- Email: [email protected]
Biological function analysis of the phosphorylation sites for Arabidopsis CAP1
Accepted Manuscript Short Communications Biological function analysis of the phosphorylation sites for Arabidopsis CAP1 Yun Zhou, Lu Zhang, Zhangyun Wu, Mengmeng Dai, Luying Li, Ling Bai, Chunpeng Song PII: DOI: Reference:
S2095-9273(17)30260-8 http://dx.doi.org/10.1016/j.scib.2017.05.017 SCIB 144
To appear in:
Science Bulletin
Received Date: Revised Date: Accepted Date:
27 February 2017 28 April 2017 8 May 2017
Please cite this article as: Y. Zhou, L. Zhang, Z. Wu, M. Dai, L. Li, L. Bai, C. Song, Biological function analysis of the phosphorylation sites for Arabidopsis CAP1, Science Bulletin (2017), doi: http://dx.doi.org/10.1016/j.scib. 2017.05.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short communication Title: Biological function analysis of the phosphorylation sites for Arabidopsis CAP1
Yun Zhou, Lu Zhang, Zhangyun Wu, Mengmeng Dai, Luying Li, Ling Bai, Chunpeng Song
Institute of Plant Stress Biology, State Key Laboratory of Cotton Biology, Department of Biology, Henan University, Kaifeng 475001, China
Correspondence to: [email protected]
Received: 2/27/17 Revised: 4/28/2017 Accepted: 5/8/2017
1
Cells need to respond successfully to ever-changing environmental conditions to maintain normal growth. This is achieved through various signal transduction cascades. Receptor-like kinases (RLKs) are involved in many aspects of the growth and development of plants. More than 600 RLKs have been identified and that are involved in various biological processes in Arabidopsis thaliana [1]. An RLK consists of an extracellular domain (the main function of which is to perceive general elicitors), a transmembrane domain, and an intracellular kinase domain that activates a signal transduction cascade mainly through serine/threonine phosphorylation. More and more attention is being paid to the abilities of RLKs to initiate downstream signal transduction. We have previously described a receptor-like kinase, [Ca2+]cyt-associated protein kinase 1 (CAP1/ERULUS), that regulates the growth of root hair tips by influencing the concentration gradients of cytoplasmic Ca2+ and reactive oxygen species in root hair cells. Deficiency of CAP1 causes mutant plants to have very short root hairs, and the inhibition of root hair growth can only be remedied by depleting ammonium from the growth medium [2]. However, the mechanism of CAP1 signal transduction has not yet been identified. CAP1 protein is a member of the Catharanthus roseus RLK1-like (CrRLK1L). The Arabidopsis thaliana CrRLK1L family has 17 members, eight of which have been characterized in detail. These are FERONIA (FER), ANXUR1 (ANX1), ANXUR2 (ANX2), HERCULES1 (HERK1), HERCULES2 (HERK2), CAP1/ERULUS, THESEUS1 (THE1), and CURVY1 (CVY1) [3]. Each CrRLK1L protein has a similar N-terminus, which contains a malectin-like domain, and a kinase domain at the C-terminus (CT) [3]. Recent experimental data have shown that CrRLK1Ls participate in different processes of growth and development, mainly by regulating cell elongation [3–5]. Similar downstream signal molecules, namely Ca2+ and reactive oxygen species, have been found to be involved in the processes required for the regulation of cell expansion by these genes [3,4,6,7]. Efforts have been made to identify the signaling events that depend on these RLKs, but it is still very difficult to uncover the detail transduction cascades involved. Only the ligand for FER has been identified, and found to be a secreted peptide called RALF1 (rapid alkalinization factor 1). The phosphorylation of FER by RALF initiates a signaling cascade that inhibits cell growth [8]. Phosphorylation is a type of protein post-translational modification that plays an 2
important role in RLKs signal transduction. Moreover, it will be helpful in understanding the functions of RLKs by identifying its actual phosphorylation site(s). Phosphopeptides in the kinase domain of the RLKs play vital roles in triggering the signal cascade, and, in at least five members of the CrRLK1L family (FER, THESEUS1, HERCULES1, At4g39110, and At2g21480), two adjacent serine (S) and threonine (T) residues in the kinase domain could be phosphorylated [5]. Conserved adjacent S and T residues, S688 and T689 (S688T689), are also present in CAP1. However, it has not yet been determined whether these are phosphorylation sites, and their biological functions are not understood [5]. Given the strong evolutionary conservation of, and similarities between, the kinase domains in members of this subfamily, it would be interesting to assess the importance of the phosphorylation of these two residues to the CAP1-modulated polar growth of root hair cells. From structural predictions
of
the
kinase
region
of
CAP1
(http://www.proteinmodelportal.org/query/uniprot/Q9FLJ8), we hypothesized that S688T689 in CAP1 might be responsible for the kinase activity of CAP1 (Figure 1a). We then investigated the biological function of their phosphorylation by analyzing site-directed mutants in vitro and in vivo. Mutational analysis of the putative phosphosites of CAP1 might help understanding the mechanism through which CAP1 regulates root hair growth to be identified. Mutating both S688 and T689 into alanine (A) or aspartic acid (D) altered these amino acids to forms that mimicked their constitutively inactive and active phosphorylated types, respectively. We then measured the autophosphorylation and substrate phosphorylation activities of expressed Glutathione S-transferase (GST)-fusion proteins in vitro and performed complementation assays in vivo to determine the importance of the phosphorylation of the two residues. First, the autophosphorylation activities of the mutated proteins were studied by expressing fusion proteins in bacteria. The CT-CAP1-GST construct that we had described previously [2], and which consisted of the coding sequence for GST fused with that of the CT of CAP1 (1549–2529 bp), was subjected to site-directed mutagenesis to generate the mutants described
above.
Plasmid
CT-CAP1-S688AT689A-GST
and
plasmid
CT-CAP1-S688DT689D-GST were constructed using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The primers used to produce 3
plasmid
CT-CAP1-S688AT689A-GST
5'-GAAGGGCATGTAGCTGCCGCGGTAAAGGGT-3'
were and
5'-
ACCCTTTACCGCGGCAGCTACATGCCCTTC-3', and the primers used to produce plasmid CT-CAP1-S688DT689D-GST were 5'-GAAGGGCATGTAGATGACGCGGTAAAGGGT-3' and 5'-ACCCTTTACCGCGTCATCTACATGCCCTTC-3'. Western blotting analysis of the expressed proteins using an anti-GST antibody (Sangon Biotech, Shanghai, China) indicated that we had produced the three desired fusion proteins (Fig. 1b left panel). In vitro assays indicated that the CT-CAP1-GST protein and the S688AT689A and S688DT689D mutant proteins underwent autophosphorylation similarly, all showing the same band shift due to phosphorylation (Fig. 1b middle panel). MEP2 (methylammonium permease 2) has been used as a substrate for CAP1 in the phosphorylation assay [2]. C-terminal tail of MEP2 has been expressed, kinase assay on MEP2 showed that all the tested protein can phosphorylate MEP2 (Figure 1b right panel). Both the mutations of S688AT689A and S688DT689D can not influence the autophosphorylation and the substrate phosphorylation of CAP1 proteins, suggesting that there are other sites also function in phosphorylation activity, except these two sites. These two residues being analyzed may be not essential for the maintenance of CAP1 phosphorylation activity, indicating that other phosphosites might be involved in the phosphorylation-triggered signal transduction by CAP1. To exclude misleading results that might be caused by the production of truncated expressed protein or the use of a heterologous expression system, we then transferred the complete cDNA of CAP1 with the site-directed mutations into cap1 mutant plants by floral dip method to obtain transgenic plants. The CAP1 overexpression construct that we had described previously [2] was used to generate two constructs that carried the desired site-directed mutations (CAP1-S688AT689A ox and CAP1-S688DT689D ox). Given that the cap1 mutant displays short root hairs, we investigated the root hair phenotype of the transgenic plants. Interestingly, even though in vitro phosphorylation was not influenced by the different mutations, the different site-directed mutations resulted in different phenotypes in the T3 transgenic seedlings in terms of root hair growth (Fig. 1c). Mutating the CAP1-S688T689 residues to their inactive form CAP1-S688AT689A did not rescue root hair growth in cap1, but the active form CAP1-S688DT689D resulted in root hairs that resembled 4
the wild type (Fig. 1c). The rescued root hair growth by CAP1-S688DT689D not by CAP1-S688AT689A demonstrated that phosphorylation of S688 and T689 are necessary for maintaining the biological function of CAP1 in terms of regulating root hair growth although there are still other sites may involve in the phosphorylation activity indicated by the in vitro phosphorylation assay. The presence of the conserved malectin-like domain in the N-terminus of members of the CrRLK1L family might indicate that there are some similarities in the perception of ligands among these proteins [3]. Phosphopeptide analyses indicated that S227 in the CAP1 malectin domain sequence INIGGDLISPK (Fig. 1a, left panel, with the peptide marked red) was phosphorylated rapidly when environmental concentrations of NO3− or NH4+ varied [9]. Considering the relationship between CAP1-controlled root hair growth and the environmental NH4+ concentration [2], it would be interesting to evaluate the biological function of the phosphorylation of S227. The role of this phosphosite in CAP1-regulated root hair growth was analyzed directly by performing in vivo experiments. The CAP1 overexpression construct [2] was subjected to site-directed mutagenesis to generate the plasmids CAP1-S227A ox and CAP1-S227D ox. The CAP1-S227A ox construct was produced using the primers 5'-ATATCGGAGGAGATTTGATTGCTCCGAAGAT-3' and 5'-CAATCAAATCTCCTCCGATATTTATCCGATG-3', and the CAP1-S227D ox construct was produced using the primers 5'-ATATCGGAGGAGATTTGATTGATCCGAAGAT-3' and 5'-TCAATCAAATCTCCTCCGATATTTATCCGAT-3'. Transformation of cap1 with either the CAP1-S227A ox or the CAP1-S227D ox plasmid resulted in wild-type-like growth of root hairs in the T3 seedlings (Figure 1c). Therefore, the recovery of root hair growth was not related to the phosphorylation of S227, which indicates that S227 phosphorylation might not play an essential role in CAP1-related root hair growth or that the underlying mechanism is more complex. Phosphorylation is the key regulatory process involved in almost all processes of plant growth and development. To improve our understanding of CAP1-mediated signaling, it is important to identify the actual phosphorylation sites involved. However, identifying phosphorylated site(s) is a major bottleneck when analyzing individual proteins [10]. Furthermore, identifying phosphorylation sites in vivo is difficult, especially when the 5
phosphorylation status of a protein is dynamic. Our results indicated that the CAP1-regulated growth of root hairs depends on the phosphorylation of amino acids S688T689, although the results of the in vitro experiments suggested that other phosphosites might participate in the modification of CAP1. The phosphorylation of S227 in the malectin domain was not essential to CAP1-regulated root hair growth, which suggests that the phosphorylation of other residues or other regulatory mechanisms might be involved in signal perception and transduction by CAP1.
Acknowledgments This work was supported by the National Natural Science Foundation of China (31570287).
Conflict of interest: The authors declare that they have no conflicts of interest.
References 1.
Shiu SH, Bleecker AB (2001) Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc Natl Acad Sci USA 98:10763–10768.
2.
Bai L, Ma XN, Zhang GZ et al (2014) A receptor-like kinase mediates ammonium homeostasis and is important for the polar growth of root hairs in Arabidopsis. Plant Cell 26:1497–1511.
3.
Nissen KS, Willats WG, Malinovsky FG (2016) Understanding CrRLK1L function: cell walls and growth control. Trends Plant Sci. 21:516–527.
4.
Wolf S, Hofte H (2014) Growth control: a saga of cell walls, ROS, and peptide receptors. Plant Cell 26:1848–1856.
5.
Lindner H, Müller LM, Boisson-Dernier A et al (2012) CrRLK1L receptor-like kinases: not just another brick in the wall. Curr. Opin. Plant Biol. 15:659–669.
6.
Bai L, Zhou Y, Ma XN et al (2014) Arabidopsis CAP1-mediated ammonium sensing required reactive oxygen species in plant cell growth. Plant Signal. Behav. 9, e29582.
7.
Boisson-Dernier A, Lituiev DS, Nestorova A et al (2013) ANXUR receptor-like kinases coordinate cell wall integrity with growth at the pollen tube tip via NADPH oxidases. 6
PLoS Biol. 11: e1001719. 8.
Haruta M, Sabat G, Stecker K et al (2014) A peptide hormone and its receptor protein kinase regulate plant cell expansion. Science 2014 343:408–411.
9.
Engelsberger WR, Schulze WX (2012) Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen-starved Arabidopsis seedlings. Plant J. 69:978–995.
10. Nühse TS, Stensballe A, Jensen ON, et al (2004) Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16:2394–2405.
Figure legend Fig. 1 The biological function of S688T689 and S227 phosphorylation in CAP1-regulated root hair growth. (a) Predicted phosphorylation sites (in blue) in CAP1. The peptides shown in red indicate the conserved malectin domain, where S227 has been found to function as a phosphorylation site [9]. S688T689 (the putative phosphorylation sites) are in the kinase domain, and the predicted structure of the kinase domain is shown on the right. (b) Western blotting results (left panel) and results of the autophosphorylation and substrate phosphorylation analyses (middle and right panels) for proteins expressed in vitro. Recombinant C-terminal CAP1 fragments (CT-CAP1-GST) and the site-directed mutants CT-CAP1-S688AT689A-GST and CT-CAP1-S688DT689D-GST were analyzed using an anti-GST antibody (left panel). C-terminal protein of MEP2 (CT-MEP2) was used for the phosphorylation assay by CAP1 proteins (right panel). Phosphorylated proteins were less mobile than the unphosphorylated proteins treated with CIAP (alkaline phosphatase), proteins treated with CIAP were served as control. Phosphorylation reactions were stopped by boiling the samples for 5 min, and the proteins were separated by SDS-PAGE. (c) Root hair growth of the wild type (Col), cap1, and the transgenic lines CAP1-S688AT689A ox, CAP1-S688DT689D ox, CAP1-S227A ox, and CAP1-S227D ox; each has two overexpression lines (named as ox-1 and ox-2). The seedlings were grown vertically on 1.2% agar MS medium for 7 d. The bar indicates 2 mm.
7
S2095-9273(17)30260-8 http://dx.doi.org/10.1016/j.scib.2017.05.017 SCIB 144
To appear in:
Science Bulletin
Received Date: Revised Date: Accepted Date:
27 February 2017 28 April 2017 8 May 2017
Please cite this article as: Y. Zhou, L. Zhang, Z. Wu, M. Dai, L. Li, L. Bai, C. Song, Biological function analysis of the phosphorylation sites for Arabidopsis CAP1, Science Bulletin (2017), doi: http://dx.doi.org/10.1016/j.scib. 2017.05.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short communication Title: Biological function analysis of the phosphorylation sites for Arabidopsis CAP1
Yun Zhou, Lu Zhang, Zhangyun Wu, Mengmeng Dai, Luying Li, Ling Bai, Chunpeng Song
Institute of Plant Stress Biology, State Key Laboratory of Cotton Biology, Department of Biology, Henan University, Kaifeng 475001, China
Correspondence to: [email protected]
Received: 2/27/17 Revised: 4/28/2017 Accepted: 5/8/2017
1
Cells need to respond successfully to ever-changing environmental conditions to maintain normal growth. This is achieved through various signal transduction cascades. Receptor-like kinases (RLKs) are involved in many aspects of the growth and development of plants. More than 600 RLKs have been identified and that are involved in various biological processes in Arabidopsis thaliana [1]. An RLK consists of an extracellular domain (the main function of which is to perceive general elicitors), a transmembrane domain, and an intracellular kinase domain that activates a signal transduction cascade mainly through serine/threonine phosphorylation. More and more attention is being paid to the abilities of RLKs to initiate downstream signal transduction. We have previously described a receptor-like kinase, [Ca2+]cyt-associated protein kinase 1 (CAP1/ERULUS), that regulates the growth of root hair tips by influencing the concentration gradients of cytoplasmic Ca2+ and reactive oxygen species in root hair cells. Deficiency of CAP1 causes mutant plants to have very short root hairs, and the inhibition of root hair growth can only be remedied by depleting ammonium from the growth medium [2]. However, the mechanism of CAP1 signal transduction has not yet been identified. CAP1 protein is a member of the Catharanthus roseus RLK1-like (CrRLK1L). The Arabidopsis thaliana CrRLK1L family has 17 members, eight of which have been characterized in detail. These are FERONIA (FER), ANXUR1 (ANX1), ANXUR2 (ANX2), HERCULES1 (HERK1), HERCULES2 (HERK2), CAP1/ERULUS, THESEUS1 (THE1), and CURVY1 (CVY1) [3]. Each CrRLK1L protein has a similar N-terminus, which contains a malectin-like domain, and a kinase domain at the C-terminus (CT) [3]. Recent experimental data have shown that CrRLK1Ls participate in different processes of growth and development, mainly by regulating cell elongation [3–5]. Similar downstream signal molecules, namely Ca2+ and reactive oxygen species, have been found to be involved in the processes required for the regulation of cell expansion by these genes [3,4,6,7]. Efforts have been made to identify the signaling events that depend on these RLKs, but it is still very difficult to uncover the detail transduction cascades involved. Only the ligand for FER has been identified, and found to be a secreted peptide called RALF1 (rapid alkalinization factor 1). The phosphorylation of FER by RALF initiates a signaling cascade that inhibits cell growth [8]. Phosphorylation is a type of protein post-translational modification that plays an 2
important role in RLKs signal transduction. Moreover, it will be helpful in understanding the functions of RLKs by identifying its actual phosphorylation site(s). Phosphopeptides in the kinase domain of the RLKs play vital roles in triggering the signal cascade, and, in at least five members of the CrRLK1L family (FER, THESEUS1, HERCULES1, At4g39110, and At2g21480), two adjacent serine (S) and threonine (T) residues in the kinase domain could be phosphorylated [5]. Conserved adjacent S and T residues, S688 and T689 (S688T689), are also present in CAP1. However, it has not yet been determined whether these are phosphorylation sites, and their biological functions are not understood [5]. Given the strong evolutionary conservation of, and similarities between, the kinase domains in members of this subfamily, it would be interesting to assess the importance of the phosphorylation of these two residues to the CAP1-modulated polar growth of root hair cells. From structural predictions
of
the
kinase
region
of
CAP1
(http://www.proteinmodelportal.org/query/uniprot/Q9FLJ8), we hypothesized that S688T689 in CAP1 might be responsible for the kinase activity of CAP1 (Figure 1a). We then investigated the biological function of their phosphorylation by analyzing site-directed mutants in vitro and in vivo. Mutational analysis of the putative phosphosites of CAP1 might help understanding the mechanism through which CAP1 regulates root hair growth to be identified. Mutating both S688 and T689 into alanine (A) or aspartic acid (D) altered these amino acids to forms that mimicked their constitutively inactive and active phosphorylated types, respectively. We then measured the autophosphorylation and substrate phosphorylation activities of expressed Glutathione S-transferase (GST)-fusion proteins in vitro and performed complementation assays in vivo to determine the importance of the phosphorylation of the two residues. First, the autophosphorylation activities of the mutated proteins were studied by expressing fusion proteins in bacteria. The CT-CAP1-GST construct that we had described previously [2], and which consisted of the coding sequence for GST fused with that of the CT of CAP1 (1549–2529 bp), was subjected to site-directed mutagenesis to generate the mutants described
above.
Plasmid
CT-CAP1-S688AT689A-GST
and
plasmid
CT-CAP1-S688DT689D-GST were constructed using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The primers used to produce 3
plasmid
CT-CAP1-S688AT689A-GST
5'-GAAGGGCATGTAGCTGCCGCGGTAAAGGGT-3'
were and
5'-
ACCCTTTACCGCGGCAGCTACATGCCCTTC-3', and the primers used to produce plasmid CT-CAP1-S688DT689D-GST were 5'-GAAGGGCATGTAGATGACGCGGTAAAGGGT-3' and 5'-ACCCTTTACCGCGTCATCTACATGCCCTTC-3'. Western blotting analysis of the expressed proteins using an anti-GST antibody (Sangon Biotech, Shanghai, China) indicated that we had produced the three desired fusion proteins (Fig. 1b left panel). In vitro assays indicated that the CT-CAP1-GST protein and the S688AT689A and S688DT689D mutant proteins underwent autophosphorylation similarly, all showing the same band shift due to phosphorylation (Fig. 1b middle panel). MEP2 (methylammonium permease 2) has been used as a substrate for CAP1 in the phosphorylation assay [2]. C-terminal tail of MEP2 has been expressed, kinase assay on MEP2 showed that all the tested protein can phosphorylate MEP2 (Figure 1b right panel). Both the mutations of S688AT689A and S688DT689D can not influence the autophosphorylation and the substrate phosphorylation of CAP1 proteins, suggesting that there are other sites also function in phosphorylation activity, except these two sites. These two residues being analyzed may be not essential for the maintenance of CAP1 phosphorylation activity, indicating that other phosphosites might be involved in the phosphorylation-triggered signal transduction by CAP1. To exclude misleading results that might be caused by the production of truncated expressed protein or the use of a heterologous expression system, we then transferred the complete cDNA of CAP1 with the site-directed mutations into cap1 mutant plants by floral dip method to obtain transgenic plants. The CAP1 overexpression construct that we had described previously [2] was used to generate two constructs that carried the desired site-directed mutations (CAP1-S688AT689A ox and CAP1-S688DT689D ox). Given that the cap1 mutant displays short root hairs, we investigated the root hair phenotype of the transgenic plants. Interestingly, even though in vitro phosphorylation was not influenced by the different mutations, the different site-directed mutations resulted in different phenotypes in the T3 transgenic seedlings in terms of root hair growth (Fig. 1c). Mutating the CAP1-S688T689 residues to their inactive form CAP1-S688AT689A did not rescue root hair growth in cap1, but the active form CAP1-S688DT689D resulted in root hairs that resembled 4
the wild type (Fig. 1c). The rescued root hair growth by CAP1-S688DT689D not by CAP1-S688AT689A demonstrated that phosphorylation of S688 and T689 are necessary for maintaining the biological function of CAP1 in terms of regulating root hair growth although there are still other sites may involve in the phosphorylation activity indicated by the in vitro phosphorylation assay. The presence of the conserved malectin-like domain in the N-terminus of members of the CrRLK1L family might indicate that there are some similarities in the perception of ligands among these proteins [3]. Phosphopeptide analyses indicated that S227 in the CAP1 malectin domain sequence INIGGDLISPK (Fig. 1a, left panel, with the peptide marked red) was phosphorylated rapidly when environmental concentrations of NO3− or NH4+ varied [9]. Considering the relationship between CAP1-controlled root hair growth and the environmental NH4+ concentration [2], it would be interesting to evaluate the biological function of the phosphorylation of S227. The role of this phosphosite in CAP1-regulated root hair growth was analyzed directly by performing in vivo experiments. The CAP1 overexpression construct [2] was subjected to site-directed mutagenesis to generate the plasmids CAP1-S227A ox and CAP1-S227D ox. The CAP1-S227A ox construct was produced using the primers 5'-ATATCGGAGGAGATTTGATTGCTCCGAAGAT-3' and 5'-CAATCAAATCTCCTCCGATATTTATCCGATG-3', and the CAP1-S227D ox construct was produced using the primers 5'-ATATCGGAGGAGATTTGATTGATCCGAAGAT-3' and 5'-TCAATCAAATCTCCTCCGATATTTATCCGAT-3'. Transformation of cap1 with either the CAP1-S227A ox or the CAP1-S227D ox plasmid resulted in wild-type-like growth of root hairs in the T3 seedlings (Figure 1c). Therefore, the recovery of root hair growth was not related to the phosphorylation of S227, which indicates that S227 phosphorylation might not play an essential role in CAP1-related root hair growth or that the underlying mechanism is more complex. Phosphorylation is the key regulatory process involved in almost all processes of plant growth and development. To improve our understanding of CAP1-mediated signaling, it is important to identify the actual phosphorylation sites involved. However, identifying phosphorylated site(s) is a major bottleneck when analyzing individual proteins [10]. Furthermore, identifying phosphorylation sites in vivo is difficult, especially when the 5
phosphorylation status of a protein is dynamic. Our results indicated that the CAP1-regulated growth of root hairs depends on the phosphorylation of amino acids S688T689, although the results of the in vitro experiments suggested that other phosphosites might participate in the modification of CAP1. The phosphorylation of S227 in the malectin domain was not essential to CAP1-regulated root hair growth, which suggests that the phosphorylation of other residues or other regulatory mechanisms might be involved in signal perception and transduction by CAP1.
Acknowledgments This work was supported by the National Natural Science Foundation of China (31570287).
Conflict of interest: The authors declare that they have no conflicts of interest.
References 1.
Shiu SH, Bleecker AB (2001) Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc Natl Acad Sci USA 98:10763–10768.
2.
Bai L, Ma XN, Zhang GZ et al (2014) A receptor-like kinase mediates ammonium homeostasis and is important for the polar growth of root hairs in Arabidopsis. Plant Cell 26:1497–1511.
3.
Nissen KS, Willats WG, Malinovsky FG (2016) Understanding CrRLK1L function: cell walls and growth control. Trends Plant Sci. 21:516–527.
4.
Wolf S, Hofte H (2014) Growth control: a saga of cell walls, ROS, and peptide receptors. Plant Cell 26:1848–1856.
5.
Lindner H, Müller LM, Boisson-Dernier A et al (2012) CrRLK1L receptor-like kinases: not just another brick in the wall. Curr. Opin. Plant Biol. 15:659–669.
6.
Bai L, Zhou Y, Ma XN et al (2014) Arabidopsis CAP1-mediated ammonium sensing required reactive oxygen species in plant cell growth. Plant Signal. Behav. 9, e29582.
7.
Boisson-Dernier A, Lituiev DS, Nestorova A et al (2013) ANXUR receptor-like kinases coordinate cell wall integrity with growth at the pollen tube tip via NADPH oxidases. 6
PLoS Biol. 11: e1001719. 8.
Haruta M, Sabat G, Stecker K et al (2014) A peptide hormone and its receptor protein kinase regulate plant cell expansion. Science 2014 343:408–411.
9.
Engelsberger WR, Schulze WX (2012) Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen-starved Arabidopsis seedlings. Plant J. 69:978–995.
10. Nühse TS, Stensballe A, Jensen ON, et al (2004) Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database. Plant Cell 16:2394–2405.
Figure legend Fig. 1 The biological function of S688T689 and S227 phosphorylation in CAP1-regulated root hair growth. (a) Predicted phosphorylation sites (in blue) in CAP1. The peptides shown in red indicate the conserved malectin domain, where S227 has been found to function as a phosphorylation site [9]. S688T689 (the putative phosphorylation sites) are in the kinase domain, and the predicted structure of the kinase domain is shown on the right. (b) Western blotting results (left panel) and results of the autophosphorylation and substrate phosphorylation analyses (middle and right panels) for proteins expressed in vitro. Recombinant C-terminal CAP1 fragments (CT-CAP1-GST) and the site-directed mutants CT-CAP1-S688AT689A-GST and CT-CAP1-S688DT689D-GST were analyzed using an anti-GST antibody (left panel). C-terminal protein of MEP2 (CT-MEP2) was used for the phosphorylation assay by CAP1 proteins (right panel). Phosphorylated proteins were less mobile than the unphosphorylated proteins treated with CIAP (alkaline phosphatase), proteins treated with CIAP were served as control. Phosphorylation reactions were stopped by boiling the samples for 5 min, and the proteins were separated by SDS-PAGE. (c) Root hair growth of the wild type (Col), cap1, and the transgenic lines CAP1-S688AT689A ox, CAP1-S688DT689D ox, CAP1-S227A ox, and CAP1-S227D ox; each has two overexpression lines (named as ox-1 and ox-2). The seedlings were grown vertically on 1.2% agar MS medium for 7 d. The bar indicates 2 mm.
7