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ISSN : 1225-5009(Print)
ISSN : 2287-772X(Online)
Flower Research Journal Vol.28 No.2 pp.54-59

Relative Humidity Influences Epicuticular Wax Load and Particulate Matter Accumulation on the Leaf Surface in Asplenium nidus

Hyun Hee Kim1, Wan Soon Kim1,2*
1Department of Environmental Horticulture, University of Seoul, Seoul 02504, Korea
2Natural Science Research Institute, University of Seoul, Seoul 02504, Korea
*Corresponding author: Wan Soon Kim Tel: +82-2-6490-2693 E-mail:
04/06/2020 12/06/2020 18/06/2020


Particulate matter (PM) has recently been considered one of the most harmful air pollutants to public health. Plants have been known to degrade and deposit particle pollutants with epicuticular wax (EW), and this capacity can be influenced by environmental conditions including relative humidity (RH). The present study examined the effects of RH on EW generation and PM deposition upon leaf surfaces within Asplenium nidus ‘Avis’. The plants were treated in growth chambers with two levels of RH (low: 30% - 40% and high: 80% - 90%) for a period of four weeks, and subsequently exposed to a 30 μg·m-3 concentration of TiO2 particles as a PM resource for 72 hours. The EW ultrastructure on the leaf surface was observed as the thin films type, which was not morphologically changed in the condition of low or high RH treatment. For four weeks of RH treatment, the fresh weight and leaf area per plant were not significant between low and high RH treatment, while dry weight was significantly higher in the high RH condition. We also found that greater amounts of EW per fresh weight, dry weight and leaf area were generated in high RH. However, the total amounts of PM deposition (surface PM + in-wax PM) of the plants were higher within the low RH treatment with a higher proportion of surface PM. In contrast the proportion of in-wax PM was 15% higher within the high RH. These results suggest that EW generation is affected by air humidity and that proportion of PM deposition in the EW layer were influenced by the amount of total wax load.



    Rapid industrial and urban growth of recent decades increased anthropogenic air pollutants. Particulate matter (PM) is particulates consisted of various solid and liquid substances and mostly emitted by anthropogenic sources including vehicle exhausts, coal burning, industrial processing and road dust. Secondary particles from nucleation, condensation or coagulation of nitrogen oxides, sulfur dioxide, ammonia, and volatile organic compounds can also generate PM (USEPA 2004). There are four fraction sizes of PM with large (10 - 100 μm), coarse (2.5 - 10 μm), fine (0.1 - 2.5 μm), and ultrafine (≤ 0.1 μm) based on aerodynamic diameter. PM smaller than 2.5 μm can cause a severe impact on human health by penetrating the respiratory tract and distributing it to various organs through blood (Nemmar et al. 2002).

    Indoor air quality is one of the most critical factors for public health since people living in the urban area spend up to 85 - 90% of the daily time indoors (Soreanu et al. 2013). Indoor air pollutants which are entered from outdoors and generated indoors by household cleaning, cooking, heaters, and household members’ activity can increase the pollution concentration level of indoors even higher than outdoors. Also, thermally insulted building for energy saving represents one of the priority concerns for human health (Bruce et al. 2002).

    Plants can improve air quality by adsorbing PM onto the surface of aerial parts. The features of plant surface such as leaf s ize, structure, cuticular w ax, leaf hair and surface roughness, increase PM adsorption capacity. Epicuticular wax (EW), the outermost coating of plant surface, consisted of lipophilic compounds and deposited on the outer surface with thin film coating or various 3D-crystal formation. Studies have shown that PM deposited on the wax layer was difficult to remove by water agitation, so organic solvents are needed to extract the wax substance and PM accumulated on the EW. The composition of EW is influenced by the abiotic environment such as light intensity (Baker 1974), air humidity (Sutter 1984) and drought stress (Bondada et al. 1996).

    Asplenium nidus ‘Avis’ is u seful indoor f oliage, recently spotlighted as indoor garden plants for green wall with its superior adaptation to low light condition. The development of heating and cooling systems has maintained a constant thermal environment indoors. However, relative humidity (RH) throughout every season was lower than the outdoor level (Bae and Chun 2009).

    This study aimed to examine the change of EW density on Asplenium nidus cultivated in different RH conditions and to determine the effect of EW density on the deposition of PM.

    Materials and Methods

    Plant materials and PM treatment

    10 cm-potted plants of Bird’s nest fern (Asplenium nidus) were used in this study. The plants were cultivated in growth chambers for four weeks with two different levels of RH (low: 30 - 40%, high: 80 - 90%) and a bowl of silicate gel was also placed in a low RH cultivation chamber for additional drying. The light intensity of 30 ± 5 μmol・m-2・s-1 was set in both growth chambers for 16/8 h photoperiod at 23℃. Plants are watered once for three days with 100 mL of tap water and pots in all chambers are covered with plastic wrap to reduce evaporation from the soil. After four weeks of RH treatment, growth parameters including fresh weight, dry weight, l eaf area and mass of wax l oad were measured. Then the plants were placed in transparent acrylic chambers (0.4 m x 0.4 m x 0.6 m) for PM exposure with 30 μg・m-3 concentration of TiO2 (US Research Nanomaterials, Inc., USA). The acrylic chambers were set inside of growth chambers with the same light condition, and stalled fans circulated TiO2 particles.

    Scanning electron microscopy (SEM)

    Leaf samples were prepared by simple air-drying without pre-treatment (Pathan et al. 2008). Two pieces (mm2) cut from the center of the lamina of adaxial and abaxial were excised from the plants cultivated in different RH for four weeks. The samples were fixed to aluminum stubs with conductive double-sided adhesive tape and air-dried in a desiccator with silicate gel at room temperature for five days. Samples also sputter-coated with platinum (Leica, Austria) and examined with a field emission scanning electron microscope (Carl Zeiss, Germany).

    Quantitative analysis of PM and EW

    The quantity of PM and EW was gravimetrically determined as described in detail by Dzierżanowski et al. (2011). The PM was d etermined in two c ategories, firstly washed with distilled water for surface PM and then washed with chloroform for in-wax PM. Surface PM and in-wax PM were denoted as sPM and wPM respectively. Three size fraction filters were used to capture particles < 10 μm (Type 91, Whatman, UK), < 2 .5 μm (Type 42, Whatman, UK) and < 1 μm (5C, ADVANTEC, Japan). Before filtration, the filters were dried (VS-1202D3 VISION SCIENTIFIC CO., LTD, Korea), stabilized for humidity and weighed (Analytical Balance MS204TS/00, METTLER TOLEDO, Switzerland). After filtration, the filters were dried, stabilized, and weighed again as above. The EW quantity was measured after filtrating chloroform in 0.2 μm PTFE membrane filters (Whatman, UK) and evaporating chloroform. The amount of PM and waxes were recalculated to μg・cm-2 after measuring the leaf area of samples (LI-3100 AREA METER, LI-COR, inc., USA). The PM was washed off form both surfaces of the leaves, but the amounts are expressed per one sided leaf area.

    Statistical analysis

    Statistical Analysis System (SAS) version 9.4 (SAS Institute Inc., USA) was used to analyze the data for one-way ANOVA (analysis of variance). The significance of differences between mean values was tested using Student’s t-test at p = 0.05.

    Results and Discussion

    Leaf micromorphology

    After four weeks of different RH treatment, leaf micromorphology was examined by FESEM. According to the classification and terminology of wax micromorphology from Barthlott et al. (1998), films type of wax was found on A. nidus (Fig. 1). This type of wax has very thin covering and not showing fissures after drying (Barthlott et al. 1998). The epidermal cells of A. nidus were a slightly convex periclinal cell. Because the samples were air-dried without any chemical fixation, some shrinkage of t he e pidermal c ells w as a lso observed. No differences were found in both adaxial and abaxial surfaces of the leaves except more stomata were observed in abaxial leaves. Even there were no micromorphology differences between low and high RH treated leaf surfaces, as A. nidus has f ilm type of epicuticular wax, the thickness of the wax layer might be influenced by RH difference.

    Plant growth responses

    After four weeks of different RH treatment, the plant growth parameters including fresh weight, dry weight and leaf area were measured. There were no significant differences in fresh weight, dry w eight, and leaf a rea during four w eeks i n different RH treatment (Table 1). In general, plant growth increased under high RH condition because the turgor pressure in epidermal cell and stomatal conductance increased. Previous studies showed that high RH increased leaf number and leaf area in Hydrangea (Codarin et al. 2006) and leaf number, leaf area and dry weight in Petunia (Hoang and Kim 2018). However, the effect of different RH on A. nidus was not significant in this study.

    Epicuticular wax load

    Epicuticular wax load per fresh weight, dry weight and leaf area were measured in control and four weeks of different RH treatment (Fig. 2). Wax load per fresh weight wax significantly higher in both low and high RH for four weeks. Wax load per leaf area were also increased in both RH conditions but the difference was not significant in low RH. There were no significant differences of wax load per dry weight in both low and high RH. The Increase rate of wax load per fresh weight is 43% in low RH and 83% in high RH, respectively. S ince t here w as n o significant difference in f resh weight, wax generation rate was h igher in high RH treatment. Wax load per dry weight was decreased in low RH but increased in high RH. Wax generation was more significant in high RH than low RH treatment. Wax load per leaf area showed a similar tendency of the wax generation with wax load per fresh weight. The increased rate of wax load per leaf area is 27% in l ow R H and 95% i n high RH. O verall, the w ax generation rate of high RH was higher than the low RH treatment. This result was in contrast to the previous study of EW production in different water conditions with a higher wax amount per leaf area in lower RH (Koch et al. 2006). As its function of transpiration barrier, the wax load increased in a dry environment to avoid uncontrolled water loss, in general. However, water stress above a certain point might act as severe drought to plant, so the plant growth and wax development process could be restricted.

    PM deposition

    Total amounts of PM on leaf surface were higher in low RH treated plants with a more significant proportion of sPM than wPM (Fig. 3). However, compared to low RH, the balance of wPM in high RH increased with 15%. This result suggested that increased wax generation in high RH treatment might lead to increased deposition of PM in the EW layer. Different size fractions of deposited PM were also measured after PM exposure. 1 - 2.5 μm of sPM and all size fractions of wPM had no significant differences between low and high RH treatment. Coarse PM (10 - 100 μm) and fine PM (2.5 - 10 μm) of sPM had significantly higher amounts in low RH. Previous studies have shown PM deposition had higher on leaf surface than in-wax. However, PM deposition could vary between plant species (Popek et al. 2013;Gawrońska and Bakera 2015). Also, Popek et al. (2013) suggested that in some species, the correlation between the amount of wax, and in-wax PM was highly positive. Therefore, the changes in the EW load in high RH treatment might influenced the proportion of in-wax deposited PM in this study.



    FESEM images of adaxial (A, B) and abaxial (C, D) leaf surfaces of Asplenium nidus 'Avis’ treated in different relative humidity for four weeks. A, C: 30 - 40% low RH; B, D: 80 - 90% high RH. Scale bars mean 1 μm.


    The epicuticular wax load of Asplenium nidus 'Avis’ treated in different relative humidity for four weeks. Control means the initial value before PM and RH treatment. Vertical bars represent standard error (n = 4). The ns and * indicate no significance and significance at p < 0.05, respectively, ANOVA.


    Total amounts of surface and in-wax particulate matter (Surface PM, In-wax PM) accumulated on leaves of Asplenium nidus 'Avis’ treated in different relative humidity for four weeks. Vertical bars represent standard error (n = 4).


    Plant growth parameters of Asplenium nidus 'Avis’ treated in different relative humidity for four weeks.

    Deposition of particulate matters in different size fractions on leaves of Asplenium nidus 'Avis’ treated in different relative humidity for four weeks.


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