Doi:10.1016/j.asr.2007.03.074

Advances in Space Research 39 (2007) 1176–1181 Up-regulation of expression of tubulin genes and roles of microtubules in hypergravity-induced growth modification in Arabidopsis hypocotyls Shouhei Matsumoto, Yuka Saito, Saori Kumasaki, Kouichi Soga, Kazuyuki Wakabayashi, Takayuki Hoson * Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan Received 30 September 2006; received in revised form 14 March 2007; accepted 28 March 2007 We examined the roles of microtubules in gravity-induced modification of growth and development in plants by analyzing the expres- sion levels of the a- and b-tubulin gene family and growth behavior of Arabidopsis hypocotyls treated with the microtubule-disruptingreagents colchicine, oryzalin, and propyzamide. Expression of the majority of the examined a- and b-tubulin genes was up-regulated byhypergravity at 300 g, although the extent was variable among genes, indicating that up-regulation of the expression of tubulin genes isthe universal response of Arabidopsis hypocotyls to hypergravity. Hypergravity suppressed elongation growth by decreasing the cell-wallextensibility, whereas it stimulated lateral thickening of hypocotyls. By treatment with colchicine, oryzalin, and propyzamide, the elon-gation growth was suppressed, lateral thickening was stimulated, and the cell-wall extensibility of hypocotyls decreased dose-dependentlyeven under 1 g conditions. The degree of hypergravity-induced changes decreased with increasing concentration of microtubule-disrupt-ing reagents. As a result, hypergravity affected neither the length, the thickness, nor the cell-wall extensibility of hypocotyls in the pres-ence of high concentrations of microtubule-disrupting reagents. Moreover, colchicine-treated seedlings showed helical growth even under1 g conditions, and this phenotype was intensified under hypergravity conditions. These results suggest that the up-regulation of theexpression of tubulin genes is involved in gravity-induced modification of microtubule dynamics, which may play an important rolein the resistance of plant organs to the gravitational force and maintenance of normal growth phenotype.
Ó 2007 COSPAR. Published by Elsevier Ltd. All rights reserved.
Keywords: Arabidopsis; Colchicine; Gravity; Hypergravity; Microtubules of cell-wall constituents indicated that the shoots exposedto hypergravity had thicker cell walls with modified com- Hypergravity, a gravitational force exceeding 1g, has positions and structure ( been shown to suppress elongation growth of shoot organs in various plants Cortical microtubules have been shown to reorient in response to the gravity signal. In gravitropism, cortical ). Such a suppression of growth was in general accom- microtubules in the faster-expanding convex flank were panied by a decrease in cell-wall extensibility transverse, whereas the microtubules in the concave flank ). The analysis of chemical nature showed a longitudinal orientation with respect to the longi-tudinal axis of the cell In protoplasts of Brassica hypocotyls, hypergravity stimulated the regeneration of cortical micro- Corresponding author.
E-mail address: (T. Hoson).
tubules in parallel arrays (). Also, 0273-1177/$30 Ó 2007 COSPAR. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.asr.2007.03.074 S. Matsumoto et al. / Advances in Space Research 39 (2007) 1176–1181 hypergravity increased the percentage of cells with longitu- 2. Materials and methods dinal cortical microtubules with strong fluorescence (). These results suggest that cortical microtu- 2.1. Plant material and growth conditions bules, in addition to the cell wall, are involved in gravity-induced modification of growth and development.
Seeds of Arabidopsis thaliana L. cv Columbia were ster- To understand the series of events leading to growth ilized in 2% (v/v) sodium hypochlorite solution for 1 min, modifications by the gravity signal, we analyzed the hyper- and then washed thoroughly with water. The sterilized gravity-induced changes in gene expression in Arabidopsis seeds were planted on 1.5% (w/v) agar medium in a hypocotyls by the differential display method 25 ml centrifuge tube, kept at 4 °C for two days, and ). Screening and analysis of genes, confirmed exposed to white light (5 W/m2 at seed level) for 6 h to the up-regulation of the expression of six genes by hyper- induce germination. Seeds were then grown in the dark at gravity. One of the isolated genes encoded a-tubulin 25 °C. To analyze the expression levels, we exposed the (TUA3), a component of microtubules. These data suggest plants to hypergravity at 300 g for 9 h at 25 °C in the dark that the up-regulation of the expression of tubulin genes is with a centrifuge (H-28-F; Kokusan Co., Japan). For col- involved in gravity-induced modification of the amount chicine treatment, sterilized seeds were planted on 1.5% and orientation of cortical microtubules. However, at least (w/v) agar containing 10 mM MES–KOH buffer (pH 6.0) six genes encoding a-tubulin and with different concentrations of colchicine (Wako Pure nine genes encoding b-tubulin have Chemical, Japan). For oryzalin and propyzamide treat- been identified in Arabidopsis, and the effects of gravity ment, plants that had been grown for 48 h in the dark were on the expression levels of other tubulin genes have not transferred to agar medium containing 10 mM MES–KOH been clarified yet. In the present study, we examined the buffer (pH 6.0) with various concentrations oryzalin (Dr.
involvement of up-regulation of the expression of tubulin Ehrenstofer Gmbh, Germany) or propyzamide (Wako genes in gravity-induced modification of microtubule Pure Chemical, Japan) in 0.3% dimethyl sulfoxide dynamics by comprehensive analysis of the expression of (DMSO). DMSO at 0.3% was shown not to affect growth the whole a- and b-tubulin gene family under hypergravity of seedlings. To analyze the growth behavior, we exposed conditions. We also examined the role of microtubules in the plants to hypergravity at 300 g for 24 h at 25 °C in regulation by gravity of growth and development using the dark with a centrifuge. After the treatment, we mea- sured the length of hypocotyls using a scale, and the thick- Fig. 1. Effects of hypergravity on the expression of a-tubulin genes (TUA)in Arabidopsis seedlings. Wild-type seedlings, 48 h-old, were exposed to Fig. 2. Effects of hypergravity on the expression of b-tubulin genes in 300 g conditions and grown for 9 h at 25 °C. The expression levels of Arabidopsis seedlings. Arabidopsis seedlings were grown as in The TUA1–TUA6 were determined by real time PCR. mRNAs of TUA2 and expression levels of TUB1–TUB9 were determined by real time PCR.
TUA4 or TUA3 and TUA5 were not distinguishable. The values were mRNAs of TUB2 and TUB3 were not distinguished. The values were compensated with cDNA levels. Values are means ± SE (n = 3). All values compensated with cDNA levels. Values are means ± SE (n = 3). *Mean are significantly different between 1 and 300 g treatments (P < 0.05).
values significantly different between 1 and 300 g treatments (P < 0.05).
S. Matsumoto et al. / Advances in Space Research 39 (2007) 1176–1181 ness of hypocotyls with a digital light field microscope (VB- sibility was measured with a tensile tester (Tensilon G25; Keyence, Japan). The digital light field microscope RTM-25, Toyo Baldwin, Japan) ().
was also used to measure the angle of the cortical cell line The sample was fixed between two clamps (the distance to the longitudinal axis of hypocotyls. Because the middle between the clamps was 1 mm) and stretched by lowering region of hypocotyls showed typical helical growth, we the bottom clamp at a speed of 10 mm min 1 until a load used this region for measurements of the angle of the cor- of 0.8 g was produced. The cell-wall extensibility (strain tical cell line. In the measurement, the longitudinal axis of load 1, lm g 1) was determined by measuring the rate of hypocotyls was defined as 0°. The cortical cell lines aligned the increase in load just before it reached 0.8 g.
at angles between 0 and 90° (counterclockwise from thetop) form left-handed helical growth, whereas thosealigned at angles between 0 and 90° form right-handed 2.3. Analysis of gene expression helical growth. Thus, the absolute values of the angle ofthe cortical cell line were calculated.
Arabidopsis seedlings collected were immediately frozen in liquid nitrogen and kept at 80 °C until use. Frozen 2.2. Determination of the mechanical properties of cell walls hypocotyls were ground to a fine powder. Total RNAwas extracted using RNeasy Plant Mini Kit (Qiagen, The samples of Arabidopsis seedlings were boiled in USA). Single strand cDNA was synthesized from 0.2 lg methanol for 10 min and stored in fresh methanol until of total RNA according to the instructions of the manufac- use. Before the measurement of the cell-wall extensibility, turer using a High-Capacity cDNA Archive Kit (Applied the methanol-killed seedlings were rehydrated overnight Biosystems, USA). The single strand cDNA was amplified at 4 °C with several changes of water. The cell-wall exten- using SYBR Green PCR Master Mix (Applied Biosystems) Increase in diameter (µ 10 Concentration (µM) Fig. 3. Effects of hypergravity and microtubule-disrupting reagents on the length and the diameter of Arabidopsis hypocotyls. For colchicine treatment,Arabidopsis seedlings were grown on agar medium containing different concentrations of colchicine at 1 g for 48 h at 25 °C. Seedlings were then transferredto 1 g or 300 g conditions and grown for a further 24 h at 25 °C. For oryzalin or propyzamide treatment, seedlings were grown on agar medium in theabsence of the reagents at 1g for 48 h at 25 °C. Seedlings were then transferred to agar medium containing oryzalin or propyzamide at variousconcentrations at 1 or 300 g conditions, and grown for a further 24 h at 25 °C. The length was measured using a scale. The diameter of hypocotyls wasmeasured using a microscope. Values are means ± SE (n = 20). *Mean values significantly different between 1 and 300 g treatments (P < 0.05).
S. Matsumoto et al. / Advances in Space Research 39 (2007) 1176–1181 in the ABI 7500 Real Time PCR System (Applied 3.1. Expression of tubulin genes All six a-tubulin genes (TUA) examined were expressed in hypocotyls, irrespective of gravity conditions ().
The expression level of all members increased during incu-bation for 9 h under 1 g conditions. Under hypergravity conditions at 300 g, the expression of a-tubulin genesincreased to about 150–200% of the 1 g control ().
All of the nine b-tubulin genes (TUB) examined wereexpressed in hypocotyls, irrespective of gravity condition Cell-wall extensibility (µm/g) (The expression level of all members increased dur-ing incubation at 1 g. The expression levels of b-tubulingenes, except for TUB1 and TUB6, rose to about 140–270% of the control at 300 g (The degree of up-reg-ulation of a-and b-tubulin gene expression at 30 g was 70– 80% of that at 300 g (data not shown). Thus, the up-regu- lation of the expression of tubulin genes by hypergravity is Fig. 4. Effects of hypergravity and colchicine on the cell-wall extensibilityof Arabidopsis hypocotyls. Arabidopsis seedlings were grown as in 3.2. Effects of microtubule-disrupting reagents on growth and The cell-wall extensibility was measured with a tensile tester. Values are cell-wall extensibility mean ± SE (n = 30). *Mean values significantly different between 1 and300 g treatments (P < 0.05).
Elongation growth of hypocotyls was suppressed, whereas the lateral thickening was stimulated by hypergra- In the presence of colchicine, the alignment vity at 300 g (). By treatment with the microtubule- angle was further increased by hypergravity ( disrupting reagents colchicine, oryzalin, and propyzamide,elongation growth was suppressed dose-dependently,whereas the diameter of hypocotyls was increased even under 1 g conditions. The degrees of hypergravity-inducedsuppression of elongation growth and stimulation of lateral Hypergravity increased the percentage of cells with lon- thickening decreased by increasing the concentration of the gitudinal cortical microtubules with strong fluorescence microtubule-disrupting reagents. As a result, hypergravity In Arabidopsis hypocotyls, six a- and affected neither the length nor the diameter of hypocotyls nine b-tubulin genes were expressed irrespective of gravity in the presence of the reagents at high concentrations conditions, and the expression of the majority of the exam- (Because colchicine, oryzalin, and propyzamide ined tubulin genes was up-regulated by hypergravity of had similar effects on growth, we used only colchicine in 300 g (). Although the extent of hypergravi- the following experiments.
ty-induced increase in gene expression was variable among The cell-wall extensibility of hypocotyls was decreased members, the extent of up-regulation of total a-tubulin by hypergravity at 300 g It was also decreased gene expression and that of total b-tubulin genes were by increasing the concentration of colchicine even under almost equal. At the examined stage, hypocotyl cells grow 1g conditions. The degree of hypergravity-induced changes by cell elongation, because cell division has already ceased.
in the cell-wall extensibility was decreased by increasing These data suggest that the up-regulation of the expression concentration of colchicine. Hypergravity did not affect of tubulin genes is involved in gravity-induced modification the cell-wall extensibility, in the presence of 30 lM colchi- of the amount and orientation of cortical microtubules.
When cortical microtubules were reoriented, new corticalmicrotubules were nucleated on the existing microtubules 3.3. Effects of colchicine on cell alignment ). Thus, the synthesis of new tubulinmolecule via up-regulation of the expression of a- and b- Arabidopsis hypocotyls showed left-handed helical tubulin genes may be needed to modify the orientation of growth under hypergravity conditions at 300 g cortical microtubules under hypergravity conditions. The By increasing concentration of colchicine, the align- present results also suggest that tubulin genes are inappro- ment angle of cell lines was increased under 1 g conditions priate as a constitutive standard in the study of gene S. Matsumoto et al. / Advances in Space Research 39 (2007) 1176–1181 Absolute alignment (degree) Fig. 6. Effects of hypergravity on alignment of cortical cell lines ofArabidopsis hypocotyls. Arabidopsis seedlings were grown as in andthe angle of the cortical cell line to the longitudinal axis of hypocotyls wasmeasured using a protractor. Values are means of the absolute values ofalignment angles ± SE (n = 20). *Mean values significantly differentbetween 1 and 300 g treatments (P < 0.05).
Arabidopsis hypocotyls showed left-handed helical Fig. 5. Effects of hypergravity and colchicine on morphology of Arabid-opsis hypocotyls. Arabidopsis seedlings were grown as in and the growth, derived from disordered organization of cortical surface of the middle region of hypocotyls was observed with a digital light microtubules Moreover, propyzamide field microscope. The bar denotes 100 lm.
caused twisting in elongating Arabidopsis epidermal cellseven at a low concentration (). Inthe present study, helical growth was also induced by expression, even though they have been used, because their colchicine treatment even under 1 g conditions, and was expression levels are sensitive to environmental signals such intensified under hypergravity conditions ( ). Thus, helical growth may be induced by disintegra- By treatment with the microtubule-disrupting reagents tion of cortical microtubules. These results also suggest colchicine, oryzalin, and propyzamide, elongation growth that microtubules play an important role in maintenance was suppressed dose-dependently and the lateral thickening of normal growth phenotype under hypergravity condi- was stimulated even under 1 g conditions The cell- tions. Helical growth induced by hypergravity was wall extensibility of hypocotyls was also decreased by enhanced by colchicine ), whereas hyper- increasing the concentration of colchicine even under 1 g gravity-induced suppression of elongation growth, stimu- conditions (). These results suggest that microtubules lation of lateral thickening, and decrease in the cell-wall play an important role in the resistance of plant organs to extensibility were not clearly affected ).
the gravitational force and maintenance of normal growth These phenomena suggest that helical growth is not the phenotype. Hypergravity further suppressed elongation direct cause of the modification of growth parameter growth by decreasing the cell-wall extensibility, whereas it by hypergravity.
stimulated lateral thickening. However, the degree of such changes was decreased by increasing the concentrations of reagents suggested that microtubules contribute to main- microtubule-disrupting reagents. As a result, hypergravity tenance of normal growth phenotype in plant organs.
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