Mol Genet Genomics (2014) 289:1131–1145 DOI 10.1007/s00438-014-0874-9

ORIGINAL PAPER

Genome‑wide identification and characterization of aquaporin genes (AQPs) in Chinese cabbage (Brassica rapa ssp. pekinensis) Peng Tao · Xinmin Zhong · Biyuan Li · Wuhong Wang · Zhichen Yue · Juanli Lei · Weiling Guo · Xiaoyun Huang 

Received: 26 January 2014 / Accepted: 30 May 2014 / Published online: 28 June 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Aquaporins (AQPs) are members of a superfamily of integral membrane proteins and play a significant role in the transportation of small molecules across membranes. However, currently little is known about the AQP genes in Chinese cabbage (Brassica rapa ssp. pekinensis). In this study, a genome-wide analysis was carried out to identify the AQP genes in Chinese cabbage. In total, 53 non-redundant AQP genes were identified that were located on all of the 10 chromosomes. The number of AQP genes in Chinese cabbage was greater than in Arabidopsis. They were classified into four subfamilies, including PIP, TIP, NIP, and SIP. Thirty-three groups of AQP orthologous genes were identified between Chinese cabbage and Arabidopsis, but orthologs corresponding to AtNIP1;1 and AtPIP2;8 were not detected. Seventeen groups of paralogous genes were identified in Chinese cabbage. Threedimensional models of the AQPs of Chinese cabbage were constructed using Phyre2, and ar/R selectivity filters Communicated by S. Hohmann. Electronic supplementary material  The online version of this article (doi:10.1007/s00438-014-0874-9) contains supplementary material, which is available to authorized users. P. Tao · X. Zhong (*) · B. Li · W. Wang · Z. Yue · J. Lei · X. Huang  Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China e-mail: [email protected] W. Guo  Zhejiang Institute of Communication and Media, Hangzhou 310018, China X. Huang  College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

were analyzed comparatively between Chinese cabbage and Arabidopsis. Generally, gene structure was conserved within each subfamily, especially in the SIP subfamily. Intron loss events have occurred during the evolution of the PIP, TIP, and NIP subfamilies. The expression of AQP genes in Chinese cabbage was analyzed in different organs. Most AQP genes were downregulated in response to salt stress. This work shows that the AQP genes of Chinese cabbage have undergone triplication and subsequent biased gene loss. Keywords  Aquaporin · Protein structure · Gene structure · Gene expression · Brassica rapa ssp. pekinensis

Introduction Aquaporins (AQPs) are a large superfamily of major intrinsic proteins that selectively control the flow of water and other small molecules through biological membranes (Baiges et al. 2002). AQP1 was the first discovered and plays diverse roles in the erythrocytes of mammals (Denker et al. 1988; Preston and Agre 1991). So far, hundreds of AQP genes have been identified in the plant kingdom. The number of AQP genes in plants is more diverse than in animals (Maurel et al. 2008; Ahmad et al. 2013). In plants, AQPs play extensive roles not only in seed germination (Gao et al. 1999; Ge et al. 2014), reproduction (Bots et al. 2005), stoma movement (Siefritz et al. 2004; Uehlein and Kaldenhoff 2008), photosynthesis (Uehlein and Kaldenhoff 2008; Vera-Estrella et al. 2012), and cell elongation (Higuchi et al. 1998), but also in response to diverse stresses (Luu and Maurel 2005; Peng et al. 2007; Ishibashi et al. 2011). With the boom in genome sequencing projects in plants, genome-wide analyses of AQP genes have revealed

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that there are 35 AQP genes in Arabidopsis (Johanson et al. 2001), more than 36 in maize (Chaumont et al. 2001), about 34 in rice (Sakurai et al. 2005; Nguyen et al. 2013), and 55 in Populus (Gupta and Sankararamakrishnan 2009). These AQPs are mainly divided into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs), and uncategorized X intrinsic proteins (XIPs) (Sakurai et al. 2008; Gupta and Sankararamakrishnan 2009; Ishibashi et al. 2011). AQP proteins of different subfamilies show obvious diversity in protein localization. PIPs, XIPs, and some NIPs are located in plasma membranes (Ma et al. 2006; Takano et al. 2006; Zelazny et al. 2007; Bienert et al. 2011), TIPs in the tonoplast (Liu et al. 2003), SIPs in the endoplasmic reticulum (Maeshima and Ishikawa 2008), and the remaining AQPs in other subcellular compartments (Maurel et al. 2008). Although AQP proteins of different subfamilies show differences in their primary sequences, evolutionary distances, subcellular localization, and substrate specificity, they share a conserved hourglass model with an α-helical bundle forming six transmembrane (TM) helices (H1 to H6) and two additional half-helices (loops B, LB and loops E, LE) (Fu et al. 2000; Sui et al. 2001; Savage et al. 2003; Gonen et al. 2004; Harries et al. 2004; Lee et al. 2005; Newby et al. 2008). These conserved structural elements constitute two halves of the three-dimensional (3D) structure. The first half (H1, H2, LB, and H3) and the second half (H4, H5, LE, and H6) are oppositely orientated in the membrane because of the uneven number of TM helices. LB and LE are embedded in the membrane in opposite directions and come together in the center of the membrane, where they generate the first pore constriction. Four residues of the TM helices H2, H5, and LE, situated towards the extracellular side approximately 8 Å from the NPA region, construct the second pore constriction, also known as the aromatic/arginine (ar/R) selectivity filter (Fujiyoshi et al. 2002; Gupta and Sankararamakrishnan 2009). Different AQP proteins show variations in ar/R selectivity filter that result in different pore sizes between AQP proteins (Ayadi et al. 2011). AQPs form tetramers in the cell membrane, with each monomer acting as a functional pore (Gonen and Walz 2006). Chinese cabbage (Brassica rapa ssp. pekinensis), which originates from China, is a dicotyledonous plant of the Brassicaceae family and is an important vegetable crop in China. The genome of Chinese cabbage is considered to be derived from the genome of a hexaploid ancestor with a triplicated diploid ancestral genome closely related to the A. thaliana genome (Wang et al. 2011; Cheng et al. 2013). Much evidence supports the occurrence of an ancestral whole-genome triplication prior to the species radiation of the tribe Brassiceae (Lagercrantz 1998; Lysak et al. 2005;

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Parkin et al. 2005; Wang et al. 2011). Wang et al. (2011) revealed the three subgenomes of the hexaploid ancestor based on an analysis of the draft genome sequence of B. rapa (n = 10). The three subgenomes are named the least fractionated (LF), the medium fractionated (MF1), and the most fractionated (MF2) subgenomes according to their gene density and rate of gene loss (fractionation). Brassica rapa might have undergone a two-step origin involving a tetraploidization followed by substantial genome fractionation (MF1 and MF2) and subsequent hybridization with a third, less-fractionated genome (Cheng et al. 2012, 2013; Tang and Lyons 2012; Tang et al. 2012). The Chinese cabbage genome (Chiifu-401-42) sequencing project (BrGSP) has been completed and the results have been published (Wang et al. 2011). Chinese cabbage offers a new opportunity to investigate some aspects that cannot be studied in other model annual plants (Jansson and Douglas 2007). As a well-established vegetable of the B. rapa species, Chinese cabbage is considered to be a typical representative of the A genome of Brassica. Although AQP genes have been extensively studied in Arabidopsis, poplar, maize, and rice through genome-wide analysis, our knowledge of AQP genes in Chinese cabbage is still limited, and several questions remain to be answered. For example, how many AQP genes are present in Chinese cabbage? Is the number of AQP genes increased in Chinese cabbage? What are the expression characteristics of the AQP genes of Chinese cabbage? In this study, a total of 60 new AQP genes were identified from the Chinese cabbage genome by homology and synteny analyses. Seven AQP genes were eliminated as a result of incomplete TM regions. The remaining 53 AQP genes were spread over all 10 chromosomes and were classified into four different groups (PIP, TIP, NIP, and SIP) based on the phylogenetic analysis. 3D structure analysis showed that all 53 AQPs possessed six TM helices and a seventh TM helix consisting of two half-helices (LB and LE). We also analyzed their transcriptional levels in different organs according to published data. Additionally, the expression profiles of the AQP genes in response to salt stress were investigated according to EST library in NCBI.

Materials and methods Identification and physical locations of AQP genes in Chinese cabbage Due to the Chinese cabbage genome sequence project being completed and the result being published (Wang et al. 2011), both gene sequences and protein sequences have also become available (http://brassicadb.org/brad/ index.php). Nucleotide sequences of 35 Arabidopsis AQP genes and 34 O. sativa AQP genes were obtained from

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GenBank, and they were used as probe sequences for searching AQP genes in Chinese cabbage genome. Due to no XIP genes in Arabidopsis and O. sativa genome, nucleotide sequences of additional 22 XIP genes of dicot plants (Danielson and Johanson 2008; Gupta and Sankararamakrishnan 2009) were used in database searches to obtain the potential XIPs of Chinese cabbage. The resulting genes, showing similarity in coding region and query coverage more than 75 % by Blastx analysis, were collected for further analysis. These AQP genes were mapped back to the B. rapa v1.5 genome sequence, and chromosome location image was generated using MapInspect software. Analysis of protein characteristic and phylogenetic relationship of AQP proteins in Chinese cabbage To compare AQPs’ protein characteristic of different subfamily, molecular weight (MW) and isoelectric point (pI) of AQP proteins of Chinese cabbage and Arabidopsis were analyzed using ProtParam (http://web.expasy.org/protpa ram/). To understand the evolutionary relationship of the Chinese cabbage AQP proteins, amino acid sequences of AQP proteins, along with AQP proteins of A. thaliana and O. sativa (data from NCBI), were aligned using ClustalX (version 1.83) (Chenna et al. 2003). A phylogenetic tree was constructed using neighbor-joining with MEGA v. 5.2 (Tamura et al. 2011). Distance matrices were based on the Jones-Taylor-Thornton substitution matrix for the amino acid data. One thousand bootstrap replicates were carried out to calculate the relative support for branches of the inferred phylogenetic tree. Identification of syntenic A. thaliana‑B. rapa (At‑Br) orthologs and paralogs in Chinese cabbage To obtain each syntenic At-Br orthologs and paralogs in Chinese cabbage, we searched syntenic genes between A. thaliana and B. rapa by a online tool “syntenic gene analysis” (http://brassicadb.org/brad/searchSyntenytPCK.php) (Cheng et al. 2012). The identified At-Br orthologs and paralogs were marked in the phylogenetic tree. Analysis of conserved motifs and 3D structure of AQP proteins in Chinese cabbage Conserved motif was analyzed by MEME tool (Version 4.9.1) (http://meme.nbcr.net/meme/cgi-bin/meme.cgi). The parameter settings were as follows: the optimum width of each motif ranged from 6 to 50, the maximum number of motifs to find was 20, and the other parameter settings were default values (Bailey and Elkan 1994; Bailey et al. 2006). Furthermore, all candidate AQP proteins of Chinese cabbage were checked by Pfam (http://pfam.janelia.org/).

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3D models of all AQP proteins of Chinese cabbage were constructed using Phyre2 (Kelley and Sternberg 2009) as a final quality check. Quality control and thresholds for the structural models were as followins: alignment coverage >65 % and confidence = 100 %. Those AQP proteins, not containing six TM helices and two additional half-helices, were eliminated. Gene structure analysis Gene structure of AQP genes of Chinese cabbage were predicted by GENSCAN (http://genes.mit.edu/GENSCAN.html). Gene structure was checked by alignment of coding sequence and genomic DNA sequence of each AQP gene. The sequence gaps were identified as introns of AQP genes. To show intron site, position of each intron was marked in second structure map. Gene expression and EST expression profile Raw RNA-seq data of Chinese cabbage organ expression were published (Tong et al. 2013) and downloaded from NCBI Gene Expression Omnibus (http://www.ncbi.nlm. nih.gov/geo/) under accession no. GSE43245. The expression data of 53 AQP genes of Chinese cabbage in different organs were available. The program MeV was used to produce a heat map (Saeed et al. 2003). Distance metric and Linkage method for hierarchical clustering were Pearson correlation and average linkage clustering, respectively. We considered that paralogous genes shared a similar expression pattern when gene distance was 95 %, length >200 bp and E value 80 %. The pentagram represents paralogous genes of B. rapa ssp. pekinensis. Circles represent orthologous genes from B. rapa ssp. pekinensis (black) and Arabidopsis (white). The Arabidopsis AQP genes, which have no corresponding orthologous genes in B. rapa ssp. pekinensis, are boxed. The abbreviations of species are as follows: Br B. rapa ssp. pekinensis, At A. thaliana, Os O. sativa

(the largest family), 14 into TIP, 13 into NIP, and 6 into SIP. No XIP genes were found in Chinese cabbage. Furthermore, the PIPs were clustered into two subgroups (PIP1 and PIP2), the TIPs into five subgroups (TIP1, TIP2, TIP3, TIP4, and TIP5), and the SIPs into two subgroups (SIP1 and SIP2). The AQP proteins of Chinese cabbage and Arabidopsis formed a small cluster. The PIPs, TIPs and NIPs were closely related to each other. Homologous genes corresponding to AtPIP2;8 and AtNIP1;1 were not found in Chinese cabbage (Fig. 3). Paralogous AQP genes in Chinese cabbage and orthologous AQP genes between Chinese cabbage and Arabidopsis Recently, analysis of the draft genome sequence of B. rapa (n  = 10) revealed the three subgenomes of its hexaploid ancestor (LF, MF1 and MF2) based on their gene density (Wang et al. 2011). To assess AQP gene triplication between Chinese cabbage and Arabidopsis, orthologous AQP genes were identified by searching with “syntenic genes” (http://brassicadb.org/brad/searchSyntenytPCK.php), an online tool for collinearity comparison between B. rapa

and Arabidopsis genes. Orthologous AQP genes between Chinese cabbage and Arabidopsis were identified and are shown in Fig. 3. The results revealed that there were 33 groups of orthologous AQP genes between A. thaliana and Chinese cabbage. Most of the Arabidopsis AQP genes had 0–3 syntenic orthologs in the B. rapa genome. In total, six Arabidopsis AQP genes had three corresponding orthologs in Chinese cabbage, 15 Arabidopsis AQP genes had two orthologs in Chinese cabbage, and 12 Arabidopsis AQP genes had a single ortholog in Chinese cabbage. However, AtPIP2;8 and AtNIP1;1 had no syntenic orthologs in Chinese cabbage (Supplementary File 2). Regarding the distribution of the 53 AQP genes across the three subgenomes of B. rapa, 24 AQP genes were located in the LF subgenome, 16 in the MF1 subgenome, and 13 in the MF2 subgenome (Supplementary File 2). Furthermore, paralogous AQP genes were identified in Chinese cabbage and marked on the phylogenetic tree (Fig.  3). In total, there were 17 groups of paralogous AQP genes in Chinese cabbage. The potential mechanisms involved in the evolution of the AQP gene family were elucidated by analyzing

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duplication events that may have occurred during B. rapa genome evolution. In total, 21 duplicated gene pairs were identified, including 17 segmental duplication events between chromosomes (e.g. BrPIP1;1a and BrPIP1;1b, BrPIP1;2a and BrPIP1;2b) as well as five tandem duplication events within the same chromosome (e.g. BrPIP2;2 and BrPIP2;3, BrNIP2;1a and BrNIP2;1b) (Supplementary File 2). Two tandem duplication events caused a partial loss of nucleotide sequence in four AQP genes and resulted in incomplete TM regions in their corresponding proteins (Bra005215 and Bra005216, Bra025435, and Bra025436). Duplication of AQP genes was observed in all 10 chromosomes. These results demonstrated that AQP duplication accompanied the genome triplication in Chinese cabbage. Sequence alignment and analysis of conserved domains in the AQP proteins in Chinese cabbage To analyze their conserved domains, multiple alignment of the 53 AQP protein sequences was carried out, clearly indicating that the majority of the Chinese cabbage AQP proteins had six TM helices (H1 to H6) and two halfhelices (LB and LE) (Supplementary File 3). Each AQP protein was comprised of a first half (H1, H2, LB, and H3) and a second half (H4, H5, LE, and H6). The two halves were reversely symmetrical in the orientation of the TM helices and the NPA motifs. LB and LE were the two conserved domains of the AQP proteins in Chinese cabbage. To further check for conserved regions, motif analysis was carried out with the MEME web server. The results indicated that motifs 1 (H5 and LE) and 2 (partial H2 and LB) were conserved across the PIP, TIP, and NIP subfamilies. Motifs 4, 7, and 11 were representative of PIPs, while motif 8 was representative of NIPs. Motif 15 appeared exclusively in TIPs, while motif 13 appeared exclusively in SIPs. The PIP1 and PIP2 subclasses could be divided by motif 12 (Fig. 4). Detailed sequence logos of the 20 motifs are shown in Supplementary File 4. In the motif analysis, BrPIP1;2a and BrPIP2;4c lacked motifs 5 and 6, respectively, which were conserved across the whole of the PIP and TIP subfamilies. Sequence alignment was performed for BrPIP1;2a and BrPIP2;4c, indicating that six amino acids, corresponding to Loop D (LD), were lost in BrPIP1;2a (Fig. 5a). Twenty-five amino acids corresponding to part of H1 were lost in BrPIP2;4c (Fig. 5b). Because of these fragment deletions, the conserved motif EXXXT was lost in H4 of BrPIP1;2a and H1 of BrPIP2;4c, and the histidine residue related to pH-regulated gating was deleted in LD of BrPIP1;2a (Fig. 5). Because the sequence alignment and MEME motif analysis did not correspond precisely to the TM helices region,

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Phyre2 was used to predict the 3D structures of the AQP proteins; the results are shown in Supplementary File 5. The 53 AQP proteins had a conserved hourglass model with an α-helical bundle forming six TM helices (H1 to H6) and two additional half-helices. These conserved motifs constituted two halves in the 3D structure. LB and LE came together in the center of the membrane (Supplementary File 5). Although there was fragment deletion in BrPIP1;2a and BrPIP2;4c, they both could be modeled with six TM helices, LB and LE. Comparison of NPA motifs and ar/R selectivity filters in BrAQPs and AtAQPs Homology modeling analysis demonstrated that the 3D structures of the B. rapa AQPs were highly conserved across different subfamilies. NPA motifs and the ar/R selectivity filter are known as key areas of AQP protein sequences related to channel selectivity. To examine differences in channel selectivity between BrAQPs and AtAQPs, we identified and compared NPA motifs and ar/R selectivity filters among different subfamilies of BrAQPs and AtAQPs. The results showed that all PIPs and TIPs from Chinese cabbage and Arabidopsis had a typical “NPA” in LB and LE. However, some members of the NIP family (BrNIP1;2a, BrNIP1;2b, BrNIP5;1a, BrNIP5;1b, BrNIP6;1a, BrNIP6;1b, and BrNIP7;1) showed a variable third residue in which the alanine was replaced with serine, glycine or valine. The third residue of the first NPA motif in all SIPs was threonine, cysteine, valine or leucine. Besides the NPA motifs, the ar/R selectivity filter is the other pore constriction determining channel selectivity. Different subfamily members of the BrAQPs and AtAQPs showed obvious differences in their ar/R selectivity filters. In the PIP subfamily, the ar/R selectivity filters of all BrPIPs and AtPIPs were composed of phenylalanine in H2, histidine in H5, threonine in LE1, and arginine in LE2. In the NIP subfamily in B. rapa and Arabidopsis, the four positions of the ar/R selectivity filters were occupied by tryptophan or alanine in H2, isoleucine or valine in H5, alanine or glycine in LE1, and arginine in LE2. In the TIP subfamily, the ar/R selectivity filters were constituted by histidine or asparagine in H2, isoleucine/methionine/valine in H5, glycine or alanine in LE1, and valine/arginine/cysteine in LE2. In the SIP subfamily, the ar/R selectivity filters were constituted by isoleucine/serine/valine in H2, valine/histidine/phenylalanine in H5, proline/glycine in LE1, and isoleucine/alanine in LE2. In addition, the ar/R selectivity filters of most BrAQPs from Chinese cabbage were consistent with those of the corresponding AQPs in Arabidopsis (Table 1). Only the ar/R selectivity filters of

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Fig. 4  Distribution of conserved motifs in the AQP family members of Chinese cabbage. All motifs are identified by MEME using the complete amino acid sequences of 53 AQPs of Chinese cabbage. Combined p values are indicated on the left side. The height of the motif “block” is proportional to –log (p value), truncated at the height for a motif with a p value of 1e−10. Different motifs are shown by different colors numbered 1–20. Putative fragment deletion is marked by black arrowhead. Distribution of six TM helices (H1–H6) and two additional half-helices (LB and LE) is indicated at the bottom of the figure. Detailed sequence logos of the 20 motifs are shown in Supplementary File 2

BrTIP3;2a, BrTIP3;2b and BrNIP5;1b were distinct from their corresponding homologs in Arabidopsis. BrTIP3;2a and BrTIP3;2b had methionine in place of isoleucine in

the H5 position when compared with AtTIP3;2, while BrNIP5;1b had alanine in place of glycine in the LE1 position (Table 1).

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Gene structure

Fig. 5  Multiple sequence alignment of the PIP family proteins in Chinese cabbage, indicating deletion of fragment, conserved motif and residue happen in protein sequences of BrPIP1;2a (a) and BrPIP2;4c (b). The deleted regions are marked by black arrowhead. The highly conserved motif (EXXXT) on H1 and H4 are marked by underline. The histidine residue of loop D (LD) is boxed. Gray arrowheads with number represent different TM helices. LD regions are represented by rectangle

Table 1  NPA motif and ar/R selectivity filters of AQPs from Chinese cabbage

a

  Selectivity filter residues of BrTIP3;2a/b in H5 and BrNIP5;1b in LE1 are different from those of AtTIP3;2 and AtNIP5;1, respectively. Selectivity filter residue in Arabidopsis was denoted by a single underline when discrepancy is shown between orthologous AQP genes of Chinese cabbage and Arabidopsis

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The availability of the Chinese cabbage genome enabled us to analyze and compare the gene structures of AQP genes of different subfamilies. The gene structure of all 53 B. rapa AQP genes is shown in Fig. 6. Comparative analysis showed that the exact location of introns in the AQP genes was conserved within each subfamily (Fig. 6). In general, the number of introns was specific for each subfamily. The majority of PIP genes had three introns, located in LB, Loop D (LD) and H6. The introns of BrPIP2;4a and BrPIP2;4b were located in LB, Loop C(LC) and H6, while BrPIP1;3a, BrPIP1;3b, and BrPIP1;5 had two introns located in LD and H6. Among the TIPs, eight of the 14 TIP genes had two introns in Loop A (LA) and LC, and five TIP genes had just a single intron in LC, while one TIP gene had no intron. Among the NIPs, seven genes had four introns in H1, H3, LD, and H5, while the remaining six NIP genes had three introns and showed intron loss in H3, LD, and H5. All SIP genes contained two introns that were located in H3 and LE. Comparison of AQP paralogs in Chinese cabbage showed that different paralogs shared similar gene structures (e.g. BrPIP1;1a and BrPIP1;1b; BrPIP1;2a and BrPIP1;2b; BrPIP2;5a and BrPIP2;5b).

NPA motifs

PIP subfamily  BrPIP TIP subfamily  BrTIP1;1, 1;2, 1;3  BrTIP3;1, 4;1  BrTIP2;1, 2;2, 2;3  BrTIP3;2a  BrTIP5;1 NIP subfamily  BrNIP1;2, 2;1, 4;1, 4;2  BrNIP3;1,  BrNIP5;1a  BrNIP6;1  BrNIP7;1 SIP subfamily  BrSIP1;1  BrSIP1;2  BrSIP2;1

Ar/R selectivity filter

LB

LE

H2

H5

LE1

LE2

NPA

NPA

F

H

T

R

NPA NPA NPA NPA NPA

NPA NPA NPA NPA NPA

H H H H N

I I I M (I) V

A A G A G

V R R R C

NPA NPA NPS NPA NPS

NP (A/G) NPA NPV NPV NPA

W W A A A

V I I I V

A A A (G) A G

R R R R R

NPT NPC

NPA NPA

I V

V F

P P

I I

NP(L/V)

NPA

S

H

G

A

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Fig. 6  Gene structure of the AQP genes from Chinese cabbage and Arabidopsis. Exon–intron organization of AQP genes from Chinese cabbage and Arabidopsis is depicted for PIP, TIP, NIP and SIP subfamilies. The exon–intron pattern, observed in most AQP genes within a subfamily, is indicated in the first row. In this case, only the number of AQP genes having that pattern is indicated for each plant species. For example, “Br:14/20” represents that 14 out of 20 BrPIPs share the same gene structure. For the rest members with different exon–intron organization, the AQP name is explicitly given (example: Br:2;4a in PIP subfamily). The six TM helices are shown in big black bars, while the loops B and E are shown in small gray bars. The intron positions are marked by inverted triangle

Gene expression in different organs and in response to salt stress Recently, high-resolution RNA-seq data for different organs of Chinese cabbage has been obtained and provided to NCBI (Tong et al. 2013), which helped us to analyze AQP gene expression. The gene expression data for all 53 AQP genes in Chinese cabbage was downloaded from NCBI. A heatmap was produced to show the AQP gene expression in different organs (Fig. 7). Gene sets were clustered using hierarchical clustering and the heatmap was displayed based on the transcript abundance pattern. Fifty-one AQP genes (96.2 %) were expressed in at least one organ, with 29 AQP genes (54.7 %) expressed in all organs, including 16 PIP genes, 5 TIP genes, 3 NIP genes, and 5 SIP genes. The largest number of B. rapa AQP genes was expressed in roots (46, 86.8 %), followed by leaves (41, 77.4 %) and flowers (39, 73.6 %). The fewest number of AQP genes was detected in siliques (38, 71.7 %) and stems (35, 66.0 %). Nineteen AQP genes showed higher expression in roots than other organs, while the number of genes showing the highest expression in stems, leaves, flowers, and siliques was 9, 4, 8, and 11, respectively. Of the subfamilies, PIP had the most genes (17, 85.0 %) showing high expression in the

Fig. 7  Expression profile cluster analysis of the 53 AQP genes of Chinese cabbage. The expression values of each AQP gene identified in the study are obtained from NCBI Gene Expression Omnibus (htt p://www.ncbi.nlm.nih.gov/geo/) under accession no.GSE43245 from five organs, i.e. root, stem, leaf, flower, and silique. Black Circles represent that paralogous AQP genes share similar expression patterns in Chinese cabbage

majority of organs, followed by SIP (4, 66.7 %), TIP (6, 42.9 %), and NIP (4, 30.8 %). In the PIP subfamily, except for BrPIP2;4a and BrPIP2;4c, which were rarely expressed in these organs, most PIP genes were more or less expressed in all organs. In particular, BrPIP2;7a, BrPIP2;7b, BrPIP1;2a and BrPIP1;2b showed high expression levels in all organs. BrPIP2;7a had the highest expression in siliques. In the TIP subfamily, BrTIP2;2 and BrTIP2;3 exhibited root-preferential expression. BrTIP1;1 showed high expression in all

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Table 2  EST profiles showing approximate gene expression profile as inferred from EST counts and the cDNA library sources (LIBEST_021827 whole plant cDNA library and LIBEST_021813 salt-treated whole plant cDNA library) (Lee et al. 2008) Name

EST expression profile (TPM, transcripts per million) Whole plant

Salt-treated whole plant

BrPIP1;1a BrPIP1;2a BrPIP1;2b BrPIP1;3a BrPIP1;3b BrPIP1;4 BrPIP2;1 BrPIP2;3 BrPIP2;4b BrPIP2;5a BrPIP2;6 BrPIP2;7a BrPIP2;7b BrTIP1;1 BrTIP1;2a BrTIP1;2b BrTIP2;1a BrTIP2;1b BrTIP4;1 BrNIP6;1a

200 4,800 6,800 800 800 400 100 500 100 200 100 500 1,900 1,000 100 2,000 1,800 200 500 100

0 400 1,900 0 0 100 300 0 0 0 300 400 0 0 100 1,300 0 0 0 0

BrNIP6;1b

200

0

organs. BrTIP1;2a, BrTIP1;2b, BrTIP2;1a and BrTIP4;1 showed high expression in roots, stems, leaves, and flowers. In the NIP subfamily, BrNIP6;1b was expressed in different organs and showed the highest expression in roots. BrNIP1;2a showed root-preferential expression. Expression of BrNIP2;1a or BrNIP4;1 was not detected in any organ. In the SIP subfamily, BrSIP2;1c was not expressed in flowers, stems or siliques, and rarely in roots and leaves. However, the remaining SIP genes were more or less expressed in all five organs. To examine salt-responsive expression, the EST expression profiles of the AQP genes of Chinese cabbage were studied using the corresponding EST database (whole plant cDNA library and salt-treated whole plant cDNA library of B. rapa ssp. pekinensis) with known AQP coding sequences. ESTs for 32 AQP genes were not found in the two cDNA libraries (Data not shown), suggesting that these AQP genes were not expressed or detected in whole plants or salt-treated whole plants. The remaining 21 AQP genes were detected in both or at least one cDNA library, and showed down-regulation after salt treatment except BrPIP2;1, BrPIP2;6 and BrTIP1;2a, (Table 2).

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Discussion With the rapid development of genome sequencing projects, the information hidden in plant genomes can be explored to elucidate the mechanisms regulating plant growth and development. Analysis of plant genomes can also facilitate genome and gene evolution studies. Genomewide studies of AQP genes have been reported in the higher plant model species Arabidopsis, maize, Populus, and rice (Johanson et al. 2001; Chaumont et al. 2001; Sakurai et al. 2005; Gupta and Sankararamakrishnan 2009; Nguyen et al. 2013). The Chinese cabbage genome sequencing project has been completed, and its genome data are now available. However, the AQP gene family in Chinese cabbage has not been studied. Therefore, genome-wide identification and characterization of the AQP genes in Chinese cabbage is viable and important. In this study, 53 AQP genes were identified from the B. rapa genome, including 20 genes belonging to the PIP family (13 PIPs in Arabidopsis), 14 to the TIP family (10 TIPs in Arabidopsis), 13 to the NIP family (9 NIPs in Arabidopsis), and 6 to the SIP family (3 SIPs in Arabidopsis). We did not detect any XIP genes in Chinese cabbage. Although XIP genes have been detected in dicotyledonous plants such as Lactuca sericola, Citrus sinensis, and Ricinus communis, none have been identified in Arabidopsis (Gupta and Sankararamakrishnan 2009). The number of AQP genes and members in each subfamily (PIP, TIP, NIP, and SIP) has clearly expanded in Chinese cabbage compared with Arabidopsis. In a previous study, comparative genome analysis indicated that B. rapa was derived from a paleohexaploidy event, and it has three subgenomes that share the same diploid ancestor as the model species Arabidopsis (Wang et al. 2011; Cheng et al. 2013). However, the number of AQP family members was less than three times the number of Arabidopsis AQP genes. Among the three subgenomes (LF, MF1 and MF2), 24 AQP genes were located in LF, 16 in MF1, and 13 in MF2. Loss of AQP genes was more extensive in MF1 and MF2 than in LF (Supplementary File 2), supporting the genome of the hexaploid ancestor of Chinese cabbage having experienced a two-step origin; a tetraploidization followed by substantial genome fractionation (MF1 and MF2) and subsequent hybridization with a third, less-fractionated genome (LF) (Wang et al. 2011; Tang et al. 2012; Cheng et al. 2013). Additionally, most A. thaliana AQP genes had 0–3 syntenic orthologs in the Chinese cabbage genome according to a collinearity comparison between Chinese cabbage and Arabidopsis genes (Supplementary File 2). Six Arabidopsis AQP genes (AtPIP2;4, AtPIP2;7, AtTIP2;1, AtNIP3;1, AtNIP4;1, and AtSIP2;1) had three orthologs in Chinese cabbage, 15 Arabidopsis AQP genes had two orthologs, and 12 Arabidopsis AQP genes had a single ortholog.

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According to the phylogenetic tree, AtPIP2;8 and AtNIP1:1 had no syntenic ortholog in Chinese cabbage (Fig. 3). In our opinion, these results may be derived from biased gene fractionation (gene losing) during the evolution of the AQP family in Chinese cabbage or alternatively be due to recent AQP gene duplication in the Arabidopsis line. In previous studies, genome comparison has shown that polyploid speciation was accompanied by gene loss in many eukaryotes (Wang et al. 2011; Cheng et al. 2013). In terms of protein motifs, Motif 5 in BrPIP1;2a and Motif 6 in BrPIP2;4c have been lost compared with other BrPIP1 subfamily members (Figs. 4, 5). Sequence analysis clearly indicated that BrPIP2;4c lacked the highly conserved motif EXXXT in H1. Additionally, BrPIP1;2a also lacked EXXXT in H4 and a strictly conserved histidine in LD compared with other PIPs of Chinese cabbage and Arabidopsis. The glutamates of the highly conserved motifs (EXXXTXXF/L) of H1 and H4 play a significant role in stabilizing LB and LE in the right position (Heymann and Engel 2000; Johanson and Gustavsson 2002). However, the histidine of LD in spinach aquaporin SoPIP is involved in pH-regulated PIP gating (Törnroth-Horsefield et al. 2006). Fragment deletion in the amino acid sequences of BrPIP1;2a and BrPIP2;4c in the highly conserved H1, H4, and LD regions has probably resulted in loss of water channel function. Taken together, the three subgenomes of the Chinese cabbage ancestor have undergone different levels of gene loss during their two-step origin. Additionally, some AQP genes have experienced fragment deletion during evolution, resulting in some important motifs have been deleted completely or partially. The 3D structures of the Chinese cabbage AQPs were predicted, demonstrating that the 53 BrAQPs shared a conserved model with AQPs from other organisms. The NPA motifs and ar/R selectivity filters of BrAQPs showed some discrepancies among different subfamilies (Table 1). The NPA motifs and ar/R filters of the largest PIP subfamily in Chinese cabbage and Arabidopsis were strictly conserved, suggesting that they probably transport more specific solutes than other subfamilies (Maurel 2007). In a previous study, PIP subfamily members were suggested to play major roles as efficient water channels (Wallace and Roberts 2004; Wudick et al. 2009; Ahmad et al. 2013). Although all of the TIPs of Chinese cabbage and Arabidopsis contained two identical NPA motifs in LB and LE, some of them showed differences in the ar/R selectivity filter. Both BrTIP5:1 and AtTIP5;1 had a specific ar/R selectivity filter, suggesting that they probably allow permeation of unconventional solutes (Wallace and Roberts 2004; Dynowski et al. 2008; Soto et al. 2008). In some BrNIPs and all BrSIPs, the third alanine of the NPA motif was replaced with other residues and their ar/R selectivity filters differed from those of the PIP and TIP subfamilies,

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suggesting that they could transport some novel substrates (Wudick et al. 2009; Perez Di Giorgio et al. 2014). In addition, comparative analysis of ar/R selectivity filters between BrAQPs and AtAQPs indicated that a majority of AQPs in Chinese cabbage shared the same ar/R selectivity filter type as the corresponding AQPs in Arabidopsis. This result indicates that these AQPs from Chinese cabbage facilitate the transport of the same or similar solute molecules that are transported in Arabidopsis. Only three of the 53 BrAQPs (BrTIP3;2a, BrTIP3;2b, and BrNIP5;1b) had novel ar/R selectivity filters different from those of their counterparts in Arabidopsis (Table 1). BrTIP3;2a and BrTIP3;2b had methionine in place of isoleucine in the H5 position, making their pore constriction less hydrophobic than that of AtTIP3;2. Compared with AtNIP5;1, the glycine in the LE1 position was replaced with a much larger alanine in BrNIP5;1b, suggesting that the pore of BrNIP5;1b is more narrow than that of AtNIP5;1. These results suggest that BrTIP3;2a, BrTIP3;2b, and BrNIP5;1b could be involved in the transport of novel solute molecules. Recently, the structures of AQP genes from Populus, Arabidopsis, and rice have been analyzed and compared (Gupta and Sankararamakrishnan 2009). However, structural analysis of the AQP genes of Chinese cabbage had not been carried out. Here, we compared the gene structure of AQP genes in the same AQP subfamily in Arabidopsis and Chinese cabbage (Fig. 6). Generally, the majority of AQP genes were conserved both in the number and positions of introns within each subfamily in Chinese cabbage and Arabidopsis. Intron loss was observed in conserved regions of syntenic AQP genes (PIP2;4, TIP1;1, TIP1;2, TIP1;3, TIP2;2 and TIP2;3, NIP2;1, NIP3;1, NIP5;1) between Chinese cabbage and Arabidopsis, suggesting that intron loss in these AQP genes occurred before the divergence of Chinese cabbage and Arabidopsis from their common ancestor. However, some variation was also observed in the pattern of exon–intron organization of AQP genes between Arabidopsis and Chinese cabbage. Introns loss was observed in BrPIP1;3a, BrPIP1;3b, and BrPIP1;5 of Chinese cabbage, but not in the corresponding orthologous AQP genes in Arabidopsis (Fig. 6), suggesting that the intron loss events of these AQP genes might have happened independently during the evolution of Chinese cabbage to achieve genome slimming (Petrov et al. 1996; Gupta and Sankararamakrishnan 2009). Deviations from the conserved gene structure were observed in three AQP subfamilies of Chinese cabbage (PIP, TIP and NIP). In the NIP subfamily, the second intron was lost in BrNIP2;1s and BrNIP5;1s, and the third intron lost in BrNIP3;1s. In the TIP subfamily, the first intron was lost in BrTIP1;1, BrTIP1;2s, BrTIP2;2 and BrTIP2;3, and the first and second introns lost in BrTIP1;3. In the PIP subfamily, the position of intron 2 in AtPIP2;4

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has changed as a result of the insertion of a new intron and the loss of the old intron 2 in Arabidopsis (Johanson et al. 2001). Both intron gain and loss were also observed in BrPIP2;4a, BrPIP2;4b, and BrPIP2;4c (Fig. 6). Previous studies have indicated that low intron gain rates and intron number reduction are common features of the evolution of plants (Roy and Penny 2007). We also analyzed the expression of AQP genes in different organs. The expression levels of PIP subfamily members in Chinese cabbage were higher than those of the other subfamilies. Their high expression is probably related to their significant function as efficient water channels mediating water uptake in plant cells (Jang et al. 2004; Gomes et al. 2009; Ahmad et al. 2013). Most PIP genes showed higher expression in roots than in other organs. However, BrPIP2;4b, BrPIP2;5a, and BrPIP2;5b were mainly expressed in flowers. These results were similar to results from Arabidopsis. Alexandersson et al. (2005) reported that the AQP genes of Arabidopsis are predominantly expressed in roots or flowers. In Arabidopsis, root cortex cells showed reduced hydraulic conductivity when the root-preferential AtPIP2;2 was knocked out (Javot et al. 2003). Among the TIP gene family, BrTIP1;1 showed high expression in all organs. Similar results were also found in Arabidopsis and maize (Chaumont et al. 2001; Alexandersson et al. 2005; Ahmad et al. 2013). BrTIP2;2 and BrTIP2;3 exhibited root-preferential expression in Chinese cabbage. Similar expression patterns were observed for the corresponding homologs in Arabidopsis (Schmid et al. 2005). In general, NIP subfamily members showed lower expression than the PIP and TIP subfamilies in Chinese cabbage. For example, neither BrNIP2;1a nor BrNIP4;1 transcripts were detected in any organs. BrNIP6;1b was expressed in all five organs and showed the highest expression in roots. BrNIP2;1b of Chinese cabbage showed root-preferential expression like the corresponding gene AtNIP2;1 in Arabidopsis (Schmid et al. 2005). The AtNIPs of Arabidopsis transport a diverse range of substrates, including silicic acid, boric acid, lactic acid, urea, and formamide, depending on the protein sequence (Mitani-Ueno et al. 2011). In the SIP subfamily, BrSIP2;1c was not expressed in flowers, stems or siliques, and rarely in roots and leaves. However, the remaining SIP genes were more or less expressed in all five organs. Similarly, the expression of SIP1s and SIP2;1 of Arabidopsis was also detected in all tissues and suspension-cultured cells, although their levels differed in histochemical analysis of promoter-β-glucuronidase fusions (Ishikawa et al. 2005). SIP1;1 and SIP1;2 may play roles in water transportation in the endoplasmic reticulum. However, SIP2;1 might function as an endoplasmic reticulum channel for other small molecules or ions (Ishikawa et al. 2005). The expression of some AQP genes of Chinese cabbage was distinctly different from that of the corresponding

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Arabidopsis AQP genes. For example, BrPIP2;4c and BrSIP2;1c were homologous to AtPIP2;4 and AtSIP2;1, respectively, and were barely expressed in the five organs. On the contrary, AtPIP2;4 and AtSIP2;1 showed high expression in all organs (Schmid et al. 2005; Ishikawa et al. 2005). Overall, because of obvious differences in the ploidy level and genetic background of these two closely related species, some discrepancies in gene expression existed between Chinese cabbage and Arabidopsis. We also compared the expression of 17 groups of paralogous AQP genes in Chinese cabbage. The results indicated that only four groups of paralogous genes in Chinese cabbage were clustered together (Fig. 7). Most paralogous AQP genes from the same group showed differential expression patterns, suggesting that different paralogous genes in each group play differential roles in Chinese cabbage. Paralogs are genes in the same genome created by gene duplication events that may have different functions (Thornton and DeSalle 2000). Tao et al. (2014) observed that two paralogous genes from B. oleracea L. var. capitata L. (BoKIN1 and BoKIN2) showed discrepancies not only in their expression patterns and transcript levels, but also in premRNA processing. The transcription of plant AQP genes is regulated by environmental stimuli, including drought, cold, and salinity (Hachez et al. 2006; Gomes et al. 2009). Comprehensive analyses of AQP gene expression in response to abiotic stresses have been performed in Arabidopsis (Alexandersson et al. 2005), maize (Zhu et al. 2005), and rice (Li et al. 2008). A cDNA library for whole plants and salt-treated whole plants of B. rapa ssp. pekinensis is available at NCBI (Lee et al. 2008). These data were used to investigate the expression of the AQP genes in Chinese cabbage in response to salinity stress. When roots were soaked in 250 mM NaCl solution, AQP expression showed changes at various levels. The majority of AQP genes showed down-regulation in Chinese cabbage after salt stress except for BrPIP2;1, BrPIP2;6, and BrTIP1;2a (Table 2). Similar results were observed in other plants (Jang et al. 2004; Zhu et al. 2005; Katsuhara et al. 2011). The transcription of AQP genes is reduced to prevent cell death as a result of regular dehydration and to gain time for intracellular osmotic adjustment (Ahmad et al. 2013). Additionally, continuous over-expression of a HvPIP2;1 gene enhanced the root water permeability and salt sensitivity of transgenic rice plants (Katsuhara et al. 2003). In summary, 53 AQP genes from the PIP, TIP, NIP, and SIP subfamilies were found in the Chinese cabbage genome but no XIP genes were obtained. There were 0–3 AQP paralogs in Chinese cabbage corresponding to one syntenic ortholog in Arabidopsis. All of the AQPs of Chinese cabbage shared a conserved hourglass model in 3D structures. Most AQPs of Chinese cabbage showed identical ar/R selectivity

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filter types to their corresponding orthologs in Arabidopsis. The gene structure of most AQP genes was conserved between Chinese cabbage and Arabidopsis, but some discrepancies were observed between them. The expression of AQP genes in different organs showed differences among different families. Most AQP genes showed down-regulation in Chinese cabbage after salt stress. Further research will help us gain a better understanding of the molecular mechanisms of the AQP genes in Chinese cabbage. Acknowledgments  This work was supported by the National Natural Science Foundation of China (31301788, 31372058), the Grand Science and Technology Special Project of Zhejiang Province (2012C12903-3-8), and the China Postdoctoral Science Foundation (2013M540500).

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