS particles deposited on the leaf surface deliquesced,allowing ions such as SO4

2� in the deliquescence solution to be absorbed into the leaf. The plants weregrown and exposed to AS particles for 16 days in naturally lit phytotrons. The daily mean increase in theconcentration of SO4

2� in PM2.5 by the exposure to AS particles was 22.5 mg m�3 in the phytotrons. Thegrowth and yield of the plants were significantly reduced by the exposure to AS particles. The exposureto AS particles did not affect the leaf concentrations of nitrogen and chlorophyll, but significantly reducedstomatal conductance. Therefore, stomatal closure is one of the reasons for the AS particle-induced re-ductions in the growth and yield of komatsuna plants.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

The concentration of aerosols in the atmosphere in East Asia hasincreased because of the increase in energy consumption alongwith rapid economic growth (Ohara et al., 2007; Kurokawa et al.,2013). Anthropogenic aerosols emitted by the combustion of

fossil fuels are mainly submicron particles, although the size ofaerosol particles ranges from a few nanometers to fractions of amillimeter (Hatakeyama, 2017). Submicron particles affect the at-mospheric environment not only in the region of the emissionsource, but also in other regions because they are often transportedover long distances (Andreae, 2007; Aikawa et al., 2010; Fisheret al., 2011). A simulation using a chemical transport model pre-dicted that the proportional contribution of non-sea-salt SO4

2� (nss-SO4

2e) from China to the total annual mean nss-SO42e concentration

A. Motai et al. / Atmospheric Environment 169 (2017) 278e286 279

in Japan was 50e70% (Aikawa et al., 2010). Thus, the atmosphericenvironment in Japan is relatively dominated by transboundary airpollutants such as submicron particles from the Asian continent(Shimohara et al., 2001; Takami et al., 2007; Kaneyasu et al., 2014;Hatakeyama, 2017). The main component of fine particulate matter(i.e. PM2.5) in Japan is SO4

2�, which is transported as ammoniumsulfate (Kaneyasu et al., 2014).

Submicron ammonium sulfate (AS) particles deposited on thesurface of plants may affect their physiological functions andgrowth. Therefore, several studies have focused on the effects ofsubmicron AS particles on crops and trees. Gmur et al. (1983) re-ported that exposure of pinto bean (Phaseolus vulgaris) to AS par-ticles in the range of 15e25mgm�3 for 13 days did not significantlyaffect dry mass, but led to visible foliar injury such as chlorosis.Martin et al. (1992) investigated the effects of acute exposure to ASparticles (diameter, 0.25 mm) on net photosynthetic rate of bur oak(Quercus macrocarpa), soybean (Glycine max) and maize (Zea mays).After exposure to AS particles at approximately 600 mg SO4

2� m�3

for 2 h, net photosynthetic rate was significantly reduced in maize,but not in bur oak and soybean. However, these studies wereconducted for relatively short periods with high concentrations ofparticles, which are unlikely to occur naturally. Yamaguchi et al.(2014) studied the effects of long-term exposure to submicron ASparticles at realistic levels on the growth of Japanese forest treespecies, and found that net photosynthetic rate of Cryptomeriajaponica was more sensitive to submicron AS particles than that ofFagus crenata, Castanopsis sieboldii, or Larix kaempferi. At present,however, there is no information on the effects of long-termexposure to submicron AS particles at realistic levels on thegrowth and yield of crop plants such as leafy vegetables.

Previous studies have shown that hygroscopic particles such asAS particles deliquesce on the leaf surface and establish thincontinuous films connecting the leaf surface to the stomatal cavity(Burkhardt et al., 2001, 2012). Such films are thought to contributeto the uncontrollable water loss through the stomata and adverselyaffect water relations in leaves (Burkhardt, 2010; Pariyar et al.,2013; Burkhardt and Pariyar, 2016). The results of previousstudies have suggested that long-term exposure to submicron ASparticles may adversely affect water relations in leaves and thusreduce stomatal conductance. Therefore, it is necessary to investi-gate the effects of long-term exposure to submicron AS particles atrealistic levels on the physiological functions, including the sto-matal conductance, of crop plants.

In Japan, there is a recent concern that the productivity, qualityand safety of crops are adversely affected by relatively high con-centrations of submicron AS particles although the facts on the realimpacts of the particles on crops under field conditions are still notclarified. It is considered that the deposited AS particles on the leafsurface deliquesce and influence the plants by forming thin waterfilms connecting leaf surface and interior. Leaf is very importantplant organ in crop productivity such as carbon gain by photo-synthesis (Lambers et al., 2008). Therefore, there is a possibility thatleafy vegetables are relatively susceptible to AS particles becausethe fraction of leaf to the whole-plant in leafy vegetables is rela-tively large as compared to that in the other types of crops. In Japan,komatsuna (Brassica rapa var. perviridis) is a popular leafy vegetableand all above-ground part of the plants is edible. Recently, relativelyhigh concentrations of AS particles, mainly contributed by inflowfrom the Asian continent, have been detected in Japan, especially inearly spring (Itahashi and Hayami, 2015). Because komatsunaplants is cultivated year-round in Japan, there is the possibility thatthe plants cultivated under relatively high concentrations of ASparticles reduces its growth due to the reduction of leaf net CO2uptake rate induced by stomatal closure. To date, however, therehave been no reports on the effects of submicron AS particles on the

yield and physiological functions of komatsuna plants.To investigate the effects of fine AS particles on komatsuna

plants, we first needed to optimize a leaf-washing method tomeasure the amounts of AS particles deposited on the leaf surfaceof the plants. The leaf-washing technique has been widely used tomeasure the amount of water-soluble particles on the surfaces ofplant leaves or needles (Sæbø et al., 2012; Watanabe et al., 2013;Yamaguchi et al., 2014; Burkhardt and Pariyar, 2016). However,relatively long washing time may increase the amount of ionsleaching from the leaf interior, leading to overestimates of theamount of AS particles deposited on the leaf surface. Furthermore,it is important to understand the retention time of AS particles onthe leaf surface because AS particles may deliquesce on the leafsurface and be absorbed into the leaf interior. In the present study,therefore, we determined a suitable washing time to measure theamount of AS particles deposited on the leaf surface, and then usedthis method to clarify the retention time of AS particles on the leafsurface of komatsuna plants. Then, we investigated the effects ofsubmicron AS particles at realistic levels on the growth and yield ofkomatsuna plants.

2. Materials and methods

2.1. Establishment of leaf-washing technique

An experiment was conducted to determine a suitable leafwashing time to estimate the amount of AS particles deposited onthe leaf surface of komatsuna plants. Then, this methodwas used toclarify the retention time of AS particles on the leaf surfaces of theplants.

2.1.1. Plant materials and exposure to submicron AS particlesSeeds of komatsuna (Brassica rapa L. cv. Hakkei) plants were

sown in 2-L pots (diameter: 14 cm, height: 18 cm) filled withgranular culture soil (N:P:K¼ 200:1 223:150 mg L�1). After sowing,the 8 pots were transferred into a naturally lit phytotron (approx-imately 6 m3 with an air-conditioner system, Koito Industries Co.Ltd., Kanagawa, Japan) located at Tokyo University of Agricultureand Technology (Fuchu, Tokyo, Japan). The ability of air circulationin the phytotron was 60 m3 h�1. In the phytotron, air temperatureand relative air humidity were maintained at 25.0 ± 1.0�C/18.0 ± 1.0 �C (6:00e18:00/18:00e6:00) and 70 ± 5%, respectively.The plants were carefully irrigated with tap water withoutsplashing the leaf surface to avoid washing off the deposited par-ticles. Nine days after sowing (9 DAS), the plants were thinned toone plant per pot. At 26 DAS, 4 of the 8 plants were transferred toanother phytotron and were exposed to submicron AS particles (AStreatment). The rest 4 plants were not exposed to AS particles(control treatment). After finishing the exposure to AS particles, thethird to fifth leaves of the plants placed in the phytotrons for AS andcontrol treatments were immediately washed with ultrapurewater(18.2 MU cm at 25 �C). These leaves were located at the upper partof the plant and fully expanded at 26 DAS. The AS-treated andcontrol plants were grown in the same phytotron except for theduration of the exposure to AS particles. The circulation of air andthe air-conditioner system in the phytotron for AS treatment werestopped during the exposure of the plants to AS particles, and werealso stopped for the same duration in the phytotron for controltreatment. To avoid the adverse effects of high air temperature onthe plants, the exposure to AS particles was conducted two times at19:00e20:00 and 21:00e22:00 with relatively low ambient airtemperature.

In the present study, submicron AS particles were generatedfrom 0.1% (w/v) ammonium sulfate solution using an ultrasonicnebulizer (NE-U17, Omron Healthcare Co. Ltd., Tokyo, Japan). The

A. Motai et al. / Atmospheric Environment 169 (2017) 278e286280

particles generated from the AS solution were dried using a tapeheater at 100 �C and a diffusion dryer with granular silica gels.Komatsuna plants were exposed to AS particles from approxi-mately 1.0 m above the plants with a slight downward wind pro-vided by a fan (6.0 cm in length � 6.0 cm in width � 2.5 cm inheight, 1.8 W, TUDC12Z4, Nidec Servo Corp., Gunma, Japan) whichwas attached on the outlet of the aerosol exposure system. Themaximum wind speed of the fan was 0.55 m3 min�1. To uniformlyexpose the plants to AS particles, two aerosol exposure systemswere set on a rotating table with 1 turn per minute. Immediatelyafter the exposure of the plants to AS particles, the air inside thephytotrons for AS and control treatments was cleaned up by HEPAfilter unit (BV-R01MH1L, Panasonic Ecology Systems Co. Ltd., Aichi,Japan) for 10 min. Subsequently, air circulation by the air-conditioner system in the phytotrons for AS and control treat-ments was re-started. The PM2.5 inside the phytotrons for AS andcontrol treatments were collected using a filter holder with animpactor (flow rate at 20 L min�1) for 24 h. The collected PM2.5 onthe filters were extracted into ultrapure water by ultrasonicextraction, and the concentration of SO4

2� in the washing water wasmeasured using an ion chromatography (883 Basic IC plus, Met-rohm Japan Ltd., Tokyo, Japan). The daily average concentrations ofSO4

2� in PM2.5 in the phytotrons for AS and control treatments were112.5 and 9.3 mg m�3, respectively. The difference of the concen-tration of SO4

2� in PM2.5 between AS and control treatments wasonly achieved by the exposure of the plants to submicron AS par-ticles (two times of 1 h exposure). There was no difference of at-mospheric concentration of AS particles in the phytotron betweenAS and control treatments during the other periods (non-exposureperiod). Based on these assumptions, we calculated the concen-tration of SO4

2� in PM2.5 during the exposure of the plants to ASparticles as 1.2 mg m�3.

Using an aerosol measurement system based on a differentialmobility analyzer and a particle counter (SMPS, TSI Incorporated,Shoreview, MN, USA), the mean size of AS particles can bemeasured on-line in the range of a submicron size (around300 nm). The image of AS particles deposited on an aluminumsheet (Fig. 1) was obtained using a field-emission scanning electronmicroscope (FE-SEM; JSM-7100F, JEOL Co. Ltd., Tokyo, Japan) at anaccelerating voltage of 5 kV.

2.1.2. Measurement of amount of AS particles on leaf surfaceAt 26 DAS, the amount of SO4

2� on the leaf surface of komatsunaplants was determined by a leaf washing technique with ultrapure

Fig. 1. A field-emission scanning electron micrograph of ammonium sulfate (AS)particles deposited on aluminum sheet. AS particles were highlighted by arrows. Scalebar equals 1 mm.

water (Yamaguchi et al., 2014). Immediately after finishing theexposure of the plants to submicron AS particles (i.e. 22:00), thethird to fifth leaves of the plants in AS and control treatments (4plants per treatment) were sampled. Separated three leaves perplant were placed in plastic bags and washed gently with 150 mlultrapure water. At 15 s, 30 s, 1 min, 2 min, 3 min and 6 min afterstarting the leaf washing, the washing water was sampled andfiltered through a PTFE membrane (0.20 mm, DISMIC®-13HP, ToyoRoshi Kaisha, Ltd., Tokyo, Japan). Then, the concentrations of SO4

2�

and NH4þ in the washing solution were determined by the ion

chromatography. After washing the leaves, the area of each leaf wasmeasured with an area meter (AAM-8, Hayashi Denko Co. Ltd.,Tokyo, Japan). The amounts of SO4

2� and NH4þ deposited on the leaf

surface were expressed a unit leaf area basis. The difference in theamount of SO4

2� or NH4þ on the leaf surface between AS particle-

exposed and control plants was regarded as the amount of ASparticles deposited on the leaf surface by the exposure to submi-cron AS particles.

2.1.3. Observation of submicron AS particles on leaf surfaceTo investigate the trend in the amount of AS particles deposited

onto the leaf surfaces of komatsuna plants, leaf washing was con-ducted at 0, 9, 19 and 44 h after the final exposure (HAE) to sub-micron AS particles. In this experiment, seeds of komatsuna plants(cv. Hakkei) were sown in 2-L pots filled with granular culture soil(44 pots in total). After sowing, all the pots were transferred into anaturally-lit phytotron. At 9 DAS, the plants were thinned to oneplant per pot. At 26 DAS, the half of the plants (22 plants) weretransferred to another phytotron and were exposed to AS particles(AS treatment). The rest 22 plants were not exposed to AS particles(control treatment). Growth condition and aerosol exposure systemin the phytotrons were described in section 2.1.1. Leaf washing at 0,9, 19 and 44 HAE was conducted at 22:00, 7:00, 17:00 and 18:00,respectively. The 5 plants were analyzed for each treatment at eachwashing time, thus, totally 40 plants were used. The rest 4 plants (2plants per treatment) were used for the observation of the leafsurface. The amounts of AS particles on the leaf surface of the plantsplaced in AS and control treatments were determined as describedin section 2.1.2. In this experiment, the leaves were washed for1 min. The difference in the amount of SO4

2� between AS particle-exposed and control plants was calculated at each washing time,and was regarded as the amount of SO4

2� deposited onto the leafsurface as the result of exposure to submicron AS particles. At 9 and19 HAE (1 plant per treatment at each time), submicron AS particlesdeposited on the leaf surface were observed by the FE-SEM(accelerating voltage, 5 kV). The sampled leaves were air-dried ina desiccator for at least 1 week. The specimens were mounted onstubs and then coated with platinum (Pt) with a sputter coater. Theelemental compositions of particles on the leaf surface weredetermined with an energy dispersive X-ray spectrometer (EDX;JED-2300, JEOL Co. Ltd., Tokyo Japan). This experiment had threeexperimental replicates.

2.2. Growth experiment

A growth experiment was conducted to clarify the effects ofsubmicron AS particles on the growth and yield of komatsunaplants.

2.2.1. Plant materials and growth conditionsOn 4 May 2016, seeds of komatsuna (cv. Hakkei) plants were

sown in 36 pots filled with granular culture soil. After sowing, allthe pots were transferred into a naturally-lit phytotron and grownunder the conditions described in section 2.1.1. At 9 DAS when thethird leaf had emerged, the plants were thinned to one plant per

A. Motai et al. / Atmospheric Environment 169 (2017) 278e286 281

pot. The leaf area and dry mass of leaf, stem and roots of 4 thinnedout plants were determined as initial biomass harvest. The 36 potswere transferred into 6 naturally-lit phytotrons (6 pots per phyto-tron). For 16 days from 10 to 25 DAS, the plants grown in the 3phytotrons were exposed to submicron AS particles three times perday during 5:00e6:00, 17:30e18:30 and 19:00e20:00 (AS treat-ment), and the plants in the other 3 phytotrons were left untreated(control treatment). In AS treatment, one AS particle-exposuresystem per phytotron was installed. To uniformly expose theplants to AS particles, the aerosol exposure system was set on arotating table with 1 turn per minute. The 6 plants per phytotronwere harvested at 26 DAS as the final harvest. In general, thegrowth period from sowing to harvest of komatsuna plants isapproximately 26 days under field conditions in summer in Japan.

The details of AS particle-exposure system are described insection 2.1.1. The concentration of SO4

2� during the exposure to ASparticles was not measured, but the concentration of SO4

2� in PM2.5increased by the exposure to AS particles inside the phytotrons wascalculated by using a method described below (Yamaguchi et al.,2014). The PM2.5 inside two phytotrons were collected using a fil-ter holder with an impactor (flow rate at 20 L min�1) for 24 h. Theexposure of komatsuna plants to AS particles was conducted in onephytotron and not in another phytotron. After the exposure of theplants to AS particles, exposure time and reduction in the volume of0.1% AS solution were recorded. Using these records, theoreticalexposure-induced increase in the concentration of SO4

2�

([SO42�]Theo) inside the phytotronwas calculated. The filters used for

the collection of PM2.5 from the air in the two phytotrons werewashed with ultrapure water and SO4

2� concentration in thewashing solutionwas measured using the ion chromatography. Thedifference in the amount of SO4

2� between the filters was assumedto be the actual increase in SO4

2� concentration in PM2.5 by theexposure to AS particles ([SO4

2�]Act). A significant linear relationshipwas obtained between [SO4

2�]Theo and [SO42�]Act (R2 ¼ 0.818,

p < 0.05, n ¼ 8). In the present study, exposure time and reductionin the volume of 0.1% AS solution were recorded to calculate[SO4

2�]Theo in each operation of exposure to AS particles. Using therelationship between [SO4

2�]Theo and [SO42�]Act, [SO4

2�]Act was esti-mated from [SO4

2�]Theo. For 16 days from 10 to 25 DAS, the dailyaverage concentration of SO4

2� in PM2.5 increased by the exposureto AS particles inside the three phytotrons for AS treatment was22.5 mg m�3. The difference of the concentration of SO4

2� in PM2.5between AS and control treatments was only achieved by theexposure of the plants to AS particles (three times of 1 h exposure).There was no difference of atmospheric concentration of AS parti-cles between AS and control treatments in the other periods (non-exposure period). Based on these assumptions, we calculated theconcentration of SO4

2� in PM2.5 during the exposure of the plants toAS particles as 180.0 mg m�3.

The photosynthetic photon flux density (PPFD) outside thephytotrons was monitored using a quantum sensor (LI-190SA, LI-COR Inc., Lincoln, NE, USA), and the daily mean PPFD from 0 to 25DAS was 417 mmol m�2 s�1. Light transmittance in the phytotronwas reduced to approximately 86% of ambient radiation. Komat-suna plants were carefully irrigated with tap water withoutsplashing the leaf surface to avoid washing off the AS particles.

2.2.2. Measurement of amount of AS particles on leaf surfaceAt the end of experimental period, 4 komatsuna plants per

phytotron were randomly selected for the measurement of theamount of AS particles deposited on the leaf surface by the methoddescribed in section 2.1.2 with a leaf-washing time of 1 min.

2.2.3. Measurements of growth and yieldAt the initial (9 DAS) and final harvests (26 DAS), komatsuna

plants were separated into the leaves, stem and roots. The freshweight of the above-ground part (leaves and stem) was measuredas the yield of the plants. The plant parts were dried at 80 �C for 1week and weighed. According to the method of Hunt et al. (2002),relative growth rate (RGR) of the plants during the 9e26 DAS wascalculated as follows:

RGR ¼ ðlnW2 � lnW1Þðt2 � t1Þ

where t1 and t2 are the days of initial and final harvests, respec-tively, and W1 and W2 are the whole-plant dry mass at t1 and t2,respectively. The RGR can be expressed as the product of netassimilation rate (NAR) and leaf area ratio (LAR) as follows:

RGR ¼ NAR � LAR

NAR and LAR were calculated as follows:

NAR ¼ ðW2 �W1Þ ðlnLA2 � lnLA1Þðt2 � t1Þ ðLA2 � LA1Þ

LAR ¼ ðLA2 � LA1Þ ðlnW2 � lnW1ÞðlnLA2 � lnLA1Þ ðW2 �W1Þ

where LA1 and LA2 are leaf area per plant at t1 and t2, respectively.

2.2.4. Measurements of leaf concentrations of SO42�, NH4

þ,chlorophyll and nitrogen

The leaves of komatsuna plants used for the measurements ofleaf concentrations of SO4

2�, NH4þ, chlorophyll and nitrogenwere the

same as those used for the leaf washing described in section 2.2.2.To determine the leaf concentrations of SO4

2�, NH4þ, chlorophyll and

nitrogen, the third leaf washed with ultrapure water was sampledat the end of experimental period (26 DAS). After leaf washing,three and two leaf disks (1.75 cm2 per disk) per washed leaf weresampled for the measurements of SO4

2� and NH4þ concentrations,

and chlorophyll concentration, respectively. The disks wereimmediately frozen in liquid nitrogen and stored at �80 �C untilanalysis.

To determine the leaf concentrations of SO42� and NH4

þ, thestored leaf samples were homogenized to a fine powder in liquidnitrogen with a mortar and pestle before adding 3 ml ultrapurewater. The homogenate was centrifuged at 16,000 g for 10 min at4 �C. The supernatant was filtered through an Amicon Ultra-0.5Centrifugal Filter (MWCO, 3000 Da; Merck Millipore Ltd, Darm-stadt, Germany). The concentrations of SO4

2� and NH4þ were

measured by the ion chromatography and were expressed on a unitleaf area basis.

Chlorophyll was extracted from the stored leaf samples in 6 mldimethyl sulfoxide (DMSO) in a test tube. The samples were incu-bated for 60 min at 65 �C in a water bath in the dark, and then theabsorption of the extract solution was measured at 664.9 nm and648.2 nm with a spectrophotometer (UV mini 1240, Shimadzu Co.,Kyoto, Japan). The chlorophyll concentration was calculated ac-cording to the method of Barnes et al. (1992) and was expressed ona unit leaf area basis.

To measure the leaf nitrogen concentration, the dried third leafwas ground finely and the nitrogen concentration was measuredwith a CN analyzer (MT-700, Yanaco Co. Ltd., Tokyo, Japan). Thenitrogen concentration in the leaf was expressed on a unit leaf areabasis.

2.2.5. Measurement of stomatal conductanceStomatal conductance to H2O of the third leaf of komatsuna

Fig. 2. The amount of SO42� on the leaf surface and wash-off rate of SO4

2� from the leafsurface of komatsuna plants as a function of leaf washing time. Each symbol representsthe mean of four determinations. The asterisk shows significant difference betweenammonium sulfate (AS) and control treatments (Student's t-test; *p < 0.05, **p < 0.01).Vertical bars indicate standard deviation.

A. Motai et al. / Atmospheric Environment 169 (2017) 278e286282

plants was measured during 16e18 DAS with a leaf porometer (SC-1, Decagon Device Inc., Pullman, WA, USA). Because the direction ofexposure of the plants to submicron AS particles was from above,stomatal conductance was measured on the adaxial surface of thethird leaf (gad). Stomatal density of third leaf of the plants wasmeasured in the preliminary experiment by the method of Hiroseet al. (1992). The average stomatal densities (±standard deviation,n ¼ 9) on the adaxial and abaxial leaf surfaces were 127 ± 23 and265 ± 43 mm�2, respectively. The PPFD on the adaxial leaf surfacewas simultaneously monitored during the gad measurements witha light meter (LI-250A, LI-COR Inc., Lincoln, NE, USA). To obtain gadunder various light intensities, gad was measured during5:00e18:00. Measurements of stomatal conductance were con-ducted under the normal growth conditions in the phytotrons. TheAS or control treatment had 3 chamber replications. In eachchamber, 2 plants were randomly selected and measured gad fourtimes per plant, thus, 24 determinations were obtained in eachtreatment.

2.3. Statistical analysis

Statistical analyses were performed with the IBM® SPSS®

Advanced Statistics 19 software package. Student's t-test was usedto identify difference between AS and control treatments. The PPFDwas ln-transformed and the regression line between PPFD and gadwas obtained prior to analysis. A standardizedmajor axis techniquewas applied using (S)MATR software (Version 2; see http://www.bio.mq.edu.au/ecology/SMATR/) to test the effects of submicronAS particles on the slopes and intercepts of the regression lines. Apermutation test and ANOVA in (S)MATR were used to test thehomogeneity of slopes and intercepts, respectively. When no sig-nificant difference was observed between the slopes of theregression lines, we applied a common slope and analyzed thedifferences in the intercepts of the regression lines.

3. Results

3.1. Determination of the amount of SO42� on leaf surface by leaf

washing

Estimated amount of SO42� on the leaf surface of komatsuna

plants tended to increase with washing time in both AS and controltreatments (Fig. 2a). The estimated amount of SO4

2� on the leafsurface of the plants in AS treatment was significantly higher thanthat in control treatment at all washing time. Wash-off rate of SO4

2�

from the leaf surface was significantly higher for the plants in AStreatment than for those in control treatment at 15 s and 30 swashing time (Fig. 2b). With longer washing times (�1 min), thewash-off rates of SO4

2� did not differ significantly between AS andcontrol treatments.

3.2. Time trend of the amount of SO42� deposited on leaf surface

The amount of SO42� deposited on the leaf surface of komatsuna

plants had decreased by approximately 60% at 9 HAE (Fig. 3). After 9HAE, the rate of the decrease in the amount of SO4

2� on the leafsurface became slower, and the amount of SO4

2� was decreased byapproximately 20% at 44 HAE.

Fig. 4 shows FE-SEM micrographs of AS particles deposited onthe leaf surface of komatsuna plants, and X-ray spectra that wereobtained from the particles and the leaf surface by point analyses.The high Pt peaks were due to the Pt pre-treatment using thesputter coater before the observations. At 9 HAE, the spectrum fromthe particles showed a relatively high S peak compared with Cl, K orCa peaks (Fig. 4b), although the spectrum from the leaf surface (as

the background) showed a relatively low S peak compared with Cl,K or Ca peaks (Fig. 4c). At 19 HAE, the spectrum from the particlesdid not show a relatively high S peak compared with Cl, K or Capeaks (Fig. 4e and f).

3.3. Effects of submicron AS particles on the amount of SO42� and

NH4þ on leaf surface, leaf components, stomatal conductance and

growth of komatsuna plant

At the final harvest, the amount of SO42� on the leaf surface of

komatsuna plants in AS treatment tended to be higher than that incontrol treatment, although the difference was not significant(Table 1). NH4

þ could not be detected on the leaf surface of theplants.

Table 2 shows the effects of submicron AS particles on leaf area,dry mass of plant organs, whole-plant dry mass, ratio of root drymass to shoot dry mass (R/S) and yield of komatsuna plants at thefinal harvest. Leaf area, leaf and stem dry mass, whole-plant drymass and yield of the plants grown in AS treatment were signifi-cantly lower than those of the plants grown in control treatment.There were no significant effects of submicron AS particles on root

Fig. 3. Time trend of relative amount of SO42� on the leaf surface of komatsuna plants.

The amount of SO42� on the leaf surface was calculated by subtracting the amount of

SO42� on the leaf surface in control treatment from that in ammonium sulfate (AS)

treatment. Relative amount of SO42� was expressed by regarding the amount of SO4

2� at0 h after the final exposure to AS particles (HAE) as 1.0 (the absolute value at 0 HAEwas approximately 0.880 nmol cm�2). Each symbol represents the mean of threeexperimental replicates. Vertical bars indicate standard deviation.

A. Motai et al. / Atmospheric Environment 169 (2017) 278e286 283

dry mass and the R/S. The exposure of the plants to submicron ASparticles resulted in reduction in RGR and NAR, but in increase inLAR (Table 3).

As shown in Table 4, the exposure to submicron AS particles didnot significantly affect the concentrations of NH4

þ, chlorophyll andnitrogen in the leaves of komatsuna plants.

The relationship between gad and PPFD of komatsuna plants at16e18 DAS is shown in Fig. 5. There was no significant difference inthe slope of the regression lines between AS particle-treated andcontrol plants. However, the intercept of the regression line in ASparticle-treated plants was significantly lower than that in controlplants.

4. Discussion

4.1. Determination of the amount of AS particles deposited on leafsurface of komatsuna plants

When estimating the amount of SO42� deposited on the leaf

surface of komatsuna plants by leaf washing, the estimated amounttended to increase with washing time irrespective of treatments(Fig. 2a). When a leaf-washing technique is used to measure theamount of water-soluble particles on the leaf surface, the measuredvalue may include ions leached from the leaf interior (Lindberg andLovett, 1985; Rea et al., 2000). Rea et al. (2000) reported that dryparticles deposited on the surface of tree foliage were washed offrapidly, whereas ions leached slowly and continuously from thefoliage interior during the entire washing period. In the presentstudy, the wash-off rate of SO4

2� from the leaf surface was signifi-cantly higher for komatsuna plants in AS treatment than for thosein control treatment at 15 s and 30 s washing time (Fig. 2b). Becausethe control plants were not artificially exposed to AS particles,significantly higher wash-off rate of SO4

2� from the plants in AStreatment than that from the plants in control treatment withwashing times of <1 min was considered to represent the wash-offof AS particles deposited on the leaf surface. On the other hand, thewash-off rates of SO4

2� with longer washing times (>1 min) did notdiffer significantly between AS and control treatments. It indicated

that the ions washed off during longer washing times were leachedfrom the leaf interior (Fig. 2b). These results indicated that0.5e1.0 min was a suitable washing time to measure the amount ofSO4

2� deposited on the leaf surface of komatsuna plants using theleaf-washing technique, because almost all AS particles werewashed-off from the leaf surface and there was only a small in-fluence of ions leaching from the leaf interior.

At 9 HAE, the shape of particles observed on the leaf surface ofkomatsuna plants was similar to that of AS particles deposited onan aluminum sheet (Figs.1 and 4a), and the X-ray spectrum analysisindicated that the particle included S compounds (Fig. 4b and c).From these results, the particles observed on the leaf surface at 9HAE were regarded as the AS particles generated by the exposuredevice. Conversely, the X-ray spectrum from the particles at 19 HAEindicated that most of the particles hardly included S compounds(Fig. 4e and f). Hygroscopic particles such as AS particles deliquesceon the leaf surface, and resulting the formation of thin AS filmsbecause transpiration may enhance relative humidity in the leafboundary layer (Burkhardt, 2010). If deposited AS particles deli-quesced on the leaf surface of komatsuna plants and more homo-genously distributed to form thin AS films, it is considered that theS peaks obtained by EDX analysis become smaller. On the otherhand, it is known that the deliquesced particles establish liquidwater films on the stomatal walls that connect the leaf surface tothe interior, and water and/or ions can be transported through thisconnection (Burkhardt, 2010). Furthermore, ions in the solution onthe leaf surface can enter the leaf interior through the cuticle andstomatal pores (Eichert and Goldbach, 2008). The reduction in SO4

2�

due to the stomatal absorption is seemed to consist with the resultsof the quantitative analysis of the amount of AS particles depositedon the leaf surface of komatsuna plants (Fig. 3). From these results,it is considered that the lower S peak obtained by EDX analysis at 19HAE was attributed to the formation of thin AS films on the leafsurface and the stomatal absorption of SO4

2� into the leaf interior ofthe plants.

It was reported that the absorption of solution from the leafsurface into the leaf interior was faster when stomata are open(Eichert and Goldbach, 2008). Therefore, we assumed the rate ofthe decrease in the amount of SO4

2� deposited on the leaf surface ofkomatsuna plants would be higher during the light period (9e19HAE) than during the dark period (0e9 HAE), because the stomatalaperture is greater and transpiration rate is higher during the lightperiod than during the dark period. However, we could not obtainresults to confirm this assumption (Fig. 3). This indicates that theepidermal transpiration of komatsuna plants during the darkperiod was sufficient to induce deliquescence of AS particlesdeposited on the leaf surface. Furthermore, the amphistomatousleaves of the plants may have contributed to high absorption rate ofSO4

2� into the leaf interior, because the absorption rate of deli-quescent salt solution is known to be higher on stomatous leafsurfaces than on astomatous leaf surfaces (Will et al., 2012). Fromthese results, we conclude that the retention time of submicron ASparticles deposited on the leaf surface of komatsuna plants is lessthan 19 h.

4.2. Effects of submicron AS particles on growth and yield ofkomatsuna plants

In the growth experiment, the daily average concentration ofSO4

2� in PM2.5 increased by the exposure to submicron AS particleswas 22.5 mg m�3 for 16 days from 10 to 25 DAS. The monthlyaverage concentration of SO4

2� in PM2.5 is usually lower than10 mg m�3 in Japan (Chatani et al., 2011), but the concentration insome areas facing the Asian continent can exceed 20 mg m�3

(Kaneyasu et al., 2014). In the growth experiment, therefore,

Fig. 4. Field-emission scanning electron micrographs of ammonium sulfate (AS) particles deposited on the leaf surface of komatsuna plants and X-ray spectra from the spotshighlighted by arrows at 9 (a, b, c) and 19 (d, e, f) hours after the final exposure to AS particles (HAE). b and e; X-ray spectra from particles at 9 and 19 HAE, respectively. e and f; X-ray spectra from the leaf surface at 9 and 19 HAE, respectively. Scale bars equal 1 mm.

Table 1Amount of SO42� and NH4þ on the leaf surface of komatsuna plants at the finalharvest.

Treatment SO42

(nmol cm�2)NH4

þ

(nmol cm�2)

Control 4.05 (0.06) n.d.AS 6.22 (2.25) n.d.

t etest n.s. e

Each value is the mean of three chamber replicates, and the standard deviation isshown in parentheses.Student's t-test: n.s., not significant.n.d.: not detectable.

A. Motai et al. / Atmospheric Environment 169 (2017) 278e286284

komatsuna plants were exposed to realistic levels of submicron ASparticles.

The amount of SO42� deposited on the leaf surface of komatsuna

plants after a single exposure to submicron AS particles wasapproximately 0.375 nmol cm�2 in the growth experiment. How-ever, no significant increase in the amount of SO4

2� on the leafsurface by the exposure to AS particles was detected (Table 1). Theamount of SO4

2� deposited on the leaf surface decreased over time,

and so there was very little SO42� on the leaf surface at 19 HAE

(Fig. 3). Therefore, it is possible that most of submicron AS particlesdeposited on the leaf surface of komatsuna plants were absorbedinto the leaf interior because leaf washing was conducted at least11 h after the final exposure to AS particles.

The growth and yield of komatsuna plants were significantlyreduced by the exposure to submicron AS particles for 16 days(Table 2). It indicates that submicron AS particles can reduce thegrowth and yield of komatsuna plants in Japan, especially thosecultivated in areas facing the Asian continent where relatively highatmospheric concentrations of submicron AS particles weredetected. According to the growth analysis (Table 3), the ASparticle-induced reduction in the RGR of the plants was mainlyattributed to a decrease in the NAR. Therefore, the AS particle-induced reduction in the whole-plant dry mass was due to thedecrease in net carbon assimilation rate per unit leaf area.

The leaf net carbon assimilation rate is mainly limited by CO2diffusion from the atmosphere into the leaf interior and into thecarboxylation site in the chloroplasts, and biochemical reactions inthe chloroplasts (Lambers et al., 2008). Yamaguchi et al. (2014)reported that net photosynthetic rate in the previous-year

Table 2Effects of submicron ammonium sulfate (AS) particles on leaf area, dry mass of each plant organ, whole-plant dry mass, ratio of root dry mass to shoot dry mass (R/S) and yieldof komatsuna plants at the final harvest.

Treatment Leaf area (cm2) Dry mass (g) R/S (g g�1) Yield (g)

Leaf Stem Roots Whole-plant

Control 891 (24) 3.14 (0.07) 1.08 (0.07) 0.81 (0.05) 5.03 (0.18) 0.19 (0.01) 57.1 (1.8)AS 836 (11) 2.76 (0.08) 0.94 (0.01) 0.74 (0.06) 4.44 (0.10) 0.20 (0.02) 51.5 (1.8)

t etest * ** * n.s. ** n.s. *

Each value is the mean of three chamber replicates, and the standard deviation is shown in parentheses.Student's t-test: *p < 0.05, **p < 0.01, n.s., not significant.

Table 3Effects of submicron ammonium sulfate (AS) particles on relative growth rate (RGR),net assimilation rate (NAR) and leaf area ratio (LAR) of komatsuna plants during the9e26 days after sowing.

Treatment RGR NAR LAR

(g g�1 day�1) (g m�2 day�1) (m2 kg�1)

Control 0.286 15.3 18.7AS 0.279 14.2 19.7

Table 4Effects of submicron ammonium sulfate (AS) particles on the concentrations ofSO42�, NH4þ, nitrogen (Narea) and chlorophyll (Chl aþb) in the third leaf ofkomatsuna plants at the final harvest.

Treatment SO42- NH4

þ Narea chl aþb

(mmol cm�2) (nmol cm�2) (mmol cm�2) (mg cm�2)

Control 1.88 (0.15) 13.0 (0.6) 14.8 (0.7) 44.8 (2.0)AS 1.74 (0.01) 15.4 (4.4) 14.8 (1.5) 46.5 (1.5)

t etest n.s. n.s. n.s. n.s.

Each value is the mean of three chamber replicates, and the standard deviation isshown in parentheses.Student's t-test: n.s., not significant.

Fig. 5. Relationship between stomatal conductance of adaxial surface of the third leaf(gad) of komatsuna plants and ln-transformed photosynthetic photon flux density(PPFD; mmol m�2 s�1) at 16e18 days after sawing. Permutation test and ANOVA wereused to test the homogeneity of slopes and intercepts of regression lines, respectively(*p < 0.05; n.s.; not significant). Because no significant difference in the slopes wasdetected between ammonium sulfate (AS) and control treatments, we used a commonslope.

A. Motai et al. / Atmospheric Environment 169 (2017) 278e286 285

needles of Cryptromeria japonica seedlings was significantlyreduced by long-term exposure to submicron AS particles becauseof a decrease in the concentration of ribulose 1,5-bisphosphatecarboxylase/oxygenase (Rubisco). In general, net photosyntheticrate or concentration of Rubisco is strongly related to the nitrogenconcentration in leaves (Makino et al., 1984; Evans, 1989). In thepresent study, however, the exposure to submicron AS particlesdid not significantly affect the concentrations of NH4

þ, chlorophylland nitrogen in the leaves of komatsuna plants (Table 4). There-fore, we consider that the AS particle-induced reduction in netcarbon assimilation in the leaves was not attributable tobiochemical changes of the plants.

Leaf net carbon assimilation is limited by CO2 diffusion from theatmosphere into the leaf interior. In the present study, there was nosignificant difference in the slopes of the regression lines betweenAS particle-treated and control komatsuna plants (Fig. 5). However,the intercept of the regression line of AS particle-treated plants wassignificantly lower than that of control plants (Fig. 5). It indicatesthat the gad of AS-treated plants was lower than that of controlplants at the same PPFD level. A reduction in stomatal conductancedue to plugging of stomata by loam powders (super-micron size)was reported as a physical effect of particles on cucumber andkidney bean plants (Hirano et al., 1995). In the present study,however, plugging of stomata by submicron AS particles was un-likely to occur because AS particles were hygroscopic and wouldcollapse after absorbing water in the stomatal cavity. On the otherhand, deliquesced particles may form thin water films connectingthe leaf surface and interior, leading to an increase in minimumepidermal conductance (gmin) (Burkhardt, 2010; Burkhardt andPariyar, 2014). An increase of gmin indicates increased water lossthat could not be controlled by stomatal closure, and that maycause a reduction of midday water potential in crop plants (Pariyaret al., 2013). From these reports, it is considered that submicron ASparticles deposited on the leaf surface of komatsuna plants affectedplant water relations and stomatal conductance by enhancing un-controllable water loss. In our previous study, the increase of gmin inthe leaves of komatsuna plants was observed by approximately thesame amount of AS particles deposited on the leaf surface as thatobserved in the present study (data not shown). Thus, it is possiblethat the exposure to submicron AS particles enhanced the uncon-trollable water loss from the leaves of komatsuna plants, leading todehydration and reduced stomatal conductance during the daytime(Fig. 5). The results of this study indicate that the exposure tosubmicron AS particles reduced the growth of komatsuna plantsbecause of a decrease in the leaf net carbon assimilation rate, whichoccurred as a result of a decrease in stomatal conductance and thediffusional limitation of CO2. Further research is required to explorethe mechanisms by which submicron AS particles reduced thestomatal conductance of komatsuna plants. To the best of ourknowledge, however, this is the first report of submicron AS par-ticles reducing stomatal conductance during the daytime to nega-tively affect the growth and yield of Asian crop plants.

A. Motai et al. / Atmospheric Environment 169 (2017) 278e286286

5. Conclusions

In the present study, most of the submicron AS particlesdeposited on the leaf surface of komatsuna plants were washed-offafter 1 min of washing with ultrapure water. The amount of par-ticulate SO4

2� deposited on the leaf surface of komatsuna plantsdecreased over time, and few AS particles remained on the leafsurface at 19 h after the final exposure to AS particles. The growthand yield of komatsuna plants was significantly reduced by theexposure to submicron AS particles for 16 days from 10 to 25 DASwhen the daily mean increase in the concentration of SO4

2� in PM2.5by the exposure to AS particles was 22.5 mg m�3. The exposure tosubmicron AS particles did not affect leaf concentrations of chlo-rophyll and nitrogen, but stomatal conductance during the daytimewas significantly reduced. Therefore, submicron AS particle-induced reductions in the growth and yield of komatsuna plantswere attributed to a decrease in the leaf net assimilation rate due tostomatal closure. Further studies on the effects of submicron ASparticles on growth, yield and water relations are needed to un-derstand the risks to the productivity, quality and safety of Asianleafy vegetables.

Acknowledgements

The authors thank the members of Izuta and Watanabe Labo-ratories of Tokyo University of Agriculture and Technology for theirvaluable suggestions and technical support. This research did notreceive any specific grant from funding agencies in the public,commercial or not-for-profit sectors.

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