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. 2024 Oct 9;8(10):e70003. doi: 10.1002/pld3.70003

Systematic expression analysis of cysteine‐rich secretory proteins, antigen 5, and pathogenesis‐related 1 protein (CAP) superfamily in Arabidopsis

Megumi Matsuzawa 1, Takumi Nakayama 1, Masa H Sato 1,✉, Tomoko Hirano 1,✉
PMCID: PMC11464146  PMID: 39385761

Abstract

The Cysteine‐rich secretory proteins (CRISPS), Antigen 5 (Ag5), and Pathogenesis‐related 1 (PR‐1) protein (CAP) superfamily members are found in multiple eukaryotic organisms, including yeasts, animals, and plants. Although one of the plant CAP family genes, PR‐1 is known to respond to pathogen infection in plants, the functions of other CAP family genes in Arabidopsis remain largely unknown. In this study, we conducted a comprehensive analysis of the similarities, loci, and expression patterns of 22 Arabidopsis CAP genes/proteins, providing a clue to elucidate their molecular functions. According to the promoter‐β‐glucuronidase (GUS) analysis, members of the Arabidopsis CAP family were expressed in various young tissues or organs, such as root and shoot meristems, reproductive tissues, and particularly at the lateral root initiation site before the formation of the lateral root primordium, with distinct expression specificity. In particular, CAP51, CAP52, and CAP53 were specifically expressed in the cortical cells at the lateral root developing regions, suggesting that these genes may function in lateral root development. Thus, the expression patterns of Arabidopsis CAP family genes suggest that CAP family proteins may have certain function in the expressed organs or tissues in Arabidopsis plant.

Keywords: antigen 5 (Ag5), Arabidopsis, cysteine‐rich secretory proteins (CRISPS), gene expression pattern, pathogenesis‐related 1 (PR‐1) 

1. INTRODUCTION

The cysteine‐rich secretory proteins (CRISPS), antigen 5 (Ag5), and pathogenesis‐related 1 (PR‐1) protein (CAP) superfamily members are widely distributed across the kingdoms of life, including bacteria, fungi, plants, and animals. The CAP superfamily proteins, comprising more than 3400 members from 1189 species, are defined by the presence of four conserved motifs of CAP1, CAP2, CAP3, and CAP4 domains, although the other sequences vary among species. Most members of CAP family proteins have the N‐terminal secretion signal sequence (Gaikwad et al., 2020; Gibbs et al., 2008).

From the protein structure and the localization data of CAP family proteins, several functions of the family proteins have been suggested in some organisms. For example, the CAP proteins found as an excretory/secretory (ES) product of helminths and known as venom allergen‐like proteins (VALs or VAPs) are specifically upregulated during their infection, suggesting they have a suppressor function of the host immune response (Wilbers et al., 2018). In mammals, CAP proteins are expressed in the reproductive organs, in immune cells and tumors, or during embryogenesis, suggesting a function in autocrine or paracrine signaling in mammals (Choudhary & Schneiter, 2012; Gibbs et al., 2008).

In plants, the plant‐type CAP family proteins, PR‐1 have been reported. For example, overexpression of the PR‐1 homolog enhanced plant resistance to heavy metals and pathogen stress in tobacco (Sarowar et al., 2005). The C‐terminal 11‐amino acid peptide fragment (CAPE) derived from tomato PR1b increases resistance to the bacterial pathogen Pseudomonas syringae pv. Tomato DC3000 and reduces larval growth of Spodoptera litura (Chen et al., 2014), and a C‐terminal conserved 11‐amino‐acid peptide (AtCAPE1) derived from the salinity‐upregulated gene PROAtCAPE1 (At4g33730) negatively regulates salt stress tolerance in Arabidopsis (Chien et al., 2015). In a model plant, Arabidopsis thaliana, a member of the CAP proteins is known as AtPR‐1 (At2g14610.1), which is one of the pathogenesis‐related proteins (PRs) classified into 22 family genes (Van Loon et al., 2006). AtPR‐1 is highly expressed by pathogens, insects, and chemical treatments in pathogen‐infected leaves and accounted for 10% of the total protein (Cornelissen et al., 1986) and was reported to have antifungal and antibacterial activities (Lincoln et al., 2018; Niderman et al., 1995; Rauscher et al., 1999). Thus, while the functions of a few members of CAP family proteins or genes have been reported in plants, the localization and functional analysis of most Arabidopsis CAP protein members remain poorly characterized.

In this study, as a first step toward understanding the molecular function of CAP family genes/proteins in Arabidopsis, we systematically renamed 22 Arabidopsis CAP genes/proteins based on their phylogenetic relationships and comprehensively investigated their expression patterns under normal growth conditions using a promoter: β‐glucuronidase (GUS) reporter system.

2. RESULTS

2.1. Systematic nomenclature of 22 CAP family proteins of A. thaliana

Although the phylogenetic relationships and structural features of several predicted Arabidopsis PR‐1 type CAP family proteins have been reported previously (Van Loon et al., 2006), they have not yet been systematically named. Therefore, based on the previously proposed phylogenetic relationship of Arabidopsis CAP family genes (Van Loon et al., 2006), we systematically named the Arabidopsis CAP family proteins as follows: CAP11 (At1g01310.1), CAP12 (At3g09590.1), CAP13 (At5g02730.1), CAP21 (At4g31470.1), CAP22/AtCAPE2 (At4g25780.1), CAP23/AtCAPE6 (At4g30320.1), CAP24/AtCAPE4 (At4g25790.1), CAP25/AtCAPE5 (At5g57625.1), CAP31 (At4g33710.1), CAP32/AtCAPE8 (At5g26130.1), CAP33 (At1g50050.2), CAP34 (At1g50060.1), CAP35/AtCAPE7/basic PR‐1 (PRB1) (At2g14580.1), CAP36/AtCAPE9/PR1 (At2g14610.1), CAP37/AtCAPE3 (At4g33720.1), CAP38 (At3g19690.1), and CAP39 (At4g07820.1), CAP41/AtCAPE1 (At4g33730.1), CAP51/PR‐1 homolog (PRH1) (At2g19990.1), CAP52 (At2g19970.1), CAP53 (At2g19980.1), and CAP61 (At5g66590.1) (Figure 1 and Table S1). We confirmed that all Arabidopsis CAP proteins possess 20–30 residues of the secretion signal sequence at the N‐terminus and four conserved domains, CAP1; [G/D/E/R] [H/R] [F/Y/W] [T/V/S] [Q/A] [L/I/V/M] [L/I/V/M/A] W x x [S/T/N], CAP2; [L/I/V/M/F/Y/H] [L/I/V/M/F/Y] x C [N/Q/R/H/S] Y x [P/A/R/H] x [G/L] N [L/I/V/M/F/Y/W/D/N], CAP3; H N x x R, and CAP4; G [E/Q] N [I/L/V] (Van Loon et al., 2006) (Figure 1).

FIGURE 1.

FIGURE 1

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Phylogenetic tree and full‐length alignment of the 22 Arabidopsis CAP protein sequences. The 50% more conserved residues are highlighted in a yellow box. The amino acid identity and conserved residues between the 22 CAP proteins are shown by the bar graph and the amino acid above the alignment. The signal peptide and conserved CAP motifs (CAP1–4) are underlined below the alignment.

The CAP family genes are widely distributed across all Arabidopsis chromosomes and several CAP genes form gene clusters: CAP33 (At1g50050) and CAP34 (At1g50060) on chromosome 1; CAP51 (At2g19990), CAP52 (At2g19979), and CAP53 (At2g19980) on chromosome 2; CAP22 (At4g25780) and CAP24 (At4g25790) on chromosome 4; and CAP31 (At4g33710), CAP37 (At4g33720), and CAP41 (At4g33730) on chromosome 4 (Figure 2).

FIGURE 2.

FIGURE 2

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Chromosomal distribution of 22 Arabidopsis CAP genes (black; gene name) is indicated on the chromosome (gray horizontal bars). The genomic position and orientation of the CAP genes (red; gene name and AGI code) are indicated by the pale yellow arrow lines on the genome (gray horizontal bars), and their introns are indicated by pale yellow thin lines. The promoter region is indicated by the black arrows.

Cis‐element analysis in the Arabidopsis CAP genes revealed that six different cis‐element motifs, such as “Auxin responsive element (AuxRE),” “Myc transcription factors recognition motif (MYC),” “Myb transcription factors binding motif (MYB),” “GT‐1 cis‐acting element responsive to pathogen and salt stress (GT‐1),” “low temperature responsive element (LTRE),” and “TGACG responsive motif responsive to methyl jasmonate (TGACG‐motif),” exist within the 2.0 kbp upstream regions of CAP family genes (Figure 2). According to the comprehensive cis‐element analysis of Arabidopsis promoter regions (Maruyama et al., 2012), there is no clear enrichment of the above cis‐elements related to the various stress or phytohormone responses within the 2‐kbp upstream region of the CAP family genes (Figure S1).

2.2. Analysis of promoter activity of CAP genes of A. thaliana

Next, we generated the Arabidopsis transcriptional reporter lines expressing a β‐glucuronidase (GUS)‐mNeonGreen fusion protein under the control of approximately 2.5‐kbp 5′‐upstream regions of 22 Arabidopsis CAP genes to study the promoter activities of 22 CAP genes in different organs or tissues. These Arabidopsis promoter‐reporter lines allowed monitoring of the expression profiles of the CAP genes at the tissue level by GUS staining and at the cellular level spatiotemporally by fluorescence live imaging.

2.3. Detection of the GUS activity in the root tip

In the root tip, the GUS activity of CAP11, CAP21, CAP33, CAP34, CAP52, and CAP61 was detected in the cells of the meristematic zone; that of CAP11, CAP24, CAP33, CAP34, and CAP52 was detected in the columella; and that of CAP33, CAP34, and CAP52 was detected in the lateral root cap (Figure 3a,c,e). In the mature root region, the GUS activity of CAP23, CAP24, CAP25, and CAP41 was detected in epidermis, especially in root hair, whereas the GUS activity of CAP21, CAP22, CAP31, and CAP61 was detected in endodermis (Figure 3a,c,e).

FIGURE 3.

FIGURE 3

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GUS staining of the transgenic lines expressing GUS‐mNeonGreen under the promoter of each CAP gene (pCAP::GUS‐mNeonGreen) in 9‐day‐old seedling, root tip, root hair, lateral root, shoot apex, and cotyledon (a,c,e), sepal, receptacle, petal, stamen, stigma, and silique (b,d,f). Bars = 100 μm.

2.4. Detection of the GUS activity in leaf and shoot

In the shoot apex during the vegetative growth phase, strong GUS activity of CAP11, CAP31, CAP33, CAP34, CAP35, CAP36, CAP39, and CAP41 was detected at the position of the stipules, whereas the GUS activity of CAP23, CAP24, CAP25, CAP51, and CAP52 was slightly detected in the same region (Figure 3a,c,e). The GUS activity of CAP22, CAP31, and CAP41 was detected in the veins of young rosette leaves, while the GUS activity of CAP61 was detected in the mesophyll of rosette leaves and cotyledons (Figure 3a,c,e). In contrast, the GUS activity of CAP53 was observed only in the guard cells of rosette leaves and cotyledons (Figure 3e and Figure S2).

2.5. Detection of the GUS activity in flower organs

In flower organs, CAP11, CAP22, CAP38, CAP53, and CAP35 were slightly expressed in mature pollen and anthers, while CAP21 and CAP31 were slightly expressed only in the pollen grains. The expression of CAP12 was observed in pollen tubes; CAP31, CAP51, and CAP52 were expressed at the receptacle, while CAP61 was expressed in the stamen filament, style, and sepal (Figure 3b,d,f).

2.6. Expression profile of CAP genes in lateral roots

The lateral roots that emerge from the pericycle cell layer penetrate the endodermal, cortical, and epidermal tissues during emergence in Arabidopsis (Péret et al., 2009). CAP51/PRH1 has been reported to be expressed in the cortical cells overlying the developing lateral root primordia at early stages of lateral root development, suggesting it is likely involved in the lateral root development under the control of AUXIN RESPONSE FACTOR7 (ARF7)‐mediated auxin signaling (Zhang et al., 2020).

Like CAP51/PRH1, the GUS activity of CAP52 and CAP53 was observed at the lateral root initiation site (Figure 3e), although the GUS expression of CAP52 was also observed in the ovules (Figure 3f). Furthermore, we monitored the time course of the expression of CAP51/PRH1, CAP52, and CAP53 using the mNeonGreen fluorescence during lateral root formation. The mNeonGreen fluorescence was observed using confocal laser microscopy every 2 h from 20 to 44 h after the induction of lateral root formation via the gravistimulation of roots. We confirmed that CAP51, CAP52, and CAP53 were expressed in the cortex cells bordering the lateral root primordial cells. Notably, the fluorescence of CAP51 and CAP52 seemed to appear at the cortical tissues before the formation of the lateral root primordium (Figure 4a,b and Movies S1 and S2), whereas that of the CAP53 line seemed to appear after the emergence of the lateral root primordium (Figure 4c and Movie S3).

FIGURE 4.

FIGURE 4

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Confocal time‐lapse images at 20, 24, 26, 28, 30, and 44 h time points of the root bending region after root gravistimulation for 20 h in the transgenic line expressing GUS‐mNeonGreen driven by the CAP51 promoter (a; pCAP51::GUS‐mNeonGreen), CAP52 promoter (b; pCAP52::GUS‐mNeonGreen), or CAP53 promoter (c; pCAP53::GUS‐mNeonGreen). Black arrows indicate the lateral root primordia. Bars = 100 μm.

2.7. Expression analysis of CAP genes from the publicly available data

We compared our promoter GUS analysis with the expression data from publicly available transcriptome data (Figure S3) or the e‐Northerns Expression Browser database (Toufighi et al., 2005) (Figure S4). In root, the strong expression of CAP34 and CAP61 or CAP34 and CAP21 was shown based on publicly available transcriptome data (De Luis Balaguer et al., 2017; Krishnamurthy et al., 2018) or the e‐Northerns Expression Browser database (Figure S4). In lateral root, the strong expression of CAP51 and CAP52 was shown based on publicly available transcriptome data (Bellande et al., 2022; Hurný et al., 2020; Serrano‐Ron et al., 2021). In shoot apical meristem, the strong expression of CAP11, CAP12, CAP35, and CAP61 or CAP22 was shown based on publicly available transcriptome data (Mandel et al., 2016) (Figure S3) or the e‐Northerns Expression Browser (Figure S4). Collectively, our promoter GUS analysis results were consistent with these organ‐level transcriptome data.

3. DISCUSSION

We investigated the tissue‐ and cell‐specific expression of all Arabidopsis CAP family genes using the promoter‐reporter analysis using GUS‐mNeonGreen fusion reporter protein and found that CAP family genes were expressed in various tissues and organs with different expression patterns, suggesting that Arabidopsis CAP genes have distinct functions in various tissues and organs (Figure 5). In addition, CAP genes were commonly expressed during the initial or early stages of tissue or organ development.

FIGURE 5.

FIGURE 5

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Summary of the tissue‐specific expression pattern of CAP genes based on GUS staining in this study. Expression levels are indicated by red gradients (bottom).

The first discovery of plant CAP proteins, pathogenesis‐related protein 1 (PR‐1) was detected in tobacco leaves after infection with the tobacco mosaic virus (Cornelissen et al., 1986; Van Loon & Van Kammen, 1970) and C‐terminal 11 amino acid peptide fragments derived from tomato or Arabidopsis function in defense against the bacterial pathogen and tolerance to salt stress (Chen et al., 2014; Chien et al., 2015). In this study, the expression of PR‐1/CAP36 (At2g14610) was low in almost all tissues and only in the stipules under normal growth conditions, suggesting that it may only function under pathogen infection conditions.

On the other hand, the CAP 51/PRH1 product participates in the development of lateral roots under the control of the ARF7 signaling pathway (Zhang et al., 2020). In this study, we found similar expression patterns and different expression timing of the cluster genes CAP51/PRH1, CAP52, and CAP53 during lateral root development.

The functions of most CAP family genes are still largely unknown, except for a few CAP family genes that have been reported to be involved in abiotic and biotic stress responses and lateral root development in Arabidopsis. This study provides a clue to the elucidating of the molecular mechanisms of CAP family proteins in plants.

4. MATERIAL AND METHODS

4.1. Plant material and growth conditions

A. thaliana ecotype Columbia (Col‐0) was used as the wild type (WT) in all experiments. Arabidopsis seeds were surface sterilized and germinated on 1/2 Murashige‐Skoog (MS) 1.2% agar plates. The plants were grown on tilted plates at 45° in a growth chamber with a 16 h light/8 h dark photoperiod at 22°C. The 6‐day‐old seedlings were used for the time‐course expression pattern analysis of the lateral roots. The 9‐day‐old seedlings were used for the GUS assay of the shoots and roots. The plants were transferred to soil, grown under white light with a 16 h light/8 h dark photoperiod at 22°C, and used for GUS assay of the flower at stage 15 and silique at stages 17 and 18.

4.2. Plasmid constructs and generation of the promoter‐reporter transgenic plants

In each of the Arabidopsis CAP family genes, the promoter region comprises more than 1.5‐kbp upstream region from the start codon of each CAP gene. Promoter sequences were generated by amplifying the Arabidopsis genome, and cloning into the pENTR entry vector was confirmed by DNA sequencing. Subsequently, the promoter sequences were subcloned into Arabidopsis expression vector pGWB533‐GUS‐mNeonGreen (Hirano et al., 2023) using the LR reaction to generate transgenic Arabidopsis plants expressing GUS‐mNeonGreen under their promoters. The binary plasmids were transformed into Col‐0 plants using the Agrobacterium tumefaciens‐mediated floral dip method. The primers used are listed in Table S2.

4.3. Histochemical GUS assays

For each transgenic plant, at least five independent lines were selected, and 9‐day‐old seedlings, flowers at stage 15, and siliques at stages 17 and 18 were used for the GUS assay. After samples were immersed in cold 90% acetone for 15 min at room temperature, the acetone was removed from the sample. After being washed twice with GUS buffer (50 mM sodium phosphate, pH 7.2, 0.5 mM K3Fe(CN)6, and 0.5 mM K4Fe(CN)6), the samples were vacuum infiltrated in GUS staining buffer (0.5 mg/ml 5‐bromo‐4‐chloro‐3‐indoxyl‐b‐D‐glucuronide, cyclohexylammonium salt (X‐Gluc)/dimethylformamide and 0.1% (v/v) Triton X‐100) for 2 min and incubated at 37°C overnight. After being rinsed twice with 70% ethanol and incubated in 70% ethanol for more than 2 h at room temperature, the samples were observed using a zoom microscope (ZEISS) (Koizumi et al., 2009).

4.4. mNeonGreen fluorescence observations

Fluorescence and differential interference contrast (DIC) images were obtained using a Leica TCS SP8 laser scanning confocal microscope. For the detection of mNeonGreen, the excitation wavelength was set at 488 nm, and the emission wavelength was 493–550 nm. The captured images were processed using Leica LAS X software.

4.5. Time‐course expression pattern analysis during the LRP formation

Lateral root initiation is induced mechanically by gravitropic curvature at the site of bending (Ditengou et al., 2008). Then, after rotating the 6‐ or 7‐day‐old seedlings by 90° on 1/2 MS medium with 0.8% agar in a glass bottom dish (AS ONE) for 20 h, the root bending region was observed at 20, 24, 26, 28, 30, and 44 h using a Leica TCS SP8 laser scanning confocal microscope.

4.6. Identification of promoter sequences and alignment of amino acid sequences for 22 Arabidopsis CAP genes/proteins

The promoter regions, genome sequences, amino acid sequences, and five prime untranslated regions (5′UTR) of 22 Arabidopsis CAP genes/proteins were obtained from The Arabidopsis Information Resource (https://www.arabidopsis.org/). Full‐length amino acid sequences were aligned using the SnapGene software and ClustalOmega method. Signal peptide sequences of CAP proteins were also identified using SignalP 3.0 (https://services.healthtech.dtu.dk/services/SignalP-3.0/).

4.7. Promoter cis‐regulatory elements analysis of Arabidopsis CAP genes

The 2‐kbp upstream region from the start codon of each CAP gene was analyzed by online analysis software PLACE (https://www.dna.affrc.go.jp/PLACE/?action=newplace) (Higo et al., 1999) and PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al., 2002).

4.8. Expression analysis of Arabidopsis CAP genes by bioinformatics databases

The expression patterns of 21 Arabidopsis CAP genes were predicted using e‐Northerns w. Expression Browser (https://bar.utoronto.ca/affydb/cgi-bin/affy_db_exprss_browser_in.cgi) (Toufighi et al., 2005) in the Bio‐Analytic Resource for Plant Biology (BAR) (https://bar.utoronto.ca/).

AUTHOR CONTRIBUTIONS

Tomoko Hirano and Masa H. Sato conceived and designed this study. Megumi Matsuzawa performed almost all study experiments. Megumi Matsuzawa and Takumi Nakayama generated the transgenic lines. Megumi Matsuzawa, Tomoko Hirano, and Masa H. Sato analyzed the data. Tomoko Hirano and Masa H. Sato wrote the manuscript and supervised the study.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing financial interests.

Supporting information

Data S1. Supporting Information.

PLD3-8-e70003-s002.pdf (325.4KB, pdf)

Table S1. The name of 22 Arabidopsis CAP genes/proteins.

PLD3-8-e70003-s007.xlsx (15.7KB, xlsx)

Table S2. The primer list for 22 Arabidopsis CAP promoter sequences.

PLD3-8-e70003-s004.xlsx (14.9KB, xlsx)

Movie S1. Time‐lapse movie of mNeonGreen fluorescence in the line expressing GUS‐mNeonGreen under the CAP51 promoter [pCAP51::GUS‐mNeonGreen] during lateral root formation was constructed from images taken at 20, 24, 26, 28, 30, and 44 h after gravistimulation.

Download video file (110.5KB, avi)

Movie S2. Time‐lapse movie of mNeonGreen fluorescence in the line expressing GUS‐mNeonGreen under the CAP52 promoter [pCAP52::GUS‐mNeonGreen] during lateral root formation was constructed from images taken at 20, 24, 26, 28, 30, and 44 h after gravistimulation.

Download video file (163.7KB, avi)

Movie S3. Time‐lapse movie of mNeonGreen fluorescence in the line expressing GUS‐mNeonGreen under the CAP53 promoter [pCAP53::GUS‐mNeonGreen] during lateral root formation was constructed from images taken at 20, 24, 26, 28, 30, and 44 h after gravistimulation.

Download video file (121.1KB, avi)

Figure S1. Diagram showing the 5'UTR and cis‐regulatory elements of the 2 kbp upstream region from the start codon of each CAP gene. The black boxes indicate the downstream region from the start codon of CAP genes. The legends of 5'UTR and cis‐regulatory elements are shown below the diagram.

PLD3-8-e70003-s009.pdf (603.8KB, pdf)

Figure S2. An enlarged image of the cotyledon shown in Figure 4e in the lines expressing GUS‐mNeonGreen under the CAP53 promoter. Bar = 100 μm.

PLD3-8-e70003-s001.pdf (1.1MB, pdf)

Figure S3. Expression levels of 22 Arabidopsis CAP genes in the root meristem, shoot apex, and lateral root are shown as a heatmap (red gradients) based on transcriptome data from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) (bottom). The phylogenetic tree of the 22 Arabidopsis CAP genes is also shown at the top of the diagram.

PLD3-8-e70003-s006.pdf (457.2KB, pdf)

Figure S4. The expression of 21 Arabidopsis CAP genes except CAP25 (At5g57625) at various tissues (top), and each tissue (bottom) shown as a heatmap by using the online software e‐Northerns w. Expression Browser (Toufighi et al., 2005). The color codes, tissue types, and growth stages are shown at the bottom of each heatmap.

PLD3-8-e70003-s010.pdf (623.2KB, pdf)

ACKNOWLEDGMENTS

This work was supported by the Japan Society for the Promotion of Science, a Grant‐in‐Aid for Scientific Research (B) (Grant Number 23H02177) to Masa H. Sato, and by the Precursory Research for Embryonic Science and Technology (PRESTO) Grant Number JPMJPR20D5, Japan, to Tomoko Hirano.

Matsuzawa, M. , Nakayama, T. , Sato, M. H. , & Hirano, T. (2024). Systematic expression analysis of cysteine‐rich secretory proteins, antigen 5, and pathogenesis‐related 1 protein (CAP) superfamily in Arabidopsis . Plant Direct, 8(10), e70003. 10.1002/pld3.70003

Contributor Information

Masa H. Sato, Email: mhsato@kpu.ac.jp.

Tomoko Hirano, Email: thirano@kpu.ac.jp.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1. Supporting Information.

PLD3-8-e70003-s002.pdf (325.4KB, pdf)

Table S1. The name of 22 Arabidopsis CAP genes/proteins.

PLD3-8-e70003-s007.xlsx (15.7KB, xlsx)

Table S2. The primer list for 22 Arabidopsis CAP promoter sequences.

PLD3-8-e70003-s004.xlsx (14.9KB, xlsx)

Movie S1. Time‐lapse movie of mNeonGreen fluorescence in the line expressing GUS‐mNeonGreen under the CAP51 promoter [pCAP51::GUS‐mNeonGreen] during lateral root formation was constructed from images taken at 20, 24, 26, 28, 30, and 44 h after gravistimulation.

Download video file (110.5KB, avi)

Movie S2. Time‐lapse movie of mNeonGreen fluorescence in the line expressing GUS‐mNeonGreen under the CAP52 promoter [pCAP52::GUS‐mNeonGreen] during lateral root formation was constructed from images taken at 20, 24, 26, 28, 30, and 44 h after gravistimulation.

Download video file (163.7KB, avi)

Movie S3. Time‐lapse movie of mNeonGreen fluorescence in the line expressing GUS‐mNeonGreen under the CAP53 promoter [pCAP53::GUS‐mNeonGreen] during lateral root formation was constructed from images taken at 20, 24, 26, 28, 30, and 44 h after gravistimulation.

Download video file (121.1KB, avi)

Figure S1. Diagram showing the 5'UTR and cis‐regulatory elements of the 2 kbp upstream region from the start codon of each CAP gene. The black boxes indicate the downstream region from the start codon of CAP genes. The legends of 5'UTR and cis‐regulatory elements are shown below the diagram.

PLD3-8-e70003-s009.pdf (603.8KB, pdf)

Figure S2. An enlarged image of the cotyledon shown in Figure 4e in the lines expressing GUS‐mNeonGreen under the CAP53 promoter. Bar = 100 μm.

PLD3-8-e70003-s001.pdf (1.1MB, pdf)

Figure S3. Expression levels of 22 Arabidopsis CAP genes in the root meristem, shoot apex, and lateral root are shown as a heatmap (red gradients) based on transcriptome data from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) (bottom). The phylogenetic tree of the 22 Arabidopsis CAP genes is also shown at the top of the diagram.

PLD3-8-e70003-s006.pdf (457.2KB, pdf)

Figure S4. The expression of 21 Arabidopsis CAP genes except CAP25 (At5g57625) at various tissues (top), and each tissue (bottom) shown as a heatmap by using the online software e‐Northerns w. Expression Browser (Toufighi et al., 2005). The color codes, tissue types, and growth stages are shown at the bottom of each heatmap.

PLD3-8-e70003-s010.pdf (623.2KB, pdf)

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