Therefore, studies involving the use of sound as a trigger have been recognized as fringe science. However, recent findings using cutting-edge technology, quality control for hertz and decibel levels, and the integration of big data have helped change the viewpoint about this field as it has entered the realm of generally accepted science Gagliano et al.
We now believe that plants can indeed benefit from sound through their mechanosensory machinery. Many studies have already demonstrated sound-induced phenotypic changes and possible sound signaling pathways in model and crop plants. In this review, we discuss how plants generate and respond to sound and how sound can be used to improve plant growth and plant resistance against biotic and abiotic stresses. Here, we propose that sound is an emerging physical trigger in plants beyond chemical triggers, such as plant hormones and other immune activators which have been used to improve plant health.
To understand how plants respond to sound, we need a new framework beyond chemical compound-based signal initiation and responses in plants. We therefore classified the steps involved in this multi-layered process from the emission of sound by plants to the altered phenotypes observed after the plant has recognized the sound information. This basic knowledge helps us elucidate how sound signals trigger changes in plants in nature. It was long thought that plants do not make sounds.
Although humans cannot perceive sound from plants, recent studies using small, highly sensitive sound receivers have surprisingly demonstrated that plants indeed make spontaneous sounds and even release sound emissions from their xylem Borghetti et al.
Since the xylem is a water-transporting system in plants, transpiration, and re-hydration occur in xylem vessels. Transpiration produces tension in xylem vessels, and simultaneously, gas bubbles cavitation are produced in xylem vessels during transpiration. Indeed, gas bubbles adhering to vessels may produce sound in plants Laschimke et al. It is reported that when transpiration decreases, audible sound is released and transpiration increases, ultrasonic emission is released Ritman and Milburn, Also, the fact that plants emit ultrasonic vibration has been disputed, but it has recently been confirmed that ultrasonic vibration of 20— kHz is emitted by connecting a sensor directly to the plant stem that has been barked Laschimke et al.
Increasing studies also suggest that tension in the xylem is the cause of this sound in plants. However, whether plants use this ultrasonic sound for their communication remains to be elucidated. In addition to sound produced by plants, the idea that insects also produce sounds is widely accepted because we often hear sounds such as bees buzzing, insects chewing, and flies buzzing.
How do the sounds of insects affect plants? Specific frequencies of bee buzzing facilitate the pollination of flowers, since these sounds induce the release of pollen from plant anthers De Luca and Vallejo-Marin, In addition, insect chewing serves as an alarm signal to plants. Recorded insect chewing sounds induce the production of chemicals related to plant defense in Arabidopsis , such as glucosinolate and anthocyanin Appel and Cocroft, Collectively, these findings suggest that plants respond to insects through sound, sometimes serving as warning signals or beneficial signals to the plant.
Sound production and perception in plants. A Sound production. Additionally, gas bubbles produced in xylem vessels during transpiration may produce sound Laschimke et al. B Sound Perception. Although there are no visible alterations, transcriptional and translational changes occur in plants exposed to sound vibrations.
Levels of mechano-stimulus responsive, signaling-related, redox homeostasis, and defense-related transcripts are changed in sound-exposed plants Ghosh et al. However, the specific organs or proteins used for sound perception have not yet been identified.
How can plants perceive sound and thereby respond to specific stress stimuli without a hearing organ? The roots of Zea mays were reported to bend toward sound with a frequency of — Hz among the tested frequencies of 0— Hz in the hydroponic system Gagliano et al. Even small environmental stimuli such as touch or wind alter the transcriptional levels of plants. A recent study described commonalities and differences between responses to sound and mechanical vibrations at the gene expression level.
Expression of some genes e. This supports the notion that sound vibrations provide a special stimulus to plants, unlike mechanical vibrations. In addition, sound vibration increased the rate of growth by changing the cell metabolism of yeast, but reduced biomass production. Theses result imply that sound affects the cell level rather than the specific structure of the organism Aggio et al.
Here, we focus on recent findings about plant responses to sound treatment based on transcriptome and proteomics technology Figure 1B. Although sound is not a visible or chemical stimulus, plants exposed to sound a physical force produce increasing amounts of mRNA Ghosh et al. Indeed, two genes, the fructose 1,6-bisphosphate aldolase ald and Rubisco small sub-unit rbcS genes, which play critical roles in photosynthesis, were specifically induced in rice following and Hz sound treatment Jeong et al.
Continuous exposure to sound is thought to enhance plant growth by promoting CO 2 fixation Uematsu et al. These findings can be attributed to sound-mediated photosynthesis-related gene expression and increased CO 2 fixation. A similar study showed that the expression of genes in the Gene Ontology categories mechano-stimulus responsive, signaling related, redox homeostasis, biosynthesis, and defense increased in response to exposure to Hz sound waves in Arabidopsis Ghosh et al.
These results imply that sound vibrations provide a stimulus to plants. More extensive research is needed on the function of the identified genes and the signaling network.
In fact, plant hormone signaling networks are already beginning to be elucidated. Plant hormones typically regulate plant cellular processes and orchestrate most aspects of plant physiology including plant growth, flowering, ripening, senescence, and defense responses Hou et al.
Recent studies showed that, in Arabidopsis , treatment with Hz sound induces the production of the growth-related hormones indoleacetic acid IAA and gibberellin GA 3 and the defense-related hormones salicylic acid SA and jasmonic acid JA Ghosh et al. Although the optimal sound treatment differs depending on the plant species, such sound-induced hormonal changes might increase plant growth and provide strong resistance against biotic or abiotic stress.
A recent study reported that plant roots can respond to environmental sound Gagliano et al. Specifically, Pisum sativum roots locate water by actively growing toward flowing water belowground Gagliano et al. This implies that plants also respond to natural sound in the environment. As mentioned above, plants appear to perceive sound, as they exhibit transcriptional and hormonal changes in response to sound wave treatment.
Next, we provide an overview of the implications of sound wave treatment in the field or growth room. Based on this information, we discuss the expansion of the use of sound in modern agriculture and plant biology. Exposing plants to sound activates plant innate immunity and more specifically elicits representative SA and JA defense signaling pathways similar to those observed in response to different chemical triggers Ghosh et al. Meta-analyses have demonstrated the occurrence of sound-mediated plant protection through the activation of the systemic immune response in crop plants such as pepper, cucumber, tomato, and strawberry Hou et al.
These ions might serve as secondary messengers upon exposure to environmental stress, thereby enhancing plant resistance against microbial pathogens. Arabidopsis calmodulin-like 38 CML38 gene, which encodes a calcium-binding messenger protein, is upregulated in response to sound treatment in Arabidopsis leaves Ghosh et al. In addition, membrane architecture changes in response to sound treatment, which may facilitate the movement of signaling components related to defense responses Mishra et al.
In addition to biotic stress responses, sound treatment increases plant tolerance to abiotic stresses such as drought.
For example, rice exposed to 0. Water deficiency is first detected in the plant root, and drought stress signaling is transmitted to the shoot through the xylem. Since membrane architecture changes in response to sound treatment, the plant is better able to absorb water in situations where water is lacking.
Furthermore, among hormones, ABA is the most important regulator of the plant response to abiotic stress, especially osmotic stress Kim et al. Consequentially, sound waves may be involved in both abiotic and biotic stress responses through the regulation of various plant hormones. Sound waves as a plant stimulant and protectant. Artificial sound treatment can elicit various effects in plants.
First, enhancement of seed germination and plant growth. Sound promotes plant growth by regulating the plant growth hormones indoleacetic acid IAA and gibberellin Bochu et al. Second, induction of plant defense responses against pathogens. Sound pretreatment enhances plant immunity against subsequent pathogen attacks by activating the plant defense hormones salicylic acid SA and jasmonic acid JA Hassanien et al.
Third, induction of abiotic stress tolerance. For instance, sound treatment triggers drought tolerance by changing the elasticity and flexibility of the cell wall, which affects the ability of plants to absorb water Jeong et al.
Fourth, perturbation of ripening. Sound treatment disrupts the ripening of tomato fruit. Ethylene production is delayed by down-regulation of ethylene biosynthesis and expression of signaling-related genes Kim et al. Fifth, enhancement of the photosynthetic capacity. Plant gene responses to frequency-specific sound signals. Breeding Effect of six different acoustic frequencies on growth of cowpea Vigna unguiculata during its seedling stage.
Keli, S. The effects of alternative stress on the thermodymical properties of cultured tobacco cells. Acta Bio. Li, B. Effect of sound wave stress on antioxidant enzyme activities and lipid peroxidation of Dendrobium candidum. Influence of ultrasonic stimulation on the growth and proliferation of Oryza sativa Nipponbare callus cells. Ultrason Sono. Effects of an audible sound frequency on total amino acids and major free alcohol-soluble amino acids of Rideau wheat grains.
Responses on photosynthesis and variable chlorophyll fluorescence of Fragaria ananassa under sound wave. Energy Procedia A highly regulated enzyme with multiple physiological functions.
The Wave Theory of Sound. New York. Acoustical Society of America. Qi, L. Influence of sound wave stimulation on the growth of strawberry in sunlight greenhouse. AICT , pp Qin, Y. Biochemical and physiological changes in plants as a result of different sonic exposures. Ultrasonics The Sound of Music and Plants.
Rooke, A. Physiological aspects of cadmium and lead toxic effects on higher plants. Structure and function of plasma membrane ATPase. Rev Plant Physiol. A pilot study of the effect of audible sound on the growth of Escherichia coli.
Method for enhancing germination. Patent number. Sistrunk, M. Plant Cell 6: Spillane, M. Brave new waves. TCI for Plants. Subramanian, S. A study of the effect of music on the growth and yield of paddy. Madras Agr. Takahashi, H. Growth promotion by vibration at 50 Hz in rice and cucumber seedlings. Plant Cell Physiol. A unified hypothesis of mechanoperception in plants. Application of acoustic frequency technology to protected vegetable production.
Chinese Soc. Tompkins, P. The secret life of plants. Harper collins. Uchida, A. Effects of mechanical vibration on seed germination of Arabidopsis thaliana L Heynh.
Touch-regulated genes are marked in red color. Lee et al. Twenty-three touch regulated genes were also up-regulated by SV treatments, which are marked as mechanostimulus responsive genes in this group Fig. Thus, those genes are mentioned in the other groups as well.
Among the up-regulated genes, four were noted to function in cell signaling. Total thirteen genes that comprise a variety of TFs constitute this group. Four of the up-regulated genes were noted to function in cell redox homeostasis. At3g, encoding a member of thioredoxin TRX superfamily, showed the highest up-regulation by all the treatments.
Many differentially expressed genes fall into this group. TPS8 encodes a type II trehalosephosphatase synthase, which is involved in biosynthesis of trehalose, an osmoprotectant TCH4 involves in cell wall biogenesis through xyloglucan metabolism 1. Cytochrome P family members are very important for various metabolic process, like hormone, alkaloids, terpeniods and glucosinolates Many defense related genes were also differentially expressed after SV treatments.
Chitin is known as pathogen-associated molecular patterns PAMP which sensitize the plant immune system NHL3 , a positive regulator of plant defense, was up-regulated by SV treatment The genes in this group are involved in diverse cellular functions.
At1g EXL1 , which is involved in plant growth during carbon starvation 27 , was highly up-regulated by all treatments. At4g DIC2 belongs to mitochondrial carrier protein family and involved in membrane transport At4g EXO , which is involved in cell expansion of Arabidopsis leaves, was upregulated by all Hz Genes, which are not defined by any GO biological process, are placed in this group.
Among them At2g was highly induced by all SV treatments whereas At4g, At5g, At1g and At4g were down-regulated. Seven mechanostimulus responsive genes are also categorized in this group Fig.
A brassinosteriod BR responsive gene EXO , which encodes for an extracellular protein involved in cell expansion 30 , was highly up-regulated by all treatments.
Additionally, MYB77 is also involved in auxin signaling pathway by directly interacting with auxin response factors ARFs Furthermore, RAV1 is known as a negative regulator of abscisic acid ABA signaling during seed germination and early seedling stage Additionally, two more time points 0.
Expression of total seventeen genes was validated Fig. These seventeen genes are involved in diverse function. For a quick reference, the fold change based on microarray results and functions of these seventeen genes are mentioned in Table 1. The expression of the rest of the genes declined, in large, to control levels.
Error bar indicates the standard error of means from four biological replications. S3 ; Tables S4 and S5. Differentially expressed proteins between control and treatment were classified by using online GO platform.
Among the differentially expressed proteins, most proteins turned out to be involved in carbohydrate metabolism and photosynthesis Supplementary Fig.
The third largest group is constituted by stress and defense related proteins. As suggested by GO analysis, many of the differentially expressed proteins are either plastidic or apoplastic in localization Supplementary Fig. Further, the differential expression of proteins having antioxidant function was most significant Supplementary Fig. Fold changes and functional annotations of differentially expressed proteins are available in supplementary Tables S6 and S7 , respectively.
For the ease of understanding, we categorize the differentially expressed proteins on the basis of their function into following categories Fig. Classification is marked by uppercase letter. Functional annotations of proteins are mentioned in Supplementary Table S7. RuBisCO is the most important enzyme involved in the first major step of carbon fixation. It consists of two types of protein subunits, the large chain and the small chain. Glutamate-glyoxylate aminotransferase 1 GGAT , which is an important enzyme in photosynthetic carbon fixation cycle, photorespiration and metabolism of major amino acids, was up-regulated by all treatments, largely, at every time points.
Several glyceraldehydephosphate dehydrogenase GAPDH proteins were also significantly up-regulated. Similarly, cysteine synthase CS and S-adenosylmethionine synthase SAMS , enzymes involved in biosynthesis of cysteine and methionine, respectively, were strongly up-regulated by SV treatment. These enzymes are interlinked in glutamate, glutamine and glycine biosynthesis during photorespiratory nitrogen cycle.
There are two types of ATPase transporters. V-type is located in vacuole, while F-type is located in chloroplast and mitochondria Maintaining redox homeostasis is of utmost importance for cellular survival both under normal conditions and different environmental stresses.
APX also showed significant up-regulation at many time points. MDAR was up-regulated by all treatments, in large, at each time points. Nucleoside diphosphate kinase NDK is an important enzyme playing role in nucleotide metabolism.
Chloroplast stem-loop binding protein CSP41, endoribonuclease , critical for regulation of chloroplastic transcription and translation 35 , were up-regulated by all treatments. ATP sulfurylase ATPS , the first enzyme of the sulfate assimilation pathway 18 , was also up-regulated by all treatments. Uroporphyrinogen decarboxylase UROD , an important enzyme linked to chlorophyll synthesis pathway by virtue of its function in heme biosynthesis 37 , was strongly up-regulated by SV treatments.
Several enzymes related to protein metabolism, folding and degradation were also differentially expressed after SV treatments. Alteration in hormonal levels is modulated by several synergistic and antagonistic upstream events and thus hormonal interplay comes later in a signaling cascade. We noted significant changes in the levels of all the phytohormones, except ABA. Beneficial effects of SV on plant physiology leading to enhanced growth, development and disease resistance are well established by many previous reports 8 , However, there is evidently no study addressing the detailed molecular events triggered by SV to substantiate these observations.
Being prompted by this lacuna in our knowledge, we aimed at analyzing the global transcriptomic, proteomic and phytohormonal changes stimulated by SV in Arabidopsis. When an environmental cue incites a plant, several intercellular events occur in a cascading fashion. All these changes collectively aid plants to bring in favorable adjustments.
In our study, we strikingly noted differential regulation of many molecular components associated with these events in Arabidopsis exposed to SV. Interestingly, our microarray results revealed huge change in the expression profile of genes associated with these signature cellular events providing a glimpse of the overall signaling cascade triggered by SV in plants. Moreover, a large number of genes involved in hormonal signaling were also up-regulated.
In a previous study, a direct connection between calcium transients and SV-mediated enhancement in callus growth was established through pharmacological studies This highlighted the importance of calcium in SV-mediated responses. Calcium acts as a second messenger which facilitates a signaling cascade. Interestingly, we noted remarkable up-regulation of CML38 , encoding calmodulin-like protein. CML is a calcium-binding messenger protein that facilitates downstream signaling In light of the previous studies, our result thus suggests a possible involvement of the second messenger calcium in signaling of SV stimulus.
It is argued that being pressure waves, SV influences cells mechanically 5. Thus, there may be a crosstalk between the signal transduction mechanism of sound and mechanical stimuli. It was suggested that calcium signaling plays an important role in perception of mechanical stimuli, like touch Perturbation by external stimuli causes elevation of ROS, which participates in cellular signaling This is further substantiated by few available studies, where enhancement in activities of several ROS scavenging enzymes were noted in plant calli and seedlings exposed to SV 13 , It thus appears that ROS signaling is possibly an important player for SV-mediated molecular changes in the plant cell.
Active transport of important molecules across cell membrane is a prerequisite to maintain a healthy physiological state within a cell. Among them F-type showed consistent up-regulation by various Hz. Hamilton et al. Considering that some ion channels have mechanosensing property, activation of ion transporters may have a possible role in the SV-mediated cellular responses.
Stimulation by an external factor is likely to bring modifications in proteome related to primary metabolism in plants In previous studies, long term audible-SV treatment has been shown to promote plant growth 8 , which indicates that sound brings positive changes in photosynthesis and sugar metabolism. Corroboratively, SV has been noted previously to increase photosynthetic rate and chlorophyll fluorescence in strawberry plant Accelerated growth also fits well with enhanced carbon metabolism.
In line with this, we noted that SV altered expression of several enzymes involved in light reaction, Calvin cycle, glycolysis and TCA cycle, with majority of them being up-regulated Figs 4 and 5. In a previous study, Jeong et al. RuBisCO is the key regulatory enzyme of C3 carbon fixation and its up-regulation may thus reflect the enhanced photosynthetic state of plants after SV exposure. Contrastingly, RBCL protein was up-regulated in our study.
Furthermore, we did not find the up-regulation of RBCS protein. The important thing to notice here is that RBCL is encoded by chloroplast genome whereas RBCS is nuclear encoded; contrasting expression of these subunits suggests that SV can trigger different signaling events between cell organelle.
RuBisCO can adopt either carboxylase photosynthesis or oxygenase photorespiration activity depending on CO 2 concentration Increased accumulation of carbonic anhydrase noted in our study thus possibly helps to maintain CO 2 concentration favorable for enhanced photosynthesis. Besides RuBisCO, we also noticed strong up-regulation of LHCB2 transcript, which encodes a light harvesting complex playing critical role in providing energy required for photolysis.
While LHCB protein mainly showed down-regulation, it is in agreement to the previous findings FNR is involved in production of NADPH, which is used as reducing power by various metabolic process like Calvin cycle, amino acid biosynthesis, and lipid biosynthesis Thus an overall shift in the physiological state which is more favorable for efficient photosynthesis is suggested by our results.
Previous research indicates that sound can increase soluble sugar in chrysanthemum root and Dendranthema morifolium callus 53 , In this study, up regulation of TPS8 , which is involved in trehalose metabolism, also indicates the change of carbohydrate metabolism upon sound. This observation is important to note as soluble sugar itself act as a signaling molecule for activation of hormonal crosstalk and oxidative pentose phosphate OPP pathway-mediated ROS scavenging Glycolytic-TCA enzymes e.
Increased expression of the aforementioned glycolytic enzymes suggests efficient breakdown of complex sugar for gaining useful energy to fuel cellular activities. This energy, in large, feeds linked metabolic pathways like amino acid metabolism. Clearly, rerouting metabolic pathway to bring adjustments favorable to an external stimulus requires ample energy.
In cellular milieu, the molecular power that can readily be utilized is ATP. In plant cell, ATP is produced by both photophosphorylation in the chloroplast and oxidative phosphorylation in the mitochondrion, which are linked to light reaction, glycolysis and TCA cycle.
Hence increase in ATP synthase, which eventually may results in increased ATP synthesis, could be one of the reasons behind acceleration of metabolic pathways.
Like other external stimuli, SV has also been noted to increase soluble protein contents in plant cells 53 , Also, to account for the increased levels of several proteins noted in our study, synthesis of amino acids directly from stem pathway or through inter conversion is a prerequisite.
In corroboration to this, strong up-regulation of various amino acid biosynthetic enzymes was also noted in our proteome result Figs 4 and 5. Furthermore, several enzymes related to protein metabolism, folding and degradation were also differentially expressed. Among the up-regulated pool, two important enzymes ATP sulfurylase and cysteine synthase showed remarkable up-regulation.
ATP sulfurylase is one of the key enzymes for sulfur metabolism 18 , which is responsible for the synthesis of important amino acids, cysteine and methionine.
Further, cysteine is an important amino acid for the production of various sulfur containing defense molecules in plants, like sulfur-rich proteins SRPs , phytoalexins and glucosinolates Increased synthesis of sulfur containing amino acids may thus be a reason behind the increased disease resistance noted in SV-treated plants in earlier studies 3.
Plant growth and development in response to an external cue is tightly regulated by phytohormones. In previous studies, SV were noted to bring alteration in hormonal levels 57 , In the present study, SV exposure resulted into alterations in endogenous phytohormonal levels Supplementary Fig.
Interestingly, SA showed accumulation at all three time points. It is important to note here that SA and JA are antagonistic with each other Comparing the levels of all hormones, SA showed high accumulation at all-time points after SV exposure. We discussed in the foregoing text that there exist a possible crosstalk between the touch and sound mechanosensing. Response to mechanical stimuli is itself partly mediated by ethylene and other hormones like auxin, ABA and JA Notably, touch and other mechanical stimuli showed reduced growth phenotype, which is strongly correlated with mechano-stimulated up-regulation of the negative growth regulator ABA However, the studies available hitherto suggest growth enhancement in plants upon exposure to SV 8.
The important points emerging here are: 1 SV exposure appears to have less impact on ABA biosynthesis compared to other hormones, 2 besides the possible crosstalk between the signaling of touch and sound, plant perceives SV as an ecologically distinct stimulus and comes up with responses tailored accordingly. Thus, in the future course, detailed hormonal studies are required to establish strong correlation between SV and hormonal modulation.
Based on the results obtained in our study, we conclude with a model depicting the effect of SV on plant cell Fig. How a plant cell is sensitized mechanically, is still unknown. However, it is majorly believed that plant cell wall and membrane interface has an important role to play in perception of mechanical stimulus 5 , Upon mechanical disturbance, cytoskeleton coordinates with and opens various stretch activated ion channels 5 , It has already been noted that SV affect cell wall and membrane microstructure, thereby resulting in an overall increase cell membrane tension 8 , 60 , 61 , In response to SV, XTH could function as an important player for alteration of elasticity of cell wall.
Thus, it can be hypothesized that SV is perceived at cell wall-cell membrane interface, which leads to the activation of membrane-bound mechanosensitive ion channels. Thereafter, signals are transduced into the cell in which calcium binding CMLs and various kinases play critical role. Sidewise, ROS are generated which act as signaling molecule for various complex metabolic processes.
Huge changes in the transcriptome and proteome thus occur, which eventually affect several vital processes, like photosynthesis, glycolysis, amino acid metabolism and sulfur metabolism. These changes together with hormonal adjustments result in enhanced growth and defense. Combining previous reports and our analysis we can predict sound perception mechanism by cytoskeleton-plasma membrane-cell wall CPMCW interface.
Dotted boxes indicate representative transcripts or proteins identified by our experiment. Arrow marks indicate activation. Employing high throughput transcriptomic, proteomic and hormonal analyses, we revealed the global cellular changes taking place in a plant exposed to SV.
Interestingly, our results go hand in hand with several previous reports on effect of SV in plants, providing strong scientific basis to this least explored area of plant biology. Indeed, there are many more facets of plant acoustics left to be explored in upcoming research. The Adobe Audition version 3. To prevent the mechanical vibrations during the SV treatments, the speaker and plants were placed on different shelves.
After SV treatment, plants were shifted to growth room and samples were harvested at requisite time points for transcriptomic, proteomic and hormonal analyses. For transcriptomic analysis, samples at 0, 0. For a better representation of treatment method and sample harvesting time, a schematic diagram has been shown in Supplementary Fig.
Primers used are listed in Supplementary Table S8. Threshold of 0.
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