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Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles
Journal of Integrative Agriculture 2014, 13(8): 1634-1639
August 2014
RESEARCH ARTICLE
Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles LI Jian-ping1, 2, 3, HOU Pei1, ZHENG Xu1, 3, SONG Mei-fang1, SU Liang1 and YANG Jian-ping1 1
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China Institute of Nuclear and Biotechnology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, P.R.China 3 Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 2
Abstract The phytochrome gene family, which is in Arabidopsis thaliana, consists of phytochromes A-E (phyA to phyE), regulates plant responses to ambient light environments. PhyA and phyB have been characterized in detail, but studies on phyC to phyE have reported discrepant functions. In this study, we show that phyD regulates the Arabidopsis gravitropic response by inhibiting negative gravitropism of hypocotyls under red light condition. PhyD had only a limited effect on the gravitropic response of roots in red light condition. PhyD also enhanced phyB-regulated gravitropic responses in hypocotyls. Moreover, the regulation of hypocotyl gravitropic responses by phyD was dependent upon the red light fluence rate. Key words: phytochrome D, gravitropism, Arabidopsis thaliana
INTRODUCTION In response to the detection of various environmental signals such as light, gravity, touch, and humidity, the absorption of water and nutrients and the rate of photosynthesis are altered in plants, resulting in a change in plant growth (Miyo 2010). Light is an important environmental influence on plant growth and development; plant photoreceptors detect the quality and direction of light, and plant growth is adjusted in response. Phytochromes are the primary photoreceptors involved in the regulation of red/far-red light-induced responses. Arabidopsis has five phytochromes, phyA to phyE. Phylogenetic analysis suggests that these phytochromes can be clustered into four subfamilies: phyA, phyB/D, phyC, and phyE (Sharrock et al. 2003). PhyA, a light-liable phytochrome, mediates responses to far-red light; phyB is light stable and is dominant in responses to red light (Franklin et al.
2003; Monte et al. 2003). Within the light-stable-phytochrome family, phyB, phyD and phyE act redundantly to control multiple physiological responses, including leaf expansion, hypocotyl development and flowering time (Aukerman et al. 1997; Franklin et al. 2003). Gravity is an important environmental cue for modulating the directional growth of plants. Generally, plant shoots grow upward (i.e., negative gravitropism) and roots grow downward (i.e., positive gravitropism) (Blancaflor and Masson 2003; Bhalerao et al. 2004) tropic responses. Both gravity and light can affect the gravitropic growth of plants. For example, dark-grown hypocotyls of Arabidopsis grow upward, but seedlings grown in red or far-red light grow in random directions (Liscum and Hangarter 1993; Poppe et al. 1996; Robson and Smith 1996; Hennig et al. 2002). Red and far-red light can also influence gravitropic responses in roots (Lu and Feldman 1997; Takano et al. 2001). Thus, gravitropic responses in plants are also modulated by signals from light-activated photoreceptors (Correll and Kiss 2002; Whippo and Hangarter 2003; Blakeslee
Received 9 May, 2013 Accepted 28 August, 2013 LI Jian-ping, E-mail: [email protected]; Correspondence YANG Jian-ping, Tel/Fax: +86-10-82105859, E-mail: [email protected]
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60607-3
Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles
et al. 2004), and roles in light-regulated gravitropism have been identified for several phytochromes. PhyA is the primary photoreceptor involved in far-red light-regulated gravitropic responses of both roots and hypocotyls in Arabidopsis (Parks and Spalding 1999; Hennig et al. 2002). Both phyA and phyB influence the gravitropic responses of maize roots and Arabidopsis hypocotyls (Liscum and Hangarter 1993; Liu and Iino 1996; Poppe et al. 1996; Robson and Smith 1996; Parks and Spalding 1999). PhyC also participates in the regulation of negative gravitropic and phototropic responses of hypocotyles (Kumar et al. 2008). In this study we compared the gravitropic responses of phytochrome mutants with their wild-type (WT) in Arabidopsis thaliana to explore the role of phyD in gravitropic responses in both of roots and hypocotyls and to discover how phytochromes inhibit hypocotyl negative gravitropism.
conducted a time-course analysis of root and hypocotyl growth. We compared the gravitropic curvatures of roots and hypocotyls of phyD single mutant seedlings with those of WT plants (Landsberg erecta (Ler) background) by re-orientating Petri dishes from the vertical to the horizontal position under red light (20.0 μmol m-2 s-1). There were no significant differences in gravritropism between the roots of the phyD mutant and the WT seedlings, with the exception of the 15-h time point (Fig. 1-A). In contrast, the gravitropic curvature of hyrpocotyls was inhibited in red light-grown phyD mutant seedlings relative to the WT seedlings, especially at 3 and 24 h (Fig. 1-B).
PhyB and phyD synergistically regulate the hypocotyl gravitropic response under red light Previous studies have demonstrated the genetic interactions between phytochrome signallings and gravitropic responses and have suggested that phyB plays a predominant role in the regulation of gravitropic growth (Poppe et al. 1996; Robson and Smith 1996; Kumar et al. 2008). Given that phyB and phyD are closely related phylogenetically, we speculated that they could interact in
RESULTS Phy D mediates red light-regulated hypocotyl gravitropism To determine the role of phyD in gravitropism, we B Ler
phyD
PHYD RT-PCR
Curvature (degree)
A
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Fig. 1 Curvature of roots and hypocotyls in wild-type (Landsberg erecta, Ler) and phyD mutant seedlings of Arabidopsis thaliana grown under red light (20.0 μmol m-2 s-1). A, analysis of RT-PCR and Western blot of phyD mutant and the WT seedlings. B, roots. C, hypocotyls. Time-course studies following 90° re-orientation of the Petri dishes. Error bars indicate standard deviations. Time points of mutants that show a significant difference (P<0.05) relative to the WT at the corresponding time points are indicated *. The same as below.
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
LI Jian-ping et al.
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controlling gravitropic growth of Arabidopsis. First, we investigated the gravitropic response of WT, phyB and phyD single mutants and phyB phyD double mutant seedlings grown under red light by determining the growth orientation of roots and hypocotyls as the degree from vertical (Fig. 2-A-E). The growth angles from vertical were markedly less for the roots of the phyB and phyB phyD mutants compared with the angles for the phyD mutant and WT roots (Fig. 2-B, C, D, E; dark), and the root angles did not differ between WT and phyD mutants (Fig. 2-B, C; dark). These results are consistent with previous studies (Liscum and Hangarter 1993; Correll et al. 2003; Correll and Kiss 2002, 2005) and suggest an important role of phyB in root gravitropic responses.
Hypocotyl gravitropism showed similar results, except there was also a difference between the angles of phyD and WT hypocotyls (Fig. 2-B and C; grey). Moreover, a slight yet significant decrease was observed in the angle from vertical for the hypocotyls of phyB phyD double mutant seedlings compared with phyB single mutant seedlings (Fig. 2-D and E; grey). To confirm these results and further understand the kinetics of hypocotyl gravitropism, we performed timecourse studies by determining the change in curvature of light-grown seedlings stimulated by re-orientating the Petri dishes by 90° in red light. As expected, there was no difference in root curvature between phyD and WT, whereas the roots of phyB and phyB phyD had less
phyD
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Fig. 2 Scatter diagram of hypocotyl and root orientation with respect to the gravity vector for Ler and mutant A. thaliana seedlings (n=160) that were grown vertically under red light (20.0 μmol m-2 s-1). © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles
curvature than those of phyD and WT (Fig. 3-A). The curvature of the hypocotyls was significantly less in both phyB and phyB phyD compared with WT seedlings, with significant reductions in curvature at 9, 12, 15, and 24 h in phyB single mutant seedlings and at 3, 9, 12, 15, and 24 h in phyB phyD double mutant seedlings (Fig. 3-B). There was a slight difference in hypocotyl curvature between phyD single mutant and WT seedlings at 3, 9 and 24 h (Fig. 3-B).
Fluence rate of red light affects the gravitropic response in hypocotyls Light has been proven to be an important factor in gravitropic growth through interactions with phytochrome signalling. In previous studies, gravitropism was shown to be impaired by red light (Britz and Galston 1982; Woitzik and Mohr 1988). To characterize the effect of light, we measured a fluence rate response curve. Arabidopsis WT and phyB, phyD and phyB phyD mutants were grown in
Curvature (degree)
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continuous red light at fluence rates of 0 (darkness), 5.0, 20.0, 50.0, 100.0, and 200.0 μmol m-2 s-1 for 24 h (Fig. 4). With increases in the red light fluence rate from 5.0 to 20.0 to 50.0 μmol m-2 s-1, the curvature of the hypocotyls accelerated. However, reduced acceleration of the curvature was observed at 100 and 200 μmol m-2 s-1. The loss of phyB in Arabidopsis resulted in continuous inhibition of gravitropism in the hypocotyl at all fluence rates, and phyD acted synergistically with phyB at a fluence rate of 20.0 μmol m-2 s-1. Interestingly, a significant difference in gravitropic growth between the phyD mutant and WT was found only at a fluence rate of 20.0 μmol m-2 s-1. These results indicate that the effect of red light on hypocotyl gravitropism is fluence rate-dependent.
DISCUSSION Phytochromes have been shown to play a role in gravitropism (Liscum and Hangarter 1993). Both phyA and phyB are involved in controlling the gravitropic response of maize roots and the gravitropic orientation and inhibition of elongation of Arabidopsis hypocotyls (Poppe et al. 1996; Robson and Smith 1996; Lu and Feldman 1997; Parks and Spalding 1999). Most of these previous studies were based on observations of mutant phenotypes. The present study used a single phyD mutant and a double phyB phyD mutant. The results for the curvature of both roots and hypocotyls in these mutants revealed that phyD attenuated negative gravitropic growth in hypocotyls under red light and that phyB and 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.00
Ler phyB phyD phyB phyD
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Fig. 3 Curvature of roots and hypocotyls of Ler and phyB, phyD and phyB phyD mutant seedlings of A. thaliana grown under red light (20.0 μmol m-2 s-1). A, time-course of root curvature after 90° re-orientation of Petri dishes (n 20 for each time point). B, timecourse of hypocotyl curvature after 90° re-orientation of Petri dishes (n 20 for each time point).
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Fluence rate (µmol m-2 s-1)
Fig. 4 Effect of fluence rate on red light-regulated gravitropic growth of hypocotyls. Ler and phyB, phyD, and phyB phyD mutant seedlings of A. thaliana were grown on vertically orientated plates under red light of different fluence rates (0, 5.0, 20.0, 50.0, 100.0, and 250 μmol m-2 s-1) for 24 h. The hypocotyl curvature was measured and plotted vs. the fluence rate.
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
LI Jian-ping et al.
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phyD may act synergistically to regulate this response. Recent studies have also shown that both phyC and phyD mediate positive phototropism in hypocotyls and stems under blue light (Correll et al. 2003) and inhibit red lightbased positive phototropism in roots (Correll and Kiss 2005). PhyB and phyE have been reported to attenuate gravitropism in inflorescence and stems in Arabidopsis (Kumar and Kiss 2006). Thus, the entire phytochrome family appears to be involved the regulation of tropic responses, and phytochromes may interact with each other or with other photoreceptors such as cryptochromes to regulate tropic responses. Evidences have shown that phyD plays an appurtenant role in the inhibition of hypocotyl elongation (Aukerman et al. 1997; Franklin et al. 2003) while root elongation from phyD were similar to WT for both dark- and lightgrown seedlings (Correll and Kiss 2005). In our studies, we also demonstrated that phyD plays a role in hypocotyl gravitropic responses but not in root. However, the regulation of gravitropism by light is one of the less-understood light responses and it is not also clear how phytochromes inhibit gravitropism, based on previous studies (Correll and Kiss 2005; Kumar et al. 2008). Phytochromes (phyA and phyB especially) may directly be involved in regulating the inhibition of hypocotyl elongation and gravitropism but may have not a direct effect of modulating the root gravity signaling cascade through the elongation process. In addition, the crosstalk of the various auxins and phytochormes also determine the elongation processes and tropisms (Marchant et al. 1999; Nagashima et al. 2008; Tajagasgu et al. 2009).
CONCLUSION By analysing the phenotypes of phyB, phyD and phyB phyD mutants of Arabidopsis, we demonstrated that phyD is involved in the inhibition of negative gravitropism of hypocotyls regulated by red light. Under red light condition, root growth in response to gravity did not differ significantly between WT and phyD, whereas the curvature of phyD hypocotyls was less than that of WT. Comparisons of the angles from vertical among the roots and hypocotyls of the phyB, phyD and phyB phyD mutants highlighted the role of phyD in red light-regulated gravitropic growth. PhyD also enhanced
phyB-regulated gravitropic responses in hypocotyls, which was confirmed by the time course of hypocotyl curvature. The present results show that phytochromes regulate gravitropic responses in hypocotyls under red light in a fluence rate-dependent manner.
MATERIALS AND METHODS Plant materials and growth conditions The Landsberg erecta (Ler) ecotype of A. thaliana was used in these studies. Seeds were surface sterilized in 30% bleach with 0.01% (v/v) Triton X-100 for 15 min, rinsed five times in sterile double-distilled water, and stored in water at 4°C for 3 d. Then the seeds were sown on Murashige and Skoog medium with 1% (w/v) sucrose, 0.05% MES (pH 5.7) and 0.9% (w/v) agar in square Petri dishes (5 mm×150 mm) and irradiated with white light for 24 h to promote seed germination.
Gravitropism experiments To test the gravitropic responses of the seedlings, the germination-induced plates were placed vertically in red light (20.0 μmol m-2 s-1) and seedlings grew along the surface of the agar for 96 h at 22°C. Seedlings were photographed and the angle of growth away from gravity was measured relative to the vertical position for hypocotyls and roots using Image J program (ver. 1.41, Fukaki et al. 1997, http://rsb.info.nih. gov/ij/). To measure the effect of light on hypocotyl and root gravitropic curvature, the germination-induced dishes were grown vertically under darkness for 4 d, and then the seedlings were transferred to new assay plates, which were placed in darkness or red light and rotated 90°. The seedlings were photographed at 0, 1, 2, 3, 6, 9, 12, 15, and 24 h of unilateral light, respectively. Curvature was defined as the change in angle from the starting point. Organs that curved in the direction of the gravity vector were assigned positive angles, and organs that curved away from the direction of the gravity vector were assigned negative angles (Kiss et al. 1996). Experiments were repeated three times and values are reported as the mean±SE.
Acknowledgements
We thank Dr. Keara A. Franklin, University of Bristol, for providing the seeds of phyB, phyD and phyB phyD mutants. This work was financially supported by funds from the Genetically Modified Organisms Breeding Major Projects of China (2011ZX08010-002), the National Natural Science Foundation of China (30871438 and 31170267) and the Natural Science Foundation of Xinjiang, China (2012211B49).
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles
References
Aukerman M J, Hirschfeld M, Wester L, Weaver M, Clack T, Amasino R M, Sharrock R A. 1997. A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing. The Plant Cell, 9, 1317-1326. Bhalerao R, Morita M T, Tasaka M. 2004. Gravity sensing and signaling. Current Opinion in Plant Biology, 7, 712-718. Blancaflor E B, Masson P H. 2003. Plant gravitropism. Unraveling the ups and downs of a complex process. Plant Physiology, 133, 1677-1690. Blakeslee J J, Bandyopadhyay A, Peer W A, Makam S N, Murphy A S. 2004. Relocalization of the PIN1 auxin efflux facilitator plays a role in phototropic responses. Plant Physiology, 134, 28-31. Briggs W R, Christie J M. 2002. Phototropins 1 and 2: versatile plant blue-light receptors. Trends Plant Science, 7, 204-210. Britz S J, Galston A W. 1982. Light-enhanced perception of gravity in stems of intact pea seedlings. Planta, 154, 189-192. Correll M J, Coveney K M, Raines S V, Mullen J L, Hangarter R P, Kiss J Z. 2003. Phytochromes play a role in phototropism and gravitropism in Arabidopsis roots. Advances in Space Research, 31, 2203-2210. Correll M J, Kiss J Z. 2002. Interactions between gravitropism and phototropism in plants. Journal of Plant Growth Regulation, 21, 89-101. Correll M J, Kiss J Z. 2005. The roles of phytochromes in elongation and gravitropism of roots. Plant Cell Physiology, 46, 317-323. Fukaki H, Fujisawa H, Tasaka M. 1997. The RHG Gene is involved in root and Hypocotyl gravitropism in Arabidopsis thaliana. Plant Cell Physiology, 38, 804-810. Franklin K A, Praekelt U, Stoddart W M, Billingham O E, Halliday K J, Whitelam G C. 2003. Phytochromes B, D, and E act redundantly to control multiple physiological responses in Arabidopsis. Plant Physiology, 131, 13401346. Hennig L, Stoddart W M, Dieterle M, Whitelam G C, Schafer E. 2002. Phytochrome E controls light-induced germination of Arabidopsis. Plant Physiology, 128, 194-200. Kiss J Z, Wright J B, Caspar T. 1996. Gravitropism in roots of intermediate-starch mutants of Arabidopsis. Plant Physiology, 97, 237-244. Kumar P, Kiss J Z. 2006. Modulation of phototropism by phytochrome E and attenuation of gravitropism by phytochromes B and E in inflorescence stems. Physiologia Plantarum, 127, 304-311. Kumar P, Montgomery C E, Kiss J Z. 2008. The role of phytochrome C in gravitropism and phototropism in Arabidopsis thaliana. Functional Plant Biology, 35, 298-305. Liscum E, Hangarter R P. 1993. Genetic evidence that the redabsorbing form of phytochrome B modulates gravitropism
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in Arabidopsis thaliana. Plant Physiology, 103, 15-19. Liu Y J, Iino M. 1996. Phytochrome is required for the occurrence of time-dependent phototropism in maize coleoptiles. Plant, Cell & Environment, 19, 1379-1388. Lu Y T, Feldman L J. 1997. Light-regulated root gravitropism: a role for, and characterization of, a calcium/calmodulindependent protein kinase homolog. Planta, 203, S91-S97. Marchant A, Kargul J, May S T, Muller P, Delbarre A, PerrotRechenmann C, Bennett M J. 1999. AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues. EMBO Journal, 18, 2066-2073. Miyo T M. 2010. Directional gravity sensing in gravitropism. Annual Review of Plant Physiology and Plant Molecular Biology, 61, 705-720. Monte E, Alonso J M, Ecker J R, Zhang Y, Li X, Young J, Austin-Phillips S, Quail P H. 2003. Isolation and characterization of phyC mutants in Arabidopsis revealscomplex crosstalk between phytochrome signaling pathways. The Plant Cell, 15, 1962-1980. Nagashima A, Suzuki G, Uehara Y, Saji K, Furukawa T, Koshiba T, Sekimoto M, Fujioka S, Kuroha T, Kojima M, Sakakibara H, Fujisawa N, Okada K, Sakai T. 2008. Phytochromes and cryptochromes regulate the differential growth of Arabidopsis hypocotyls in both a PGP19dependent and a PGP19-independent manner. The Plant Journal, 53, 516-529. Parks B M, Spalding E P. 1999. Sequential and coordinated action of phytochromes A and B during Arabidopsis stem growth revealed by kinetic analysis. Proceedings of the National Academy of Sciences of the United States of America, 96, 14142-14146. Poppe C, Hangarter R P, Sharrock R A, Nagy F, Schafer E. 1996. The light-induced reduction of the gravitropic growth-orientation of seedlings of Arabidopsis thaliana (L.) Heynh. is a photomorphogenic response mediated synergistically by the farred-absorbing forms of phytochromes A and B. Planta, 199, 511-514. Robson P R H, Smith H. 1996. Genetic and transgenic evidence that phytochromes A and B act to modulate the gravitropic orientation of Arabidopsis thaliana hypocotyls. Plant Physiology, 110, 211-216. Sharrock R A, Clack T, Goosey L. 2003. Differential activities of the Arabidopsis phyB/D/E phytochromes in complementing phyB mutant phenotypes. Plant Molecular Biology, 52, 135-142. Takano M, Kanegae H, Shinomura T, Miyao A, Hirochika, H, Furuya M. 2001. Isolation and characterization of rice phytochrome A mutants. The Plant Cell, 13, 521-534. Whippo C W, Hangarter R P. 2003. Second positive phototropism results from coordinated co-action of the phototropins and cryptochromes. Plant Physiology, 132, 1499-1507. Woitzik F, Mohr H. 1988. Control of hypocotyl gravitropism by phytochrome in a dicotyledonous seedling (Sesamum indicum L.). Plant Cell Environment, 11, 663-668. (Managing editor WANG Ning) © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
August 2014
RESEARCH ARTICLE
Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles LI Jian-ping1, 2, 3, HOU Pei1, ZHENG Xu1, 3, SONG Mei-fang1, SU Liang1 and YANG Jian-ping1 1
Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China Institute of Nuclear and Biotechnology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, P.R.China 3 Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 2
Abstract The phytochrome gene family, which is in Arabidopsis thaliana, consists of phytochromes A-E (phyA to phyE), regulates plant responses to ambient light environments. PhyA and phyB have been characterized in detail, but studies on phyC to phyE have reported discrepant functions. In this study, we show that phyD regulates the Arabidopsis gravitropic response by inhibiting negative gravitropism of hypocotyls under red light condition. PhyD had only a limited effect on the gravitropic response of roots in red light condition. PhyD also enhanced phyB-regulated gravitropic responses in hypocotyls. Moreover, the regulation of hypocotyl gravitropic responses by phyD was dependent upon the red light fluence rate. Key words: phytochrome D, gravitropism, Arabidopsis thaliana
INTRODUCTION In response to the detection of various environmental signals such as light, gravity, touch, and humidity, the absorption of water and nutrients and the rate of photosynthesis are altered in plants, resulting in a change in plant growth (Miyo 2010). Light is an important environmental influence on plant growth and development; plant photoreceptors detect the quality and direction of light, and plant growth is adjusted in response. Phytochromes are the primary photoreceptors involved in the regulation of red/far-red light-induced responses. Arabidopsis has five phytochromes, phyA to phyE. Phylogenetic analysis suggests that these phytochromes can be clustered into four subfamilies: phyA, phyB/D, phyC, and phyE (Sharrock et al. 2003). PhyA, a light-liable phytochrome, mediates responses to far-red light; phyB is light stable and is dominant in responses to red light (Franklin et al.
2003; Monte et al. 2003). Within the light-stable-phytochrome family, phyB, phyD and phyE act redundantly to control multiple physiological responses, including leaf expansion, hypocotyl development and flowering time (Aukerman et al. 1997; Franklin et al. 2003). Gravity is an important environmental cue for modulating the directional growth of plants. Generally, plant shoots grow upward (i.e., negative gravitropism) and roots grow downward (i.e., positive gravitropism) (Blancaflor and Masson 2003; Bhalerao et al. 2004) tropic responses. Both gravity and light can affect the gravitropic growth of plants. For example, dark-grown hypocotyls of Arabidopsis grow upward, but seedlings grown in red or far-red light grow in random directions (Liscum and Hangarter 1993; Poppe et al. 1996; Robson and Smith 1996; Hennig et al. 2002). Red and far-red light can also influence gravitropic responses in roots (Lu and Feldman 1997; Takano et al. 2001). Thus, gravitropic responses in plants are also modulated by signals from light-activated photoreceptors (Correll and Kiss 2002; Whippo and Hangarter 2003; Blakeslee
Received 9 May, 2013 Accepted 28 August, 2013 LI Jian-ping, E-mail: [email protected]; Correspondence YANG Jian-ping, Tel/Fax: +86-10-82105859, E-mail: [email protected]
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60607-3
Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles
et al. 2004), and roles in light-regulated gravitropism have been identified for several phytochromes. PhyA is the primary photoreceptor involved in far-red light-regulated gravitropic responses of both roots and hypocotyls in Arabidopsis (Parks and Spalding 1999; Hennig et al. 2002). Both phyA and phyB influence the gravitropic responses of maize roots and Arabidopsis hypocotyls (Liscum and Hangarter 1993; Liu and Iino 1996; Poppe et al. 1996; Robson and Smith 1996; Parks and Spalding 1999). PhyC also participates in the regulation of negative gravitropic and phototropic responses of hypocotyles (Kumar et al. 2008). In this study we compared the gravitropic responses of phytochrome mutants with their wild-type (WT) in Arabidopsis thaliana to explore the role of phyD in gravitropic responses in both of roots and hypocotyls and to discover how phytochromes inhibit hypocotyl negative gravitropism.
conducted a time-course analysis of root and hypocotyl growth. We compared the gravitropic curvatures of roots and hypocotyls of phyD single mutant seedlings with those of WT plants (Landsberg erecta (Ler) background) by re-orientating Petri dishes from the vertical to the horizontal position under red light (20.0 μmol m-2 s-1). There were no significant differences in gravritropism between the roots of the phyD mutant and the WT seedlings, with the exception of the 15-h time point (Fig. 1-A). In contrast, the gravitropic curvature of hyrpocotyls was inhibited in red light-grown phyD mutant seedlings relative to the WT seedlings, especially at 3 and 24 h (Fig. 1-B).
PhyB and phyD synergistically regulate the hypocotyl gravitropic response under red light Previous studies have demonstrated the genetic interactions between phytochrome signallings and gravitropic responses and have suggested that phyB plays a predominant role in the regulation of gravitropic growth (Poppe et al. 1996; Robson and Smith 1996; Kumar et al. 2008). Given that phyB and phyD are closely related phylogenetically, we speculated that they could interact in
RESULTS Phy D mediates red light-regulated hypocotyl gravitropism To determine the role of phyD in gravitropism, we B Ler
phyD
PHYD RT-PCR
Curvature (degree)
A
ACTIN
C
HSP90
Curvature (degree)
α-phyD Western blot
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90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0
Root
Ler phyB phyD phyB phyD 0
5
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Time (h) -90.0 -80.0 -70.0 -60.0 -50.0 -40.0 -30.0 -20.0 -10.0 0.0
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Time (h)
Fig. 1 Curvature of roots and hypocotyls in wild-type (Landsberg erecta, Ler) and phyD mutant seedlings of Arabidopsis thaliana grown under red light (20.0 μmol m-2 s-1). A, analysis of RT-PCR and Western blot of phyD mutant and the WT seedlings. B, roots. C, hypocotyls. Time-course studies following 90° re-orientation of the Petri dishes. Error bars indicate standard deviations. Time points of mutants that show a significant difference (P<0.05) relative to the WT at the corresponding time points are indicated *. The same as below.
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
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controlling gravitropic growth of Arabidopsis. First, we investigated the gravitropic response of WT, phyB and phyD single mutants and phyB phyD double mutant seedlings grown under red light by determining the growth orientation of roots and hypocotyls as the degree from vertical (Fig. 2-A-E). The growth angles from vertical were markedly less for the roots of the phyB and phyB phyD mutants compared with the angles for the phyD mutant and WT roots (Fig. 2-B, C, D, E; dark), and the root angles did not differ between WT and phyD mutants (Fig. 2-B, C; dark). These results are consistent with previous studies (Liscum and Hangarter 1993; Correll et al. 2003; Correll and Kiss 2002, 2005) and suggest an important role of phyB in root gravitropic responses.
Hypocotyl gravitropism showed similar results, except there was also a difference between the angles of phyD and WT hypocotyls (Fig. 2-B and C; grey). Moreover, a slight yet significant decrease was observed in the angle from vertical for the hypocotyls of phyB phyD double mutant seedlings compared with phyB single mutant seedlings (Fig. 2-D and E; grey). To confirm these results and further understand the kinetics of hypocotyl gravitropism, we performed timecourse studies by determining the change in curvature of light-grown seedlings stimulated by re-orientating the Petri dishes by 90° in red light. As expected, there was no difference in root curvature between phyD and WT, whereas the roots of phyB and phyB phyD had less
phyD
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Fig. 2 Scatter diagram of hypocotyl and root orientation with respect to the gravity vector for Ler and mutant A. thaliana seedlings (n=160) that were grown vertically under red light (20.0 μmol m-2 s-1). © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles
curvature than those of phyD and WT (Fig. 3-A). The curvature of the hypocotyls was significantly less in both phyB and phyB phyD compared with WT seedlings, with significant reductions in curvature at 9, 12, 15, and 24 h in phyB single mutant seedlings and at 3, 9, 12, 15, and 24 h in phyB phyD double mutant seedlings (Fig. 3-B). There was a slight difference in hypocotyl curvature between phyD single mutant and WT seedlings at 3, 9 and 24 h (Fig. 3-B).
Fluence rate of red light affects the gravitropic response in hypocotyls Light has been proven to be an important factor in gravitropic growth through interactions with phytochrome signalling. In previous studies, gravitropism was shown to be impaired by red light (Britz and Galston 1982; Woitzik and Mohr 1988). To characterize the effect of light, we measured a fluence rate response curve. Arabidopsis WT and phyB, phyD and phyB phyD mutants were grown in
Curvature (degree)
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continuous red light at fluence rates of 0 (darkness), 5.0, 20.0, 50.0, 100.0, and 200.0 μmol m-2 s-1 for 24 h (Fig. 4). With increases in the red light fluence rate from 5.0 to 20.0 to 50.0 μmol m-2 s-1, the curvature of the hypocotyls accelerated. However, reduced acceleration of the curvature was observed at 100 and 200 μmol m-2 s-1. The loss of phyB in Arabidopsis resulted in continuous inhibition of gravitropism in the hypocotyl at all fluence rates, and phyD acted synergistically with phyB at a fluence rate of 20.0 μmol m-2 s-1. Interestingly, a significant difference in gravitropic growth between the phyD mutant and WT was found only at a fluence rate of 20.0 μmol m-2 s-1. These results indicate that the effect of red light on hypocotyl gravitropism is fluence rate-dependent.
DISCUSSION Phytochromes have been shown to play a role in gravitropism (Liscum and Hangarter 1993). Both phyA and phyB are involved in controlling the gravitropic response of maize roots and the gravitropic orientation and inhibition of elongation of Arabidopsis hypocotyls (Poppe et al. 1996; Robson and Smith 1996; Lu and Feldman 1997; Parks and Spalding 1999). Most of these previous studies were based on observations of mutant phenotypes. The present study used a single phyD mutant and a double phyB phyD mutant. The results for the curvature of both roots and hypocotyls in these mutants revealed that phyD attenuated negative gravitropic growth in hypocotyls under red light and that phyB and 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.00
Ler phyB phyD phyB phyD
Curvature (degree)
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-10.0
Ler phyB phyD phyB phyD
Hypocotyl
Fig. 3 Curvature of roots and hypocotyls of Ler and phyB, phyD and phyB phyD mutant seedlings of A. thaliana grown under red light (20.0 μmol m-2 s-1). A, time-course of root curvature after 90° re-orientation of Petri dishes (n 20 for each time point). B, timecourse of hypocotyl curvature after 90° re-orientation of Petri dishes (n 20 for each time point).
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0
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Fluence rate (µmol m-2 s-1)
Fig. 4 Effect of fluence rate on red light-regulated gravitropic growth of hypocotyls. Ler and phyB, phyD, and phyB phyD mutant seedlings of A. thaliana were grown on vertically orientated plates under red light of different fluence rates (0, 5.0, 20.0, 50.0, 100.0, and 250 μmol m-2 s-1) for 24 h. The hypocotyl curvature was measured and plotted vs. the fluence rate.
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
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phyD may act synergistically to regulate this response. Recent studies have also shown that both phyC and phyD mediate positive phototropism in hypocotyls and stems under blue light (Correll et al. 2003) and inhibit red lightbased positive phototropism in roots (Correll and Kiss 2005). PhyB and phyE have been reported to attenuate gravitropism in inflorescence and stems in Arabidopsis (Kumar and Kiss 2006). Thus, the entire phytochrome family appears to be involved the regulation of tropic responses, and phytochromes may interact with each other or with other photoreceptors such as cryptochromes to regulate tropic responses. Evidences have shown that phyD plays an appurtenant role in the inhibition of hypocotyl elongation (Aukerman et al. 1997; Franklin et al. 2003) while root elongation from phyD were similar to WT for both dark- and lightgrown seedlings (Correll and Kiss 2005). In our studies, we also demonstrated that phyD plays a role in hypocotyl gravitropic responses but not in root. However, the regulation of gravitropism by light is one of the less-understood light responses and it is not also clear how phytochromes inhibit gravitropism, based on previous studies (Correll and Kiss 2005; Kumar et al. 2008). Phytochromes (phyA and phyB especially) may directly be involved in regulating the inhibition of hypocotyl elongation and gravitropism but may have not a direct effect of modulating the root gravity signaling cascade through the elongation process. In addition, the crosstalk of the various auxins and phytochormes also determine the elongation processes and tropisms (Marchant et al. 1999; Nagashima et al. 2008; Tajagasgu et al. 2009).
CONCLUSION By analysing the phenotypes of phyB, phyD and phyB phyD mutants of Arabidopsis, we demonstrated that phyD is involved in the inhibition of negative gravitropism of hypocotyls regulated by red light. Under red light condition, root growth in response to gravity did not differ significantly between WT and phyD, whereas the curvature of phyD hypocotyls was less than that of WT. Comparisons of the angles from vertical among the roots and hypocotyls of the phyB, phyD and phyB phyD mutants highlighted the role of phyD in red light-regulated gravitropic growth. PhyD also enhanced
phyB-regulated gravitropic responses in hypocotyls, which was confirmed by the time course of hypocotyl curvature. The present results show that phytochromes regulate gravitropic responses in hypocotyls under red light in a fluence rate-dependent manner.
MATERIALS AND METHODS Plant materials and growth conditions The Landsberg erecta (Ler) ecotype of A. thaliana was used in these studies. Seeds were surface sterilized in 30% bleach with 0.01% (v/v) Triton X-100 for 15 min, rinsed five times in sterile double-distilled water, and stored in water at 4°C for 3 d. Then the seeds were sown on Murashige and Skoog medium with 1% (w/v) sucrose, 0.05% MES (pH 5.7) and 0.9% (w/v) agar in square Petri dishes (5 mm×150 mm) and irradiated with white light for 24 h to promote seed germination.
Gravitropism experiments To test the gravitropic responses of the seedlings, the germination-induced plates were placed vertically in red light (20.0 μmol m-2 s-1) and seedlings grew along the surface of the agar for 96 h at 22°C. Seedlings were photographed and the angle of growth away from gravity was measured relative to the vertical position for hypocotyls and roots using Image J program (ver. 1.41, Fukaki et al. 1997, http://rsb.info.nih. gov/ij/). To measure the effect of light on hypocotyl and root gravitropic curvature, the germination-induced dishes were grown vertically under darkness for 4 d, and then the seedlings were transferred to new assay plates, which were placed in darkness or red light and rotated 90°. The seedlings were photographed at 0, 1, 2, 3, 6, 9, 12, 15, and 24 h of unilateral light, respectively. Curvature was defined as the change in angle from the starting point. Organs that curved in the direction of the gravity vector were assigned positive angles, and organs that curved away from the direction of the gravity vector were assigned negative angles (Kiss et al. 1996). Experiments were repeated three times and values are reported as the mean±SE.
Acknowledgements
We thank Dr. Keara A. Franklin, University of Bristol, for providing the seeds of phyB, phyD and phyB phyD mutants. This work was financially supported by funds from the Genetically Modified Organisms Breeding Major Projects of China (2011ZX08010-002), the National Natural Science Foundation of China (30871438 and 31170267) and the Natural Science Foundation of Xinjiang, China (2012211B49).
© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.
Arabidopsis Phytochrome D Is Involved in Red Light-Induced Negative Gravitropism of Hypocotyles
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