(Lycopersicon Esculentum) and Mung Bean (Vigna Radiata) Seedlings

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 SWANDJAJA1 , Rizkita Rachmi ESYANTI1 , KHAIRURRIJAL2), 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

㸦Received June 24th, 2013㸧

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 gravity, the statolith mass concentrated at the distal part of the statocytes, following the vector of gravity. The experiment Plant growth and development are affected by several proposed that clinorotation disrupted root cap cells structure. abiotic factors such as light, temperature and water quantity. The constant rotation of clinostat caused the cell wall in the However, nearly upon germination, another physical factor columella cells deteriorated, and so reduced the amount of called gravity, affected the growth of root and shoot to orient visible amyloplasts. Several results also showed that the seedling correctly in space for the survival of the newly sedimentation of amyloplasts affected auxin movement. developed seedling. One of the most important mechanisms Until recently, the complex signal transduction cascade that for the survival of the germinating seedling is the growth of regulates the differential cell elongation induced by the root, which guarantees the young plant a rapid supply of gravitropic and phototropic remains unclear. A general model water and minerals. On the other hand, the shoot grows of root/shoot gravitropic and phototropic response is based on upward to tract sunlight. Since plants have evolved growing the model of the asymmetric redistribution of auxin that under the constant stimulus of gravity, its presence is one of causes a differential cellular elongation on opposite flanks of the most important requirements for their growth and spatial 1) the central elongation zone. The hypothesis was proposed by orientation . Studies on the effects of a weightlessness and 6) Cholodny–Went . The downward bending of roots and the hypergravity environment on plants have focused mainly on upward bending of shoots in multicellular plants to response investigations of the gravitropic response 2). gravity is the result from induction of unequal redistribution of In higher plants, the gravity detector called statolith, are auxin within the elongation zone, which is correlated with an believed to be sedimentation of amyloplast in the cells called accumulation of elevated auxin levels along the lower side. In statocyte3). Amyloplast displacement in statocyte is sufficient roots, high concentration of auxin inhibits cell growth and to promote organ-tip curvature4). However, signal transduction elongation, thus a downward curvature is the consequence. In of amyloplast sedimentation into a physiological signal in the contrast, the upward bending in shoots is induced due to the statocyte is not fully understood. growing stimulation effect of auxin, resulting in an upward curvature6). The characteristic of unequal polar auxin movement induced by gravity in the early growth stages of The modulation of the statolith of clover seedlings in etiolated Pisum sativum epicotyls has been studied7). The normal gravity and microgravity simulated by a clinostat has results suggested that gravity was indeed important for 5) been reported before . The results suggested that in normal inducing asymmetrical accumulation of auxin during the

Th_5 Trans. JSASS Aerospace Tech. Japan Vol. 12, No. ists29 (2014) negative gravitropic response of the epicotyls. significant asymmetry found in other tissues17), supported the Auxin regulates cell division, elongation and differentiation. research done much earlier by Went6). Therefore, it plays critical role in plant growth and For this experiment, microgravity condition was simulated 8) development . While regulating cell division and elongation, on 3D clinostat. The verification for the clinostat was done by auxin is basipetally transported into and out of the cell by growing tomato (Lycopersicon esculentum) seeds and the protein transporter. The influx and efflux of the auxin is effect of clinorotation on plant growth was observed in mung believed to be involved in many physiological responses, bean (Vigna radiata) growth. Both experiments were then including phototropism and gravitropism. Auxin bioassay in verified by auxin concentration analysis and statolith position curved Coleus stem showed uneven auxin distribution. The observation. convex side of the stem had less auxin than the concave side9). Harrison and Pickard observed auxin asymmetry in tomato 2. Materials and Methods hypocotyl10). The results suggested that the upper part of tomato hypocotyl had higher auxin level at the curvature site The 3D clinostat (Fig. 1) was made in Institut Teknologi compare to the lower side. The ratio became larger as it was Bandung. The verification was conducted by observing nearing the curvature point. This result suggested that the tomato seeds growth on the 3D clinostat in comparison to curvature could only happen if the ratio of the auxin level is ground control. large enough. Aside from gravitropism, plant seedlings also respond to water and light. The response is known respectively as hydroptopism and phototropism11,12). 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 gravitropism13). 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 Fig. 1. Three dimensional (3D) clinostat made for microgravity also influences hydrotropic response. Research in radish root simulation. showed that moisture differential led to amyloplasts degradation, though it was formed again once the root reached 2.1 Seeds and seedlings preparation agar medium. Water stress also reduced gravitropic response For clinostat verification, the tomato seeds were sterilized 14) in radish root without reduction in elongation growth . with 15% of chlorox for 5 minutes, then rinse three times with Wild-type Arabidopsis seedlings treated with inhibitors to sterilized water. The sterilized seeds were then incubated nullify auxin influx, efflux, or response, and their responses to overnight in sterile water. After incubation, the seeds were 15) either gravitropic or hydrotropic stimuli was observed . Their placed on plain agar medium and incubated in dark room until analysis showed that polar auxin transport was needed in the shoots reach 3 cm in height (± 7 days). Half of the gravitropic response, whereas other auxin response played prepared seedlings were placed on 3D-clinostat (8-10 rpm) definite role in giving hydrotropic response. Even though the that simulated microgravity for 2-3 days, the rest were treated mechanism remained unclear, both amyloplasts and auxin as control. All experiments were conducted in light and dark react to gravity and moisture, and give different response at condition. the same time. In the second experiment, mung beans were sterilized by As the seedlings respond to gravitropism and hydrotropism, 70% ethanol for several seconds. The seeds were then rinsed it also responds to phototropism. The relation between root in sterilized water several times to ensure no ethanol left on gravitropism and phototropism in Arabidopsis has been the surface of seeds coat. The seeds were left overnight in 16) observed . They proposed that gravitropic response of 30oC closed water bath. After 24 hours, the seedlings were Arabidopsis was stronger in some position of wild-type root placed on the aluminium mesh inside a clear chamber (Fig. 2). grown for some time. The result indicated that root The chambers were covered with black cloth, one of the phototropism significantly influenced the time course of chambers was placed on the 3D clinostat (8-10 rpm) for 3 gravitropic response. Other experiment with pea seedling days, while other chamber was placed in normal gravity as suggested that during phototropism and gravitropism, auxin in control. the epidermal cells asymmetrically distributed, but no

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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.

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.

2.2. 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. 2.3. Statolith observation The roots of the samples were extracted and sliced into a Fig. 3. Picture A and B are shoot and root of tomato seedlings in normal very thin segment longitudinally with the help of cassava pith gravity, while C and D are the shoot and root of tomato seedlings after 3 as matrix. The fresh sectioned slide was then stained with days rotation on a 3D clinostat. freshly made I2KI. The sample was observed under stage microscope directly. A B C 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 D E F 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). Fig. 4. Tomato seedlings grew in normal gravity (A: 0 hour, B: 3 days In normal light condition, shoot of the tomato seedlings hours and C: 6 days) and the seedlings that were grown on the 3D treated on clinostat showed the same results, but the effect was clinostat (D: 0 hour, E: 3 days and F: 6 days) under normal light not as strong as in the dark (Fig. 4). The bending angle of the condition. 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 The data showed that the lower part of the curvature in the phototropic response in tomato seedlings was probably hypocotyl of the seedlings treated on 3D clinostat had less stronger than its gravitropic response. Hence, the next concentration of auxin (811.3911 µg/g) compare to the upper gravitropism experiment for tomato seeds should be part (1905.017 µg/g). To prove that phototropic response in conducted in dark condition. tomato seedlings was stronger than its gravitropic response, Arabidopsis thaliana produced longer hypocotyl after further experiment in the dark covered tube with a hole was clinorotation compare to control18). The result was confirmed conducted (Fig. 6). The results showed that the seedling bend again in Lepidium sativum L., which showed that the growth to the source of the light (red cross) both in ground control of the seedling was promoted in microgravity compared to and clinostated sample. The auxin analysis of the samples, control plant19). This experiment showed no significant both grew on ground and on clinostat, indicated that in the differences both in plant length and dry weight (data not response to the source of light (phototropism) the auxin level shown). However, the auxin analysis in tomato hypocotyls of the upper part is less than the lower part. This result is used for clinostat verification showed there was significant consistent with the auxin level analysis that was done to the

Th_7 Trans. JSASS Aerospace Tech. Japan Vol. 12, No. ists29 (2014) clinostated sample in light condition. All results supported Went-Cholodny theory6), 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.

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 Fig. 9. The position of the statolith (blue arrows) that surrounding the conducted for the observation of the effect of clinorotation to nucleus (n) in the columella cell of the mung bean grew on clinostat for Vigna radiata (mung bean) seedlings growth. Mung bean was three days. used as tomato replacement because its root was bigger to enable statolith observation in the root cap. The position of the Experiment with the mung bean seedlings showed some statolith in the root cap of clinorotated sample was compared bending response (Fig. 10). However, as it responded to the to ground control. The results showed that statolith from the loss of gravity cue due to clinorotation, the roots also grew to ground control cells positioned at the bottom of the columella the wet rock wool at the chamber, which might indicate its cells (Fig. 7). The statolith grouped together at the bottom of growth toward the water source. This phenomenon was the statocytes, following the vector of the gravity as it was probably suggested that hydrotropic response maybe stonger stated by others5,13,20,21). than gravitropic response in mung bean. The moisture gradient The statolith in the root cap of the mung bean seedlings caused by water evaporation during the experiment might treated on 3D clinostat was not easily observed (Fig. 8). The trigger the degradation of the amyloplast in the columella cells, 5,23) statolith was not grouped at the distal part of the cell, instead it which caused the root lost its ability to detect gravity . 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 before5).

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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 Fig. 10. The root of the mung bean directed to the wet rock wool while hydrotropism influenced the root growth stronger than it was showing a bending response due to clinorotation. gravitropism.

Mung bean root that was placed on clinostat bent irregularly, Acknowledgements therefore the root could be divided according to the diagram showed (Fig. 11), according to the curvation of the root tip. This research was funded by ASAHI Glass Foundation The results of the analysis showed that in normal gravity, the grant to Dr. Rizkita R. Esyanti. It was conducted in National auxin was evenly distributed. On the other hand, under Institute of Aeronautics and Space (LAPAN) Bandung with microgravity condition, the concentration of auxin was higher the help from Prof. Chunaeni Latief and Ary Ginaldi. The (1112.875 µg/g) in the upper part of the curvation point of the authors also greatly thanked Indra Wahyudin Fathona, root segment compare to the lower part (498.160 µg/g). graduated student from Master of Physics Department in Institut Teknologi Bandung, to his contribution in designing the 3D-clinostat.

References

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