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First published online February 5, 2008; 10.1105/tpc.107.052670 The Plant Cell 20:353-366 (2008) © 2008 American Society of Plant Biologists An Inositol Polyphosphate 5-Phosphatase Functions in PHOTOTROPIN1 Signaling in Arabidopis by Altering Cytosolic Ca2+[W]
a Shanghai Institutes for Biological Science–University of California Berkeley Center of Molecular Life Sciences, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 20032 Shanghai, China 2 Address correspondence to hwxue{at}sibs.ac.cn.
Inositol polyphosphate 5-phosphatase (5PTase) is a key enzyme in the phosphatidylinositol metabolic pathway, which plays critical roles in a number of cellular processes in plants. Our previous work implicated the role of 5PTase13, which encodes a WD40-containing type II 5PTase, in hormone-mediated cotyledon vein development. Here, we show that 5PTase13 is also involved in blue light responses in Arabidopsis thaliana. Compared with that in darkness, the expression of 5PTase13 was suppressed by blue light irradiation, and disruption of the gene resulted in shortened hypocotyls and expanded cotyledons. Genetic analysis showed that 5PTase13 acted independently from CRYPTOCHROME1 and CONSTITUTIVE PHOTOMORPHOGENIC1 but interacted functionally with PHOTOTROPIN1 (PHOT1). The expression level of 5PTase13 was significantly enhanced in phot1 single or phot1 phot2 double mutants under blue light, and suppression of 5PTase13 expression rescued the elongated hypocotyls in the phot1 or phot1 phot2 mutants. Further analysis showed that the blue light–induced elevation of cytosolic Ca2+ was inhibited in the phot1 mutant but enhanced in the 5pt13 mutant, suggesting that 5PTase13 antagonizes PHOT1-mediated effects on calcium signaling under blue light.
Plants have evolved a number of mechanisms for responding to a broad spectrum of light through activation of specific sets of photoreceptors (Franklin et al., 2005  ; Wang, 2005). Recent studies have characterized the phytochrome family of red/far-red light receptors (Schepens et al., 2004; Kim et al., 2005; Rockwell et al., 2006), the cryptochrome (CRY) family of blue light receptors (Lin and Todo, 2005; Li and Yang, 2007), and the phototropin (PHOT) family of blue light receptors (Kimura and Kagawa, 2006).
Arabidopsis thaliana mutants cry1 and cry2 exhibit elongated hypocotyls, and the CRY1 and CRY2 gene products have been identified as blue light receptors that mediate light-dependent inhibition of hypocotyl elongation. Studies have also shown that CRY1 is negatively regulated by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1; a photomorphoregenesis repressor) (Ang et al., 1998
The phototropin proteins carry two domains: a C terminus–localized Ser/Thr kinase domain and an N terminus–localized LOV domain (Kasahara et al., 2002
Calcium, a universal second messenger, is involved in diverse cellular functions and is crucial in cell responses to a variety of stimuli (Luan et al., 2002
Phosphatidylinositols play crucial roles in multiple developmental processes, including hormone effects (Lin et al., 2004
Through genetics approaches, several 5PTases, especially type I members, have been functionally characterized in Arabidopsis, revealing their diverse functions in various aspects of plant growth and development. Disruption of 5PTase1 increased sensitivity to abscisic acid treatment (Berdy et al., 2001
Blue Light Suppresses 5PTase13 Expression and 5pt13 Mutants Display Shortened Hypocotyls and Expanded Cotyledons in Response to Blue Light Irradiation Our previous studies have shown that 5PTase13 expression is differentially regulated under light and dark (Lin et al., 2005 ). To further investigate the effects of different light spectra on 5PTase13 gene expression, we examined 5PTase13 transcripts under different light spectra through quantitative RT-PCR analysis. As shown in Figure 1A
, expression of 5PTase13 was higher in dark-grown seedlings compared with those grown under light. Histochemical analysis of transgenic plants harboring the 5PTase13 promoter-β-glucuronidase (GUS) reporter gene confirmed that expression of 5PTase13 in hypocotyls was inhibited by light at the level of transcription (see Supplemental Figure 1 online).
We further analyzed seedling growth of a 5PTase13 knockout mutant, 5pt13, under various light conditions and observed a small but significant change in blue light–induced hypocotyl shortening in mutant compared with wild-type seedlings. Although the 5pt13 seedlings showed shorter hypocotyls under both dark and light conditions, the hypocotyl shortening phenotype of the mutant was most significant under blue light (Figure 1B). In addition, 5pt13 mutant cotyledons were smaller than the wild type under red light but larger under blue light (Figure 1C, left and middle panels). Under white light or far-red light, there were no significant differences between the mutant and wild type. These results suggest that the 5pt13 mutant may have altered light responses, especially with regard to blue light. Overexpression of 5PTase13 in 5pt13 plants complemented the mutant phenotype under blue light (Figure 1D, left panel; see Supplemental Figure 2A online), indicating that the changes in blue light responses in the mutant were a result of disruption in the 5PTase13 gene. In addition, transgenic wild-type plants that overexpressed 5PTase13 displayed longer hypocotyls under blue light (see Supplemental Figure 2B online; Figure 1D, right panel), further confirming the effects of 5PTase13 on hypocotyl elongation under blue light. More detailed analysis of hypocotyl lengths revealed no significant difference between the wild type and the 5pt13 mutant under white light at various fluence rates (5, 10, 20, and 60 µmol/m2/s; Figure 1E, left panel). The 5pt13 mutant seedlings had significantly shorter hypocotyls under blue light at the fluence rates of 8 or 20 µmol/m2/s but not at lower or higher fluence rates (Figure 1E, right panel). In addition, enlarged cotyledons were observed under blue light at different fluence rates (1, 8, or 20 µmol/m2/s; Figure 1C, right panel). Taken together, these results suggest that 5PTase13 participates in blue light–mediated regulation of seedling growth under a specific range of blue light fluence rates.
Consistent with the above observations, analysis of transcription profiling revealed altered expression of many light-regulated genes in 5pt13 mutant seedlings compared with those in wild-type seedlings under white light (chip hybridization was described in Lin et al., 2005
5PTase13 Acts Independently from CRY1-COP1 but Interacts Functionally with PHOT1 Disruption of 5PTase13 led to changes in blue light responses, including a change in hypocotyl growth. As a blue light receptor, CRY1 plays a key role in the regulation of hypocotyl elongation. Interestingly, COP1, an E3 ligase, regulates blue light response by interacting directly with CRY1. In addition, both COP1 and 5PTase13 contain WD40 repeat domains, which are involved in protein–protein interaction. These observations prompted us to test if there might be functional interactions between 5PTase13 and the other two proteins. We tested for protein–protein interactions using a yeast two-hybrid system and found that 5PTase13 did not interact with either CRY1 or COP1 (Figure 2A ). Further genetic analysis of a 5pt13 cry1 double mutant showed that, except for a small proportion (3 to 5%) of seedlings that exhibited long hypocotyls (cry1 mutants) or shortened hypocotyls (5pt13 mutants), the majority (>90%) of seedlings had hypocotyls of an intermediate length (Figure 2B), indicating functional independence of CRY1 and 5PTase13. Additionally, the 5pt13 cop1 double mutant had a similar phenotype to cop1 (Figure 2C), suggesting that COP1 either acts downstream of 5PTase13 or they have no functional relationship.
In addition to CRY1 and COP1, other photoreceptors, such as PHOT1 and PHOT2, have been identified as functioning in blue light responses (Briggs and Christie, 2002 ; Briggs, 2007; Cho et al., 2007). PHOT1 is involved in blue light–mediated hypocotyl growth inhibition through stimulating an increase in [Ca2+]cyt (Folta et al., 2003). Because the expression level of 5PTase13 is regulated by blue light, we decided to measure 5PTase13 expression in the phot1 and phot2 single mutants and the phot1 phot2 double mutant to determine if these blue light receptors are involved in the regulation of 5PTase13 expression. As shown in Figure 3A
, under blue light, the transcript level of 5PTase13 in the phot1 or phot1 phot2 double mutants was much higher than that in the wild-type plants, increasing 25- and 12-fold, respectively, whereas no significant change was detected in the phot2 mutant (Figure 3A, left). This result indicates that PHOT1 may be the major blue light receptor mediating blue light–induced repression of 5PTase13 gene expression.
Further studies employing genetic epistatic analysis showed that under blue light, phot1 5pt13 double mutant plants exhibited shortened hypocotyls similar to 5pt13 (Figure 3B), suggesting that 5PTase13 may act downstream of PHOT1 and serve as a negative regulator in blue light signaling. In addition, phot1 single mutant or phot1 phot2 double mutant seedlings with reduced levels of 5PTase13 (due to transgenic expression of antisense RNA) exhibited shortened hypocotyls under blue light (Figures 3C and 3D, right panels), similar to the phot1 5pt13 double mutant seedlings. By contrast, overexpressing 5PTase13 in phot1 seedlings enhanced the long hypocotyl phenotype of the phot1 mutant under blue light (Figure 4A ), further supporting the hypothesis that 5PTase13 functions downstream of PHOT1 as a negative regulator of the blue light response. Similar transgenic analysis with phot2 showed no affect on the hypocotyl length of phot2 (Figure 4B), indicating there may not be a functional link between PHOT2 and 5PTase13. Additionally, the yeast two-hybrid assay revealed no physical interaction between PHOT1 and 5PTase13 (see Supplemental Figure 3 online).
Deficiency of 5PTase13 Results in Increased [Ca2+]cyt We hypothesize that inositol phosphate metabolism may provide a common link between 5PTase function and calcium fluctuations. It is generally believed that 5PTase13 functions in calcium signaling processes by regulating the level of Ins(1,4,5)P3 and/or Ins(1,3,4,5)P4, the two potent calcium-mobilizing molecules (Zhong et al., 2004 ; see Supplemental Figure 4 online). Indeed, our experiments indicated that Ins(1,4,5)P3 and calcium levels appeared to be increased in the 5pt13 seedlings (Figure 5A
; see Supplemental Figure 5 online).
To determine a possible connection between calcium levels and blue light responses in the 5pt13 mutant, we tested the effects of exogenous Ca2+ and its specific chelator EGTA on hypocotyl elongation. Whereas exogenous Ca2+ (10 to 20 mM) did not affect the hypocotyl lengths of the wild type or 5pt13 under blue light, this treatment resulted in shortened hypocotyls of phot1, phot2, and phot1 phot2 mutants (see Supplemental Figure 6 online). The increased [Ca2+]cyt in the 5pt13 mutant resulted in an altered responses to EGTA. In the hypocotyl assay, exogenous EGTA at low concentrations (100 µM) induced hypocotyl elongation in wild-type plants under blue light, especially under a higher fluence rate of blue light (35% longer, 20 µmol/m2/s), and suppressed the elongation when higher concentrations (3 mM EGTA) were used (Figure 5B, top panel). However, 5pt13 mutant hypocotyls displayed reduced sensitivity to EGTA, and hypocotyl length was minimally affected by high concentrations of EGTA (Figure 5B, bottom panel). Further analysis showed that in the presence of a high concentration of EGTA (500 µM), the hypocotyl length of 5pt13 was also significantly increased under white light (a 1.5-fold increase; Figure 6A ) and blue light (a 1.6-fold increase; Figure 6E) but not under red light, far-red light, or dark (Figures 6B to 6D). This result indicates that blue light and white light normally inhibit hypocotyl growth through increased [Ca2+]cyt; the addition of EGTA under these light conditions reduced calcium elevation, thereby promoting hypocotyl elongation. The observation that the 5pt13 mutant was less sensitive to a high concentration of EGTA (>2 mM) is consistent with the finding that 5PTase13 deficiency leads to an increased calcium elevation in the mutant.
To further test this idea, we also included the phot1 and phot2 mutants in the EGTA hypocotyl assay. Elongation of hypocotyls in the phot1 and phot2 single mutants and phot1 phot2 double mutants, compared with that of 5pt13, was not significantly altered by addition of exogenous EGTA (Figure 6E), further supporting the notion that increased [Ca2+]cyt of 5pt13 may be a critical factor influencing the response of hypocotyls to blue light.
Previous studies have shown that RPT2 transduces signals downstream of PHOT1 and is involved in the phototropic response and stomatal opening (Sakai et al., 2000
5PTase13 Regulates Blue Light Response by Increasing Ins(1,4,5)P3 and [Ca2+]cyt
In addition, seedlings overexpressing 5PTase13 appear to have reduced [Ca2+]cyt under blue light (Figure 7B). These results indicate that 5PTase13 deficiency results in higher levels and longer durations of [Ca2+]cyt elevation upon blue light irradiation, whereas phot1 seedlings have lower levels and a shorter duration of [Ca2+]cyt elevation relative to the wild type. We thus propose that 5PTase13 is involved in the blue light–stimulated increase in [Ca2+]cyt and functions downstream of PHOT1 in blue light signaling (Figure 7C).
Phosphatidylinositols regulate calcium fluctuations and other cellular functions. The 5PTases dephosphorylate Ins(1,4,5)P3, Ins(1,3,4,5)P4, or PtdIns(4,5)P2, and often serve as terminators for calcium signaling. In this study, we show that Arabidopsis 5PTase13 plays a role in the regulation of blue light responses. In particular, the expression of 5PTase13 was suppressed by blue light, whereas a deficiency of 5PTase13 resulted in a hypersensitive response to blue light in the hypocotyl assay. Disruption of 5PTase13 rescued the hypocotyl phenotype in the phot1 mutant but not in the cry1 mutant, indicating that 5PTase13 functions downstream of PHOT1 in blue light signaling. The 5pt13 mutant displayed enhanced [Ca2+]cyt, whereas phot1 exhibited reduced [Ca2+]cyt in response to blue light irradiation, leading to a model that PHOT1 suppresses 5PTase13 activity, thereby controlling calcium levels, which in turn regulate hypocotyl elongation.
Our study indicates that PHOT1 mediates blue light suppression of 5PTase13, as this suppression was not evident in the phot2 mutant. A previous study using microarray analysis (Ohgishi et al., 2004
Much effort has been focused on the identification of signaling components downstream of blue light receptors and upstream of phenotypic effects, such as inhibition of hypocotyl growth. Several studies have shown that blue light induces elevation of [Ca2+]cyt, and mutations in genes encoding blue light receptors, such as PHOT1 or PHOT2, have compromised calcium elevation, leading to changes in photomorphogenesis (Babourina et al., 2002 How does PHOT1 regulate the activity of 5PTase13? Our study provides evidence that 5PTase13 is regulated by blue light through PHOT1, at least in part, at the transcriptional level. Our quantitative RT-PCR analysis indicated that 5PTase13 mRNA levels were downregulated by blue light. The finding that the 5PTase13 promoter-GUS reporter negatively responds to blue light confirmed the notion that regulation occurred at the level of transcription. However, we cannot exclude the possibility that 5PTase13 also might be regulated by PHOT1 through other mechanisms, such as direct modification of enzyme activity. The protein–protein interaction by yeast two-hybrid assay did not detect a physical interaction between PHOT1 and 5PTase13 (see Supplemental Figure 3B online); however, the kinase–substrate interaction was often weak and transient, and further studies are needed to determine if 5PTase13 is in fact a substrate of PHOT1.
Blue light suppression of 5PTase13 reveals a double-negative step in blue light signaling, and such negative-negative regulation steps appear to be a common feature of plant signal transduction. For example, similar double-negative steps are found in ethylene signal transduction where ethylene receptors negatively regulate the downstream CONSTITUTIVE TRIPLE RESPONSE1 kinase, which in turn inhibits ethylene responses (Stepanova and Alonso, 2005
Several major questions remain to be addressed, including how 5PTase13 regulates calcium, and what the function of calcium is in cell elongation. While little information is available regarding calcium action in cell growth upon blue light irradiation, previous studies have built a connection between 5PTase13 and calcium fluctuations in the cell. At least several inositol phosphates molecules, especially Ins(1,4,5)P3 and Ins(1,3,4,5)P4, have been shown to mediate calcium fluxes across cell membranes. Such transport processes can include release of calcium from intracellular stores or influxes from the apoplast through inward calcium channels. Intracellular calcium is stored in the vacuole, which harbors ligand-gated calcium channels or Ins(1,4,5)P3 receptors. High levels of Ins(1,4,5)P3 activate these calcium channels and then release calcium to the cytosol. The phosphate group at the 5' position of the inositol ring is thought to be important for ligand specificity of calcium channels, and dephosphorylation at the 5' position by 5PTases could reduce the level of active ligands, leading to the closure of calcium channels and termination of the signaling process. Therefore, the 5PTases function as terminators for calcium signaling, which might be initiated by various signals, including blue light, as demonstrated here. In this context, earlier studies have identified both plasma membrane channels and intracellular calcium stores that are involved in blue light signaling (Harada et al., 2003
5PTase13 expression was similarly suppressed by both blue and red light compared with darkness. However, 5pt13 mutants exhibited shortened hypocotyls and larger cotyledons under blue light but smaller cotyledons under red light (Figure 1C). The phytochrome A–specific signaling intermediate SPA1 (Hoecker and Quail, 2001
Plant Growth and Light Conditions Arabidopsis thaliana (Columbia ecotype) seeds were surface sterilized with 20% bleach for 15 min and washed four times with sterile water and planted aseptically on agar medium containing Murashige and Skoog (1962) salts and 2% sucrose (w/v). In some cases, the medium was supplemented with 25 µg/mL of hygromycin, 15 µg/mL of basta, or 25 µg/mL of kanamycin to screen the resistant transgenic plants or homozygous mutants. After 48 h of vernalization, seedlings were grown at 22°C with a 16-h-white light/8-h-dark cycle, followed by continued growth under the same conditions or transfer to blue light, red light- or far-red light (continuous light) with different fluence rates for 5 d. Exogenous EGTA or calcium treatment was performed by growing the seedlings on MS medium supplemented with different concentrations of EGTA or calcium chloride for 5 d.
All experiments involving blue light, red light, or far-red light illumination were performed in an E-30 LED chamber (Percival) using the blue diodes ( Hypocotyl length and cotyledon area were measured using the E-ruler software and statistically analyzed. Photos were taken by an SMZ 800 stereoscope (Nikon) equipped with a Nikon digital Coolpix 995 camera. Statistical analyses were performed using Excel tools as described in the figure legends. All experiments were performed in triplicate (n > 30).
Mutant Confirmation and Plant Crosses
GUS Histochemical Staining
Yeast Two-Hybrid Analysis The DNA fragment encoding N-terminal and WD40 repeats of 5PTase13 was amplified by primers 5pt13-14 (5'-CGCGGATCCCGATGGATTCGCTAATTATC-3'; BamHI site underlined) and 5pt13-15 (5'-ACGCGTCGACGGGTCCTGGAGATGTCAC-3'; SalI site underlined) and that encoding the 5PTase13 catalytic domain was amplified by primers 5pt13-16 (5'-GGACAACATAATCCGAACGGA-3') and 5pt13-17 (5'-ACGCGTCGACTCTCGAGTGTCTTCGCACC-3'; SalI site underlined). Resulting fragments were cloned into the pGBKT7 vector and confirmed by restriction enzyme digestion and DNA sequencing. The DNA fragment encoding the LOV domain of PHOT1 was amplified by primers PHOT1-l1 (5'-CGGAATTCATGGAACCAACAGAAAAACCA-3'; EcoRI site underlined) and PHOT1-l2 (5'-CGGGATCCTGGTGTCATGTTGGCATCAG-3'; BamHI site underlined) and that encoding the PHOT1 kinase domain was amplified by primers PHOT1-k1 (5'-CGGAATTCGAGGATTTATGGGCAAACCA-3'; EcoRI site underlined) and PHOT1-k2 (5'-CGGGATCCAACATTTGTTTGCAGATCTTCT-3'; BamHI site underlined). Resulting fragments were cloned into the pGADT7 vector and confirmed by restriction enzyme digestion and DNA sequencing. All combinations of prey and bait constructs were cotransformed into the yeast strain AH109 through the small-scale LiAc yeast transformation method (Clontech).
Interaction analysis, including growth marker-based selection and measurement of the β-galactosidase activity, were essentially performed as described previously (McNellis et al., 1996
Coimmunoprecipitation Analysis
Constructs and Plant Transformation
RT-PCR and Quantitative Real-Time PCR Analysis
Quantitative real-time RT-PCR analysis was performed with the RotorGene 3000 (Corbett Research) using a SYBR green detection protocol (SYBR Premix Ex Taq System; TaKaRa). The product amounts were determined by the method of comparative
Measurement of [Ca2+]cyt
Fluorescence imaging of [Ca2+]cyt was performed according to Legue et al. (1997)
Measurement of Ins(1,4,5)P3 Content
Expression, Purification, and Biochemical Characterization of Recombinant 5PTase13
Enzymatic analyses were performed by incubation of purified protein (1.5 µg) at 37°C overnight in solutions containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 2 mM MgCl2, supplemented with either 20 mM Ins(1,4,5)P3 and 0.02 µCi [3H]Ins(1,4,5)P3 (10 mM), or 20 mM Ins(1,3,4,5)P4 and 0.016 µCi [3H]Ins(1,3,4,5)P4 (10 mM). The reactions were terminated by heating at 95°C for 5 min (Sanchez and Chua, 2001
Microarray Hybridization and Analysis
Accession Numbers
Supplemental Data
This work was supported by the National Natural Science Foundation of China (Grants 30425029 and 30421001), by the Shanghai municipal government (Grant 055407072), and by a grant from the National Science Foundation (to S.L.). We thank Hong-Quan Yang (Institute of Plant Physiology and Ecology, Chinese Academy of Sciences) for providing seeds of phot1, phot2, phot1 phot2, cry1, and cop1. We also thank Alex A.R. Webb and Mark Tester (University of Cambridge, Cambridge, UK) for the gifts of Wassilewskija ecotype and Columbia ecotype harboring CaMV35S:aequorin and Jian-Ben Gu (Institute of Plant Physiology and Ecology, Chinese Academy of Sciences) for the luminometer measurement of [Ca2+]cyt.
1 These authors contributed equally to this work.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Hong-Wei Xue (hwxue{at}sibs.ac.cn).
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.107.052670 Received May 5, 2007; Revision received November 22, 2007. accepted January 21, 2008.
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ABSTRACT
INTRODUCTION
45 s) compared with wild-type seedlings (
max = 469 nm), red diodes (
-first-strand synthesis kit; Toyobo), and PCR amplification was performed by specific primers 5pt13-7 (5'-TCCGAACGGAATTGTCTCAG-3') and 5pt13-8 (5'-GCAACATCAAGATCTCCAACA-3'). The Arabidopsis ACTIN8 gene, amplified by primers actin-1 (5'-GATGCTGATGACATTCAACCT-3') and actin-2 (5'-GAAGTGAGAAACCCTCATAG-3'), was used as a positive internal control. 
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