The Effect of Clinorotation to the Growth of Tomato (Lycopersicon esculentum) and Mung Bean (Vigna radiata) Seedlings

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1 Trans. JSASS Aerospace Tech. Japan Vol. 12, No. ists29, pp. Th_5-Th_10, 2014 Topics The Effect of Clinorotation to the Growth of Tomato (Lycopersicon esculentum) and Mung Bean (Vigna radiata) Seedlings (9mm) By Leonita SWANDJAJA 1), Rizkita Rachmi ESYANTI 1), KHAIRURRIJAL 2), Fenny M. DWIVANY 1) and Chunaeni LATIEF 3) K(5mm) RR 1) School of Life Sciences and Technology, Institut Teknologi Bandung, Bandung, West Java, Indonesia 2) Physics Department of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung, West Java, Indonesia 3) Laboratory of Atmospheric Technology, National Institute of Aeronautics and Space, Bandung, West Java, Indonesia Received June 24th, 2013 Plant growth and development are affected by abiotic factors such as light, temperature, water and gravity. Gravity ensures primary shoot grows upward towards sunlight to optimize photosynthesis, while the primary root grows downward into the soil to find water and mineral supply. Plants with impaired gravity response are poorly fit for survival in nature, since the roots may not be able to absorb the nutrient and the shoot may not be able to track sunlight. In the first study, the tomato seedlings on agar medium were treated on clinostat in light and dark condition. In dark, the tomato seedlings on the clinostat responded by bending their shoot and coiled their root. In the light condition, the shoot bending and root coiling were reduced significantly compare to the plants grew in the dark after seven days in clinorotation, which might indicate that phototropic response was stronger than gravitropic response in tomato seedlings. The mung bean on hanging mesh was tested on clinostat without light. Under this condition, instead of coiling, the root grew staight to the wet rockwool. The condition might indicate that mung bean seedling has stronger hidrotropic response compare to gravitropic response, as moisture gradient may trigger statolith degradation in columella cells. Key Words: Microgravity, 3D Clinostat, Gravitropism, Tomato, Mung Bean 1. Introduction Plant growth and development are affected by several abiotic factors such as light, temperature and water quantity. However, nearly upon germination, another physical factor called gravity, affected the growth of root and shoot to orient the seedling correctly in space for the survival of the newly developed seedling. One of the most important mechanisms for the survival of the germinating seedling is the growth of the root, which guarantees the young plant a rapid supply of water and minerals. On the other hand, the shoot grows upward to tract sunlight. Since plants have evolved growing under the constant stimulus of gravity, its presence is one of the most important requirements for their growth and spatial orientation 1). Studies on the effects of a weightlessness and hypergravity environment on plants have focused mainly on investigations of the gravitropic response 2). In higher plants, the gravity detector called statolith, are believed to be sedimentation of amyloplast in the cells called statocyte 3). Amyloplast displacement in statocyte is sufficient to promote organ-tip curvature 4). However, signal transduction of amyloplast sedimentation into a physiological signal in the statocyte is not fully understood. The modulation of the statolith of clover seedlings in normal gravity and microgravity simulated by a clinostat has been reported before 5). The results suggested that in normal gravity, the statolith mass concentrated at the distal part of the statocytes, following the vector of gravity. The experiment proposed that clinorotation disrupted root cap cells structure. The constant rotation of clinostat caused the cell wall in the columella cells deteriorated, and so reduced the amount of visible amyloplasts. Several results also showed that sedimentation of amyloplasts affected auxin movement. Until recently, the complex signal transduction cascade that regulates the differential cell elongation induced by gravitropic and phototropic remains unclear. A general model of root/shoot gravitropic and phototropic response is based on the model of the asymmetric redistribution of auxin that causes a differential cellular elongation on opposite flanks of the central elongation zone. The hypothesis was proposed by Cholodny Went 6). The downward bending of roots and the upward bending of shoots in multicellular plants to response gravity is the result from induction of unequal redistribution of auxin within the elongation zone, which is correlated with an accumulation of elevated auxin levels along the lower side. In roots, high concentration of auxin inhibits cell growth and elongation, thus a downward curvature is the consequence. In contrast, the upward bending in shoots is induced due to the growing stimulation effect of auxin, resulting in an upward curvature 6). The characteristic of unequal polar auxin movement induced by gravity in the early growth stages of etiolated Pisum sativum epicotyls has been studied 7). The results suggested that gravity was indeed important for inducing asymmetrical accumulation of auxin during the Copyright 2014 by the Japan Society for Aeronautical and Space Sciences and ISTS. All rights reserved. Th_5

2 Trans. JSASS Aerospace Tech. Japan Vol. 12, No. ists29 (2014) negative gravitropic response of the epicotyls. Auxin regulates cell division, elongation and differentiation. Therefore, it plays critical role in plant growth and development 8). While regulating cell division and elongation, auxin is basipetally transported into and out of the cell by protein transporter. The influx and efflux of the auxin is believed to be involved in many physiological responses, including phototropism and gravitropism. Auxin bioassay in curved Coleus stem showed uneven auxin distribution. The convex side of the stem had less auxin than the concave side 9). Harrison and Pickard observed auxin asymmetry in tomato hypocotyl 10). The results suggested that the upper part of tomato hypocotyl had higher auxin level at the curvature site compare to the lower side. The ratio became larger as it was nearing the curvature point. This result suggested that the curvature could only happen if the ratio of the auxin level is large enough. Aside from gravitropism, plant seedlings also respond to water and light. The response is known respectively as hydroptopism and phototropism 11,12). Hydrotropism is caused by moisture differential, while phototropism is caused by the light, mainly the sunlight. Under natural situations, all these abiotic stimuli interact with each other and with gravity to control plant growth, explaining a concise description of such interactions in an evaluation of gravitropism 13). It was proposed that the root growth orientation, both on ground and under microgravity condition, was probably controlled by hydrotropic response in the absence or reduction of gravity. Whilst sedimentation of amyloplasts affects auxin distribution in columella cells during gravitropic response, it also influences hydrotropic response. Research in radish root showed that moisture differential led to amyloplasts degradation, though it was formed again once the root reached agar medium. Water stress also reduced gravitropic response in radish root without reduction in elongation growth 14). Wild-type Arabidopsis seedlings treated with inhibitors to nullify auxin influx, efflux, or response, and their responses to either gravitropic or hydrotropic stimuli was observed 15). Their analysis showed that polar auxin transport was needed in gravitropic response, whereas other auxin response played definite role in giving hydrotropic response. Even though the mechanism remained unclear, both amyloplasts and auxin react to gravity and moisture, and give different response at the same time. As the seedlings respond to gravitropism and hydrotropism, it also responds to phototropism. The relation between root gravitropism and phototropism in Arabidopsis has been observed 16). They proposed that gravitropic response of Arabidopsis was stronger in some position of wild-type root grown for some time. The result indicated that root phototropism significantly influenced the time course of gravitropic response. Other experiment with pea seedling suggested that during phototropism and gravitropism, auxin in the epidermal cells asymmetrically distributed, but no significant asymmetry found in other tissues 17), supported the research done much earlier by Went 6). For this experiment, microgravity condition was simulated on 3D clinostat. The verification for the clinostat was done by growing tomato (Lycopersicon esculentum) seeds and the effect of clinorotation on plant growth was observed in mung bean (Vigna radiata) growth. Both experiments were then verified by auxin concentration analysis and statolith position observation. 2. Materials and Methods The 3D clinostat (Fig. 1) was made in Institut Teknologi Bandung. The verification was conducted by observing tomato seeds growth on the 3D clinostat in comparison to ground control. Fig. 1. Three dimensional (3D) clinostat made for microgravity simulation. 2.1 Seeds and seedlings preparation For clinostat verification, the tomato seeds were sterilized with 15% of chlorox for 5 minutes, then rinse three times with sterilized water. The sterilized seeds were then incubated overnight in sterile water. After incubation, the seeds were placed on plain agar medium and incubated in dark room until the shoots reach 3 cm in height (± 7 days). Half of the prepared seedlings were placed on 3D-clinostat (8-10 rpm) that simulated microgravity for 2-3 days, the rest were treated as control. All experiments were conducted in light and dark condition. In the second experiment, mung beans were sterilized by 70% ethanol for several seconds. The seeds were then rinsed in sterilized water several times to ensure no ethanol left on the surface of seeds coat. The seeds were left overnight in 30 o C closed water bath. After 24 hours, the seedlings were placed on the aluminium mesh inside a clear chamber (Fig. 2). The chambers were covered with black cloth, one of the chambers was placed on the 3D clinostat (8-10 rpm) for 3 days, while other chamber was placed in normal gravity as control. Th_6

3 L. SWANDJAJA et al.: The Effect of Clinorotation to the Growth of Tomato and Mung Bean Seedlings Fig. 2. Mung bean seedlings (green) was placed in a clear container, between alumunium mesh (blue) with wet rockwool (yellow), was put on 3D clinostat without light. auxin level asymmetry in the hypocotyl curvature compare to control (Fig. 5). The data showed that in ground control, the concentration of the auxin in both upper and lower part of the hypocotyl showed no significant difference. However, the tomato seedlings treated on the clinostat in light condition grew to the direction of the light source (Fig. 6), which may show a possibility that the phototrophic response is stronger than the gravitropic response Auxin analysis The shoot (tomato) and root (mung bean) sample from both treatments was collected for auxin concentration analysis. The samples from ground control treatment was divided longitudinally while the sample treated on 3 D clinostat was divided based on the convex and the concave side of the curvature. The samples were then extracted with methanol. The extract was analyzed using high performance liquid chromatography (HPLC) with IAA as standard Statolith observation The roots of the samples were extracted and sliced into a very thin segment longitudinally with the help of cassava pith as matrix. The fresh sectioned slide was then stained with freshly made I 2 KI. The sample was observed under stage microscope directly. 3. Results and Discussion The verification of the clinostat was conducted by observing the growth of tomato seedlings. The first verification was done in dark condition, hence the seedlings were etiolated (Fig. 3). The root of the seedlings grew in normal condition (control) showed no abnormality, as the shoot grew straightly upward while the root downward (Fig. 3A and B). Whereas, the shoot and root of seedlings treated on clinostat, lost their direction and curved irregularly even coiled (Fig. 3C and D). In normal light condition, shoot of the tomato seedlings treated on clinostat showed the same results, but the effect was not as strong as in the dark (Fig. 4). The bending angle of the shoot after 3 days of clinorotation, even after 6 days, was not as sharp as the one grew in the dark. This result proposed that phototropic response in tomato seedlings was probably stronger than its gravitropic response. Hence, the next gravitropism experiment for tomato seeds should be conducted in dark condition. Arabidopsis thaliana produced longer hypocotyl after clinorotation compare to control 18). The result was confirmed again in Lepidium sativum L., which showed that the growth of the seedling was promoted in microgravity compared to control plant 19). This experiment showed no significant differences both in plant length and dry weight (data not shown). However, the auxin analysis in tomato hypocotyls used for clinostat verification showed there was significant Fig. 3. Picture A and B are shoot and root of tomato seedlings in normal gravity, while C and D are the shoot and root of tomato seedlings after 3 days rotation on a 3D clinostat. A B C D E F Fig. 4. Tomato seedlings grew in normal gravity (A: 0 hour, B: 3 days hours and C: 6 days) and the seedlings that were grown on the 3D clinostat (D: 0 hour, E: 3 days and F: 6 days) under normal light condition. The data showed that the lower part of the curvature in the hypocotyl of the seedlings treated on 3D clinostat had less concentration of auxin ( µg/g) compare to the upper part ( µg/g). To prove that phototropic response in tomato seedlings was stronger than its gravitropic response, further experiment in the dark covered tube with a hole was conducted (Fig. 6). The results showed that the seedling bend to the source of the light (red cross) both in ground control and clinostated sample. The auxin analysis of the samples, both grew on ground and on clinostat, indicated that in the response to the source of light (phototropism) the auxin level of the upper part is less than the lower part. This result is consistent with the auxin level analysis that was done to the Th_7

4 clinostated sample in light condition. All results supported Went-Cholodny theory 6), auxin is transported to lower part of the hypocotyl to avoid sunlight. High level of auxin at the bottom part of the shoot will induce cell elongation and make cell grow longer than the cells at the upper part, so that the shoot bend upward. Trans. JSASS Aerospace Tech. Japan Vol. 12, No. ists29 (2014) g Fig. 7. The statolith (blue arrows) position of the mung bean root cap in normal gravity under 300x and 1200x magnification. Fig. 5. Auxin level (µg/g) asymmetry in tomato hypocotyl in ground control plant compare to the seedlings grew on 3D clinostat in light condition (L), dark condition (D) and with a slight opening in the black cloth (V). Fig. 8. The statolith (blue arrows) position of the mung bean root cap after 3 days rotation on 3D clinostat under 300x and 1200x magnification. Fig. 6. Tomato seedling on ground control (A) and on clinostat (B), covered with black cloth with an opening at the crossed part. Following clinostat verification, the experiment was conducted for the observation of the effect of clinorotation to Vigna radiata (mung bean) seedlings growth. Mung bean was used as tomato replacement because its root was bigger to enable statolith observation in the root cap. The position of the statolith in the root cap of clinorotated sample was compared to ground control. The results showed that statolith from the ground control cells positioned at the bottom of the columella cells (Fig. 7). The statolith grouped together at the bottom of the statocytes, following the vector of the gravity as it was stated by others 5,13,20,21). The statolith in the root cap of the mung bean seedlings treated on 3D clinostat was not easily observed (Fig. 8). The statolith was not grouped at the distal part of the cell, instead it formed several groups that were dispersed in the cytoplasm. In several cells, the statolith located around the nucleus at the proximal part of the cell (Fig. 9) as it was also stated by other experiment before 5). Fig. 9. The position of the statolith (blue arrows) that surrounding the nucleus (n) in the columella cell of the mung bean grew on clinostat for three days. Experiment with the mung bean seedlings showed some bending response (Fig. 10). However, as it responded to the loss of gravity cue due to clinorotation, the roots also grew to the wet rock wool at the chamber, which might indicate its growth toward the water source. This phenomenon was probably suggested that hydrotropic response maybe stonger than gravitropic response in mung bean. The moisture gradient caused by water evaporation during the experiment might trigger the degradation of the amyloplast in the columella cells, which caused the root lost its ability to detect gravity 5,23). Th_8

5 L. SWANDJAJA et al.: The Effect of Clinorotation to the Growth of Tomato and Mung Bean Seedlings Fig. 10. The root of the mung bean directed to the wet rock wool while it was showing a bending response due to clinorotation. Mung bean root that was placed on clinostat bent irregularly, therefore the root could be divided according to the diagram showed (Fig. 11), according to the curvation of the root tip. The results of the analysis showed that in normal gravity, the auxin was evenly distributed. On the other hand, under microgravity condition, the concentration of auxin was higher ( µg/g) in the upper part of the curvation point of the root segment compare to the lower part ( µg/g). dispersed in the cytoplasm. In several cells, the statolith positioned around the nucleus, at the proximal part of the cells. The analysis of auxin confirmed that bending response was caused by auxin asymmetry distribution. The root of the mung bean seedlings bend due to clinorotation, but the root still grew to the direction of the water, suggested that hydrotropism influenced the root growth stronger than gravitropism. Acknowledgements This research was funded by ASAHI Glass Foundation grant to Dr. Rizkita R. Esyanti. It was conducted in National Institute of Aeronautics and Space (LAPAN) Bandung with the help from Prof. Chunaeni Latief and Ary Ginaldi. The authors also greatly thanked Indra Wahyudin Fathona, graduated student from Master of Physics Department in Institut Teknologi Bandung, to his contribution in designing the 3D-clinostat. References Fig. 11. Auxin (µg/g) distribution in mung bean root grew on normal gravity (control) compare to microgravity simulation (3D clinostat). The statolith position changed from its original position at the distal of the columella cells, as clinorotation (Fig. 8-9) was probably disrupted the F-actin arrangement in the cells 24). The disarrangement of the F-actin will lead to the changes in the auxin distribution pattern 25). Auxin will follow statolith movement and localized at the lower part of the root. In the meantime, the auxin in the shoot will also move to the lower part of the shoot following the position of the center of gravity. High concentration of auxin at the bottom part of the root will inhibit the cell elongation, hence, the size of the cell at the bottom part were shorter than the upper part of the root, which made the root bend downward 6). 4. Conclusion The verification of the clinostat was conducted by growing tomato seedlings. The tomato seedlings showed bending response due to rotation of the clinostat which then altered the cue of the gravity. The orientation of seedlings growth and its auxin level indicated that phototropism was stronger than gravitropism. The effect of clinorotation was further observed in the growth of mung bean seedlings. 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