Low-fluence blue light-induced phosphorylation of Zmphot1 mediates the first positive phototropism.

Phototropin1 (phot1) perceives low- to high-fluence blue light stimuli and mediates both the first and second positive phototropisms. High-fluence blue light is known to induce autophosphorylation of phot1, leading to the second positive phototropism. However, the phosphorylation status of phot1 by low-fluence blue light that induces the first positive phototropism had not been observed. Here, we conducted a phosphoproteomic analysis of maize coleoptiles to investigate the fluence-dependent phosphorylation status of Zmphot1. High-fluence blue light induced phosphorylation of Zmphot1 at several sites. Notably, low-fluence blue light significantly increased the phosphorylation level of Ser291 in Zmphot1. Furthermore, Ser291-phosphorylated and Ser369Ser376-diphosphorylated peptides were found to be more abundant in the low-fluence blue light-irradiated sides than in the shaded sides of coleoptiles. The roles of these phosphorylation events in phototropism were explored by heterologous expression of ZmPHOT1 in the Arabidopsis thaliana phot1phot2 mutant. The first positive phototropism was restored in wild-type ZmPHOT1-expressing plants; however, plants expressing S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 showed significantly reduced complementation rates. All transgenic plants tested in this study exhibited a normal second positive phototropism. These findings provide the first indication that low-fluence blue light induces phosphorylation of Zmphot1 and that this induced phosphorylation is crucial for the first positive phototropism.

Introduction

Light is one of the main stimuli for plants, and plants have evolved several photoreceptors to perceive and respond to different wavelengths of light. Phototropin (phot) is a plant blue light receptor that mediates multiple blue light-dependent physiological responses (Briggs ; Christie ). Angiosperms generally have two phot isoforms, phot1 and phot2, which contain two light–oxygen–voltage-sensing (LOV) domains and a Ser/Thr protein kinase domain (Christie ). Phots were first identified as major blue light receptors associated with phototropism, which allows plants to orient their bodies to efficiently capture light (Khurana and Poff, 1989; Liscum and Briggs, 1995). The initial signaling step of the phototropic response involves the perception of blue light by phots and the subsequent asymmetric distribution of auxin [indole-3-acetic acid (IAA)], resulting in the differential growth of tissues and subsequent bending (Briggs, 2014; Liscum ; Fankhauser and Christie, 2015). Classical physiological studies, mainly on monocot coleoptiles, have identified two types of phototropic responses, the first and second positive phototropisms, which occur in response to low and high total fluences, respectively (Iino, 1988; Whippo and Hangarter, 2006; Briggs, 2014). The first positive phototropism induced by a pulse irradiation of blue light is a bell-shaped dose-dependent response depending on the total light fluence. This is followed by a transition period in which no visible curvature occurs. Then, the time-dependent second positive phototropism occurs in response to prolonged irradiation with blue light. These phototropisms have been reported not only in monocots, but also in dicots such as Arabidopsis thaliana (Konjevic ; Haga and Sakai, 2012), indicating that the underlying mechanisms associated with phototropism responses are conserved in angiosperms.

Phot1 mediates phototropic responses to low- to high-fluence blue light (the first and second positive phototropisms), while phot2 predominantly mediates the response to high-fluence blue light, the second positive phototropism (Sakai ). Light irradiation causes the activation of phot1 to induce its autophosphorylation. Several Ser and Thr residues have been identified as phosphorylation sites in the phot1s of A. thaliana, Avena sativa, and Zea mays. However, only the phosphorylation sites in phot1 under high-fluence blue light, which induces the second positive phototropism, have been detected in vivo (Salomon ; Inoue ; Sullivan ; Boex-Fontvieille ; Deng ; Christie ; Walley ). To date, light-activated phosphorylation of phot1 has not been detected in vivo at the low fluence level that induces the first positive phototropism (Christie and Murphy, 2013; Briggs, 2014). The threshold and saturation fluence required for the first positive phototropism have been reported to be one order of magnitude lower than that required to induce detectable phosphorylation of phot1. For example, phosphorylation of membrane-associated proteins, including Zmphot1, has been observed in maize coleoptiles irradiated with blue light at ~10 µmol m−2, which corresponds to one order higher fluence than that inducing the first positive phototropism of maize coleoptiles (Palmer ). The asymmetric phosphorylation of membrane-associated proteins (e.g. Asphot1) in oat coleoptiles has been detected in vivo, also with two orders higher fluence than that which induces the first positive phototropism (Salomon ). The reason for the apparent discrepancy between the fluences required for first positive phototropism and phosphorylation of phot1 is still unknown (Christie and Murphy, 2013; Briggs, 2014). In previous studies, the phosphorylation of phot1 induced by low-fluence blue light was consistent with the Bunsen–Roscoe law of reciprocity correlating with the first positive phototropism, and the action spectrum for the phosphorylation reaction was the same as that for the first positive phototropism. These results suggested that there is a strong correlation between the low blue light-induced phosphorylation of phot1 and the first positive phototropism (Christie and Murphy, 2013; Briggs, 2014). In this context, studies on the phosphorylation status of phot1 after low-fluence blue light irradiation that induces the first positive phototropism are warranted.

In a series of earlier studies, we revealed that the region within the top 2 mm of the maize coleoptile is the site of the IAA biosynthesis (Mori ; Nishimura ), the perception site of blue light for the first positive phototropism, and the region in which IAA becomes asymmetrically distributed in response to unilateral low-fluence blue light irradiation (Matsuda ). The finding that ZmPHOT1 is highly expressed in coleoptiles further supported that its encoded protein is involved in the perception of blue light for the first positive phototropism in coleoptiles (Suzuki ). Atphot1, an ortholog of Zmphot1, is considered to act as the main photoreceptor for the first positive phototropism in A. thaliana (Sakai ), suggesting that Zmphot1 is also involved in the first positive phototropism in maize. Together, these findings suggested that the signal transduction pathway of the first positive phototropism of maize coleoptiles might be mediated by a series of Zmphot1-dependent protein phosphorylation events in the top 2 mm of the coleoptile. However, no phosphorylation of Zmphot1 in the maize coleoptile tip (0–3 mm) was detected by comparing the mobilities of Zmphot1 with or without low-fluence blue light irradiation in electrophoretic mobility shift assays (Suzuki ), and no phosphorylation of Zmphot1 (114 kDa protein) in the coleoptile tips was detected in an in vivo phosphorylation assay using [γ-32P]ATP after low-fluence blue light irradiation (Palmer ). The methods used in those studies may not have been sensitive enough to detect very low levels of phosphorylated proteins.

In this study, to determine the phosphorylation status of Zmphot1 under the first positive phototropic conditions by a highly sensitive procedure, we conducted a phosphoproteomic analysis by LC-MS/MS combined with selected reaction monitoring (SRM) using extracts from the top 3 mm portion of maize coleoptiles. Low-fluence blue light (LBL; 0.33 µmol m−2 s−1 for 8 s) or high-fluence blue light (HBL; 10 µmol m−2 s−1 for 10 min) was applied unilaterally to maize coleoptiles as the first and second positive phototropic conditions, respectively. The LBL treatment significantly increased the phosphorylation level of Ser291 in Zmphot1. In addition, Ser291-phosphorylated and Ser369Ser376-diphosphorylated peptides were found to be abundant in the LBL-irradiated side than in the shaded sides of coleoptiles. These phosphorylation sites are located in the hinge region between the LOV1 and LOV2 domains of Zmphot1. Wild-type (WT) and mutated [Ser(s) substituted with Ala] ZmPHOT1 genes were introduced into the A. thaliana phot1phot2 double mutant to evaluate their complementation effects. These results provide the first indication that LBL, which induces the first positive phototropism, leads to the phosphorylation of Ser residues in Zmphot1, and that this LBL-induced phosphorylation is essential for the first positive phototropism.

Materials and methods

Plant materials and growth conditions

Seeds of Zea mays L. cv. ‘Golden Cross Bantam 70’ (Sakata Seed Co., Kanagawa, Japan) were rinsed in running tap water at 25 °C for 16–20 h, and then germinated on moistened paper towels at 25 °C for 2.5 d under red light (2 µmol m−2 s−1) as previously described (Suzuki ). Seeds of A. thaliana (ecotype Columbia) were sown in soil (Professional soil No. 2; Daio Chemicals Co., Tokyo, Japan) or on 0.8% agar (Wako Pure Chemical Industries, Osaka, Japan). The seeds were incubated at 4 °C for 3–5 d and then at 22 °C under a 16 h white light (80 µmol m−2 s−1):8 h dark photoperiod.

Sample preparation for phosphoproteomic analysis

Maize coleoptiles of 2.5-day-old seedlings were irradiated unilaterally with blue light (ISL-150X150-BB LED; CCS, Kyoto, Japan) for 8 s at 0.33 µmol m−2 s−1 (2.64 µmol m−2) (LBL) or for 10 min at 10 µmol m−2 s−1 (HBL) (Supplementary Fig. S1 at JXB online). The fluence was measured with a LI190SA quantum photometer (Li-Cor, Lincoln, NE, USA) and recorded with a LI-1000 data logger (Li-Cor). During treatments, the coleoptiles were incubated under red light (2 µmol m−2 s−1). The top 3 mm of the coleoptile tips was collected under the same red light conditions and immediately frozen in liquid nitrogen with or without cutting vertically into the irradiated half and the shaded half. The coleoptiles were incubated in 20% (w/v) trichloroacetic acid (TCA) on ice for 30 min and centrifuged at 1100 g at 0 °C for 10 min to remove the TCA solution. After washing with a 10% (w/v) TCA solution and centrifuging at 1100 g at 0 °C for 10 min to remove the TCA solution, the coleoptile samples were homogenized with 8 M urea in 0.1 M Tris–HCl (pH 8.5). The pH of the extracts was adjusted to 8 with 1.5 M Tris–HCl. The crude extracts (~500 µg total protein) were diluted four times with water and digested with lysyl endopeptidase (Wako Pure Chemical Industries) at 37 °C for 18 h. The extracts were further digested with trypsin (TRTPCK; Worthington Biochemical Corporation, Lakewood, NJ, USA) at 37 °C for 18 h. The digested phosphopeptides were enriched using the Titansphere Phos-TiO Kit (GL Sciences, Tokyo, Japan) according to the manufacturer’s protocol.

Determination of received fluence at irradiated and shaded sides of coleoptiles

The blue light fluences of LBL were measured as the received fluence on the irradiated side of maize coleoptiles. To detect fluence on the shaded side of coleoptiles, 2.5-day-old maize coleoptiles were cut in half vertically, and one half was placed over the quantum photometer. The blue light fluence transmitted through the halved coleoptile was measured with a LI190SA quantum photometer (Li-Cor) and a LI-1000 data logger (Li-Cor).

Phosphoproteomic analysis by LC-MS

The phosphopeptides were analyzed using a direct nanoflow LC system (Taoka ) coupled with a Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). For global proteomic analysis, the mass spectrometer was operated in data-dependent tandem MS (MS/MS) mode with the top 10 parent ions selected from each survey scan. The survey scans were acquired using an orbitrap mass analyzer over an m/z range of 300–1500 with a resolution of 35 000. The target value was 1.0×106. The 10 most intense peaks with a charge state of 2–4 were fragmented in the HCD collision cell with 25% normalized collision energy. The tandem mass spectrum was acquired with the orbitrap mass analyzer at a resolution of 17 500 (at m/z 200). The ion selection threshold was 8.3×103 counts, while the maximum allowed ion accumulation times were 60 ms for full MS scans and 60 ms for MS/MS scans. The dynamic exclusion duration was set at 20 s.

For SRM, MS analysis was conducted in target-MS/MS mode. The SRM event employed an orbitrap resolution of 17 500 (at m/z 200), a target automatic gain control value of 2×105, and a maximum injection time of 120 ms. The precursor ion of each targeted peptide (Supplementary Files) was isolated using a 3 m/z unit window. Samples were fragmented with 25% normalized collision energy.

The MS/MS data were processed with the MASCOT algorithm (version 2.3.02; Matrix Science Ltd, London, UK) to assign peptides using the Zea_mays.AGPv3.27.pep.all_2015_06_.fasta sequence database with the following parameters: variable modification parameters, phosphorylation (Ser, Thr, and Tyr) and oxidation (Met); maximum missed cleavage, 3; peptide mass tolerance, ±15 ppm; peptide charges, +2 to +4; and MS/MS tolerance, ±0.05 Da. The MS/MS signal assignments were manually confirmed. All peptide search results were extracted using STEM software (Shinkawa ). The MS data, including peak heights, were processed with Xcalibur software (Thermo Fisher Scientific).

Vector construction and transformation of A. thaliana

The ZmPHOT1 coding sequence (GRMZM2G001457) was amplified by PCR using gene-specific primers (Supplementary Table S2), PrimeSTAR GXL DNA polymerase (Takara Bio, Inc., Shiga, Japan), and cDNA synthesized from total RNA extracted from maize coleoptiles as previously described (Suzuki ). The amplicon was cloned into the pDONR/Zeo vector (Thermo Fisher Scientific) according to the BP reaction described in the manufacturer’s protocol to generate the pDONR/Zeo vector harboring WT-ZmPHOT1. The WT-ZmPHOT1 sequence was altered by PCR with specific primers (Supplementary Table S2) to introduce amino acid substitutions into the encoded protein, and the remaining template WT-ZmPHOT1 was digested with DpnI (Toyobo, Osaka, Japan), according to the manufacturer’s recommended protocol. After confirming the accuracy of the sequences, each pDONR vector was recombined with the pGWB502 binary vector (Nakagawa ) by the LR reaction described in the manufacturer’s protocol (Thermo Fisher Scientific) to obtain the Cauliflower mosaic virus (CaMV) 35S promoter::WT-ZmPHOT1 or mt-ZmPHOT1 (i.e. mutated ZmPHOT1) constructs. These vectors were introduced into Agrobacterium tumefaciens GV3101 (pMP90) cells (Koncz and Schell, 1986). An A. thaliana phot1phot2 double mutant line obtained by crossing the phot1 (Salk_146058) and phot2 (Salk_142275) single mutants (Alonso ; Zhang ) was transformed with each construct using the floral-dip method (Clough and Bent, 1998). Transgenic A. thaliana seeds were collected from T3 or T4 plants and used in subsequent experiments.

Measurement of phototropic responses of A. thaliana seedlings

The phototropic stimulation was applied using a slightly modified version of the method described by Haga and Sakai (2012). The A. thaliana seeds were placed on 1.5% (w/v) agar medium containing half-strength Murashige and Skoog salts (Wako Pure Chemical Industries) and then incubated at 4 °C for 3–5 d in the dark. The seeds were exposed to red light at 10 µmol m−2 s−1 for 3–4 h to induce germination and then incubated at 22 °C in the dark. Two-day-old etiolated seedlings were irradiated with red light at 10 µmol m−2 s−1 for 4 min and then incubated for 2 h in the dark. The seedlings were subsequently unilaterally irradiated with blue light (ISL-150X150-BB LED) at 0.017 µmol m−2 s−1 for 1 min (log0) and then for 3 h in the dark to induce the maximum first positive phototropism. The fluence response of pulse-induced phototropism was investigated under a range of blue light fluences from log−3 to log3 (0.000017 µmol m−2 s−1 for 1 min to 17 µmol m−2 s−1 for 1 min). To induce the second positive phototropism, seedlings were unilaterally irradiated with blue light at 0.17 µmol m−2 s−1 for 3 h. All seedlings were photographed using an a6000 digital camera (Sony, Tokyo, Japan) equipped with a SEL30M35 micro lens (Sony). Phototropic curvature was measured with Lenaraf220b.xls (Vector Japan, Tokyo, Japan).

Results

Identification of blue light-induced protein phosphorylation in maize coleoptiles

We conducted global phosphoproteomic analyses of maize coleoptiles irradiated with LBL or HBL to clarify the corresponding proteomic changes. Maize seedlings were unilaterally irradiated with LBL (0.33 µmol m−2 s−1 for 8 s; 2.64 µmol m−2) or HBL (10 µmol m−2 s−1 for 10 min) as the first and second positive phototropic conditions, and the top 3 mm portion of coleoptiles was collected for phosphoproteomic analysis (Supplementary Fig. S1A).

First, we compared the signal intensities derived from phosphoproteins between the non-irradiated (control) and LBL-irradiated samples. A total of 896 and 845 proteins were identified in the control and LBL-irradiated samples, respectively. However, there were no significant differences in the number of identified peptides between the control and LBL-irradiated samples (Supplementary Table S3A). Secondly, we compared the identified proteins between the control and HBL-irradiated samples (1154 and 1245 proteins, respectively). The peptides derived from Zmphot1 (GRMZM2G001457) and polyubiquitins (GRMZM2G409726, GRMZM2G419891, and GRMZM2G118637) were detected with >10 peptides and at 10-fold higher levels in HBL-irradiated samples than in the control (Supplementary Table S3B). All seven Zmphot1-derived peptides were phosphorylated (Supplementary Table S3C). In contrast, peptides derived from polyubiquitins were non-phosphorylated, although phosphopeptides were enriched before the LC-MS/MS analyses. These non-phosphorylated peptides might have been detected due to an abundance of polyubiquitins in HBL-irradiated coleoptiles. Analyses of the Zmphot1-derived peptides revealed five phosphorylation sites in Zmphot1 (S265, S291, S369, S376, and S753) (Fig. 1). Four of these phosphorylation sites (S265, S291, S369, and S376) were located in the linker region between the LOV1 and LOV2 domains, while the other site (S753) was located in the activation loop of the kinase domain (Fig. 1). This is the first report of S265 and S753 as phosphorylation sites in Zmphot1. Although the phosphorylated S291, S369, and S376 sites have been detected in maize vegetative and reproductive tissues previously (Walley ), the function of these phosphorylation sites and the effect of light on their phosphorylation status was unknown. Because Zmphot1 presumably works in the first positive phototropism of maize coleoptiles (Suzuki ), LBL may also increase the phosphorylation level of Zmphot1. It is possible that the LBL-induced Zmphot1 phosphorylation level was lower than the detection limit of the global phosphoproteomic analysis used here. Therefore, we conducted additional analyses to investigate the effects of LBL irradiation on phosphorylation of these five Ser residues in Zmphot1.

Fig. 1.

Identified phosphorylation sites in Zmphot1. Identified phosphopeptides derived from Zmphot1 are shown. Phosphorylated Ser residues identified in this study are indicated in bold font. (This figure is available in color at JXB online.)

Low-fluence blue light increased the phosphorylation level of Zmphot1

To determine which amino acid residues in the Zmphot1 polypeptide are specifically or preferentially phosphorylated by LBL and/or HBL treatments of maize coleoptiles, an SRM assay involving five Zmphot1 derived-phosphorylated peptides was conducted by LC-MS/MS (Supplementary Table S1; Supplementary Fig. S1B). The SRM analysis was completed in triplicate or quadruplicate for each peptide using maize coleoptiles treated with LBL/HBL irradiation or untreated controls. Three monovalent ions from each phosphopeptide were selected and used to compare relative intensities (Supplementary Table S1; Supplementary Fig. S2). Interestingly, phosphorylation at S291 in Zmphot1 was significantly induced by LBL irradiation and further induced by HBL irradiation (Fig. 2A). The relative amount of monophosphorylated (S369) E363-R382 peptide decreased in HBL-treated coleoptiles (Fig. 2B), while that of the diphosphorylated (S369 and S376) peptide increased considerably in response to HBL (Fig. 2C). We did not detect any S376-monophosphorylated peptide. These results suggested that S369 in Zmphot1 is phosphorylated regardless of the light stimuli, whereas phosphorylation of S376 depends on light stimuli. In addition, the possibility could not be excluded that dephosphorylation of S369-monophosphorylated peptide is induced by blue light irradiation. Phosphorylation of S265 and S753 was induced only by HBL irradiation (Fig. 2D, E). Previously, we reported that the amount of Zmphot1 protein in coleoptiles does not change after irradiation with LBL or HBL (Suzuki ), and the same amount of total proteins from all samples was subjected to the SRM assay in the present study. Therefore, these findings indicated that the phosphorylation level of Zmphot1 was increased by LBL and HBL irradiation, but the abundance of Zmphot1 did not change.

Fig. 2.

Quantification of selected reaction monitoring of phosphopeptides from Zmphot1. (A–E) Monitored peptides are indicated at the top of each panel. Signal intensities from high-fluence blue light (HBL: 10 µmol m−2 s−1 for 10 min) were set to 100. Signal intensities for control samples (No: no irradiation) and samples irradiated with low-fluence blue light (LBL: 0.33 µmol m−2 s−1 for 8 s) are shown relative to signal intensity for HBL-irradiated samples. Data are from three or four biological replicates. Error bars indicate SEs. Asterisks indicate significant differences between control and blue light-treated samples (Student’s t-test; **P<0.01; ***P<0.001). Inset in (A), magnified relative intensity for control and LBL-irradiated samples.

The unilateral irradiation of oat coleoptiles by relatively LBL (30 µmol m−2), but still higher than that required to induce the first positive phototropism, reportedly leads to the asymmetric phosphorylation of membrane-associated proteins (e.g. Asphot1) between the irradiated and shaded sides of coleoptiles (Salomon ). Therefore, we speculated that Zmphot1 is also asymmetrically phosphorylated in maize coleoptiles unilaterally irradiated with LBL. To assess this possibility, we first measured the light fluence on the irradiated and shaded sides of maize coleoptiles to monitor the asymmetric fluence situation in LBL-irradiated coleoptiles. The fluence on the shaded side was approximately half of that on the irradiated side (Fig. 3A). Next, we conducted the SRM assay for Zmphot1 phosphopeptides in the irradiated and shaded sides of maize coleoptiles irradiated with LBL. The S291-phosphorylated and S369S376-diphosphorylated peptides were relatively more abundant in the irradiated half of coleoptiles than in the shaded half (Fig. 3B, C). The abundance of S291-phosphorylated peptide in the irradiated side was greater than that of S369S376-diphosphorylated peptide. These results implied that the asymmetric distribution of phosphorylated Zmphot1 in coleoptiles is induced by unilateral LBL irradiation.

Fig. 3.

Differences in received fluences (A) and phosphorylation of S291 and S376 in Zmphot1 (B and C) between irradiated and shaded sides of LBL-irradiated maize coleoptiles. (A) Fluence at irradiated and shaded sides of LBL-irradiated maize coleoptiles. Data are from three independent measurements. Error bars indicate SEs. Asterisks indicate significant difference between irradiated and shaded sides (Student’s t-test; P<0.001). (B, C) Selected reaction monitoring of phosphopeptides obtained from the irradiated or shaded half of LBL-irradiated maize coleoptiles. Signal intensities of S291-K303 (B) and E363-R382 (C) Zmphot1 peptides from the irradiated or shaded half of LBL-treated coleoptiles are shown relative to signal intensity of HBL-irradiated samples. The signal intensities of HBL-irradiated samples were set as 100. Data are from three or four biological replicates; error bars indicate SEs. Phosphopeptides were more abundant on the irradiated half than on the shaded half of coleoptiles [Student’s t-test; P=0.25 for (B), P=0.05 for (C)].

Involvement of S291, S369, and S376 phosphorylation in Zmphot1 in the first positive phototropism

Because LBL affected the phosphorylation levels of S291, S369, and S376 in Zmphot1 (Figs 2, 3B, C), we hypothesized that these phosphorylation sites are important for the LBL-induced first positive phototropism. To analyze the functional roles of the LBL-affected phosphorylation of Zmphot1, we introduced WT-ZmPHOT1 or mt-ZmPHOT1 constructs into the A. thaliana phot1phot2 double mutant, which has defective in blue light-dependent phototropism (Zhang ). In the mt-ZmPHOT1 constructs, three LBL-affected phosphorylation sites, S291, S369, and S376, and two non-phosphorylation sites as negative controls, S282 and S294, were substituted for Ala residues (see Table 1). Three independent T3 or T4 lines of WT-ZmPHOT1-expressing plants (WT10-1, 13-1-1, and 16-8) and two or three independent T3 lines of each mt-ZmPHOT1 (S282A7-1 and 11-3; S291A2-7, 8-3, and 10-3; S294A12-3 and 18-2; S369AS376A2-8, 7-1, and 11-3; S369A11-2 and 15-6; and S376A1-1 and 16-2) were selected based on their mRNA expression levels (Supplementary Fig. S3). In the study, transgenic lines with a high, moderate, and/or low mRNA expression level were selected to monitor the possible expression level-dependent phenotype. However, for transgenic lines expressing S294A-ZmPHOT1, two independent lines with moderate expression were selected, since a line with a high mRNA expression level was not detected among the produced transgenic lines. Most of the selected transgenic lines showed moderate or high expression levels of Zmphot1 proteins in 2-day-old etiolated seedlings (Supplementary Fig. S4), suggesting that these substitutions did not affect the stability of Zmphot1 in the cells of transgenic A. thaliana. Slight or little expression of Zmphot1 was detected in four out of 17 selected lines (S291A8-3, S369AS376A11-3, S369A15-6, and S376A16-2). In transgenic plants expressing S291A-, S282A-, S294A-, or S396A-Zmphot1, protein expression levels were mostly proportional to expression levels of mRNA. Such a proportional relationship between protein and mRNA expression levels was not detected in transgenic plants expressing WT-, S369AS376A-, or S376A-Zmphot1, although we cannot explain why these discrepancies arose in these transgenic plants. Several previous studies have shown that heterologously expressed phot1 or photoreceptors in transgenic A. thaliana could restore phototropic responses, even when their expression levels were lower than the detection limit of western blot analysis (Christie ; Doi ; Kanegae ; Kanegae and Kimura, 2015). Thus, in the present study, we used all 17 transgenic lines for subsequent experiments to monitor the functional roles of WT- and mt-Zmphot1s in phototropic responses to LBL.

Table 1.

List of transgenic Arabidopsis thaliana expressing various Zmphot1 proteins

Name of transgenic plant line	Expressed Zmphot1	Partial amino acid sequences of expressed Zmphot1 (282–294 and 363–379)
WT	Wild-type Zmphot1	282       294  363         379 SRNNTLKRKSQES...EDPLLDSDDERPDSFDD
S282A	Substitution of non- phosphorylation site	ARNNTLKRKSQES...EDPLLDSDDERPDSFDD
S291A	Substitution of LBL-induced phosphorylation site	SRNNTLKRKAQES...EDPLLDSDDERPDSFDD
S294A	Substitution of non- phosphorylation site	SRNNTLKRKSQEA...EDPLLDSDDERPDSFDD
S369AS376A	Dual substitutions of highly phosphorylated sites in LBL-irradiated side of coleoptiles	SRNNTLKRKSQES...EDPLLDADDERPDAFDD
S369A	Substitution of highly phosphorylated site in LBL-irradiated side of coleoptiles	SRNNTLKRKSQES…EDPLLDADDERPDSFDD
S376A	Substitution of highly phosphorylated site in LBL-irradiated side of coleoptiles	SRNNTLKRKSQES...EDPLLDSDDERPDAFDD

Substituted amino acids (alanine) are shown in bold.

In etiolated A. thaliana seedlings, a pulse irradiation of blue light reportedly induces the first positive phototropism with a curvature of almost 40° (Haga and Sakai, 2012). Using blue light with the same fluence, we investigated the first positive phototropism of the etiolated hypocotyl of transgenic A. thaliana. The phototropic curvatures of the WT and the phot1phot2 double mutant of A. thaliana were 37° and 2°, respectively (Fig. 4). The impaired phototropic curvature of the phot1phot2 double mutant was recovered to ~31–25° when WT-ZmPHOT1 was expressed under the control of the 35S promoter. However, heterologous expression of S291A-ZmPHOT1 in the phot1phot2 double mutant did not recover phototropic curvature in three independent transgenic lines, S291A2-7, 8-3, and 10-3, with curvature degrees of 13, 6, and 8°, respectively (Fig. 4). Importantly, the complementation effect was not observed even when the S291A-Zmphot1 protein was highly expressed in the phot1phot2 double mutant (line S291A2-7 in Fig. 4 and Supplementary Fig. S4A). When S369AS376A-ZmPHOT1 was expressed in the double mutant, the phototropic curvature was restored by approximately half, to 16° and 21° curvature in the S369AS376A2-8 and S369AS376A7-1 lines, respectively, although one of the three transgenic lines, S369AS376A11-3, exhibited only a slight complementation effect with phototropic curvature of 6° (Fig. 4). This difference might be explained by the expression levels of heterologously expressed S369AS376A-Zmphot1, since S369AS376A2-8 and S369AS376A7-1 seedlings expressing S369AS376A-Zmphot1 at high levels showed greater recovery of curvature than did S369AS376A11-3, which barely expressed heterologous Zmphot1 (Supplementary Fig. S4A). S369A-ZmPHOT1 or S376A-ZmPHOT1 with a single mutation showed reduced complementation effects compared with that of WT-ZmPHOT1 (Supplementary Fig. S5A), and the reduction of phototropic curvatures of S369A-ZmPHOT1 and S376A-ZmPHOT1 was similar to that of the S369AS376A-ZmPHOT1 double mutations (Fig. 4; Supplementary Fig. S5A), indicating that both phosphorylation sites are involved in the first positive phototropic curvature. We also examined the effects of substituting non-phosphorylated Ser residues, S282 or S294, located near S291 in Zmphot1 as negative controls. The phototropic curvature of transgenic A. thaliana seedlings was recovered by the expression of S282A-ZmPHOT1 or S294A-ZmPHOT1 (Fig. 4). Taken together, the results of these complementation assays indicated that mutations in S291, S369, and S376 in Zmphot1 reduced recovery of the first positive phototropism in the phot1phot2 double mutant, and that these phosphorylation sites in Zmphot1 are related to the first positive phototropism.

Fig. 4.

Effects of heterologous expression of WT-ZmPHOT1 and mt-ZmPHOT1 constructs on the first positive phototropism in the Arabidopsis thaliana phot1phot2 double mutant. (A) Dark-grown seedlings were irradiated with unilateral blue light at 0.017 µmol m−2 s−1 for 60 s and then incubated in darkness for 3 h. Data are mean ±SE (17–75 seedlings). Different letters on bars indicate significant differences according to Tukey’s test (P<0.05). (B) Representative images of the first positive phototropism.

Differences in maximum fluence level between Atphot1 and Zmphot1 for first positive phototropism

It has been estimated that the fluence levels required to induce maximum phototropic curvatures differ between Atphot1 and Zmphot1 because A. thaliana seedlings and maize coleoptiles showed maximum phototropic curvature at a fluence of approximately log0 and log1, respectively (Iino, 1988; Haga and Sakai, 2012). Therefore, the fluence responses of pulse-induced phototropism of WT A. thaliana seedlings expressing endogenous Atphot1 and transgenic phot1phot2 A. thaliana seedlings heterologously expressing ZmPHOT1 were investigated in a range of blue light fluences from log−3 to log3 (Fig. 5). Both WT and transgenic A, thaliana seedlings showed bell-shaped dose-dependent curvature responses. Notably, WT A. thaliana seedlings and transgenic seedlings heterologously expressing WT-Zmphot1 showed maximum responses at log0 and log1, respectively, consistent with the maximum responses of WT A. thaliana seedlings and maize coleoptiles at these log values. Next, we investigated the fluence response of pulse-induced phototropism of transgenic phot1phot2 A. thaliana seedlings heterologously expressing S291A-ZmPHOT1, because S291 was significantly phosphorylated by LBL irradiation which induces the first positive phototropism of maize coleoptiles (Fig. 2A). Compared with WT-ZmPHOT1-expressing A. thaliana, seedlings of the S291A-ZmPHOT1-expressing lines 2-7 and 8-3 showed reduced phototropic curvature by ~60% and 30% at a fluence of log1 WT10-1. These results supported that S291 in Zmphot1 is one of the key amino acids which is phosphorylated by LBL and functions in the first positive phototropism.

Fig. 5.

The first positive phototropic curvature of WT-ZmPHOT1- or ZmPHOT1S291A-expressing transgenic Arabidopsis thaliana in response to various fluences of blue light. Two-day-old dark-grown seedlings were unilaterally irradiated with blue light at fluences of log−3 to log3 for 1 min. Black line, wild-type A. thaliana, Colombia-0; gray solid line, phot1phot2 double mutant of A. thaliana heterologously expressing WT-ZmPHOT1(10-1); gray dotted lines, phot1phot2 double mutant of A. thaliana heterologously expressing ZmPHOT1S291A(2-7) and ZmPHOT1S291A(8-3). Error bars indicate SEs. n=17–123.

Non-involvement of S291A, S369A, and S376A phosphorylation in Zmphot1 in the second positive phototropism

Next, we investigated the second positive phototropism induced by continuous blue light irradiation. The phototropic curvature of WT A. thaliana seedlings was almost 80°, while the phot1phot2 double mutant did not exhibit any phototropism (Fig. 6). The impaired second positive phototropism was fully recovered in transgenic seedlings expressing WT-ZmPHOT1. In addition, transgenic seedlings expressing S291A- or S369AS376A-ZmPHOT1 also exhibited restored second positive phototropism. Although S369AS376A11-3 showed reduced complementation of the first positive phototropism (Fig. 4), the second positive phototropism was completely restored (Fig. 6). S369A- or S376A-ZmPHOT1-expressing A. thaliana displayed the same complementation effects (Supplementary Fig. S5B). We confirmed similar second positive phototropic responses in transgenic plants expressing non-phosphorylated Ser substituted with Ala, S282A-ZmPHOT1 or S294A-ZmPHOT1 (Fig. 6). Thus, the phosphorylation of S291, S369, and S376 in Zmphot1 was not essential for the second positive phototropism in etiolated transgenic A. thaliana seedlings.

Fig. 6.

Effects of heterologous expression of WT-ZmPHOT1 and mt-ZmPHOT1 constructs on the second positive phototropism in the Arabidopsis thaliana phot1phot2 double mutant. (A) Dark-grown seedlings were irradiated with unilateral blue light at 0.17 µmol m−2 s−1 for 3 h. Data are mean ±SE (16–51 seedlings). Different letters indicate significant differences (Tukey’s test, P<0.05). (B) Representative images of the second positive phototropism.

Discussion

Protein phosphorylation is an important post-translational modification that regulates protein activities and interactions with other cellular components by inducing conformational changes. Quantitative phosphoproteomic research has evolved with the development of improved enrichment procedures as well as advances in relevant instruments, strategies, and software (Rigbolt and Blagoev, 2012). Recently, 6227 phosphoproteins were identified via phosphoproteomic, transcriptomic, and proteomic analyses using 23 tissues spanning maize vegetative and reproductive stages (Walley ). In the present study, we conducted a global phosphoproteomic analysis and identified phosphoproteins in maize coleoptiles irradiated with LBL and HBL as well as in non-irradiated coleoptiles (Supplementary Table S3). These phosphoproteomic data will be useful for characterizing the effects of protein phosphorylation as it relates to maize development and physiological responses to environmental stimuli, including light-responsive signaling pathways.

Focusing on the phosphorylation status of Zmphot1 depending on the blue light irradiation, we identified five phosphorylation sites in Zmphot1 from maize coleoptiles exposed to HBL (10 µmol m−2 s−1 for 10 min) (Fig. 1; Supplementary Table S3C). Our results were consistent with those of a recent study in which HBL irradiation (20 µmol m−2 s−1 for 20 min) altered the phosphorylation level of Zmphot1 at several sites (Deng ). However, the changes in Zmphot1 phosphorylation profiles in response to LBL (0.33 µmol m−2 s−1 for 8 s), which induces the first positive phototropism of maize coleoptiles, had not been characterized previously (Palmer ; Christie and Murphy, 2013; Briggs, 2014). Although phot1 is necessary for the first positive phototropism (Sakai ), the discrepancy between required fluences for phosphorylation of phot1 and the first positive phototropism had remained unresolved, despite several studies (Christie and Murphy, 2013; Briggs, 2014). In the present study, we used the SRM assay, a more sensitive procedure than those used in previous studies, and observed for the first time that LBL treatment of maize coleoptiles significantly induced phosphorylation of S291 in Zmphot1 (Fig. 2). Thus, it is likely that the discrepancy had come from insufficient sensitivity in the phosphorylation measurement. The elevated phosphorylation level of S291 was consistent with the results of an earlier study of S300 in Asphot1a with lower fluence blue light in vitro (Salomon ) (Fig. 7). In addition, asymmetric distributions of S291-phosphorylated and S369S376-diphosphorylated Zmphot1 were observed in coleoptiles after unilateral LBL irradiation (Fig. 3B, C). Compared with the heterologous expression of WT-ZmPHOT1, S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 did not rescue the deficient first positive phototropism of the A. thaliana phot1phot2 double mutant (Figs 4, 5). These results indicated that LBL-induced phosphorylation of Zmphot1 is critically important for the first positive phototropism.

Fig. 7.

Comparison of deduced amino acid sequences of Zmphot1, Asphot1a, Asphot1b, Osphot1, and Atphot1 with identified phosphorylation sites. Orange boxes, LOV1 and LOV2 domains; green box, protein kinase domain. Phosphorylation sites identified in previous reports, Asphot1a (Salomon ), Atphot1 (Inoue ; Sullivan ; Boex-Fontvieille ; Deng ), and Zmphot1 (Walley ), and this study are highlighted with several colors according to the fluence of irradiated light. Light blue, low-fluence blue light; dark blue, high-fluence blue light; light gray, no information available; dark gray, no irradiation or darkness. Numbers on residues are phosphorylated Ser detected in this study. Phosphorylation sites in Asphot1 were determined by in vitro phosphorylation assays. Sites in other plants were detected in vivo by MS.

LBL-induced phosphorylation of Zmphot1 appeared to be important for the first positive phototropism, but not the second positive phototropism, since heterologous expression of S291A-ZmPHOT1 or S369AS376A-ZmPHOT1 in the phot1phot2 double mutant rescued the defective second positive phototropism (Fig. 6). The first positive phototropism is induced by a pulse irradiation of blue light, while the second positive phototropism is induced by continuous light irradiation, namely HBL. It is likely that the phosphorylation profiles and status of Zmphot1 differ between the HBL-induced second positive phototropism and the LBL-induced first positive phototropism, with additional phosphorylation events during the second positive phototropism affecting the function of Zmphot1. In fact, HBL irradiation increased the phosphorylation of S265 and S753 as well as S291 and S376 (Figs 1, 2, 7), implying that the fluence-dependent phosphorylation of phot1 is associated with diverse biochemical functions in different physiological responses (Christie ).

Exposure to light reportedly leads to conformational changes in phots (Christie ). In the dark, phot kinase activity is inhibited by the LOV2 domain, whereas light irradiation causes the LOV2 and kinase domains to separate, resulting in increased kinase activities (Matsuoka and Tokutomi, 2005; Okajima ). Light-induced conformational changes at the N- and C-termini of the LOV2 domain may enable phots to undergo structural changes (Aihara ; Kashojiya ; Oide ). In addition, the LOV1 domain has also been suggested to contribute to the blue light-dependent conformational changes (Oide ), and to act as an attenuator of the activation of the kinase by LOV2 (Matsuoka and Tokutomi, 2005; Oide ). Although those structural studies were conducted with HBL, the LBL-induced phosphorylation of Zmphot1 described herein may contribute to structural changes in Zmphot1. Because S291, S369, and S376 are located in the hinge region between the LOV1 and LOV2 domains of Zmphot1 (Figs 1, 7), it is possible that the LBL-induced increases in phosphorylation levels in the hinge region alter the three-dimensional position of the LOV domains relative to the kinase domain, potentially leading to increased kinase activity. Several phosphorylation sites have been detected in the hinge region of phot1 proteins of A. thaliana (Inoue ; Sullivan ; Boex-Fontvieille ; Deng ), A. sativa (Salomon ), and Z. mays (Walley ) (Fig. 7). However, the importance of these phosphorylation sites for phot-mediated responses has not been verified, except that the phosphorylation of S350, S375, and S410 in the hinge region of Atphot1 has been shown to be essential for the binding of 14-3-3 proteins but not for physiological responses (Inoue ; Sullivan ; Christie ). Notably, our results showed that the phosphorylation of S291, S369, and S376 in the hinge region of Zmphot1 is important for the first positive phototropism of transgenic A. thaliana (Fig. 4). In addition, our analyses revealed that the corresponding Ser residues S291 and S376 are conserved in phot1s of A. sativa (S300 and S388), Oryza sativa (S300 and S388), and A. thaliana (S362 and S450) (Fig. 7). It is interesting that S450 in Atphot1 was reported to be phosphorylated after higher fluence blue light irradiation than our HBL at 20 μmol m−2 for 60 min (Deng ). Although LBL-induced phosphorylation of Atphot1 has not been investigated, it is possible that Atphot1 could be phosphorylated by lower fluence blue light. In addition, an in vitro phosphorylation assay which requires additional irradiation using oat coleoptiles irradiated with blue light in vivo showed that S274 and S300 in the hinge region of Asphot1a are phosphorylated by lower fluence blue light as well as S27 and S30 at the N-terminus (Salomon ) (Fig. 7). Studies on phosphorylation in the hinge region of phot1s in response to LBL will provide new insights into the role(s) of this protein in LBL perception.

The importance of higher fluence blue light (100 μmol m−2 s−1 for 1 min) to induce phosphorylation of S849 and S851 in the activation loop of the kinase domain of Atphot1 has been demonstrated (Inoue ). The Ser residues corresponding to S849 and S851 in the kinase domain of Atphot1 were found to be conserved in all phot1s aligned in Fig. 7. Although the phosphorylation of these Ser residues in Zmphot1 was not detected either in this study or elsewhere (Walley ), it is possible that the irradiation of maize coleoptiles with higher fluence light than that of our HBL conditions could induce further phosphorylation of Zmphot1. In this study, we identified HBL-induced phosphorylation of S753 in the activation loop of the kinase domain of Zmphot1 (Figs 1, 2, 7). This phosphorylation event may play a role in regulating Zmphot1 activity, but further studies are needed to elucidate the role of S753 phosphorylation.

It has been hypothesized that the asymmetric distribution of phot1 phosphorylation levels between the irradiated and shaded sides of plant shoots functions as signal perception of the light stimulus (Christie and Murphy, 2013). Our data indicated that the light intensity on the irradiated side of coleoptiles was double that on the shaded side when the coleoptiles were unilaterally irradiated with LBL. The S291- and S369S376-diphosphorylated peptides were more abundant in the LBL-irradiated side of maize coleoptiles than in the shaded side (Fig. 3). These findings implied that unilateral irradiation of coleoptiles with LBL results in asymmetric distribution of S291- and S369S376-diphosphorylated Zmphot1 in maize tissue just after irradiation. In our previous studies, we detected lateral movement of IAA in the coleoptile top region between 10 min and 20 min after unilateral LBL irradiation. In addition, the asymmetric IAA distribution was more pronounced ~30 min after unilateral LBL irradiation, with IAA contents ~1.6-fold higher in the irradiated half than in the shaded half of coleoptiles (Matsuda ). The correlation between Zmphot1 phosphorylation and IAA abundance strongly suggested that the LBL-induced lateral distribution of Zmphot1 phosphorylation across the coleoptile leads to the lateral distribution of the IAA. Additional research is required to elucidate the signal transduction pathway linking the light perception by Zmphot1 to the asymmetric distribution of IAA in maize coleoptiles and the subsequent phototropic bending.

Supplementary data

Supplementary data are available at JXB online.

Table S1. Target peptide sequences derived from Zmphot1 for selected reaction monitoring.

Table S2. Details of primers used in this study.

Table S3. List of identified phosphopeptides in control maize coleoptiles or coleoptiles irradiated with low-fluence blue light (LBL) or high-fluence blue light (HBL).

Fig. S1. Schematic documentation of experimental designs for global phosphoproteomic analyses (A) and selected reaction monitoring assay for Zmphot1-derived phosphopeptides (B).

Fig. S2. Example of selected reaction monitoring analysis.

Fig. S3. Expression of WT-ZmPHOT1 or mt-ZmPHOT1 constructs in transgenic Arabidopsis thaliana.

Fig. S4. Expression of Zmphot1 proteins in 2-day-old etiolated transgenic Arabidopsis thaliana seedlings.

Fig. S5. Effect of heterologous expression of ZmPHOT1S369A or ZmPHOT1S376A on the first and second positive phototropisms in the Arabidopsis thaliana phot1phot2 double mutant.

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