This article was downloaded by: [New York University] On: 04 June 2015, At: 19:11 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Plant Signaling & Behavior Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kpsb20
PINOID functions in root phototropism as a negative regulator a
Ken Haga & Tatsuya Sakai
b
a
Department of Human Science and Common Education; Nippon Institute of Technology; Saitama, Japan b
Graduate School of Science and Technology; Niigata University; Niigata, Japan Published online: 03 Jun 2015.
Click for updates To cite this article: Ken Haga & Tatsuya Sakai (2015) PINOID functions in root phototropism as a negative regulator, Plant Signaling & Behavior, 10:5, e998545, DOI: 10.1080/15592324.2014.998545 To link to this article: http://dx.doi.org/10.1080/15592324.2014.998545
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
SHORT COMMUNICATION Plant Signaling & Behavior 10:5, e998545; May 2015; © 2015 Taylor & Francis Group, LLC
PINOID functions in root phototropism as a negative regulator Ken Haga1,* and Tatsuya Sakai2 1
Department of Human Science and Common Education; Nippon Institute of Technology; Saitama, Japan; 2Graduate School of Science and Technology; Niigata University; Niigata, Japan
Keywords: AGC kinase, arabidopsis, PINOID, phototropism, root
Downloaded by [New York University] at 19:11 04 June 2015
Abbreviations: PIN, PIN-FORMED; PID, PINOID; WAG, WAVY ROOT GROWTH.
The PINOID (PID) family, which belongs to AGCVIII kinases, is known to be involved in the regulation of auxin efflux transporter PIN-FORMED (PIN) proteins through changes in the phosphorylation status. Recently, we demonstrated that the PID family is necessary for phytochrome-mediated phototropic enhancement in Arabidopsis hypocotyls and that the downregulation of PID expression by red-light pretreatment results in the promotion of the PIN-mediated auxin gradient during phototropic responses. However, whether PID participates in root phototropism in Arabidopsis seedlings has not been well studied. Here, we demonstrated that negative root phototropic responses are enhanced in the pid quadruple mutant and are severely impaired in transgenic plants expressing PID constitutively. The results indicate that the PID family functions in a negative root phototropism as a negative regulator. On the other hand, analysis with PID fused to a yellow fluorescent protein, VENUS, showed that unilateral blue-light irradiation causes a lower accumulation of PID proteins on the shaded side than on the irradiated side. This result suggests that the bluelight-mediated asymmetrical distribution of PID proteins may be one of the critical responses in phototropin-mediated signals during a negative root phototropism. Alternatively, such a transverse gradient of PID proteins may result from gravitropic stimulation produced by phototropic bending.
Phototropism is induced by unilateral blue-light irradiation through blue-light photoreceptor phototropin-mediated signaling.1 On the other hand, it is well-known that red-light pretreatment enhances phototropic responses through phytochrome signaling in addition to being necessary for the appearance of the second positive phototropism.2 Although several studies have reported molecular events induced by red-light pretreatment, the molecular mechanisms that participate in phytochrome-mediated phototropic enhancement remain unclear.3 Previously, we showed that PINOID (PID), which belongs to AGCVIII kinases and regulates the phosphorylation status of PIN-FORMED (PIN) proteins,4-6 is partially involved in phytochrome-mediated phototropic enhancement.7 Because the PID family consists of four members in Arabidopsis (PID, PID2, WAVY ROOT GROWTH 1 (WAG1), and WAG2),8 careful studies of phototropic responses using the pid quadruple mutant were necessary to clarify the roles of the PID family in the phytochrome-mediated regulation of phototropism. Very recently, we reported that phytochrome-mediated phototropic enhancement of hypocotyl phototropism is specifically impaired in the pid-14 (Salk_049736) pid2 (Sail_269_G07)
wag1 (Salk_002056C) wag2 (Salk_070240) quadruple mutant in Arabidopsis seedlings. The promotion of auxin gradients by red-light pretreatment is also severely attenuated in the quadruple mutant.9 Therefore, we concluded that the PID family is a critical component for the phytochrome-mediated enhancement of hypocotyl phototropism, but not for the phototropin signaling pathway. Furthermore, constitutive expression of PID attenuated the pulse-induced first positive phototropism, and red-light pretreatment reduced the expression of PID proteins in addition to the upregulation of PIN proteins. This indicates that the PID family functions in hypocotyl phototropism as a negative regulator. However, it remains unclear whether the PID family is also involved in root phototropism under our experimental conditions. Arabidopsis seedlings clearly show a negative root phototropism induced by prolonged unilateral irradiation of strong blue light (e.g., 100 mmol m–2 s–1 for 24–48 h).10 In addition to a positive root gravitropism, a negative root phototropism is thought to be involved in determination of direction of root growth in the soil to escape stressful conditions such as dryness in nature. To clarify the roles of the PID family in a negative root
*Correspondence to: Ken Haga; Email: [email protected] Submitted: 10/31/2014; Revised: 11/22/2014; Accepted: 11/24/2014 http://dx.doi.org/10.1080/15592324.2014.998545 The citation for the original article: Haga K, Hayashi K, Sakai T. PINOID AGC kinases are necessary for phytochrome-mediated enhancement of hypocotyl phototropism in Arabidopsis. Plant Physiol 2014; 166: 1535-45; PMID: 25281709; http://dx.doi.org/10.1104/pp.114.244434
www.tandfonline.com
Plant Signaling & Behavior
e998545-1
Downloaded by [New York University] at 19:11 04 June 2015
phototropism, we investigated the phototropic responses in the pid quadruple mutant we prepared9 and in transgenic plants harboring the cauliflower mosaic virus 35S promoter-driven PID cDNA gene (35S::PID). Two-day-old dark-grown seedlings were irradiated unilaterally with blue light at 100 mmol m–2 s–1 for 24 h (Fig. 1A) and the phototropic curvatures of the roots were measured (Fig. 1B). To avoid drought stress caused by a long blue-light irradiation, the seedlings were grown along the surface of vertically oriented agar medium.10,11 Under such conditions, the wild-type roots showed negative phototropic curvatures of around ¡30 . On the other hand, the pid quadruple mutant displayed a slight enhancement of phototropic curvatures (Fig. 1B), while the transgenic plants harboring 35S::PID did not exhibit clear phototropic responses. These results indicate that the PID family is involved in a negative root phototropism as a negative regulator. Because it is known that phototropin 1 is a major photoreceptor for a negative root phototropism,10 it appears that the PID family functions in the phototropin signaling pathway. We further investigated the effects of blue-light irradiation on changes in the distribution of PID proteins using a yellow fluorescent protein, VENUS. The dark-grown transgenic plants harboring the VENUS-fused PID gene driven by its own promoter (PID::PID–VENUS) were irradiated with unilateral blue light at 100 mmol m–2 s–1 for 24 h and the fluorescent signals were observed. When the seedlings were not treated with blue light, the fluorescent signals were evenly distributed on both sides of the epidermal cells around root elongation zones (Fig. 1C and 1D, left side). Interestingly, blue-light irradiation changed the symmetrical distribution of the fluorescent signals (Fig. 1C, right side). The phototropic stimulation reduced the fluorescent signals on the shaded side compared with those on the irradiated side (Fig. 1D, right side). The results indicate that blue light somehow influences the distribution of PID proteins. Therefore, it is possible that the asymmetrical distribution of PID proteins influences the phosphorylation status of PIN proteins on the shaded and irradiated sides, leading to changes in auxin distribution. Alternatively, uneven accumulation of PID proteins may result from gravitropic stimulation produced by a negative root phototropism because the seedlings were treated with a longer irradiation of blue light. Further studies are necessary to clarify the roles of the PID family in a negative root phototropism in Arabidopsis seedlings. Previously, Zhang et al. (2013)12 reported that a negative root phototropism is slightly impaired in the wag1 wag2 pid triple mutant. According to their results, it was expected that the pid quadruple mutant would show defects in the root phototropism. However, the present study showed that the phototropic responses are enhanced in the quadruple mutant (Fig. 1B). Although PID2 may play some critical roles in a negative root phototropism, different experimental conditions, such as the blue-light intensity and duration of blue light used for the induction of phototropic bending, probably influence the physiological responses. Furthermore, we detected an asymmetrical distribution of PID proteins during negative root curvatures, while Zhang et al. (2013)12 did not observe such gradients, although blue light reduced the levels of PID proteins.12 This may also be
e998545-2
Figure 1. Negative root phototropism in the pid quadruple mutant (quad) and the transgenic plants harboring the cauliflower mosaic virus 35S promoter-driven PID gene (35S::PID). Two-day-old dark-grown seedlings were grown along the surface of agar medium oriented vertically. Columbia (Col), the pid-14 (Salk_049736) wag1 (Salk_002056C) wag2 (Salk_070240) pid2 (Sail_269_G07) quadruple mutant, and transgenic plants harboring 35S::PID (35S) were used in this study. Dark-grown seedlings were irradiated with unilateral blue light (BL) continuously at 100 mmol m–2 s–1 for 24 h. (A) Representative pictures of a negative root phototropism in Col, the pid quadruple mutant (quad), and the transgenic seedlings harboring 35S::PID. Blue arrow indicates direction of blue light irradiation. Black bar, 5 mm. (B) Negative phototropic curvatures in the roots. The data shown are the means § SE from 16 to 23 seedlings. Asterisks show statistically significant differences between Col and the quadruple mutant or the transgenic plants (*p < 0.05, ***p < 0.001). (C) Representative confocal micrographs during a negative root phototropism in the transgenic plants harboring PID–VENUS. Blue arrow indicates direction of blue light irradiation. White bar, 100 mm. (D) Distribution of fluorescence derived from PID–VENUS between the irradiated and shaded sides of the roots with or without unilateral BL irradiation. The distribution was calculated as the percentage of the signal intensity obtained from the two sides. The data shown are the means § SE from eight seedlings.
caused by differences in experimental conditions. To resolve such discrepancies, further investigations are necessary in the near future. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Plant Signaling & Behavior
Volume 10 Issue 5
Acknowledgements
Funding
We thank the Arabidopsis Biological Resource Center for providing the pid-14 (Salk_049736), wag1 (Salk_002056C), wag2 (Salk_070240), and pid2 (Sail_269_G07) mutants. We also thank Professor R. Offringa (Leiden University) and Professor M. Heisler (European Molecular Biology Laboratory) for providing the 35S::PID seeds and PID::PID–VENUS seeds, respectively.
This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (No. 22570058), a Grantin-Aid for Scientific Research on Innovative Areas “Plant Environmental Sensing” (No. 23120510) from The Ministry of Education, Culture, Sports, Science, and Technology, Japan (to T.S.), and by KAKENHI (No. 24657027), a Grant-in-Aid for Challenging Exploratory Research (to K.H.).
Downloaded by [New York University] at 19:11 04 June 2015
References 1. Sakai T, Haga K. Molecular genetic analysis of phototropism in Arabidopsis. Plant Cell Physiol 2012; 53:1517-34; PMID:22864452; http://dx.doi.org/ 10.1093/pcp/pcs111 2. Iino M. Phototropism in higher plants. In D H€ader, M Lebert, eds, Photomovement: ESP Comprehensive Series in Photosciences, Vol 1. Elsevier, Amsterdam, The Netherlands, 659-811; http://dx.doi.org/10.1016/ S1568-461X(01)80027-2 3. Briggs WR. Phototropism: some history, some puzzles, and a look ahead. Plant Physiol 2014; 164:13-23; PMID:24399823; http://dx.doi.org/10.1104/ pp.113.230573 4. Michniewicz M, Zago MK, Abas L, Weijers D, Schweighofer A, Meskiene I, Heisler MG, Ohno C, Zhang J, Huang F, et al. Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux. Cell 2007; 130:1044-56; PMID:17889649; http://dx.doi:org/10.1016/j.cell.2007.07.033 5. Huang F, Zago MK, Abas L, van Marion A, Ampudia CSG, Offringa R. Phosphorylation of conserved PIN
www.tandfonline.com
6.
7.
8.
9.
motifs directs Arabidopsis PIN1 polarity and auxin transport. Plant Cell 2010; 22:1129-42; PMID:20407025; http://dx.doi.org/10.1105/ tpc.109.072678 Zourelidou M, Absmanner B, Weller B, Barbosa IC, Willige BC, Fastner A, Streit V, Port SA, Colcombet J, de la Fuente, van Bentem S, et al. Auxin efflux by PINFORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID. Elife 2014; 3:e02860; PMID:24948515; http://dx.doi. org/10.7554/eLife.02860 Haga K, Sakai T. PIN auxin efflux carriers are necessary for pulse-induced but not for continuous light-induced phototropism in Arabidopsis. Plant Physiol 2012; 160:763-76; PMID:22843667; http://dx.doi.org/ 10.1104/pp.112.202432 Galvan-Ampudia CS, Offringa R. Plant evolution: AGC kinases tell the auxin tale. Trends Plant Sci 2007; 12:541-7; PMID:18024140; http://dx.doi.org/ 10.1016/j.tplants.2007.10.004 Haga K, Hayashi K, Sakai T. PINOID AGC kinases are necessary for phytochrome-mediated enhancement
Plant Signaling & Behavior
of hypocotyl phototropism in Arabidopsis. Plant Physiol 2014; 166:1535-45; PMID:25281709; http://dx. doi.org/10.1104/pp.114 10. Sakai T, Wada T, Ishiguro S, Okada K. RPT2: a signal transducer of the phototropic response in Arabidopsis. Plant Cell 2000; 12:225-36; PMID:10662859; http:// dx.doi.org/10.1105/tpc.12.2.225 11. Haga K, Sakai T. Differential roles of auxin efflux carrier PIN proteins in hypocotyl phototropism of etiolated Arabidopsis seedlings depend on the direction of light stimulus. Plant Sig Behav 2013; 8:1-3; PMID:23104115; http://dx.doi.org/10.4161/ psb.22556 12. Zhang K-X, Xu H-H, Yuan T-T, Zhang L, Lu Y-T. Blue-light-induced PIN3 polarization for root negative phototropic response in Arabidopsis. Plant J 2013; 76: 308-21; PMID:23888933; http://dx.doi.org/10.1111/ tpj.12298
e998545-3
Plant Signaling & Behavior Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kpsb20
PINOID functions in root phototropism as a negative regulator a
Ken Haga & Tatsuya Sakai
b
a
Department of Human Science and Common Education; Nippon Institute of Technology; Saitama, Japan b
Graduate School of Science and Technology; Niigata University; Niigata, Japan Published online: 03 Jun 2015.
Click for updates To cite this article: Ken Haga & Tatsuya Sakai (2015) PINOID functions in root phototropism as a negative regulator, Plant Signaling & Behavior, 10:5, e998545, DOI: 10.1080/15592324.2014.998545 To link to this article: http://dx.doi.org/10.1080/15592324.2014.998545
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
SHORT COMMUNICATION Plant Signaling & Behavior 10:5, e998545; May 2015; © 2015 Taylor & Francis Group, LLC
PINOID functions in root phototropism as a negative regulator Ken Haga1,* and Tatsuya Sakai2 1
Department of Human Science and Common Education; Nippon Institute of Technology; Saitama, Japan; 2Graduate School of Science and Technology; Niigata University; Niigata, Japan
Keywords: AGC kinase, arabidopsis, PINOID, phototropism, root
Downloaded by [New York University] at 19:11 04 June 2015
Abbreviations: PIN, PIN-FORMED; PID, PINOID; WAG, WAVY ROOT GROWTH.
The PINOID (PID) family, which belongs to AGCVIII kinases, is known to be involved in the regulation of auxin efflux transporter PIN-FORMED (PIN) proteins through changes in the phosphorylation status. Recently, we demonstrated that the PID family is necessary for phytochrome-mediated phototropic enhancement in Arabidopsis hypocotyls and that the downregulation of PID expression by red-light pretreatment results in the promotion of the PIN-mediated auxin gradient during phototropic responses. However, whether PID participates in root phototropism in Arabidopsis seedlings has not been well studied. Here, we demonstrated that negative root phototropic responses are enhanced in the pid quadruple mutant and are severely impaired in transgenic plants expressing PID constitutively. The results indicate that the PID family functions in a negative root phototropism as a negative regulator. On the other hand, analysis with PID fused to a yellow fluorescent protein, VENUS, showed that unilateral blue-light irradiation causes a lower accumulation of PID proteins on the shaded side than on the irradiated side. This result suggests that the bluelight-mediated asymmetrical distribution of PID proteins may be one of the critical responses in phototropin-mediated signals during a negative root phototropism. Alternatively, such a transverse gradient of PID proteins may result from gravitropic stimulation produced by phototropic bending.
Phototropism is induced by unilateral blue-light irradiation through blue-light photoreceptor phototropin-mediated signaling.1 On the other hand, it is well-known that red-light pretreatment enhances phototropic responses through phytochrome signaling in addition to being necessary for the appearance of the second positive phototropism.2 Although several studies have reported molecular events induced by red-light pretreatment, the molecular mechanisms that participate in phytochrome-mediated phototropic enhancement remain unclear.3 Previously, we showed that PINOID (PID), which belongs to AGCVIII kinases and regulates the phosphorylation status of PIN-FORMED (PIN) proteins,4-6 is partially involved in phytochrome-mediated phototropic enhancement.7 Because the PID family consists of four members in Arabidopsis (PID, PID2, WAVY ROOT GROWTH 1 (WAG1), and WAG2),8 careful studies of phototropic responses using the pid quadruple mutant were necessary to clarify the roles of the PID family in the phytochrome-mediated regulation of phototropism. Very recently, we reported that phytochrome-mediated phototropic enhancement of hypocotyl phototropism is specifically impaired in the pid-14 (Salk_049736) pid2 (Sail_269_G07)
wag1 (Salk_002056C) wag2 (Salk_070240) quadruple mutant in Arabidopsis seedlings. The promotion of auxin gradients by red-light pretreatment is also severely attenuated in the quadruple mutant.9 Therefore, we concluded that the PID family is a critical component for the phytochrome-mediated enhancement of hypocotyl phototropism, but not for the phototropin signaling pathway. Furthermore, constitutive expression of PID attenuated the pulse-induced first positive phototropism, and red-light pretreatment reduced the expression of PID proteins in addition to the upregulation of PIN proteins. This indicates that the PID family functions in hypocotyl phototropism as a negative regulator. However, it remains unclear whether the PID family is also involved in root phototropism under our experimental conditions. Arabidopsis seedlings clearly show a negative root phototropism induced by prolonged unilateral irradiation of strong blue light (e.g., 100 mmol m–2 s–1 for 24–48 h).10 In addition to a positive root gravitropism, a negative root phototropism is thought to be involved in determination of direction of root growth in the soil to escape stressful conditions such as dryness in nature. To clarify the roles of the PID family in a negative root
*Correspondence to: Ken Haga; Email: [email protected] Submitted: 10/31/2014; Revised: 11/22/2014; Accepted: 11/24/2014 http://dx.doi.org/10.1080/15592324.2014.998545 The citation for the original article: Haga K, Hayashi K, Sakai T. PINOID AGC kinases are necessary for phytochrome-mediated enhancement of hypocotyl phototropism in Arabidopsis. Plant Physiol 2014; 166: 1535-45; PMID: 25281709; http://dx.doi.org/10.1104/pp.114.244434
www.tandfonline.com
Plant Signaling & Behavior
e998545-1
Downloaded by [New York University] at 19:11 04 June 2015
phototropism, we investigated the phototropic responses in the pid quadruple mutant we prepared9 and in transgenic plants harboring the cauliflower mosaic virus 35S promoter-driven PID cDNA gene (35S::PID). Two-day-old dark-grown seedlings were irradiated unilaterally with blue light at 100 mmol m–2 s–1 for 24 h (Fig. 1A) and the phototropic curvatures of the roots were measured (Fig. 1B). To avoid drought stress caused by a long blue-light irradiation, the seedlings were grown along the surface of vertically oriented agar medium.10,11 Under such conditions, the wild-type roots showed negative phototropic curvatures of around ¡30 . On the other hand, the pid quadruple mutant displayed a slight enhancement of phototropic curvatures (Fig. 1B), while the transgenic plants harboring 35S::PID did not exhibit clear phototropic responses. These results indicate that the PID family is involved in a negative root phototropism as a negative regulator. Because it is known that phototropin 1 is a major photoreceptor for a negative root phototropism,10 it appears that the PID family functions in the phototropin signaling pathway. We further investigated the effects of blue-light irradiation on changes in the distribution of PID proteins using a yellow fluorescent protein, VENUS. The dark-grown transgenic plants harboring the VENUS-fused PID gene driven by its own promoter (PID::PID–VENUS) were irradiated with unilateral blue light at 100 mmol m–2 s–1 for 24 h and the fluorescent signals were observed. When the seedlings were not treated with blue light, the fluorescent signals were evenly distributed on both sides of the epidermal cells around root elongation zones (Fig. 1C and 1D, left side). Interestingly, blue-light irradiation changed the symmetrical distribution of the fluorescent signals (Fig. 1C, right side). The phototropic stimulation reduced the fluorescent signals on the shaded side compared with those on the irradiated side (Fig. 1D, right side). The results indicate that blue light somehow influences the distribution of PID proteins. Therefore, it is possible that the asymmetrical distribution of PID proteins influences the phosphorylation status of PIN proteins on the shaded and irradiated sides, leading to changes in auxin distribution. Alternatively, uneven accumulation of PID proteins may result from gravitropic stimulation produced by a negative root phototropism because the seedlings were treated with a longer irradiation of blue light. Further studies are necessary to clarify the roles of the PID family in a negative root phototropism in Arabidopsis seedlings. Previously, Zhang et al. (2013)12 reported that a negative root phototropism is slightly impaired in the wag1 wag2 pid triple mutant. According to their results, it was expected that the pid quadruple mutant would show defects in the root phototropism. However, the present study showed that the phototropic responses are enhanced in the quadruple mutant (Fig. 1B). Although PID2 may play some critical roles in a negative root phototropism, different experimental conditions, such as the blue-light intensity and duration of blue light used for the induction of phototropic bending, probably influence the physiological responses. Furthermore, we detected an asymmetrical distribution of PID proteins during negative root curvatures, while Zhang et al. (2013)12 did not observe such gradients, although blue light reduced the levels of PID proteins.12 This may also be
e998545-2
Figure 1. Negative root phototropism in the pid quadruple mutant (quad) and the transgenic plants harboring the cauliflower mosaic virus 35S promoter-driven PID gene (35S::PID). Two-day-old dark-grown seedlings were grown along the surface of agar medium oriented vertically. Columbia (Col), the pid-14 (Salk_049736) wag1 (Salk_002056C) wag2 (Salk_070240) pid2 (Sail_269_G07) quadruple mutant, and transgenic plants harboring 35S::PID (35S) were used in this study. Dark-grown seedlings were irradiated with unilateral blue light (BL) continuously at 100 mmol m–2 s–1 for 24 h. (A) Representative pictures of a negative root phototropism in Col, the pid quadruple mutant (quad), and the transgenic seedlings harboring 35S::PID. Blue arrow indicates direction of blue light irradiation. Black bar, 5 mm. (B) Negative phototropic curvatures in the roots. The data shown are the means § SE from 16 to 23 seedlings. Asterisks show statistically significant differences between Col and the quadruple mutant or the transgenic plants (*p < 0.05, ***p < 0.001). (C) Representative confocal micrographs during a negative root phototropism in the transgenic plants harboring PID–VENUS. Blue arrow indicates direction of blue light irradiation. White bar, 100 mm. (D) Distribution of fluorescence derived from PID–VENUS between the irradiated and shaded sides of the roots with or without unilateral BL irradiation. The distribution was calculated as the percentage of the signal intensity obtained from the two sides. The data shown are the means § SE from eight seedlings.
caused by differences in experimental conditions. To resolve such discrepancies, further investigations are necessary in the near future. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Plant Signaling & Behavior
Volume 10 Issue 5
Acknowledgements
Funding
We thank the Arabidopsis Biological Resource Center for providing the pid-14 (Salk_049736), wag1 (Salk_002056C), wag2 (Salk_070240), and pid2 (Sail_269_G07) mutants. We also thank Professor R. Offringa (Leiden University) and Professor M. Heisler (European Molecular Biology Laboratory) for providing the 35S::PID seeds and PID::PID–VENUS seeds, respectively.
This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (No. 22570058), a Grantin-Aid for Scientific Research on Innovative Areas “Plant Environmental Sensing” (No. 23120510) from The Ministry of Education, Culture, Sports, Science, and Technology, Japan (to T.S.), and by KAKENHI (No. 24657027), a Grant-in-Aid for Challenging Exploratory Research (to K.H.).
Downloaded by [New York University] at 19:11 04 June 2015
References 1. Sakai T, Haga K. Molecular genetic analysis of phototropism in Arabidopsis. Plant Cell Physiol 2012; 53:1517-34; PMID:22864452; http://dx.doi.org/ 10.1093/pcp/pcs111 2. Iino M. Phototropism in higher plants. In D H€ader, M Lebert, eds, Photomovement: ESP Comprehensive Series in Photosciences, Vol 1. Elsevier, Amsterdam, The Netherlands, 659-811; http://dx.doi.org/10.1016/ S1568-461X(01)80027-2 3. Briggs WR. Phototropism: some history, some puzzles, and a look ahead. Plant Physiol 2014; 164:13-23; PMID:24399823; http://dx.doi.org/10.1104/ pp.113.230573 4. Michniewicz M, Zago MK, Abas L, Weijers D, Schweighofer A, Meskiene I, Heisler MG, Ohno C, Zhang J, Huang F, et al. Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux. Cell 2007; 130:1044-56; PMID:17889649; http://dx.doi:org/10.1016/j.cell.2007.07.033 5. Huang F, Zago MK, Abas L, van Marion A, Ampudia CSG, Offringa R. Phosphorylation of conserved PIN
www.tandfonline.com
6.
7.
8.
9.
motifs directs Arabidopsis PIN1 polarity and auxin transport. Plant Cell 2010; 22:1129-42; PMID:20407025; http://dx.doi.org/10.1105/ tpc.109.072678 Zourelidou M, Absmanner B, Weller B, Barbosa IC, Willige BC, Fastner A, Streit V, Port SA, Colcombet J, de la Fuente, van Bentem S, et al. Auxin efflux by PINFORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID. Elife 2014; 3:e02860; PMID:24948515; http://dx.doi. org/10.7554/eLife.02860 Haga K, Sakai T. PIN auxin efflux carriers are necessary for pulse-induced but not for continuous light-induced phototropism in Arabidopsis. Plant Physiol 2012; 160:763-76; PMID:22843667; http://dx.doi.org/ 10.1104/pp.112.202432 Galvan-Ampudia CS, Offringa R. Plant evolution: AGC kinases tell the auxin tale. Trends Plant Sci 2007; 12:541-7; PMID:18024140; http://dx.doi.org/ 10.1016/j.tplants.2007.10.004 Haga K, Hayashi K, Sakai T. PINOID AGC kinases are necessary for phytochrome-mediated enhancement
Plant Signaling & Behavior
of hypocotyl phototropism in Arabidopsis. Plant Physiol 2014; 166:1535-45; PMID:25281709; http://dx. doi.org/10.1104/pp.114 10. Sakai T, Wada T, Ishiguro S, Okada K. RPT2: a signal transducer of the phototropic response in Arabidopsis. Plant Cell 2000; 12:225-36; PMID:10662859; http:// dx.doi.org/10.1105/tpc.12.2.225 11. Haga K, Sakai T. Differential roles of auxin efflux carrier PIN proteins in hypocotyl phototropism of etiolated Arabidopsis seedlings depend on the direction of light stimulus. Plant Sig Behav 2013; 8:1-3; PMID:23104115; http://dx.doi.org/10.4161/ psb.22556 12. Zhang K-X, Xu H-H, Yuan T-T, Zhang L, Lu Y-T. Blue-light-induced PIN3 polarization for root negative phototropic response in Arabidopsis. Plant J 2013; 76: 308-21; PMID:23888933; http://dx.doi.org/10.1111/ tpj.12298
e998545-3
