Impaginato 373 Adv. Hort. Sci., 2020 34(4): 373­380 DOI: 10.13128/ahsc­8109 Mechanical rubbing of tomato intern­ ode influence stem growth, improve tensile strength but negatively impact flavonoid levels S. Sabina, M.N. Jithesh (*) School of Sciences, Department of Biotechnology, Jain (Deemed‐to‐be University), Jayanagar, 560 011 Bangalore, India. Key words: Lignin deposition, Lycopersicon esculentum, mechanical induced stress, phenolic compounds, tomato. Abstract: Agricultural crops are exposed to different environmental stress fac­ tors on a daily basis. Mechanical induced stress (MIS) caused due to contact rubbing, bending, transplantation and spraying of water results in altered growth in plants (Thigmomorphogenesis). The present study was conducted by inducing mechanical stress (gentle rubbing) on the third internode of tomato (Lycopersicon esculentum Mill.) by pressing with thumb and index finger (30 sec) for 14 consecutive days. At the end of the stress period, marked morpho­ logical differences included significant reduction in plant height and decreased internodal length of the rubbed third internode as well as the neighboring fourth internode. Histochemical staining of the stem cross section of stressed plants showed intense color indicating lignin deposition. Study of biochemical response post internode stress showed an increase in total phenol content but lower flavonoid contents. Stress induction also resulted in modification of bio­ mechanical characteristics like tensile strength, elastic modulus and breaking force. Studies on the effect of mechanical perturbations in plants has gained attention because of its implication in fundamental processes of organogene­ sis/morphogenesis and their potential as an innovative means of controlling plant growth. 1. Introduction In the natural habitat, plants are challenged by various biotic and abi­ otic stress factors. Mechanical Induced Stress (MIS) is one such form of stress that occurs inevitably in plants on a daily basis. MIS is a term used in cases of either a damage or physical injury due to bending, shaking and rubbing experienced by plants as a result of wind exposure, rain, animal movement and agricultural practices (Biddington, 1986). Being sessile, plants do not possess the luxury of relocating from the stressful source and are persistently challenged by these unfavorable environmental con­ ditions. Plants are also known to sense the force of stimuli that can range from subtle effects like touching or rubbing on leaves to more intense (*) Corresponding author: narayanan.jithesh@gmail.com Citation: SABINA S., JITHESH M.N., 2020 ­ Mechanical rub‐ bing of tomato internode influence stem growth, improve tensile strength but negatively impact flavonoid levels ­ Adv. Hort. Sci., 34(4): 373­380 Copyright: © 2020 Sabina S., Jithesh M.N. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Author contribution statement: JMN conceived the experiments and SS conduc­ ted the experiments. JMN and SS contributed to the analysis, interpretation and writing of manu­ script. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 19 February 2020 Accepted for publication 23 September 2020 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(4): 373­380 374 ones like the damage caused by herbivores (Chehab et al., 2009). Morphogenic responses due to rubbing or touch are termed Thigmomorphogenesis (Jaffe, 1973). Among the most visible morphological thig­ momorphogenesis response common to many plant species includes decrease in shoot elongation, cou­ pled with increased radial dimension of cortex cell (Telewski and Jaffe, 1986; Braam, 2005; Potocka and Szymanowska­Pulka, 2018). Plant cell wall is the first barrier against external mechanical forces. Lignin is one of the main structural components of the plant cell wall and regulation of lignin metabolism is of prime importance to plant growth (Zheng et al., 2017). Apart from lignin, flavonoids are among the most crucial phenolic compounds involved in various protective roles in plants, such as attractants, feeding deterrents and even, oxidative stress protection (Treutter, 2005; Mierziak et al., 2014). While, stress induced biochemical changes in lignin are known, effect of mechanical stimulation on flavonoid levels are scarce. Regulation of plant growth to achieve essential agronomic traits such as crop yield and plant height, are important prerequisites for agricultural indus­ tries. Marketability of many important ornamental and horticultural crops increases with compact height and a strong stem as these facilitates packag­ ing and also, transportation of post­harvest produce (Babalar et al., 2016; Bornke and Rochsch, 2018). The knowledge on force response of stem structures is critical as it incorporates approaches to test bio­ mechanical properties involving tensile strength and elastic modulus. These tests besides having economic implication, is also an immense tool for crop improvement (Shah et al. , 2017). Tomato, (Lycopersicon esculentum Mill.) is one of the most cultivated vegetable crops belonging to Solanaceae family (Schwarz et al., 2014). Global tomato produc­ tion accounts for 170 million tonnes, comprising 75% fresh and 25% processed material for industrial uses (Costa and Heuvelink, 2018). Due to its short dura­ tion and relative ease of cultivation, tomato produc­ tion has become one of the principal horticultural industries (Costa and Heuvelink, 2018). In this study, we experimentally determined whether stress on the internode by rubbing can lead to thigmomorphogenetic effects, leading to mechani­ cal stress resistance in tomato. It is hypothesized that lignin accumulation due to mechanical perturbations positively regulates higher tensile strength in plant stem. Therefore, the purpose of this study was to establish physiological effects of mechanical stimula­ tion of the 3rd internode on tomato stem growth characteristics and consequently, its mechanical properties in plants. 2. Materials and Methods Plant material and growth condition Seeds of tomato (Solanum lycopersicum Mill.) belonging to a semi determinate variety (Solanum lycopersicum Mill. cv. Arka Vikas) were obtained from Indian Institute of Horticultural Research (IIHR, Bengaluru, India). Germinated seedlings were trans­ planted to fresh pots (one plant per pot) in green house (12o56‘7” N, 77o35‘3” E) and maintained in moist soil under natural light condition (14 hrs of light and 10 hrs of dark) at 27­28oC. Relative humidity was maintained around 61­70% in the green house. All the experiments were conducted between 10.00 AM ­ 2.00 PM to avoid early and late diurnal response. Application of mechanical stimulation Mechanical stress stimulus was applied to five week old plants, henceforth referred to as internode stress. Internode stress was applied by rubbing the 3rd internode for 14 days, using thumb and index fin­ ger for 30s (Depege et al., 1997). Plant growth parameters like height and internode length were measured after the application of internode stress for 14 days. The mean total height and internodal length (3rd and 4th internode) of 20 plants were recorded and compared with control plants. Total phenolic compound content Total phenolic content in leaves of plants after application of stress were determined by Folin­ Ciocalteu method as described by Marinova et al. (2005). About 1 gram of fresh leaf was collected by excising the third leaf through random selection from the 20 pots of both control and stressed plants. Evaluation of phenol content was made for each of the independent triplicates (n=3) from 1g of pooled leaf samples for both control and treated plants. Each sample was macerated in 25 ml of methanol for 24 hr with occasional shaking. 250 ml of the extracted solution was mixed with 750 ml of methanol and 1mL of Folin­Ciocalteu reagent was added. After 5 min, 1 mL of Na2CO3 (20%) was mixed to the solution. After 30 min of incubation in dark, absorbance was mea­ sured at 765 nm using a UV­Vis Spectrophotometer (Shimadzu Inc. Japan) against a blank sample. A stan­ Sabina and Jithesh ‐ Thigmorphogenesis in tomato after mechanical stimulation 375 dard Gallic acid curve was constructed by preparing dilutions of a standard solution of Gallic acid. Total phenol was calculated and was expressed as Gallic Acid Equivalents per gram of fresh weight (GAE g­1 of FW). Total flavonoid content Aluminium chloride calorimetric method was fol­ lowed for the determination of the total flavonoid content in leaf samples based on the methodology described by Dewanto et al. (2002). About 1 gram of fresh leaf was collected by excising the third leaf through random selection from the 20 pots of both control and stressed plants. Evaluation of flavonoid content was made for each of the independent tripli­ cates (n=3) from 1 g of pooled leaf samples for both control and treated plants. Each sample was macer­ ated with 70% methanol and kept for 24 hours with occasional shaking. The extracted sample solution (250 mL) was separately mixed with 75 mL of 5% NaNO2 followed by incubation for 5 min. After incu­ bation, 10% AlCl3 (150 mL) was added followed by 500 mL of 1 M Sodium hydroxide. Total volume was made up to 2.5 ml using distilled water. After the incuba­ tion period for 30 min, absorbance was measured at 510 nm against a blank using UV­Vis spectropho­ tometer (Shimadzu Inc. Japan). Quercetin standard curve was constructed by preparing dilutions of a standard solution of Quercetin for the quantification of total flavonoids. The total flavonoid was calculated and was expressed as quercetin equivalent per gram of fresh weight (QE g­1 of FW) (Lin and Tang, 2007). Lignin staining and quantification analysis To evaluate how MIS affected lignin deposition, histochemical analysis of the stem sections of control and stressed plants was performed using wiesner reagent. The amount of 2% of fresh phloroglucinol staining solution for lignin staining was prepared in 95% ethanol. Fresh stem sections from the neigh­ bouring 4th internode were immobilized on a sliced potato surface. Multiple homogeneous thin freehand sections were obtained by drawing a sharp razor blade with smooth stokes repeatedly to ensure uni­ formity. The sections were further checked by stere­ omicroscope in order to remove thick and also dam­ aged sections. The most uniform sections were picked carefully and transferred to petri plates con­ taining water. Uniform sections of control and treat­ ed plants were transferred to petriplates and immersed in phloroglucinol stain. After 24 hours, sec­ tions were transferred to a clean glass slide, to which a drop of concentrated HCl was added (Phloroglucinol­HCl stain). The slides were then gen­ tly warmed over a Bunsen burner for 20 seconds for HCl evaporation. A drop of glycerol was added to these sections after which a cover slide was placed over it gently. The slides were then observed under a microscope Labomed Vision 2000 (Inc. India) at 10X magnification (Mitra and Loque, 2014). Lignin quantification analysis was conducted through ImageJ software (ver 1.52p). An eyepiece graticule (scale bar) was used to set the scale mea­ surements for the sections of control and treatment. These images were further processed through a pro­ cess of spatial resolution information in ImageJ using the calibration set by the graticule image. To start with the quantification analysis, images were dupli­ cated. One of the images was converted from 8 bits to grayscale. Following this action, pixel information pertaining to the pixel colour, gets converted into brightness measurement. By selecting the command: image>adjust>threshold, images to be quantified was highlighted in pixels within the threshold range speci­ fied. Next, by choosing the command: analyse>set measurement, the area and limit to threshold was selected. This enabled to measure within the thresh­ old pixel range for control and treated images. Finally, the function, analyze­measure was used to read the area covered with stain. Percentage of stained area was calculated by: Stained area/Total area X 100 (Beziat et al., 2016). Determination of tensile strength Determination of biomechanical stem properties was done after 14 days of stress application. Plants of 10 cm length were defoliated after the experiment and five stem segments devoid of leaves and roots were randomly chosen for analysis from stressed and control plants. These stem segments were then kept in an oven (40­50°C) for approximately 1 hour to rid of excessive water content. They were then clamped in the Universal testing machine (MTS­Mechatronics, Inc, Ichalkaranji, India). The following biomechanical traits were determined (Shah et al., 2017): (1) Tensile strength (MPa) is a measure of the maxi­ mum stress that the material can withstand without being elongated or stretched. (2) Elastic modulus (MPa) represents a ratio of stress applied and strain, the material exhibits (degree to which the material resists deformation in response to an applied force). (3) Breaking force (N) measures the maximum force that the material can bear before its experiences mechanical stress failure. Adv. Hort. Sci., 2020 34(4): 373­380 376 Statistical analysis Statistical analysis for the results obtained were carried out using Student’s t test with level of signifi­ cance set at P<0.05. Coefficient of variation (CV) is a useful measure to determine dispersion in a variable and can be extremely informative to study variation in different phenotypic traits in a study. Coefficient of variation for the morphological traits obtained was calculated as follows: (Standard deviation/Average) x 100. Pearson correlation coefficient was performed to study the level of association between two variables, pair­wise. All these analysis was performed using GraphPad Prism (version.8). 3. Results Effect of internode stress on plant height and intern‐ odal length of tomato Application of mild mechanical stimuli in the internodes of treated plants resulted in reduction in plant height whereas control plants showed normal growth characteristics (Fig. 1). Internode stress led to 27.4% reduction of total plant height when com­ pared to control plants, a significant reduction at P<0.0001 (Fig. 2 a). Effect of internode stress affected the elongation of 3rd internode, the site of stress application. Interestingly, internode stress also resulted in significant difference in elongation of the neighbouring 4th internode. Results from the present finding showed that elongation of stressed internode was inhibited by 47% and 44% for 3rd and 4th intern­ ode respectively at P<0.0001 in comparison to con­ trol plants that were not stressed (Fig. 2 b). Coefficient of variation (CV) studies The results presented in Table 1 revealed that the mean plant height in control plants was 27.5 cm with a range between 25.2­30.5 cm. In contrast, treat­ ments resulted in mean plant height of 20.01 with range between 18.0­22.8 cm. In case of treated plants, the highest CV was observed for 3rd internode (16.64%), the site of mechanical stimuli followed by the 4th internode (14.8%). In comparison, in control conditions, coefficient of variation (CV) of 5­7% was observed for the traits measured, confirming lower variation within population. Clearly, internode stress Table 1 ­ Range of group means, mean values, standard deviation, standard error mean and coefficient of variation of internode length and total height of control and treated plants * Values in the table mentioned for n=20. Descriptive statistics for control and treated plants SDV= Standard deviation; SEM= Standard error of mean; CV= Coefficient of variation. Fig. 1 ­ Lycopersicon esculentum Mill cv. Arka vikas after 14 days of application of mechanical stimuli by rubbing the 3rd internode. Left, control plants and right, treated plants. Fig. 2 ­ Total height of control and internode stressed plants (a) and length of third and fourth internode of control and internode stressed plants (b). The vertical bars indicate standard errors. The values correspond to the mean ± SE, where n=20. Statistical analysis was performed by Student’s t­test for total height and internode length. Asterisks indicate mean values significantly differ at P<0.05 (**** indicate P <0.0001). Traits Control Treatment Range Mean SDV SEM CV (%) Range Mean SDV SEM CV (%) 3rd Internode 3.6­4.5 3.9 0.287 0.064 7.3 1.5­2.6 2.1 0.349 0.078 16.64 4th Internode 5.2­6.3 5.6 0.298 0.066 5.3 2.5­3.8 3.1 0.46 0.103 14.8 Total Height 25.2­30.5 27.5 1.9 0.429 6.9 18­22.8 20.01 1.097 0.245 5.48 Sabina and Jithesh ‐ Thigmorphogenesis in tomato after mechanical stimulation 377 lead to higher variability for the 3rd and 4th internode than in normal conditions as indicated by these results. Effect of internode stress on total phenol and flavonoid content Mechanical stimuli resulted in an increase of 25% total phenolic content in leaves, when compared to control plants, a significant increase at P = 0.0001 (Fig. 3 a). In contrast, flavonoid content in leaves of treated plants showed a reduction of 18% in compar­ ison to control plants, a significant decrease at P = 0.0013 (Fig. 3 b). Effect of internode stress on lignin deposition and tensile strength Histochemical analysis by staining revealed that the intensity of colour was visibly lower in control plants (Fig. 4 a). On the other hand, analysis revealed intense colouration (red/magenta) in stressed plants (4th internode) indicating increased lignin deposition in xylem bundles and confirming alteration in lignin deposition in the plants with internode stress (Fig. 4 b). Lignin quantification through ImageJ analysis revealed a 45% enhancement in lignified area for sec­ tions from treated plants. On the other hand, sec­ tions from control samples covered only 22% of ligni­ fied area (Fig. 4 c). Application of mechanical stimuli also led to increased mechanical strength as indicated by the various mechanical properties shown in figure 5. The mean tensile strength recorded a significant increase of 113% in treated plants, when compared to control plants at P<0.05 (Fig. 5 a). Other mechanical proper­ ties including elastic modulus, and breaking force were also significantly higher when compared to the control plants. The elastic modulus displayed by treated plants observed was 658.4 MPa, a highly sig­ nificant increase (P<0.0001) compared to control plants (Fig. 5 b). The mean breaking force for treated plants recorded was 21.13 N, in comparison to con­ trol plants, which was lower (9.8 N), a significant dif­ ference at P= 0.0007 (Fig. 5 c). Fig. 3 ­ Total phenol content expressed as mg of gallic acid equi­ valent per gram of fresh weight (GAE g­1 FW) after 48hrs of internode stress (a) and total flavonoid content expressed as mg of Quercetin Equivalent per gram of fresh weight (QE g­1 FW) after 48hrs of internode stress (b). Standard error is indicated by vertical bars. The values correspond to the Mean ± SE, where n=3. Statistical analysis was performed by Student’s t test. Asterisks indicate Mean values significantly differ from control at P <0.05 (** indicate P = 0.0013 and *** indica­ te P = 0.0001). Fig. 4 ­ Microscopic view of stained cross sections after 14 days of rubbing of the third internode for control (a) interno­ de stressed (b) and graph showing quantification of lignin analysis by ImageJ software (c). Ep­epidermis, Xy­ xylem (lignin deposition) and Pi­pith. Scale bar = 100 µm. Fig. 5 ­ Biomechanical properties of the stem measured through tensile test: tensile strength (a), elastic modulus (b) and breaking force (c). Standard errors are indicated by verti­ cal bars. The values correspond to the Mean ± SE, where n=5. Asterisks indicate Mean values significantly differ at P<0.05 (*** indicate P=0.0007 and **** indicate P<0.0001). 378 Adv. Hort. Sci., 2020 34(4): 373­380 Correlation studies among biomechanical trait The correlation coefficient among tensile strength, breaking force and plant height for both control and treated plants are reported in Table 2. Plants treated with internode stress showed a highly significant positive correlation (R = 0.999) among the two important mechanical properties for stem, ten­ sile strength and breaking force. However, the corre­ lation for these traits in control plants remained non­ significant. The correlation of total height of plant with respect to tensile strength and breaking force demonstrated a significant negative correlation for both treatment and control plants. Plants treated with internode stress showed a slightly higher nega­ tive correlation (R= ­0.97) than control plants (R= ‐ 0.91) between tensile strength and total height. A scatter plot of these associated traits clearly high­ lighted a negative slope of correlation after exposure to mechanical stimuli (Fig. 6). 4. Discussion and Conclusions In response to mechanical stimuli, plants respond by altering their growth rate and morphology (Chehab et al., 2009). In this communication, applica­ tion of internode stress was shown to be effective in reducing the overall height of the plant. Effect of brushing in reducing leaf size and stem elongation has been demonstrated in Lycopersicon esculentum (Mill.) (Johjima et al., 1992). In Arabidopsis thaliana (Linn.), inhibition of stem elongation in the inflores­ cence was also shown to be prominently affected due to mechanical perturbation (Victor and Rowe, 2011). A higher coefficient of variation in treated plants shown in this study demonstrated that the most relevant effect of internode stress treatment was to bring growth alteration by causing reduction in internode elongation for both the 3rd and the 4th internode. In comparison to the treatment, coeffi­ cient of variation in control plants was lower, reveal­ ing insignificant random variation within a phenotyp­ ic trait. Hence, reduction in internodal length clearly indicates that mechanical stress response is a gener­ alized response affecting both the rubbed internode as well as neighboring young internode. Plants do not possess specialized cells to mount a response to Mechanical Induced Stimuli (MIS) in nature (Börnke and Rocksch, 2018). Individual cells in the vicinity of local tissue that is mechanically stimulated may pos­ sess the property of mechanoperception and to transduce this signal to an unperturbed distal tissue either by hormonal or electrical signals (Erner et al., 1980). Depege et al., (1997) suggested that the trans­ duction of the signal was electrical occurring through the involvement of intercellular Ca2+ modulated by calmodulin gene expression. Moulia et al. (2015) fur­ ther proposed that local mechanosensing and the origin of ionic currents can be explained through the participation of mechanosensitive ionic channels or stretch­activated channels (SAC). Under our experimental conditions, while the phenolic contents increased, flavonoid content was reduced by application of mechanical stress. In plants, the phenylpropanoid metabolism and also the lignin biosynthetic pathway are stimulated by envi­ ronmental stress adaptation (Petersen et al., 1999). Saidi et al. (2009) reported an increase of all the major enzymes involved in the phenylpropanoid pathway with mechanical stimulation in tomato. Consequently, an increase in phenolic content and Asterisk indicates statistical significance, at P=0.12(NS), 0.033(*), 0.002(**) and <0.0001(****), NS= non significance difference. Table 2 ­ Linear correlation coefficient between different bio­ mechanical characters in control and treated plants. Correlation of morphological trait with biomechanical characteristics Traits Total plant height Breaking force Control Tensile Strength ­0.91 * 0.7 NS Total Plant Height ­0.94 * Treated plants Tensile Strength ­0.97 ** 0.999 **** Total Plant Height ­0.95 ** Fig. 6 ­ Scatter plots of tensile strength and total height measu­ red on treated plants (green) and control plants (red). The scatter plot indicates a significant negative correla­ tion between two variables with a correlation coefficient of r=­0.9, (P<0.0001). Sabina and Jithesh ‐ Thigmorphogenesis in tomato after mechanical stimulation 379 enrichment of lignin observed in this study is consis­ tent with these reports. However, biosynthesis of lignin pathway and flavonoid are highly co­regulated in plants, where repression of one pathway, redirects the metabolites into the other pathway (Besseau et al., 2007). In the phenylpropanoid pathway, p­ coumaroyl CoA is the branch point in the metabolic route leading towards biosynthesis of either one of them (Yeh et al., 2014). In plants, accumulation of flavonoid is affected as the common flavonoid­lignin pathway is diverted towards utilization of support mechanism for biosynthesis of lignin (Besseau et al., 2007). A reduction in flavonoid content observed in this study therefore, points to the proposition that lignin and flavonoid may be competing for the same substrates leading to a decrease in flavonoid synthe­ sis. MIS increased stem resistance to tensile forces in this study. The modification in these parameters is relatively fast as the experiment lasted for duration of only 14 days. In plants, important biomechanical traits for instance, higher resistance to tensile and breaking forces leads to the acquisition of a hard­ ened phenotype (Schoelynck et al., 2015; Shah et al., 2017). Cell wall rigidification due to accelerated ligni­ fication was shown to result in reduced internodal elongation after mechanical stress application in tomato (Saidi et al., 2009). Further, reinforcement of cellulose microfibrils by lignins was also reported to increase resistance to tensile forces (Genet et al., 2005). This unique thigmomorphogenetic response in plants is crucial to withstand repeated external stress and mechanical forces (Jaffe et al., 1984; Biddington, 1986). Moreover, plant height was shown to be nega­ tively correlated with tensile strength and breaking force in this study. Plant lodging, an important trait observed in plants is shown to occur as a conse­ quence of permanent displacement of plant stem by stem buckling (Kendall et al., 2017). Reduction in plant height and internodal length has been shown to improve lodging tolerance in crops, an important trait for crop improvement (Peng et al., 2014). Notably, introduction of dwarfing genes was an important outcome of the Green revolution, reducing lodging susceptibility by reducing plant height (Spielmeyer et al., 2002). In conclusion, we observed that besides modifying growth, mechanical stimulation due to rubbing resulted in increased lignin deposition and also enhanced tensile strength leading to overall gain in the physical strength of the plant stem. Further, our results provide new insights on the lignin­flavonoid pathway after MIS and demonstrate the plasticity of the biochemical response. The ability to sense and respond to mechanical stimulation by thigmomor­ phogenesis needs to be explored, as it holds great potential for commercial application in horticulture for regulating plant growth without the use of chemi­ cal regulators (Börnke and Rocksch, 2018). Acknowledgements We are grateful to Jain (Deemed to be University) for the infrastructure and also facilities provided to the authors. 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