<h2>Urban dust load impact on photosynthesis and growth of <em>Sophora japonica</em> L. seedlings</h2><p>Le Bao<sup>1, 2</sup>, Keming Ma<sup>1*</sup>, Laiye Qu<sup>1</sup>, ShuangZhang<sup>1</sup>, Lin Lin<sup>1, 2</sup></p><p><sup>1</sup>State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, P. R. China.</p><p><sup>2</sup>University of Chinese Academy of Science, Beijing 100049, P. R. China.</p><p>*Corresponding author: Email: mkm@ rcees.ac.cn; Tel/Fax: 86-10-62849104</p><p> </p><p>Abstract:</p><p>Greenspace provides many ecosystem services in maintaining urban environmental quality, it plays an important role in absorbing particulate matters (PM) from urban atmosphere, but the PM might also be harmful to urban plants hence lower the ecological services of greenspace. Road dust is a main source of air PM pollution in city, but it is not clear how the dust load and dusting time of urban road dust impact on the photosynthesis and growth of urban plants.We designed a dusting experiment of 21 days to examine the variation of pigments, gas exchange, height increment, and biomass of <em>S</em><em>. </em><em>japonica</em> seedlings (the most popular tree in Beijing) under 5 treatments of urban road dust loads in Beijing. The results showed that along with the rising of dust load, chlorophylls, net photosynthetic rate, stomatal conductance and transpiration rate had logarithmic dropping; root-shoot ratio of biomass logarithmicly increased and height increment had a linear decline. Net photosynthetic rate, stomatal conductance and transpiration rate varied with power functions that decreased fast in initial dusting time, and then reduced slowly or even increased. Chlorophyll content and photosynthesis were not influenced at dust loads that below maximum value of street<em> S. japonica</em>;biomass allocation was not impacted until about 6 times of maximum <em>S. japonica</em> foliar dust in street. We can declare that current dust detaining for urban trees like<em> S</em><em>. </em><em>japonica</em> do not affect their regulating and supporting services. Relevant knowledge would benefit the management of urban greenspace.</p><p> </p><p>Keywords:</p><p>Road dust; dust load;photosynthesis; pigments; biomass.</p><h2>1 Introduction</h2><p>The emission of particulate matter (PM) gives rise to the decline of environmental quality in cities worldwide. Plants can absorb PM from atmosphere to leaf surface, even keep PM in leaf wax(<a href="#_ENREF_19">Terzaghi et al., 2013</a>). Consequently plants could reduce the airborne particulate matters in urban area(<a href="#_ENREF_11">Nowak et al., 2006</a>). On account of the height and structure, tree species are more efficient in dust retention comparing with shrub or grass(<a href="#_ENREF_18">Tallis et al., 2011</a>).</p><p>The primary source of PM is mainly from the traffic (<a href="#_ENREF_18">Tallis et al., 2011</a>), its impacts on photosynthesis and growth of urban plants may reduce the ecosystem services of urban greenspace. Road dust can cause a series of negative impacts on plant physiology and growth, and some of those impacts closely relate to ecosystem services of urban plants, as lowering photosynthetic pigments(<a href="#_ENREF_15">Prajapati and Tripathi, 2008</a>; <a href="#_ENREF_16">Prusty et al., 2005</a>), and bringing down the net photosynthetic rate and stomatal conductance(<a href="#_ENREF_2">Chaturvedi et al., 2013</a>).Though there is a common view that dust can restrain growth of plants, biomass or height performance was researched in cement dust and ore dust studies (<a href="#_ENREF_6">Kuki et al., 2009</a>; <a href="#_ENREF_7">Kuki et al., 2008</a>; <a href="#_ENREF_12">Ots et al., 2011</a>), road dust was not concerned.</p><p>As for the impact of dust load on plants, loam, fly ash and ore dust impacts were studied by means of artificial simulation method. Pigments and biomass increased and then deceased with dust loads of fly ash (<a href="#_ENREF_17">Singh and Siddiqui, 2003</a>). Net photosynthetic rate of cucumber and kidney bean dropped with dust loads of Kanto loam(<a href="#_ENREF_5">Hirano et al., 1995</a>).Some linear regression equations were also set up between cotton photosynthetic rate, dry weight and dust concentration of loam, and found that photosynthetic rate and dry weight fluctuated and then decreased(<a href="#_ENREF_1">Armbrust, 1986</a>).Stem height of <em>Sophora tomentosa</em> significantly declined when soil iron dust load elevated; but <em>Schinus terebinthifolius</em> did not significantly rise with dust load(<a href="#_ENREF_6">Kuki et al., 2009</a>). Biomass of<em>S.tomentosa </em>increased and then deceased with the rising of iron dust load(<a href="#_ENREF_6">Kuki et al., 2009</a>). Since conclusions of those studies were different from each other, and dust type may influence the dust load effects on plants, it is absolutely needed to detect the response of plant physiology and growth to road dust load gradient in terms of urban green space maintenance under the rapid development of urbanization.</p><p><em>Sophora japonica</em> L. is a native tree species that is widely distributed in North China. It is an dominate street tree species planted in Beijing, whichaccounts for 81% of the street trees in urban area of Beijing (<a href="#_ENREF_20">Zheng and Zhang, 2011</a>). Its growth plays an important role in adjusting the air quality and keeping the health of urban environment. Photosynthetic and growth response of <em>S. japonica</em> to road dust may be a good indicator of traffic pollution impact.</p><p> To understand the effects of road dust on the growth and physiological characteristics of <em>S. japonica</em>, we set up an control experiment by artificially loaded road dust collected from the surface street of Beijing. We suppose that with the increase of dust load, <em>S. japonica</em> seedlings will generate less photosynthetic pigments and gas exchange, leading less accumulation of biomass. The detail purposes of our work were todetect: 1. Whether <em>S. japonica </em>seedlings are stressed significantly by the mean and max dust load of street ? And how would the seedlings perform by the dust load, especially by the maximum dust load? 2. Are there some trends in variations of photosynthesis and growthalong with rising dust load and dusting time? Relevant knowledge would benefit the management of urban greenspace.</p><p> </p><h2>2 Materials and methods</h2><h3>2.1 Plant material and road dust collection</h3><p>Seed germination was performed under greenhouse conditions at the Beijing Forestry University Forest Science Co,Ltd. in April 2012. <em>S. japonica </em>seedlings were obtained by the germination of seeds provided by the nursery of Qingyuan County in Hebei Province. The seeds were soaked with 85-90 °C water for 24hours, and then germinated in nursery trays when the seeds swelled. If the seeds did not swell properly, we repeated soaking once or twice. 20 days after emergence, the seedlings were transferred to plasticpots containing cultivation soil (sand+ peat+ vermiculite in a proportion of 2:2:1). Each pot contained one plant. In August 2012, seedling heights were between 30-50cm, and the crown widths were about 30cm.</p><p>The road dust used in experiment was collected from the surface of some busy traffic streets between the 2nd Ring and 3rd Ring Roads of Beijing. The road dust was sieved with nylon fine screen to get the particles smaller than 106?m in diameter(<a href="#_ENREF_1">Armbrust, 1986</a>).</p><h3>2.2 Maximum dust load experiment and treatment setting</h3><p>The amount of dust load were referred the result of Dai et al (<a href="#_ENREF_3">Dai et al., 2012</a>). The seedlings were irrigated and their leaves were washed 3 days before artificial dusting.We placed plastic wrap on the soil surface to avoid thedust retention.Road dust (diameter<106 ?m) was applied by using a dust sprayer until leaf surface could not accommodate dust any more. The maximum applied dust (A<sub>m</sub><sub>ax</sub>) was then recorded. Leaves of 10 replications were collected to count the maximum dust load (D<sub>m</sub><sub>ax</sub>). The result of maximum dust load experiment was shown in Table1, the average of maximum dust load D <sub>max</sub> was 16.97 g·m<sup>-2</sup>, corresponding to the maximum applied dust of 73.08 g/pot.</p><p>In September 2012, the dusting experiment was operated including 5 treatments, and each treatment has 9 replicate seedlings. In our previous study, dust load of <em>S. japonica</em> in streetwas significantly higher than that of <em>S. japonica</em> in park, and dust load of street tree <em>S. japonica </em>had a maximum value of 2.812 g·m<sup>-2</sup> and average value of 0.683 g·m<sup>-2</sup> (<a href="#_ENREF_3">Dai et al., 2012</a>). Therefore, we regarded the average dust load of street tree asA<sub>1</sub> treatment, and according to the ratio of maximum applied dust and maximum dust load, A<sub>1</sub>=0.683A<sub>m</sub>/D<sub>m</sub>=2.94 (g/pot). Likewise, we regarded the maximumdust load of street tree as A<sub>2</sub> treatment, A<sub>2</sub>=2.812A<sub>m</sub>/D<sub>m</sub>=12.11 (g/pot). We defined half of maximum dust load as A<sub>3 </sub>treatment, A<sub>3</sub>=1/2A<sub>m</sub>=36.53(g/pot). Maximum dust load was regarded as A<sub>4</sub> treatment, A<sub>4</sub>=73.08(g/pot). We took A<sub>0</sub> treatment with no dust as contrast.The dust load amounts of treatments were shown in Table2.</p><p>The seedlings were irrigated and the leaves were washed 3 days before dusting as then treated as the amount of dust shown in table 2. The seedlings were kept stable for 21 days and the irrigation water was only applied to soil surface directly by tubule. Immediately after dusting, 3 seedlings per treatment were cut down to test the actual dust load. Whole leaves from each seedling were washed in distilled water and ultrasonic oscillated for 3 min, and then vacuum filtered through pre-weighed filter membrane(?=0.45µm)(<a href="#_ENREF_4">FreerSmith et al., 1997</a>). Leaf area of those washed leaves was measured by WinFOLIA software after dried. The actual dust loads were similar to expected dust loads with a proximity of r=0.998, so we use actual dust loads when curve fitting in this article.</p><h3>2.3Growth, pigments, and Gas exchange measurements</h3><p>The heights of seedlings were measured before dusting and after 21 days of dusting. When the experiment finished, 3 seedlings of each treatment were excavated and washed using water. The roots, stems, and leaves were separated and oven dried at 80 ?for at least 24 hours to obtain the dry weight biomass.</p><p>The second, third and fourth leaves from the top of each sprig were collected and put into liquid nitrogen for 3 seedlings ineach treatment. Fresh leaf was extracted with 95% ethanol and the absorbance of the leaf pigment extract were measured separately at 470, 649, and 665 nm. Chlorophyll and carotenoid were calculated according to the specific absorption coefficients of Lichtenthaler (<a href="#_ENREF_8">Lichtenthaler, 1987</a>).</p><p>Gas exchange parameters of 4 fully expanded healthy leaves were measured using a Portable Photosynthesis System LI-6400 for each seedling, and 3 seedlings for each treatment. Net photosynthesis rate, stomatal conductance, and transpiration rate were measured before dusting and after 2, 4, 6, 12, 16, 20 days of dusting. All the measurements were operated between 8:00 am and 10:00 am of the day.</p><h3>2.4 Statistical analyses</h3><p>Traits of different treatments were compared by LSD test of One-Way-ANOVA (Figure1, 3). Gas exchange variation of different treatments were compared by LSD test of repeated measures in GLM (Figure5). Repeated measures were also used to test the changes of gas exchange by dust loads and dusting time (Table3). Proximities of actual and expected dust loads were obtained by correlate distance analysis(Materials and methods 2.2). Gas exchange variations with dusting time were fitted by regression analysis (Table4). All of the above tests were done by SPSS 15.0. Dust load effects on traits were described by nonlinear curve fit of Origin 9.0(Figure2,4,6). Other figures were also drawn using Origin 9.0.</p><h2>3 Results</h2><h3>3.1 Dust load impact on growth</h3><p>The results showed that there were not significantly differences of total biomass, stem biomass, and root biomass of the seedlings (Figure 1 A, C, D) under the loaded dust conditions, whatever the amount of dust. However, the leaf biomasses of all 4 dust load treatments were significantly lower than control group (Figure 1B). The leaf biomass of A<sub>3</sub> treatment was the lowest, and was 37% lower than control. Shoot biomass (leaf biomass + stem biomass) of A<sub>1</sub> treatment showed significant decreased compare with control group, and was 32% lower than control (Figure 1E). Root-shoot ratio of A<sub>4</sub> treatment were significantly higher than that of control group, and about 26% higher compared to that of control(Figure 1F), which may indicted the most significantly stressed by road dust. The decrease of shoot biomass was greater than that of root biomass, which contributed to the rising of root-shoot ratio. Height increments of all 4 dust load treatments were lower than control group but not significantly (Figure1G).</p><p>Trend fitting was done to show the dust load effects on growth traits, in which root-shoot ratio and height increment had significant fitting. Root-shoot ratio had logarithmic rising trend with dust loads (Figure 2 A), which elevated fast at lower dust loads and increased slowly at higher dust loads. While, height increment had a linear downtrend (Figure 2 B).</p><h3>3.2 Dust load impact on pigment contents</h3><p>As shown in Figure3 A,B, total chlorophyll and chlorophyll a of A<sub>2</sub>, A<sub>3</sub> and A<sub>4</sub> treatments were lower than those of control. Chlorophyll b and chlorophyll a/b of  A<sub>3</sub> and A<sub>4</sub> treatments showed significant differences from control group (Figure3 C, E). Carotenoid of A<sub>4</sub> treatment was significantly lower than that of control group (Figure3 D), which may means chlorophyll a was more sensitive to dust treatment than chlorophyll b and carotenoid. A<sub>4</sub> treatment was the lowest group For all pigment parameters. When compared with control group, total chlorophyll, chlorophyll a, chlorophyll b, carotenoid, and chlorophyll a/b ratio of seedlings decreased by 69%, 74%, 63%, 46% and 29% under the maximum road dust treated condition.</p><p>Dust load effects on total chlorophyll, chlorophyll a, chlorophyll b and chlorophyll a/b ratio were shown in Figure4. Logarithmic curves were fitted to describe the relationships between chlorophylls and dust loads. Total chlorophyll, chlorophyll a, chlorophyll b and chlorophyll a/b ratio all declined fast at lower dust loads and then had a gently decline at higher dust loads.</p><h3>3.3 Dust load and dusting time impact on gas exchange</h3><strong><br /> </strong> <br /><p>Net photosynthetic rate, stomatal conductance and transpiration rate of A<sub>2</sub>, A<sub>3</sub>and A<sub>4</sub> treatments were significantly lower than those of control respectively (Figure5). Variations of net photosynthetic rate, stomatal conductance and transpiration rate with dusting time were significant differences (Table3).Net photosynthetic rate and transpiration rate decreased fast in initial 4 days, and afterwards they varied gently. Stomatal conductance kept declining rapidly during the initial 12 days, then varied smoothly.After loading dust for 20 days, the reduction of net photosynthetic rate was 7%-42%;the reduction of stomatal conductance was reduced 13%-63%; the reduction of transpiration rate was 9%-40%, respectively.At the end of dusting, the minimum value of net photosynthetic rate, stomatal conductance and transpiration rate all appeared in A<sub>4</sub> treatment group.</p><p>Dust load effects on net photosynthetic rate, stomatal conductance and transpiration rate of the 20’ th day were shown in Figure6. Logarithmic curves were fitted to describe the relationships between gas exchange parameters and dust loads. Net photosynthetic rate, stomatal conductance and transpiration rate all declined fast at lower dust loads and then had a gently decline at higher dust loads.</p><p>As for gas exchange variation with dusting time, net photosynthetic rate, stomatal conductance and transpiration rate had decrease of power functions. Decline of gas exchange parameters acceleratedwhen exponent of regression functions rise.Regression functions were shown in Table 4.</p><h2>4 Discussion</h2><p>Previous road dust studies were lack of experimental and quantified researches. Even in studies of other kinds of dusts, the effects of dust load on photosynthesis and growth were less reported (<a href="#_ENREF_1">Armbrust, 1986</a>; <a href="#_ENREF_5">Hirano et al., 1995</a>; <a href="#_ENREF_6">Kuki et al., 2009</a>; <a href="#_ENREF_17">Singh and Siddiqui, 2003</a>). Our results suggested that the photosynthesis and growth of <em>S</em><em>. </em><em>japonica</em> seedlings were influenced by road dust load, meanwhile photosynthesis was affected by dusting time. With increased amount of road dust, the photosynthesis and growth of the seedlings decreased.</p><h3>4.1 Dust load impact on<em> S. japonica</em></h3><p>As far as the dust load effect on photosynthesis,chlorophyll contents, net photosynthetic rate, stomatal conductance and transpiration rate had logarithmic dropping with dust loads in our study. When cucumber and kidney bean were dusted by Kanto loam, net photosynthetic rate dropped with dust loads (<a href="#_ENREF_5">Hirano et al., 1995</a>). Effects of fly ash on pigments of wheat showed a different consequence that pigments increased and then deceased with dust loads(<a href="#_ENREF_17">Singh and Siddiqui, 2003</a>). Armbrust(1986) tried to set up a linear regression equation between cotton photosynthetic rate and dust concentration of loam (<a href="#_ENREF_1">Armbrust, 1986</a>). In our research, we established logarithmic functions to describe the relationship between photosynthetic performance and road dust, which declined fast at lower dust loads and then had a gently decline at higher dust loads.</p><p>Transpiration rate was reduced by road dust in our study, which was similar with the results of iron ore dust research(<a href="#_ENREF_10">Neves et al., 2009</a>). Transpiration rates of dusted treatments were significantly higher than that of control in carbon black or PM studies (<a href="#_ENREF_5">Hirano et al., 1995</a>; <a href="#_ENREF_13">Pavlik et al., 2012</a>), which were opposite to our study. Thus, transpiration rate variation may be different in treatments of different kinds of dusts. Moreover, temperature and altitudealso changed the transpiration ratevariation (<a href="#_ENREF_5">Hirano et al., 1995</a>; <a href="#_ENREF_9">Naidoo and Chirkoot, 2004</a>). In our study, we obtained a decline curve of transpiration rate with road dust.</p><p>In the case of dust load effect on growth, root-shoot ratio of biomass had a logarithmic increase and height increment had a linear decline with dust loads. When road dust stress was aggravated, <em>S. japonica </em>willdistribute more biomass to root than to crown, so growth of stem height may be delayed. Thus height increment dropped with dust load. In Kuki’s research(2009), stem height of <em>S.tomentosa</em> significantly declined when soil iron dust load elevated, which was similar with our consequence; but <em>S.terebinthifolius</em> had a not significant rise with dust load(<a href="#_ENREF_6">Kuki et al., 2009</a>). Biomass increased and then deceased with the rising of dust load in wheat and <em>S.tomentosa </em>experiments (<a href="#_ENREF_6">Kuki et al., 2009</a>; <a href="#_ENREF_17">Singh and Siddiqui, 2003</a>). None of those studies established relations between growth and dust load. In Armbrust’s research(1986), biomass fluctuated and then decreased, and linear regression equation was set up to describe the cotton dry weight and dust concentration of loam (<a href="#_ENREF_1">Armbrust, 1986</a>). In our study, root-shoot ratio elevated fast at lower dust loads and increased slowly at higher dust loads; while height increment variation was a uniform decline.</p><h3>4.2 Dusting time impact on<em> S. japonica</em></h3><p>As for dust load effects with dusting time, net photosynthetic rate and stomatal conductance decreased fast in initial dusting time, then reduced slowly or even increased. It was probably because in later period leaves had an expanded growth so that dust effects reduced a little. In Pereira’s iron dust research(2009), stomatal conductancedecreased fast in initial 10 days, which was similar to ours; but net photosynthetic rate reduced only a little in 30 days (<a href="#_ENREF_14">Pereira et al., 2009</a>), which was different with our study. Cotton photosynthetic rates of 1,3,7,14 days were  compared in Armbrust’s study(<a href="#_ENREF_1">Armbrust, 1986</a>). In our study, we set up power regression functions to describe the gas exchange variations with dusting time. Decline of gas exchange parameters acceleratedwhen exponent of regression functions rise. Falling range of gas exchange parameters at top three dust loads were significantly greater than control.</p><h3>4.3 Implications to urban greenspace management</h3><p>Road dust decreased total chlorophyll content and net photosynthetic rate at dust load of 3.78 g·m<sup>-2</sup>, 9.02 g·m<sup>-2</sup> and 16.64 g·m<sup>-2</sup> in our research, and both had a logarithmic decline with dust loads. Reduction in photosynthetic performances of street trees may decline the regulating services of green space. In previous studies, researchers found that pigment content, net photosynthetic rate and stomatal conductance of roadside plants negatively correlated with dust load (<a href="#_ENREF_2">Chaturvedi et al., 2013</a>; <a href="#_ENREF_15">Prajapati and Tripathi, 2008</a>; <a href="#_ENREF_16">Prusty et al., 2005</a>) , while how many dust load could significantly affect the photosynthetic performances were not explored before. In our research, dust loads above 3.78 g·m<sup>-2</sup>significantly affect the photosynthetic performance, which was much higher than average value (0.683 g·m<sup>-2</sup>) and maximum value (2.812 g·m<sup>-2</sup> ) of street <em>S. japonica</em><em>.</em></p><p>Root-shoot ratios of seedlings were significantly affected by road dust at 16.64 g·m<sup>-2</sup>treatment, and root-shoot ratio had a significant logarithmic rise with dust loads. The increase of root-shoot ratio exhibited a change of biomass allocation between above and under-ground in road dust stress. Loss of street tree crown may influence the supporting service of green space and even threaten the survival of street trees. However, there was few research focus on how road dust affected biomass of plants. In our research, biomass allocation was not significantly affected until 16.64 g·m<sup>-2</sup>dust load, which was close to 6 times of maximum foliar dust(2.812 g·m<sup>-2</sup> ) of street <em>S. japonica</em><em>.</em></p><p>Chlorophylls are the main photosynthetic pigments that absorb and deliver light quantum in photosynthesis. Dry matter accumulation and biomass of plants were close related to the photosynthesis. According to our study, chlorophyll content and photosynthesis were not influenced at dust loads that below maximum value of street<em> S. japonica</em> ;biomass allocation was not impacted until about 6 times of maximum <em>S. japonica</em> foliar dust in street. Understanding threshold values of dust detainment will contribute to urban afforestation and promoting regulating service of green space. For <em>S. japonica</em> street trees, common amounts of urban road dust could not affect their regulating and supporting services as green space. <em>S</em><em>. </em><em>japonica</em> is a typical deciduous tree in urban afforestation of North China. Using plants like <em>S. japonica</em>as street trees or other green spaces couldbe a good method to manage the PM pollution.</p><h2>5 Conclusion</h2><p>Along with the rising of urban dust load, chlorophylls, net photosynthetic rate, stomatal conductance and transpiration rate had logarithmic dropping; root-shoot ratio of biomass logarithmicly increased and height increment had a linear decline. Net photosynthetic rate, stomatal conductance and transpiration rate varied with power functions that decreased fast in initial dusting time, and then reduced slowly or even increased. Chlorophyll content and photosynthesis were not influenced at dust loads that below maximum value of street<em> S. japonica</em>;biomass allocation was not impacted until about 6 times of maximum <em>S. japonica</em> foliar dust in street. For <em>S. japonica</em> street trees, common amounts of urban road dust could not affect their regulating and supporting services as green space. Using plants like <em>S</em><em>. </em><em>japonica</em>as street trees or other green spaces couldbe a good measure to mitigate PM pollution. Actual urban dust loads have little impact on street trees.</p><h2>Acknowledgments</h2><p>This research supported by National Natural Science Foundation of China (Project 41430638 to KMM). The authors express their appreciation to laboratory members for providing assistance in cultivation and measurements, especially to  Guorui Xu and Xiu Yuan. The authorsalso thank Beijing Forestry University Forest Science Co,Ltd. for providing greenhouse for cultivation.</p>