PLANT SIGNALING & BEHAVIOR 2016, VOL. 11, NO. 2, e1143999 (3 pages) http://dx.doi.org/10.1080/15592324.2016.1143999
ARTICLE ADDENDUM
Importance of epidermal clocks for regulation of hypocotyl elongation through PIF4 and IAA29 Hanako Shimizu, Kotaro Torii, Takashi Araki, and Motomu Endo Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
ABSTRACT
ARTICLE HISTORY
Circadian clocks adjust an organism’s environmentally relevant physiological responses.. In plants, a decentralized circadian clock system has recently been proposed. Epidermal clock function is crucial for cell elongation; thus, epidermis-specific overexpression of CCA1 caused smaller cotyledons and longer hypocotyls under 27 C, concomitant with elevated night time levels of PIF4 mRNA. However, which tissue’s clock regulates PIF4 expression is still an open question. Here we tested spatial expression patterns of PIF4 and its downstream target IAA29 with or without epidermal clock perturbation. Using an epidermal-specific expression system, we revealed that epidermal clock perturbation increases PIF4 expression in both epidermis and mesophyll. However, IAA29 expression is mainly regulated in the epidermis, implying the potential importance of epidermis for regulation of cell elongation through PIF4 and IAA29. We conclude that the circadian clock in epidermis regulates cell elongation mainly in epidermis, and there is also another inter-tissue signaling pathway from epidermis to mesophyll.
Received 14 December 2015 Revised 4 January 2016 Accepted 14 January 2016
In multicellular organisms, time information created by cellular circadian clocks should be integrated to achieve cooperative reactions to fluctuating environmental conditions. The corresponding intra- and inter-tissue signaling networks through the circadian clock are complex, and it is an important challenge to elucidate how organisms are measuring time and using time information for their responses. Recently, the plant circadian clock network has been proposed to be decentralized,1,2 and cell elongation is mainly regulated by the epidermal clock in response to ambient temperature. Overexpression of a circadian clock gene, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) disrupts its rhythmic circadian oscillation.3 We have established several transgenic lines expressing CCA1 under tissuespecific promoters to achieve tissue-specific clock disruption. Among these lines, epidermal-clock perturbation by CCA1 driven by the 3-KETOACYL-COA SYNTHASE 6 promoter (CER6)::CCA1 showed longer hypocotyls and a smaller cotyledon area. Intriguingly, this phenotype is dependent upon ambient temperature and is well correlated with PHTYOCHROME INTERACTING FACTOR4 (PIF4) expression during the night. However, as PIF4 is expressed in all leaf tissues including epidermis, mesophyll, and vasculature,4 it was largely unknown whether multiple tissues would show PIF4 regulation by the epidermal clock, or how the epidermal clock is incorporated in the clock network. To identify the target tissues of the epidermal clock, we established transgenic plants expressing b-glucuronidase (GUS) under the PIF4 promoter (PIF4::GUS) in wild type and a CER6::CCA1 background. In the PIF4::GUS line, the blue-stained GUS signal was detected in cotyledons, hypocotyls and shoot apex (Figs. 1A, B). For further dissection CONTACT Motomu Endo © 2016 Taylor & Francis Group, LLC
[email protected]
KEYWORDS
Arabidopsis; circadian clock; decentralized clock; epidermis; hypocotyl; IAA29; PIF4; temperature
of the spatial PIF4::GUS expression pattern in cotyledons, we cross-sectioned cotyledons and observed the GUS expression pattern in each tissue (Figs. 1C, D). At variance with a previous report,4 PIF4::GUS expression was limited mainly to vasculature, although a weak signal was also detected in mesophyll and epidermis. Then which tissue’s expression of PIF4 is responsible for the cell elongation? To answer this question, we next observed a PIF4::GUS/CER6:: CCA1 line grown under the same condition. The PIF4::GUS expression level was dramatically increased by the exogenous CER6::CCA1, consistent with our view that the epidermal clock is crucial for adequate PIF4 expression. A crosssection of the cotyledon showed dense GUS expression in epidermal and mesophyll cells, implying the epidermal clock suppresses epidermal and mesophyll PIF4 expression. Interestingly, GUS staining levels in vasculature appeared to be the same, compared to the PIF4::GUS line. To support this observation, we analyzed PIF4 expression levels by qPCR using isolated epidermis, mesophyll, vasculature, and whole leaf (Fig. 1E). Consistent with the GUS staining result, higher PIF4 expression levels were observed in both epidermis and mesophyll of a CER6::CCA1 seedling compared to wild type. The transcription factor PIF4 has several direct target genes,5 and one of these is INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29), which is known to be involved in hypocotyl elongation. To test whether IAA29 expression is regulated in a similar way as PIF4 in leaves, we also observed IAA29::GUS expression in cotyledons. Unlike PIF4, only weak IAA29::GUS expression was observed in epidermis (Figs. 2A, B), suggesting that increased PIF4 in mesophyll cells did not directly lead to
e1143999-2
H. SHIMIZU ET AL.
Figure 1. Effect of epidermal clock perturbation by CER6::CCA1 on the spatial expression pattern of PIF4. (A-D) Expression of PIF4::GUS in 7-day-old wild-type (A, C) and CER6::CCA1 (B, D) seedlings. Whole seedlings (A, B) and cross-sections of cotyledons (C, D) are shown. Bars = 500 mm (A, B) and 50 mm (C, D). (E) Induction of PIF4 expression in isolated tissues by epidermal clock perturbation. Gene expression levels were detected by q-PCR and the highest value was set as 1. IPP2 and APA1 were used as internal control genes.1
up-regulation of IAA29. For further confirmation of this asymmetric regulation of PIF4 and IAA29 by epidermal clock perturbation, we observed PIF4::GUS and IAA29::GUS expression in a cross-sectioned hypocotyl (Figs. 2C, D, E, F). Both PIF4:: GUS and IAA29::GUS expression were limited to the vasculature in the hypocotyl, and this was also the case in a cotyledon. When the epidermal clock was perturbed by CER6::CCA1, PIF4::GUS expression was increased both in epidermis and mesophyll, whereas induction of IAA29::GUS expression was not observed. Together, these results demonstrate an essential role of the epidermal circadian clock for PIF4 and IAA29 expression in epidermal cells of the leaf. We also showed that upregulation of PIF4 in mesophyll did not lead to induction of IAA29 in mesophyll, even though IAA29 is a direct
target of PIF4. This asymmetric regulation of PIF4 and IAA29 by the epidermal clock strongly suggests that IAA29 is not the only regulatory target of PIF4, and the PIF4 signaling pathways appear to diverge at an early step in a specific tissue. We would like to point out the similarity of cell elongation mechanisms between epidermal clock disruption and shade avoidance response. Both suppress cotyledon expansion and increase hypocotyl elongation by regulating PIF4 expression in response to environmental changes. Our results further strengthen the previous view that PIF4 has a prominent role as a node of crosstalk between light and temperature signaling.6,7 We also revealed an inter-tissue regulation of the epidermal clock connected to mesophyll PIF4 expression. This epidermisto-mesophyll signaling reinforces our “decentralized circadian
Figure 2. PIF4 and IAA29 expression patterns in cotyledon and hypocotyl. Expression of IAA29::GUS and PIF4::GUS in 7-day-old wild-type (A, C, E) and CER6::CCA1 (B, D, F) cotyledon and hypocotyl. Weak IAA29::GUS induction was observed in epidermal cells of cotyledons (A, B) but not in hypocotyls (E, F); clear PIF4::GUS induction was observed even in hypocotyl cells (C, D). Bars = 100 mm (A, B) and 50 mm (C, D, E, F).
PLANT SIGNALING & BEHAVIOR
clock network” concept. In addition, we would like to note that induction of IAA29 expression was not observed in hypocotyl epidermal cells, implying that leaf epidermis has crucial roles for hypocotyl cell elongation. Similarly, far-red light irradiation on a cotyledon but not on a hypocotyl induces auxin-responsive gene expression in a hypocotyl. 8 Although this hypothesis is still consistent with the “epidermal-growth-control model”,9 it requires some further explanation in the future.
4.
5.
6.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. 7.
References 1. Endo M, Shimizu H, Nohales MA, Araki T, Kay SA. Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature 2014; 515:419-22; PMID:25363766; http://dx.doi.org/10.1038/nature13919 2. Shimizu H, Katayama K, Koto T, Torii K, Araki T, Endo M. Decentralized circadian clocks process thermal and photoperiodic cues in specific tissues. Nat Plants 2015; 1: Article number: 15163. 3. Wang ZY, Tobin EM. Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and
8.
9.
e1143999-3
suppresses its own expression. Cell 1998; 93:1207-17; PMID:9657153; http://dx.doi.org/10.1016/S0092-8674(00)81464-6 Kumar SV1, Lucyshyn D, Jaeger KE, Al os E, Alvey E, Harberd NP, Wigge PA. Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 2012; 484:242-5; PMID:22437497; http:// dx.doi.org/10.1038/nature10928 Stokes ME, Chattopadhyay A, Wilkins O, Nambara E, Campbell MM. Interplay between sucrose and folate modulates auxin signaling in Arabidopsis. Plant Physiol 2013; 162:1552-65; PMID:23690535; http://dx. doi.org/10.1104/pp.113.215095 Koini MA, Alvey L, Allen T, Tilley CA, Harberd NP, Whitelam GC, Franklin KA. High temperature-mediated adaptations in plant architecture require the bHLH transcription factor PIF4. Curr Biol 2009; 19:40813; PMID:19249207; http://dx.doi.org/10.1016/j.cub.2009.01.046 Shin J, Anwer MU, Davis SJ. Phytochrome-interacting factors (PIFs) as bridges between environmental signals and the circadian clock: diurnal regulation of growth and development. Mol Plant 2013; 6:592-5; PMID:23589607; http://dx.doi.org/10.1093/mp/sst060 Tanaka S, Mochizuki N, Nagatani A. Expression of the AtGH3a gene, an Arabidopsis homologue of the soybean GH3 gene, is regulated by phytochrome B. Plant Cell Physiol 2002; 43:281-9; PMID:11917082; http://dx.doi.org/10.1093/pcp/pcf033 Kutschera U, Niklas KJ. The epidermal-growth-control theory of stem elongation: an old and a new perspective. J Plant Physiol 2007; 164:1395-409. PMID:17905474; http://dx.doi.org/10.1016/j.jplph.2007. 08.002
ARTICLE ADDENDUM
Importance of epidermal clocks for regulation of hypocotyl elongation through PIF4 and IAA29 Hanako Shimizu, Kotaro Torii, Takashi Araki, and Motomu Endo Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
ABSTRACT
ARTICLE HISTORY
Circadian clocks adjust an organism’s environmentally relevant physiological responses.. In plants, a decentralized circadian clock system has recently been proposed. Epidermal clock function is crucial for cell elongation; thus, epidermis-specific overexpression of CCA1 caused smaller cotyledons and longer hypocotyls under 27 C, concomitant with elevated night time levels of PIF4 mRNA. However, which tissue’s clock regulates PIF4 expression is still an open question. Here we tested spatial expression patterns of PIF4 and its downstream target IAA29 with or without epidermal clock perturbation. Using an epidermal-specific expression system, we revealed that epidermal clock perturbation increases PIF4 expression in both epidermis and mesophyll. However, IAA29 expression is mainly regulated in the epidermis, implying the potential importance of epidermis for regulation of cell elongation through PIF4 and IAA29. We conclude that the circadian clock in epidermis regulates cell elongation mainly in epidermis, and there is also another inter-tissue signaling pathway from epidermis to mesophyll.
Received 14 December 2015 Revised 4 January 2016 Accepted 14 January 2016
In multicellular organisms, time information created by cellular circadian clocks should be integrated to achieve cooperative reactions to fluctuating environmental conditions. The corresponding intra- and inter-tissue signaling networks through the circadian clock are complex, and it is an important challenge to elucidate how organisms are measuring time and using time information for their responses. Recently, the plant circadian clock network has been proposed to be decentralized,1,2 and cell elongation is mainly regulated by the epidermal clock in response to ambient temperature. Overexpression of a circadian clock gene, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) disrupts its rhythmic circadian oscillation.3 We have established several transgenic lines expressing CCA1 under tissuespecific promoters to achieve tissue-specific clock disruption. Among these lines, epidermal-clock perturbation by CCA1 driven by the 3-KETOACYL-COA SYNTHASE 6 promoter (CER6)::CCA1 showed longer hypocotyls and a smaller cotyledon area. Intriguingly, this phenotype is dependent upon ambient temperature and is well correlated with PHTYOCHROME INTERACTING FACTOR4 (PIF4) expression during the night. However, as PIF4 is expressed in all leaf tissues including epidermis, mesophyll, and vasculature,4 it was largely unknown whether multiple tissues would show PIF4 regulation by the epidermal clock, or how the epidermal clock is incorporated in the clock network. To identify the target tissues of the epidermal clock, we established transgenic plants expressing b-glucuronidase (GUS) under the PIF4 promoter (PIF4::GUS) in wild type and a CER6::CCA1 background. In the PIF4::GUS line, the blue-stained GUS signal was detected in cotyledons, hypocotyls and shoot apex (Figs. 1A, B). For further dissection CONTACT Motomu Endo © 2016 Taylor & Francis Group, LLC
[email protected]
KEYWORDS
Arabidopsis; circadian clock; decentralized clock; epidermis; hypocotyl; IAA29; PIF4; temperature
of the spatial PIF4::GUS expression pattern in cotyledons, we cross-sectioned cotyledons and observed the GUS expression pattern in each tissue (Figs. 1C, D). At variance with a previous report,4 PIF4::GUS expression was limited mainly to vasculature, although a weak signal was also detected in mesophyll and epidermis. Then which tissue’s expression of PIF4 is responsible for the cell elongation? To answer this question, we next observed a PIF4::GUS/CER6:: CCA1 line grown under the same condition. The PIF4::GUS expression level was dramatically increased by the exogenous CER6::CCA1, consistent with our view that the epidermal clock is crucial for adequate PIF4 expression. A crosssection of the cotyledon showed dense GUS expression in epidermal and mesophyll cells, implying the epidermal clock suppresses epidermal and mesophyll PIF4 expression. Interestingly, GUS staining levels in vasculature appeared to be the same, compared to the PIF4::GUS line. To support this observation, we analyzed PIF4 expression levels by qPCR using isolated epidermis, mesophyll, vasculature, and whole leaf (Fig. 1E). Consistent with the GUS staining result, higher PIF4 expression levels were observed in both epidermis and mesophyll of a CER6::CCA1 seedling compared to wild type. The transcription factor PIF4 has several direct target genes,5 and one of these is INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29), which is known to be involved in hypocotyl elongation. To test whether IAA29 expression is regulated in a similar way as PIF4 in leaves, we also observed IAA29::GUS expression in cotyledons. Unlike PIF4, only weak IAA29::GUS expression was observed in epidermis (Figs. 2A, B), suggesting that increased PIF4 in mesophyll cells did not directly lead to
e1143999-2
H. SHIMIZU ET AL.
Figure 1. Effect of epidermal clock perturbation by CER6::CCA1 on the spatial expression pattern of PIF4. (A-D) Expression of PIF4::GUS in 7-day-old wild-type (A, C) and CER6::CCA1 (B, D) seedlings. Whole seedlings (A, B) and cross-sections of cotyledons (C, D) are shown. Bars = 500 mm (A, B) and 50 mm (C, D). (E) Induction of PIF4 expression in isolated tissues by epidermal clock perturbation. Gene expression levels were detected by q-PCR and the highest value was set as 1. IPP2 and APA1 were used as internal control genes.1
up-regulation of IAA29. For further confirmation of this asymmetric regulation of PIF4 and IAA29 by epidermal clock perturbation, we observed PIF4::GUS and IAA29::GUS expression in a cross-sectioned hypocotyl (Figs. 2C, D, E, F). Both PIF4:: GUS and IAA29::GUS expression were limited to the vasculature in the hypocotyl, and this was also the case in a cotyledon. When the epidermal clock was perturbed by CER6::CCA1, PIF4::GUS expression was increased both in epidermis and mesophyll, whereas induction of IAA29::GUS expression was not observed. Together, these results demonstrate an essential role of the epidermal circadian clock for PIF4 and IAA29 expression in epidermal cells of the leaf. We also showed that upregulation of PIF4 in mesophyll did not lead to induction of IAA29 in mesophyll, even though IAA29 is a direct
target of PIF4. This asymmetric regulation of PIF4 and IAA29 by the epidermal clock strongly suggests that IAA29 is not the only regulatory target of PIF4, and the PIF4 signaling pathways appear to diverge at an early step in a specific tissue. We would like to point out the similarity of cell elongation mechanisms between epidermal clock disruption and shade avoidance response. Both suppress cotyledon expansion and increase hypocotyl elongation by regulating PIF4 expression in response to environmental changes. Our results further strengthen the previous view that PIF4 has a prominent role as a node of crosstalk between light and temperature signaling.6,7 We also revealed an inter-tissue regulation of the epidermal clock connected to mesophyll PIF4 expression. This epidermisto-mesophyll signaling reinforces our “decentralized circadian
Figure 2. PIF4 and IAA29 expression patterns in cotyledon and hypocotyl. Expression of IAA29::GUS and PIF4::GUS in 7-day-old wild-type (A, C, E) and CER6::CCA1 (B, D, F) cotyledon and hypocotyl. Weak IAA29::GUS induction was observed in epidermal cells of cotyledons (A, B) but not in hypocotyls (E, F); clear PIF4::GUS induction was observed even in hypocotyl cells (C, D). Bars = 100 mm (A, B) and 50 mm (C, D, E, F).
PLANT SIGNALING & BEHAVIOR
clock network” concept. In addition, we would like to note that induction of IAA29 expression was not observed in hypocotyl epidermal cells, implying that leaf epidermis has crucial roles for hypocotyl cell elongation. Similarly, far-red light irradiation on a cotyledon but not on a hypocotyl induces auxin-responsive gene expression in a hypocotyl. 8 Although this hypothesis is still consistent with the “epidermal-growth-control model”,9 it requires some further explanation in the future.
4.
5.
6.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. 7.
References 1. Endo M, Shimizu H, Nohales MA, Araki T, Kay SA. Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature 2014; 515:419-22; PMID:25363766; http://dx.doi.org/10.1038/nature13919 2. Shimizu H, Katayama K, Koto T, Torii K, Araki T, Endo M. Decentralized circadian clocks process thermal and photoperiodic cues in specific tissues. Nat Plants 2015; 1: Article number: 15163. 3. Wang ZY, Tobin EM. Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and
8.
9.
e1143999-3
suppresses its own expression. Cell 1998; 93:1207-17; PMID:9657153; http://dx.doi.org/10.1016/S0092-8674(00)81464-6 Kumar SV1, Lucyshyn D, Jaeger KE, Al os E, Alvey E, Harberd NP, Wigge PA. Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 2012; 484:242-5; PMID:22437497; http:// dx.doi.org/10.1038/nature10928 Stokes ME, Chattopadhyay A, Wilkins O, Nambara E, Campbell MM. Interplay between sucrose and folate modulates auxin signaling in Arabidopsis. Plant Physiol 2013; 162:1552-65; PMID:23690535; http://dx. doi.org/10.1104/pp.113.215095 Koini MA, Alvey L, Allen T, Tilley CA, Harberd NP, Whitelam GC, Franklin KA. High temperature-mediated adaptations in plant architecture require the bHLH transcription factor PIF4. Curr Biol 2009; 19:40813; PMID:19249207; http://dx.doi.org/10.1016/j.cub.2009.01.046 Shin J, Anwer MU, Davis SJ. Phytochrome-interacting factors (PIFs) as bridges between environmental signals and the circadian clock: diurnal regulation of growth and development. Mol Plant 2013; 6:592-5; PMID:23589607; http://dx.doi.org/10.1093/mp/sst060 Tanaka S, Mochizuki N, Nagatani A. Expression of the AtGH3a gene, an Arabidopsis homologue of the soybean GH3 gene, is regulated by phytochrome B. Plant Cell Physiol 2002; 43:281-9; PMID:11917082; http://dx.doi.org/10.1093/pcp/pcf033 Kutschera U, Niklas KJ. The epidermal-growth-control theory of stem elongation: an old and a new perspective. J Plant Physiol 2007; 164:1395-409. PMID:17905474; http://dx.doi.org/10.1016/j.jplph.2007. 08.002
