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Specific Sound Frequency Improves Intrinsic Water Efficiency in Rice Leaf by Imparting Changes in Stomatal Dimensions

Writer's picture: PlantHouse EnterprisePlantHouse Enterprise

Published on: 18 April 2023


ABSTRACT

Various attempts have been made to increase the production of rice that include breeding for high yielding and stress tolerant varieties, good crop management system, and increase in agricultural input in rice production. Soundwave stimulation has been demonstrated to affect plant growth thus this method can be employed in the current methods of rice production to improve yield. The aim of this study was to determine the effects of different sound wave qualities on general growth, physiological, and morphological of rice seedlings. Rice seeds of MR219 variety were grown under a glasshouse condition in a nested design with five replications and were stimulated with various sound wave frequencies. Various sound wave frequencies, which were 380, 359, 357, 353, and 350 Hz were obtained through placing the pot at varying distances (80, 160, 240, 320, and 400 cm, respectively) from the sound source, except control treatment. There were significant effects in some of the parameters, which are plant height, leaf physiology, and stomatal pore and length when treated with varying sound wave qualities. It was observed that plant can be stimulated with 380, 357, and 350 Hz soundwaves frequencies for the best photosynthetic experience. In addition, 359 Hz of sound wave stimulation resulted in high water use efficiency, and this is beneficial in improving crop performance in drought condition. Thus, it was demonstrated that sound wave stimulation method has the potential to enhance rice performance in addition to the regular agronomic practices of rice production in farmers' field.

Keywords: Leaf physiology, Oryza sativa L., photosynthesis, rice growth, rice production, sound wave stimulation, stomatal morphology


INTRODUCTION

Rice (Oryza sativa L.) is largely regarded as a vital crop owing to its prominent roles in shaping histories, cultures, diets, and economics for half of humanity (Gomez, 2001). Rice feeds more than three billion people in addition to one billion people depend on rice cultivation for their livelihood (Skinner, 2012). In 2020, the early report of the total rice production in Malaysia was around 2.3 million tonnes, and 78.4% came from the granary areas (Department of Agriculture [DOA], 2021). The government, although has taken many efforts to improve the self-sufficiency level in Malaysia, rice production has only achieved 63% self-sufficiency level with the rest being imported to meet the needs of the 33 million people in this country (Department of Statistics Malaysia [DOSM], 2021). Although rice production is gradually increasing, consumption is also increasing, requiring more rice to be imported to meet local demand. Furthermore, the gap between rice production and consumption was projected to widen from 2018 to 2026 (Omar et al., 2019). Therefore, there is an urgent need to increase local rice production to ensure food security instead of relying on imports from neighbouring countries to meet domestic consumption.

Various efforts are taken to address the issue of rice shortage, which includes breeding and introduction of varieties resistant to pests and diseases. These include varieties, such as Minghui63-Xa21 and Shanyou63-Xa21, which are resistant to bacterial blight, and Hanyou 2 and Hanyou 3, which have tolerance to abiotic stresses such as flood responses and drought. (Luo, 2010; Zhai et al., 2001). Other researchers approached the issue from different angles (Stoop et al., 2002) such as the application of various good crop management system including system of rice intensification (SRI) on infertile soils and reduced rate of irrigation while maintaining other agricultural inputs optimally (Kim et al., 2008). Traditional crop production practices involved application of more and more fertilizers and chemicals to improve performance has shown to be environmentally unfriendly. Since 2000, the rice harvested area in Malaysia has remained constant, but rice yield in Malaysia has increased due to increasing use of nitrogen (N) fertilizers (Herman et al., 2015). Therefore, new eco-friendly approaches need to be developed to ensure sustainability in crop production and environmentally friendly. Despite increased N fertilization, manipulating the photosynthesis of plants is considered a sustainable method to improve crop production. For example, rice variety MR253 showed higher photosynthetic assimilation under light saturated condition when treated with 50% N concentration compared to 100% N concentration in a hydroponic system (Herman et al., 2015). This result proved that applying more fertilizer does not necessarily improve crop performance. Similarly, it is believed that sound wave manipulation can be used to enhance photosynthesis at the leaf level, which could potentially improve plant growth and yield production in rice.

Interestingly, sound wave has been reported to affect many biological properties in plants (Hassanien et al., 2014). However, whether the effect of sound waves promotes, or limits growth depends on the frequency, intensity, and acting time of the sound wave is not well defined. Stimuli, such as sonic field, supersonic, electromagnetic field, microgravity, and mechanical vibration, have been previously shown to have effects on plants. For example, the selectively permeable cell membrane can be injured by environmental factors, such as temperature, salinity, and air pollution. The injury incurred can be thought of like 'micro-perforations' that could bring about improved penetrability of the membrane, which could be beneficial in regulating substance movement into or out of the cell. It has been shown that sound wave with a frequency of 400 Hz can improve the float of cell membrane, strengthen the mutual function between membrane's lipid and protein region (Bochu et al., 2003).

Many studies have demonstrated that music will increase plant growth. Yiyao et al. (2002) reported that plant tissues can be enhanced at a certain range of intensity and frequency of sound wave, but the effect could become the opposite when the sound field is beyond certain range. Some research also found that the acceleration mechanical wave needed to promote seed germination (about 70 m/s²) which is far more than the acceleration of gravity wave (9.8 m/s²), although this purported value cannot be entirely confirmed due to vibrational acceleration changes that happens continuously in the testing system (Uchida & Yamamoto, 2002). Thus, it is hypothesized that there is a specific sound wave quality, with respect to the varying distances from the sound source, which will be beneficial to improve rice physiologically. In addition, that varying sound wave treatments will have differing effects in altering the stomatal and epidermal properties of rice leaf that is affecting the genetics of rice plant, which could be beneficial in selection for breeding purposes. The aim of this study was to determine the effects of different sound wave qualities on the general growth, physiological, and morphological of rice seedlings.


MATERIALS AND METHODS

Experimental Site

The experiment was conducted in 2018 in a greenhouse of the Institute of Tropical Agriculture and Food Security (ITAFoS), Universiti Putra Malaysia (UPM) (GIS location: 2.98414168 N, 101.7336908 E). Ten rice seeds of MR219 variety were sown in a pot (80 mm [D] x 80 mm [H]) containing paddy field soil (Serdang series) and all the pots were arranged in a nested design with five replications (five pots) per treatment. Treatments were imposed, and growth parameters were assessed within 30 - 35 days after sowing (DAS). Standard agronomic practices such as thinning, fertilizing, weeding, and watering were performed throughout the growing period to maintain good plant growth. Thinning of the seedlings was carried out after 10 days of sowing by leaving only one healthy plant within each pot. Rice plants were fertilized from 14 DAS once a week until 35 DAS with a proprietary blend of hydroponic nutrient solution from a local garden store.


Rice plants were stimulated with different sound qualities in terms of frequency (Hz). Six treatments were imposed on the seeds and seedlings comprising five sound wave qualities and one control. Different sound qualities, which are 380, 359, 357, 353, and 350 Hz were obtained by placing the pot at varying distances (80, 160, 240, 320, and 400 cm, respectively) from the sound source with a 600-watt loudspeaker (Aviano Precision, China) with volume set to 5/10 (Figure 1). The decibel (dB) value, which is the scale of loudness (intensity of a wave) measured at these distances, is 78, 73, 69, 65, and 60 dB, respectively. The frequencies (Hz) and decibels (dB) were monitored using the FFTWave application, an Android application on mobile phones for sound monitoring. The song used for the sound wave stimulation was a Mozart instrumental song by William McColl. The control treatment received no sound wave stimulation and was in a different greenhouse. The distance between control and treated plants is approximately 28 m (Figures 1a and 1b). The condition between the two greenhouses especially the sunlight is the same because the greenhouses are in the same area and no other building close to this facility that can create shade. Sound barrier and absorber using egg cartons were installed to the left and right of the rows of plant to reduce the impact of ambient noise. The sound wave stimulation was performed every other day for a month starting from day 1 of seed sowing for four hours in the morning. The duration of the song was 5 min and was repeated during a 4 hr of stimulation period. This whole experiment was conducted only once and was not repeated.


Figure 1. a) A satellite image of two glasshouses used in the experiment and the distance between the experimental locations (about 28 m apart) of sound wave treatments and control treatment (no sound treatment), and b) the experimental layout in the glasshouse. Rice seedlings treated with sound waves were placed in a different glasshouse (Greenhouse 1) than the non-treated rice seedlings (Greenhouse 2)
Figure 1. a) A satellite image of two glasshouses used in the experiment and the distance between the experimental locations (about 28 m apart) of sound wave treatments and control treatment (no sound treatment), and b) the experimental layout in the glasshouse. Rice seedlings treated with sound waves were placed in a different glasshouse (Greenhouse 1) than the non-treated rice seedlings (Greenhouse 2)

Data Collection

Growth and appearance. Leaf number was counted manually, and the plant height was measured from the base to the tip of the highest shoot of rice plant using a measuring tape.


Leaf Physiology. Photosynthesis rate (assimilation, A400) and stomatal conductance (gsw) were performed using a portable photosynthesis system (LICOR-6400/XT, USA) to measure physiological activity of leaves. Gas exchange were measured on the middle part of leaf 5. In the leaf chamber, light intensity, temperature, humidity, and carbon dioxide (CO2) concentration were set at 1,600 µmol m⁻² s⁻¹ PAR, 25ᵒC, 60%, and 400 ppm, respectively. Intrinsic water use efficiency (iWUE) (μmol CO2 mol⁻¹ H2O) was calculated as the ratio of assimilation rate to stomatal conductance and calculated using the following formula:



Leaf Stomatal Properties. A histology analysis was conducted to determine the properties of leaf stomata. The parameters evaluated were stomatal complex area, stomatal pore area, stomatal densities, percentage stomatal file, stomatal complex length, stomatal complex width, guard cell width, and cell file width. A section of about 1 cm of the fully expanded leaf 5 was cut and placed in Carnoy's fixative that was prepared according to Puchtler et al. (1968) without any modifications. All samples were placed in the Effendorf tubes and bleached with a 15% Clorox® (Malaysia) (sodium hypochlorite) solution for 24 hr until the samples became colourless (to remove the pigments). Then the bleached samples were cut in half and were fixed on the slide with chlorohydrate solution and arabic gum and covered with cover slip.


Microscopy Analysis. Samples were observed using a LEICA DFC310 FX light microscope (United Kingdom) with a 400x magnification lens. The area of the stomatal complex, guard cell width, stomatal length, interveinal gap, and vein counting were performed using Image J software (version 1.48) (Schneider et al., 2012).


Measurement of Stomatal and Leaf Epidermal Characteristics. For stomatal complex area, the width and length of the stomata was measured. The stomatal complex in rice consists of a pair of guard cells and the area of the stomatal complex was calculated based on the measured values of stomatal complex width and length using the following formula:



The guard cell width was determined by drawing the lines from the top to the bottom of the guard cell and the length of this line was measured as the guard cell width (Figure 4b). The stomatal density (mm-²) was calculated by counting the number of stomatal complexes in between two interveinal gap areas. The percentage of stomata files was calculated by counting the number of files with at least one stoma over the total number of cell files. The stomata files were counted in between the two parallel veins (Figure 4a).


Statistical Analysis. All data were analysed in a one-way analysis of variance using SAS 9.4 software using PROC GLM. PROC MIXED was used to analyze data of parameters with missing data and non-constant variance (SAS Institute Inc., 2012). The data were subjected to normality test to check residuals for normality and constant variance (SAS Institute Inc., 2012). When there were treatment differences, the means were compared and grouped into letter groupings using Fisher's protected least significant difference (LSD) (P = 0.05) with n = 5.

Figure 2. Micrograph images of a) a typical epidermal features and stomatal patterning in a rice leaf (S stomatal files, E epidermal files), and b) a stomatal complex outline in rice leaves indicating the stomatal complex area (SCA) (surrounded by blue dashed line), guard cell width (GCW), stomatal cell width (SCW), stomatal pore area (SPA) (surrounded by red dashed line), subsidiary cells (SCs), stomatal cell width (SCW), and stomatal pore length (SPL). Scale bar = 5µm
Figure 2. Micrograph images of a) a typical epidermal features and stomatal patterning in a rice leaf (S stomatal files, E epidermal files), and b) a stomatal complex outline in rice leaves indicating the stomatal complex area (SCA) (surrounded by blue dashed line), guard cell width (GCW), stomatal cell width (SCW), stomatal pore area (SPA) (surrounded by red dashed line), subsidiary cells (SCs), stomatal cell width (SCW), and stomatal pore length (SPL). Scale bar = 5µm

RESULTS

The effects of sound wave in rice plant were evaluated on a few growth and physiological parameters including plant growth, leaf physiology and leaf stomatal properties. Each of these parameters corresponded to specific stages of rice growth, allowing a general correlation to be deduced about the vigour and potential yield of the plant. Plant growth parameters include number of leaves and plant height. Physiological assessments were always performed on the mid region of the leaf blade of fully expanded leaf 5, and these include assimilation and stomatal conductance rate. In addition, the assessment of stomata morphologies was performed using samples obtained from leaf 5.


Growth and Appearance.

Plant Height. The sound wave treatments had significantly affected plant height. The frequency of 357 Hz resulted in the highest average plant height of 43.6 cm, which is approximately 21% higher than the control (Figure 2a). It was observed that the control and 359 Hz treatment had the lowest plant heights averaging 36 and 39 cm, respectively.


Leaf Number. There was no significant difference in leaf number between any of the sound wave treatments. However, there is a tendency for the plants that received a frequency of 350 Hz to produce higher leaf number compared to other treatments (Figure 2b).


Figure 3. Mean of a) plant height and b) leaf number of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean
Figure 3. Mean of a) plant height and b) leaf number of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean

Leaf Physiology. Assimilation rate (A400), stomatal conductance (gsw), and intrinsic water use efficiency (iWUE) were used to assess the performance of leaf 5 after being stimulated with sound wave of different quality (Hz).


Photosynthesis (A400). A400 measurements showed that stimulation with sound waves of 357 and 350 Hz of led to the highest rate of photosynthesis (17.3 and 17.9 µmol CO2 m⁻² s⁻¹, respectively) compared to the other treatments (Figure 3a). Compared to the control, these two treatments increase the rate of assimilation by 39 to 43%. Intermediate assimilation rates were observed in plants receiving 380, 359 Hz sound waves, and control treatments (14.67, 17.86, and 12.48 µmol CO2 m⁻² s⁻¹, respectively). The lowest A400 value was observed in plants stimulated with 353 Hz soundwave (9.66 µmol CO2 m⁻² s⁻¹).


Stomatal Conductance (gsw) and Intrinsic Water Use Efficiency (iWUE). Significantly high gsw value were observed in treatments of 380 and 350 Hz (0.54 and 0.65 µmol H2O m⁻² s⁻¹, respectively) compared to the control (0.37 µmol H2O m⁻² s⁻¹) (Figure 3b). It is worth noting that the plants stimulated with sound waves of 359 to 353 Hz consistently produced significantly lower gsw (and comparable to the control) compared to the plants stimulated at 380 and 350 Hz. When both A400  and gsw were combined as a ratio to assess intrinsic water use efficiency (iWUE), the plants stimulated at 359, 357, and 353 Hz consistently produced significantly high iWUE (60.7, 48.3, and 48.5 µmol CO2 mol H2O⁻¹, respectively) compared to plants stimulated at 380 and 350 Hz (Figure 3c).


The iWUE for 359 Hz treatment was the highest among other treatments with an iWUE value of 60.6 µmol CO2 mol H2O⁻¹ indicating that plants at this distance have high water use efficiency and it is expected that these plants can tolerate the drought condition well. However, this treatment was not significantly different from that of plants receiving 357 and 353 Hz with iWUE values of 48.2 and 48.5 µmol CO2 mol H2O⁻¹, respectively. The control, 380 Hz, and 350 Hz showed the lowest iWUE values, namely 37.3, 25.1, and 28.0 µmol CO2 mol H2O⁻¹, respectively.

Figure 4. Mean of a) assimilation rate (A400), b) stomatal conductance (gsw), and c) intrinsic water use efficiency (iWUE) of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean
Figure 4. Mean of a) assimilation rate (A400), b) stomatal conductance (gsw), and c) intrinsic water use efficiency (iWUE) of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean

Leaf Stomatal Properties.

Effects of Sound Wave on Stomatal and Epidermal Area. Stomata are small epidermal pores on leaves that regulate the movement of CO2 and water in and out from the plants, respectively. Therefore, the attribute of stomata is crucial to ensure plants have a balanced gas exchange where they can maintain enough CO2 for carbon fixation, while minimizing water loss. The parameters quantified in this experiment included stomatal complex area (SCA), stomatal pore area (SPA), stomatal density (SD), percentage stomatal file (PSF), stomatal complex length (SCL), stomatal pore length (SPL), stomatal cell width (SCW), guard cell width (GCW), and cell file width (CFW) (Figure 2).


Stomatal Complex (Area, Length, and Width). Different frequencies of sound wave treatment have no significant effect on the SCL, SCW, and SCA (Figure 5a - 5c). However, there is a tendency for the SCA to increase for plants stimulated with a 350 Hz sound wave (314.2 µm²) compared to the control treatment (293.6 µm²) (Figure 5c).


Figure 5. Mean of a) stomatal complex length (SCL), b) stomatal complex width (SCW), and c) stomatal complex area (SCA) of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean
Figure 5. Mean of a) stomatal complex length (SCL), b) stomatal complex width (SCW), and c) stomatal complex area (SCA) of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean

Stomatal Pore (Length and Area) and Guard Cell Width. It was observed that sound wave treatment had a significant impact in altering the length (SPL) and area of the stomatal pore (SPA), but not the width of the guard cell (GCW) (Figures 6a-6c). Significantly greater pore length was observed in 350 Hz stimulated plants and in the control treatment with mean values of 16.1 and 14.8 µm, respectively. This also showed that lower frequency has a significant effect in increasing the pore length. The shortest pore length was observed in plants that receiving 353 Hz (12.19 µm) sound wave, but not significant difference from the 357, 359, and 380 Hz sound wave treatments with mean pore lengths of 12.3, 12.9, and 13.7 µm, respectively (Figure 6a). The 353 Hz sound waves stimulation produced the smallest pore length, alikeuction was approximately 8 and 27% compared to control and 350 Hz treatments, respectively.


The largest SPA was observed in the control, 380, and 350 Hz with a mean of 72.3, 87.23, and 84.63 mm², respectively. The smallest pore area was observed in plants that received 353 Hz, but this treatment is not significantly different from plants that received 380, 359, and 357 Hz. Compared to the control, sound wave treatments significantly reduced the pore area by 20-28%. The GCW was not significantly affected by different sound wave treatments; however, the control treatment tends to have wider guard cells compared to other treatments.


Figure 6. Mean of a) stomata pore length (SPL), b) stomatal pore area (SPA), and c) guard cell width (GCW) of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean
Figure 6. Mean of a) stomata pore length (SPL), b) stomatal pore area (SPA), and c) guard cell width (GCW) of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean

Stomatal Density, Percentage Stomatal File, and Cell File Width. There was no significant difference in stomatal density (SD), percent stomatal file (PSF), and cell file width (CFW) of the rice plant when exposed to different sound wave treatments (Figures 7a-7c). The mean SD ranged from 187 to 291 mm-2 with plants stimulated with 380 Hz tending to have a higher SD and those stimulated with 350 Hz tending to have lower SD. For PSF the mean was between 18 and 26%, and there is a tendency for plants stimulated with 350 Hz to increase PSF and the control treatment had a lower PSF as compared to other treatments. For CFW, the mean ranged from 11.9 to 14.65 µm with plants exposed to 359, and 353 Hz sound waves tending to have higher and lower CFW, respectively.


Figure 7. Mean of a) stomatal density (SD), b) percentage stomatal file (PSF), and c) cell file width (CFW) of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean
Figure 7. Mean of a) stomatal density (SD), b) percentage stomatal file (PSF), and c) cell file width (CFW) of seedlings grown at different frequencies measured at 80 cm distance interval from the sound wave source. Means with the same letter are not significantly different at P > 0.05 using LSD (n = 5). Error bars indicate the standard error of the mean

Correlation between Physiological and Morphological Attributes

The correlation analysis suggested that many of the stomatal morphological parameters have intermediate to strong correlations among each other, as compared to the correlation within growth/physiological parameters, as well as correlation between growth/physiological and morphological parameters (Figure 8). The above results suggested that most of the stomatal characteristics are independent of leaf growth/physiological characteristics, except for the SPA and SPL, which had significant intermediate positive relationships with gsw, a physiological parameter. Between the leaf physiological attributes, A400 shows a significant intermediate positive correlation with stomatal conductance. In addition, iWUE shows a significant high positive correlation with stomatal conductance. On the other hands, within the leaf morphological attributes, intermediate positive correlations were observed between SCA and SCW, SCL and GCW, SCL and SCW SPL and SCW, and SPA and GCW. Strong positive correlations were observed between SPL and SCA, SPA and SCA, SPA and SCW, SPA and SCA, and SPL.


Figure 8. Pearson correlation coefficients between plant leaf characteristics, leaf physiology and stomatal morphology of rice (MR219) grown at Ladang 15, UPM in 2017  Note. *, **, *** Statistically significant at P < 0.05, 0.01, and 0.001, respectively  r = Correlation coefficient  Non-star circle means not statistically significant at P > 0.05. Blue and red circles indicate positive and negative correlations, respectively. The size and colour intensity of circle indicate the strength of correlation between parameters. Large circles mean strong correlation and small circle means weak correlation  sca = Stomata complex area; gcw = Guard cell width; scw = Stomata complex width; cfw = Cell file width; scl = Stomata complex width; spl = Stomata pore length; psf = Percentage of stomatal file; spa = Stomata pore area; sd = Stomatal density; A400 = Assimilation rate (CO2 400 ppm); gsw= Stomatal conductance; iWUE = Intrinsic water use efficiency; ph = Plant height at day 28; nleaf = Number of leaf at day 28
Figure 8. Pearson correlation coefficients between plant leaf characteristics, leaf physiology and stomatal morphology of rice (MR219) grown at Ladang 15, UPM in 2017 Note. *, **, *** Statistically significant at P < 0.05, 0.01, and 0.001, respectively r = Correlation coefficient Non-star circle means not statistically significant at P > 0.05. Blue and red circles indicate positive and negative correlations, respectively. The size and colour intensity of circle indicate the strength of correlation between parameters. Large circles mean strong correlation and small circle means weak correlation sca = Stomata complex area; gcw = Guard cell width; scw = Stomata complex width; cfw = Cell file width; scl = Stomata complex width; spl = Stomata pore length; psf = Percentage of stomatal file; spa = Stomata pore area; sd = Stomatal density; A400 = Assimilation rate (CO2 400 ppm); gsw= Stomatal conductance; iWUE = Intrinsic water use efficiency; ph = Plant height at day 28; nleaf = Number of leaf at day 28

DISCUSSION

This experiment studied the effect of sound wave qualities on three different components of crop performance in rice namely plant growth, leaf physiology, and stomata morphology. In the first part of this study, the effects of sound wave on general plant growth and leaf physiological parameters were evaluated. The second part of the study evaluated the effects of sound waves on changing the morphological properties of stomata and epidermis of rice leaves. The combined results from the physiological and morphological traits of plants provide insight into how these components affect rice seedling growth.

Since ultrasound energy can increase the permeability and selectivity of cell membrane thus promote cell wall growth (Qi et al., 2010), it has been hypothesized that overall plant growth may benefit from enhanced permeability and selectivity of cell membrane. However, there is a certain value of sound wave qualities that either promote or hinder plant growth. This study identified two soundwave frequencies namely 357 and 350 Hz, which promote plant performance by significantly increased several parameters, namely assimilation rate, stomatal conductance, and plant height. The above findings suggested that an increase or decrease in these parameters might correspond to the nature of the wave propagation which has peak and trough. The two peaks could correspond to the frequency of 357 and 350 Hz or 240 and 400 cm from the sound source (high values) while the trough could correspond to frequencies of 359 and 353 Hz or 160 and 320 cm from the sound source (low values). Although not significant, other parameters also show a similar trend in sound wave propagation at slightly different distances. The above speculation needs to be proven, but a plausible explanation was that music can enhance the uptake of nutrients from the soil, resulting in better plant metabolism leading to increased growth and performance (Chowdhury et al., 2014). Sound wave also has the capability to affect stomatal movement, so this may have an important impact on leaf gas exchange capacity (Cai et al., 2014).

Physiological data also revealed interesting findings which could be used to support the earlier results in general plant growth. It was previously reported that the frequencies of 357 Hz significantly improved plant height compared to plants that did not receive sound wave stimulation. The above finding is consistent with the photosynthesis measurements where the plants stimulated with similar sound wave quality also showed significantly higher assimilation rates compared to control (Table 1).


Summary of mean comparison when compared to control treatment only. The green and red arrows indicate significantly increase or decrease, respectively. The mean comparison was performed using least significant difference (LSD) at a significant level of P < 0.05
Summary of mean comparison when compared to control treatment only. The green and red arrows indicate significantly increase or decrease, respectively. The mean comparison was performed using least significant difference (LSD) at a significant level of P < 0.05

The morphological study of stomatal and epidermal properties of rice leaf in this study included many dimensional categories of the stomata including area, frequency, length, and width. The results showed that different sound wave qualities had minimal effects on stomatal and epidermal properties, suggesting that stomatal and epidermal properties are not very responsive to different sound wave qualities. It has been reported that stomata size is inversely related to stomata density (Büssis et al., 2006; Doheny-Adams et al., 2012) and the increase in stomata density is compensated by decrease in stomata size (Büssis et al., 2006). This implies that plants with high stomata density will have small stomata, and vice versa. It has been shown that mutants with low stomata density and large size have reduced transpiration rates when grown in different CO2 concentrations (200, 450, and 1,000 ppm), and water regimes (70 and 30%) (Doheny-Adams et al., 2012), suggesting that plants with these traits may be beneficial in drought conditions where water availability is limited.

In rice leaf, one of the most important properties in photosynthesis is the guard cell width, which controls the gas and water movement in and out of the leaf. In this study, most of stomatal properties namely stomatal complex length, stomatal complex width, stomatal complex area, guard cell width, stomatal density, percentage stomatal file, and cell file width did not respond significantly to sound waves stimulation. However, significant responses were observed in stomatal pore length and area, indicating that stomatal cells have been stimulated by the sound wave which could have resulted in the differential gene expression for these traits. It was reported that the content of RNA and soluble protein increased in chrysanthemum cell culture stimulated with sound waves suggesting an alteration in gene expression (Xiujuan et al., 2003). In addition, SPA (Figure 6b), which trend was also similar to the stomatal complex area suggesting a significant correlation between these two parameters (Table 1).

Sound vibration is a mechanical stimulus that can cause thigmomorphogenetic responses in plants (Telewski, 2006). Therefore, soundwave treatments to a certain degree could possibly alter the stomatal pore size. It was documented that stomatal features including stomatal pore size influenced stomatal conductance (Fanourakis et al., 2015). An intermediate positive correlation was observed between conductance and stomatal pore length and area in this study suggesting the tendency of stomatal pore length and area in affecting stomatal conductance (Table 1).

In addition, it was found that certain high frequencies have the tendencies to reduce the length and area of the stomata, while those of plants stimulated at 350 Hz (60 dB), were unaffected compared to the control treatment. The rate of assimilation for plants stimulated with 350 Hz (60 dB) is the greatest compared to other treatments including the control (Figure 4a). The current finding contradicts those of Hou et al. (2009), who reported that when using four speakers as sound wave treatment at a different planting distance in cotton, the minimum yield was obtained in plants grown at a relatively far distance (30 m) with a sound wave intensity range of 75-110 db. Evidence from another study showed that net photosynthesis measured weekly in strawberry plants treated with sound waves of 100 dB and a frequency of 40–2,000 Hz was not significant compared to the control (no sound wave), except during the 4th sound stimulation. Although not significant, there were tendencies to improve net photosynthesis when treated with sound waves, potentially leading to higher yield. Similarly, the sound wave treatment tends to produce higher fruit number compared to the control (Meng et al., 2012). Different ranges of sound intensity, measured in decibels used for multiple frequencies in sound wave studies. As previously mentioned, the intensity of a wave is the energy carried by the wave per unit time per unit area at that point. Frequency on the other hand, measures sound quality, or the number of sound waves per second. At a given frequency, the intensity can vary depending on how much energy is carried by the sound wave. Therefore, when treating the plants with sound waves, the researcher can adjust the intensity of a specific frequency of interest.

Additionally, this study found that the stomata density of plants treated at higher frequencies (Figure 7a) tended to have smaller pore size (length and area) (Figures 6a and 5b). Although the effect between frequencies in stomata density was not significant, the response pattern for stomata density and pore size (area and length) showed a similar trend to the results reported in a study that manipulated the genetic of stomata density in Arabidopsis. It was also implied that the plants with small pore size and high stomatal density showed significantly higher water use efficiency and are very likely to adapt well to drought environment (Franks et al., 2015). In another study, rice plants with reduced stomatal density were shown to exhibit the ability to conserve water and tolerate drought while maintaining rice yield (Caine et al., 2019).


To explain the mechanism of sound waves at the cellular level, it was observed that a certain frequency of sound waves significantly changes the structure of the membrane protein and affects the permeability and fluidity of the cell membrane (Zhao et al., 2002). In addition, it was observed that the activity of plasmalemma H+ ATPase (proton pumps), which regulates biochemical and physiological processes in plant growth, increases by 19.8% in plants treated with sound waves (Yi et al., 2003). It is believed that vibrations induced by sound waves, can enhance the permeability of membranes, could be useful in regulating the movement of substances in or out of the cell, thereby enhancing plant growth. Bochu et al. (2003) showed that sound waves with a frequency of 400 Hz can improve the buoyancy of the cell membrane and strengthen the mutual function between lipid and protein regions of the membrane.


It is worth mentioning that having high photosynthetic rate alone, although sensibly means resulting in relatively more vigorous plants, is not going to be always helpful if the plant is experiencing water scarcity. In fact, there are two key findings in this study. First, if water is never an issue, rice plants can be stimulated with 380, 357, and 350 Hz soundwaves frequencies to achieve the best photosynthetic experience. Second, if water efficiency is the aim, then stimulating the rice with 359 Hz sound wave is the answer because relatively high amount of carbon is assimilated for one-unit amount of water lost. Nevertheless, it is worth noting that music comes with various sound quality thus if the same physiological improvement is to be achieved, similar sound quality in terms of hertz and decibel should be applied regardless of the music type.


CONCLUSION

The current study aimed to determine the effects of sound wave quality on rice plant characteristics, including height, stomatal properties, and physiological performances. These findings suggest that, in general, sound waves between the frequencies of 350 and 370 Hz could promote plant growth performance in terms of increased plant height. The proportionate carbon assimilation and stomatal conductance rates could be the reason behind the increase in growth, particularly at 350 Hz frequency. It was also shown that the pore dimensions of the stomata were alterable at frequencies higher than 350 Hz. The distinct significant effects of sound wave in certain parameter classes suggest its useful potential as a stimulus like many other abiotic factors, such as light and temperature. The ability of sound wave stimulus to increase yield and achieve better water use efficiency indicates alternative options are always available in improving seedlings' establishment in rice, thus potentially leading to an enhanced overall rice yield and production. Also, since rice production is water intensive, reducing the water supply in the rice can adversely affect yield. Therefore, sound wave stimulation at a specific frequency, which can change the properties of stomata, resulting in the plant using water more efficiently, allows plants to produce a higher yield with limited water availability.


ACKNOWLEDGEMENTS

The authors would like to thank Dr. Liang Juan Boo for the constructive feedback of this manuscript review.


FINANCIAL DISCLOSURE

The authors received no specific funding to support this research work and was fully self-funded by the authors.


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Mashitah Jusoh, Shairul Izan Ramlee, Faiznur Iffah Pydi, Nur Aishah Mazlan, Zulkarami Berahim, Azzami Adam Muhamad Mujab, Uma Rani Sinniah, Joanne Pei Sze Yeoh, Khalisanni Khalid and Muhammad Nazmin Yaapar*


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