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TIP aquaporins in Cyperus esculentus: genome-wide identification, expression profiles, subcellular localizations, and interaction patterns

BMC Plant Biology volume 24, Article number: 298 (2024) Cite this article

Abstract

Background

Tonoplast intrinsic proteins (TIPs), which typically mediate water transport across vacuolar membranes, play an essential role in plant growth, development, and stress responses. However, their characterization in tigernut (Cyperus esculentus L.), an oil-bearing tuber plant of the Cyperaceae family, is still in the infancy.

Results

In this study, a first genome-wide characterization of the TIP subfamily was conducted in tigernut, resulting in ten members representing five previously defined phylogenetic groups, i.e., TIP1–5. Although the gene amounts are equal to that present in two model plants Arabidopsis and rice, the group composition and/or evolution pattern were shown to be different. Except for CeTIP1;3 that has no counterpart in both Arabidopsis and rice, complex orthologous relationships of 1:1, 1:2, 1:3, 2:1, and 2:2 were observed. Expansion of the CeTIP subfamily was contributed by whole-genome duplication (WGD), transposed, and dispersed duplications. In contrast to the recent WGD-derivation of CeTIP3;1/-3;2, synteny analyses indicated that TIP4 and − 5 are old WGD repeats of TIP2, appearing sometime before monocot-eudicot divergence. Expression analysis revealed that CeTIP genes exhibit diverse expression profiles and are subjected to developmental and diurnal fluctuation regulation. Moreover, when transiently overexpressed in tobacco leaves, CeTIP1;1 was shown to locate in the vacuolar membrane and function in homo/heteromultimer, whereas CeTIP2;1 is located in the cell membrane and only function in heteromultimer. Interestingly, CeTIP1;1 could mediate the tonoplast-localization of CeTIP2;1 via protein interaction, implying complex regulatory patterns.

Conclusions

Our findings provide a global view of CeTIP genes, which provide valuable information for further functional analysis and genetic improvement through manipulating key members in tigernut.

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Background

Aquaporins (AQPs), which belong to the ancient major intrinsic protein (MIP) superfamily, constitute a family of small integral membrane proteins facilitating the fast and passive movement of water and other small solutes [1]. In higher plants, AQPs could be classified into five subfamilies on the basis of sequence similarity, i.e., plasma membrane intrinsic protein (PIP), tonoplast intrinsic protein (TIP), NOD26-like intrinsic protein (NIP), small basic intrinsic protein (SIP), and X intrinsic protein (XIP) [2,3,4]. Among them, PIPs and TIPs, which are typically located in cell and vacuolar membranes, respectively, are vital for maintaining proper cytosolic osmolarity in plant cells [5]. In contrast to only two phylogenetic groups present in the PIP subfamily, the TIP subfamily is highly diverse and five groups have been described not only in Arabidopsis (Arabidopsis thaliana (L.) Heynh) but also in rice (Oryza sativa L.), two model plants for eudicots and monocots, respectively, implying their early origin [6, 7]. Interestingly, despite the presence of ten members in both Arabidopsis and rice, their group distribution is distinct, reflecting the occurrence of three independent whole-genome duplication (WGD) events after monocot-eudicot divergence [8, 9]. Functional analyses of TIP genes have been performed in a wide range of plant species, e.g., Arabidopsis, maize (Zea mays L.), rice, wheat (Triticum aestivum L.), and tobacco (Nicotiana tabacum L.) [10,11,12,13,14,15,16,17]. In addition to water, TIPs have also been shown to transport urea, ammonia (NH3), hydrogen peroxide (H2O2), and glycerol, corresponding to diverse aromatic/arginine (ar/R) selectivity filters that determine the substrate specificity [10,11,12,13,14,15,16,17]. The crystal structure of Arabidopsis AtTIP2;1, a water and ammonia transporter, was resolved in 2016 [18]. Compared with other AQPs such as SoPIP2;1 of spinach (Spinacia oleracea L.) [19], the side chain of the conserved R residue in AtTIP2;1 is pushed to the side of the pore by an H residue located in loop C (LC), appearing as a fifth residue of an extended selectivity filter and the relatively wide pore and the polar nature of the selectivity filter allowing the ammonia permeability [18].

Tigernut (Cyperus esculentus L. var. sativus) is an herbaceous perennial C4 plant of the Cyperaceae family within the Poales order, which also includes the well-known Poaceae family [20,21,22,23]. Although most likely originating from the Mediterranean and Southwest Asia, tigernut is now widely distributed in tropical, subtropical, temperate, and even cold zones [21, 24, 25]. It has emerged as a novel oil crop that uniquely accumulates up to 35% of oil in underground tubers [26,27,28]. Despite its strong anti-adversity and wide adaptability [24], the mechanism underlying is poorly understood. Given essential roles of TIPs in plant growth, development, and stress responses [17], in the current study, we took advantage of recently available genome and transcriptome data [29, 30] to identify the complete set of TIP subfamily genes. Their gene localizations, gene structures, sequence characteristics, and evolutionary relationships were comprehensively examined and compared with that of Arabidopsis and rice, which have extensively studied [7, 10, 14]. Moreover, gene expression profiles, protein subcellular localizations, and interaction patterns were also investigated, which facilitate further functional analysis and application.

Results

Identification and classification of TIP genes in tigernut

As shown in Table 1, a total of ten TIP genes were identified from the tigernut genome and all of them were predicted by the genome annotation [30] and detected in the full-length transcriptome [28], supporting their expression. To uncover their evolutionary relationships, an unrooted phylogenetic tree from deduced polypeptides of CeTIP, OsTIP, and AtTIP genes was constructed as shown in Fig. 1a, which clusters ten CeTIP genes into five main groups, i.e., three TIP1s, three TIP2s, two TIP3s, one TIP4, and one TIP5. Among them, TIP1 and − 2 could be divided into several subgroups. Surprisingly, the group distribution in tigernut appears be more similar to Arabidopsis but distinct from rice, though tigernut and rice share a closer biological relationship and both are monocots belonging to Poales [20].

Table 1 List of ten TIP genes identified in tigernut. (AA amino acid, AI aliphatic index, At Arabidopsis thaliana, Scf scaffold, GRAVY grand average of hydropathicity, kDa kilodalton, MIP major intrinsic protein, MW molecular weight, Os Oryza sativa, TIP tonoplast intrinsic protein, TM transmembrane helix, WGD whole-genome duplication)
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Fig. 1
figure 1

Phylogenetic analysis and structural features of the TIP subfamily in tigernut, rice and Arabidopsis. a Shown is an unrooted phylogenetic tree resulting from full-length TIPs with MEGA6 (maximum likelihood method and bootstrap of 1,000 replicates), where the distance scale denotes the number of amino acid substitutions per site and the name of each group is indicated next to the corresponding clade. b Shown are the exon-intron structures. 0 and 1 indicate intron phases. c Shown is the distribution of conserved motifs among TIPs, where different motifs are represented by different color blocks as indicated and the same color block in different proteins indicates a certain motif. d Shown are the conserved residues such as dual NPA motifs and Ar/R selectivity filter. Unusual residues are marked in red. (At Arabidopsis thaliana, Ar/R aromatic/arginine, bp base pair, Ce Cyperus esculentus, H2 transmembrane helix 2, H5 transmembrane helix 5, LB loop B, LE loop E, NPA Asn-Pro-Ala, Os Oryza sativa, TIP tonoplast intrinsic protein)

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The coding sequence (CDS) length of CeTIP genes varies from 744 to 789 base pairs (bp), putatively encoding 247–262 amino acids (AA) with the molecular weight (MW) of 24.73–27.12 kilodalton (kDa) (Table 1), which is comparative to that observed in rice (i.e. 248–269 AA; 24.88–27.55 kDa) and Arabidopsis (i.e. 249–268 AA; 25.03–28.31 kDa) (Additional file 1). Whereas most members are acidic with the theoretical pI value of less than 7.00 (i.e. 5.09–6.75), CeTIP3;2 and − 5 appear to be basic (Table 1). It’s worth noting that OsTIP3;1, OsTIP3;2, OsTIP5;1, AtTIP3;1, and AtTIP5;1 were also shown to be basic (Additional file 1). Without any exception, all CeTIPs exhibit the amphipathic property with high aliphatic index (AI) values (i.e. 97.63–119.40) and grand average of hydropathicity (GRAVY) values of more than 0 (i.e. 0.537–1.005) (Table 1), which is consistent with that observed in rice and Arabidopsis (Additional file 1). Sequence similarities between CeTIP proteins vary from 51.60 to 92.43%, where 73.73–85.83%, 82.54–92.43%, and 75.39% were found within three multi-member groups, i.e., TIP1, -2, and − 3. Compared with other groups, TIP5 is relatively distinct, only sharing 51.60–59.55% sequence similarities (Additional file 2). Interestingly, except for CeTIP1;3, all other members have one to three orthologs in rice and Arabidopsis (Table 1), implying lineage or species-specific evolution after their divergence. Significantly, despite the presence of two TIP3s in all three species tested, 2:2 but not 1:1 orthologous relationships were observed (Table 1), implying their birth after speciation.

Gene localization and synteny analysis of CeTIP genes

To learn more about species-specific evolution patterns, gene localization and synteny analysis were further conducted. As shown in Fig. 2a, gene localization showed that ten CeTIP genes are unevenly distributed over seven scaffolds (Scfs), where 71.43% of them possess a single gene, one contains two, and Scf54 includes the maximum of three, i.e., CeTIP3;2, -5;1, and − 2;2. Interestingly, CeTIP5;1 and − 2;2, which share 59.55% sequence similarity at the protein level (Additional file 2), could be defined as tandem repeats spacing only 1,254 bp, whereas CeTIP1;1 and − 2;3 were characterized as transposed repeats of CeTIP2;1 (Fig. 2a). According to synteny analysis performed within tigernut, CeTIP2;1/-4;1, CeTIP2;3/-5;1, and CeTIP3;1/-3;2, which respectively exhibit 70.92%, 57.04%, and 75.39% sequence similarities at the protein level (Additional file 2), were shown to locate within syntenic blocks and thus were defined as WGD repeats (Fig. 2b). Additionally, CeTIP2;2 and − 2;3, which share 92.43% sequence similarity at the protein level (Additional file 2), were characterized as dispersed repeats, and CeTIP1;2, -1;3, and − 3;1 were also defined as dispersed repeats of CeTIP1;1 (Fig. 2a). By contrast, relatively less WGD repeats were identified in rice (i.e. OsTIP2;1/-5;1 and OsTIP4;1/-4;3) and Arabidopsis (i.e. AtTIP2;2/2;3 and AtTIP3;1/-3;2) (Fig. 2b), though both of them experienced three rounds of WGDs after monocot-eudicot divergence [8, 9]. According to inter-species syntenic analysis, six CeTIP genes were shown to have syntelogs in rice, including 1:1, 1:2, and 2:1, in striking contrast to only two found in Arabidopsis (i.e. AtTIP2;2 and AtTIP2;3 vs. CeTIP2;3) (Fig. 2b), reflecting lineage or species-specific duplication events followed by chromosomal rearrangement and gene transposition [31, 32]. These results together with BRH (best reciprocal hit)-based orthologous analysis suggest that (1) TIP2 is more likely to give rise to TIP4 and − 5 via WGDs, occurred sometime before monocot-eudicot divergence; (2) TIP2 had also diverged into two subgroup (i.e. TIP2A and -B), occurred sometime before monocot-eudicot split; (3) similar to the α WGD-derivation of AtTIP3;1/-3;2, independent expansion of TIP3 may occur sometime after the split of Cyperaceae and Poaceae, e.g., CeTIP3;1/-3;2, and OsTIP3;1/-3;2; (4) Cyperaceae-specific CeTIP1;3 may be generated sometime after Cyperaceae-Poaceae split since its orthologs were also not found in other monocots with genome sequences available in Phytozome; (5) like TIP3, -4 expansion observed in rice was derived from tandem duplication and the Poaceae-specific ρ WGD, and their orthologs were widely found in Poaceae species (Additional file 3).

Fig. 2
figure 2

Duplication events of CeTIP genes and synteny analysis within and between tigernut, rice, and Arabidopsis. a Duplication events detected in tigernut. Serial numbers are indicated at the top of each scaffold, and the scale is in Mb. Duplicate pairs identified in this study are connected using lines in different colors, i.e., tandem (shown in green), transposed (shown in purple), dispersed (shown in gold), and WGD (shown in red). b Synteny analysis within and between tigernut, rice, and Arabidopsis. Shown are TIP-encoding chromosomes/scaffolds (tigernut, pink; rice, dark green; Arabidopsis, bluish-grey) and only syntenic blocks that contain TIP genes are marked (intra-species, red; inter-species, purple). (At Arabidopsis thaliana, Ce Cyperus esculentus, Mb megabase, Os Oryza sativa, Scf scaffold, TIP tonoplast intrinsic protein, WGD whole-genome duplication)

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Analyses of exon-intron organization and conserved motifs

To gain insights into structural divergence of CeTIP genes, their gene structures and conserved motifs of deduced proteins were further investigated and compared with that of rice and Arabidopsis. As shown in Fig. 1b, all CeTIP genes possess two introns and three exons, by contrast, nearly half of TIP genes in rice and Arabidopsis harbor a single intron and AtTIP1;3 is intronless. Genes with less than two introns were found in TIP1 and − 4 of rice and TIP1 and − 2 of Arabidopsis, respectively (Fig. 1b). Without any exception, all CeTIP genes feature the typical GT/AG splice junction. Generally, intron 1 is located near the end of transmembrane helix 1 (TM1), whereas intron 2 is located just before the A/R/E/S/P/G/H codon of the LC. Whereas intron 1 is the phase 1 intron that is located within codons, intron 2 is usually the phase 0 intron that is located between codons (Fig. 1b). Nevertheless, intron 2 of both CeTIP1;1 and − 1;3 exhibits the phase 1 intron (Fig. 1b), which was shown to locate at the C-terminus of TM4 and the loop D (LD), respectively (Additional file 4).

Fifteen motifs identified using MEME are shown in Fig. 1c. Only Motifs 2 and 3, which are characterized as TM1 and LB-TM3 (Additional file 4), are shared by all 30 TIPs examined. Motifs 1 and 4–7 are also broadly present: Motif 1, which is characterized as TM5-LE-TM6, is replaced by Motif 8 in OsTIP4;1 and all TIP5s; Motif 4, which is characterized as LC-TM4-LD, is absent from OsTIP3;2 and OsTIP4;3 and replaced by Motif 9 in all TIP5s; Motif 5, which is characterized as LA-TM2, is absent from AtTIP4;1, OsTIP4;1, OsTIP4;2, and AtTIP5;1; Motif 5, which is characterized as a part of TM6, is absent from CeTIP5;1 and OsTIP5;1; Motif 5, which is located at the C-terminus, is absent from CeTIP3;2, CeTIP4;1, AtTIP2;2, AtTIP2;3, AtTIP5;1, and OsTIP5;1 and replaced by Motif 14 in CeTIP2;2, OsTIP2;1, OsTIP4;2, and CeTIP5;1. Motifs 10, 11, and 15, which are located at the N-terminus, are mainly present in TIP1s, TIP2s/TIP4s, and AtTIP3s, respectively. Motif 12, which is located at LC, is restricted to TIP3 and replaced by Motif 13 in TIP4s, AtTIP2;1, AtTIP2;2, AtTIP2;3, CeTIP2;3, and AtTIP1;1 (Fig. 1c and Additional file 4). It’s worth noting that the motif composition in TIP5s is obviously different from other groups, reflecting their sequence divergence. Nevertheless, all of them harbor one conserved MIP domain that includes six typical TMs (TM1–6), two half helixes (HB and HE), and two typical NPA motifs as observed in SoPIP2;1 [19], though the ar/R selectivity filter is variable (Fig. 1d). Among them, CeTIP1;3 exhibits the H-I-A-A variant in contrast to the H-I-A-V filter widely present in TIP1. TIP3 and − 4 usually feature the H-I-A-R filter, however, CeTIP3;1 exhibits the H-T-A-R variant that is not observed in other members. Whereas all TIP2s feature the H-I-G-R filter, CeTIP5;1 exhibits the Q-V-G-R filter that is different from that of both OsTIP5;1 (Q-V-A-R) and AtTIP5;1 (N-V-G-C). An L residue, corresponding to the L197 of SoPIP2;1 that was proven to be involved in gating [19], is only present in CeTIP4;1, OsTIP4;3, and OsTIP5;1. In most cases, this position is replaced by an I residue. An H residue, corresponding to the H197 of AtPIP2;2 that was reported to undergo gating by cytosolic pH via protonation [33], is found in CeTIP3;1 and OsTIP3;1. Interestingly, an H residue, corresponding to the H131 in the LC of AtTIP2;1 and VvTIP2;1 that is essential for ammonia permeability [18] and involved in gating by cytosolic pH [34], was found not only in TIP2s but also TIP4s, implying their similar functions.

Global expression profiles of CeTIP genes

To reveal the global expression profiles of CeTIP genes, RNA-seq data for nine tissues/developmental stages with three biological repeats each were first examined, i.e., two stages of developmental leaf (young and mature), sheath of mature leaf, root, rhizome, shoot apex, and three stages of developmental tuber (40, 80, and 120 days after sowing (DAS)). As shown in Fig. 3a, the total CeTIP transcripts were most abundant in roots, followed by rhizomes, moderate in sheaths, young leaves, and tubers of 120 and 80 DAS, and relatively low in mature leaves, tubers of 40 DAS, and shoot apexes. Interestingly, except for roots and tubers of 40 DAS, CeTIP1;1 and − 2;1 represent two dominant members in all other samples examined, contributing 82.46–95.72% of total TIP transcripts. In roots, CeTIP2;2 and − 2;3 were shown to express more than − 1;1 and − 2;1, and these four members constituted more than 91.89% of total TIP transcripts. Additionally, CeTIP1;2 and − 4;1 were also abundant in roots, whose transcripts were shown to be comparative or considerably more than that of most other samples. In shoot apexes, CeTIP4;1 represented the third most expressed member, contributing 15.02% of total TIP transcripts. By contrast, this gene was rarely expressed in leaves and sheaths. Interestingly, CeTIP3;1 appeared to be tuber-specific and highly abundant in tubers of 120 DAS, whereas CeTIP3;2 was lowly expressed in all samples examined.

Fig. 3
figure 3

Expression profiles of CeTIP genes. a Tissue-specific expression profiles of CeTIP genes. DAS40, DAS80, and DAS120 represent tubers of 40, 80 and 120 DAS. Color scale represents FKPM normalized log2 transformed counts where blue and red indicate low and high expression, respectively. b Expression profiles of CeTIP1;1 and − 2;1 during leaf development. c Diurnal fluctuation expression patterns of CeTIP1;1 and − 2;1 in mature leaf. d Expression profiles of CeTIP1;1 and − 2;1 during tuber development. For qRT-PCR analysis, CeUCE2 and CeTIP41 were used as two reference genes. Bars and error bars indicate the mean ± SD (n = 3) and uppercase letters indicate difference significance tested following Duncan’s one-way multiple-range post hoc ANOVA (P < 0.01). (Ce Cyperus esculentus, DAI days after tuber initiation, DAS days after sowing, TIP tonoplast intrinsic protein)

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Expression profiles of CeTIP genes during leaf development and diurnal fluctuation

As shown in Fig. 3a, most CeTIP genes were significantly down-regulated in mature leaves relative to young leaves, including two dominant genes CeTIP1;1 and − 2;1. However, this is the case under a natural condition. To provide a more accurate determination, plants grown in a greenhouse as described before [23] were adopted and three representative stages of developmental leaf (i.e. young, mature, and senescing) were subjected to qRT-PCR analysis. As shown in Fig. 3b, both CeTIP1;1 and − 2;1 exhibited a bell-shaped expression pattern, peaking in mature leaves, though the difference of CeTIP1;1 between young and mature leaves was not significant.

Diurnal fluctuation expression patterns of CeTIP1;1 and − 2;1 in mature leaves were further examined. As shown in Fig. 3c, since the onset of light at 8 h, their transcripts usually increased along with the extension of the light time, peaking at 16 h and 12 h, respectively, though a slight drop was observed at 12 h for CeTIP1;1. Whereas CeTIP2;1 was expressed more in light (i.e. 8 h, 12 h, 16 h, and 20 h) relative to dark (i.e. 24 h and 4 h), the transcripts of CeTIP1;1 at 8 h and 12 h were significantly lower than that in dark (Fig. 3c).

Expression profiles of CeTIP genes during tuber development

The oil-bearing tubers are derived from rhizomes and the development process includes three main stages, i.e., initiation, swelling, and maturation, spanning approximately 35 d [22]. In contrast to gradual increase of dry weight and oil during tuber development, the water content usually maintains up to 85.0% until a significant drop to less than 45.0% at the stage of maturation [22, 26, 28], implying a key role of water for tuber development and metabolism. Among three swelling stages profiled, except for CeTIP1;3 that peaked at 40 DAS, transcripts of CeTIP2;1 and − 3;1 were shown to gradually increase, whereas five genes (i.e. CeTIP1;1, -1;2, -2;2, -2;3, and − 4;1) exhibited an apparent unimodal expression pattern, peaking at 80 DAS (Fig. 3a).

For qRT-PCR analysis, seven stages were examined, i.e., 1, 5, 10, 15, 20, 25, and 35 days after tuber initiation (DAI), representing initiation, five stages of swelling, and maturation as described before [22]. As shown in Fig. 3d, CeTIP1;1 exhibited a nearly bell-shaped expression pattern, peaking at 15 DAI, which is similar to the transcriptome profiling. By contrast, a bimodal expression pattern was observed for CeTIP2;1, peaking at 10 and 25 DAI, respectively. It’s worth noting that, for both genes, transcripts at the maturation stage were significantly lower than any other stage (Fig. 3d), corresponding to relatively low water content at this stage [22].

Subcellular localization analysis

According to bioinformatic prediction using Plant-mPLoc, all CeTIP proteins were shown to localize to the tonoplast. To verify the result, two dominant members CeTIP1;1 and − 2;1 were selected for experimental confirmation. As shown in Fig. 4, green fluorescence signals of CeTIP1;1-GFP were widely detected in vacuoles of tobacco leaves, highly coinciding with the tonoplast marker AtTIP1;1-RFP [10]. By contrast, to our surprise, fluorescence signals of CeTIP2;1-GFP were only found in cell membranes, coinciding with the plasma membrane marker HbPIP2;3-RFP [35].

Fig. 4
figure 4

Subcellular localization analysis of CeTIP1;1 and − 2;1 in tobacco leaves. AtTIP1;1-RFP and HbPIP2;3-RFP were used as markers for tonoplast and plasma membrane, respectively. (Ce Cyperus esculentus, TIP tonoplast intrinsic protein)

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Protein interaction patterns

Constitutive expression and high abundance of CeTIP1;1 and − 2;1 (Fig. 3a) suggest that their encoded proteins may interact with each other. To confirm the hypothesis, bimolecular fluorescence complementation (BiFC) analysis was conducted. As shown in Fig. 5, in contrast to no signals were detected for four negative controls, green fluorescence signals were found in vacuoles of tobacco leaves co-transformed with Ecn-CeTIP1;1 and Enn-CeTIP2;1, supporting their interaction. Moreover, homologous interaction was also observed for CeTIP1;1 but not − 2;1. These results imply that CeTIP1;1 is more likely to function in homo and heteromultimer, whereas CeTIP2;1 functions in heteromultimer (Fig. 5).

Fig. 5
figure 5

BiFC-based protein interaction of CeTIP1;1 and CeTIP2;1 in tobacco leaves. Tobacco leaves transformed with pNC-BiFC-Ecn-CeTIP1;1, pNC-BiFC-Enn-CeTIP1;1, pNC-BiFC-Ecn-CeTIP2;1, or pNC-BiFC-Ecn-CeTIP2;1 were used as negative controls. (BiFC bimolecular fluorescence complementation, Ce Cyperus esculentus, TIP tonoplast intrinsic protein)

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Discussion

The tigernut genome encodes ten TIP genes with two introns

Since the first TIP gene (i.e. AtTIP1;1 orγ-TIP1) was characterized in Arabidopsis [10], its homologs have been identified in a high number of plant species, varying from eight to more than 23 members in soybean (Glycine max (L.) Merr.) [10, 16, 17, 36, 37]. In the present study, a number of ten TIP genes that is equal to that of Arabidopsis were identified from tigernut, an oil-bearing tuber plant of the Cyperaceae family, which represents a sister family to Poaceae within the Poales order [20]. The CeTIP gene amounts are comparative to or relatively less than 9–15 members present in most Poaceae plants, e.g., rice, barley (Hordeum vulgare L.), Brachypodium distachyon (L.) P. Beauv., foxtail millet (Setaria italic (L.) P. Beauv.), green foxtail (S. viridis (L.) P. Beauv.), sorghum (Sorghum bicolor (L.) Moench), and maize [7, 16, 38,39,40], Additional file 3. Nevertheless, all these genes could be assigned into five phylogenetic groups as defined in Arabidopsis, i.e., TIP1–5 [6]. Moreover, in contrast to a high number of TIP genes present in Arabidopsis and rice harboring no or a single intron, all CeTIP genes feature two introns. Constant positions of two introns in most TIP genes support independent loss of one or both introns, whereas varied intron phase and locations of intron 2 in CeTIP1;1 and − 1;3 imply the initial loss followed by gain of a new intron. Similar phenomenon was also reported for AtPIP2;4 [6].

Expansion of the CeTIP subfamily was contributed by WGD, transposed, and dispersed duplications

Gene duplication, which may originate from WGD, tandem, proximal, dispersed, and transposed duplications, is a major mechanism for acquiring new genes [41]. Increasing evidences showed that WGDs are widespread and play an important role in the diversification of angiosperms [42, 43]. For example, after monocot-eudicot split, the model eudicot Arabidopsis experienced three rounds of WGDs named γ, β, and α, respectively [8], whereas the model monocot rice experienced three WGDs known as τ, σ, and ρ, respectively [9]. The ρ WGD identified in rice is Poaceae-specific, whereas τ and σ WGDs are shared by Cyperaceae plants, which may also experience at least one recent WGD [22, 23, 44,45,46]. Wide presence of five phylogenetic groups in both monocots and eudicots [3, 4, 16, 36, 40, 47,48,49, Additional file 3] support their early diversification. Interestingly, our data from synteny analyses provides the first evidence that both TIP4 and − 5 were derived from TIP2 via WGDs, occurred sometime before monocot-eudicot divergence. As for TIP5, CeTIP2;3, CeTIP5;1, OsTIP2;1, OsTIP5;1, AtTIP2;2, and AtTIP2;3 are still located within syntenic blocks, whereas for TIP4, CeTIP2;1, CeTIP4;1, OsTIP2;2, OsTIP4;1, OsTIP4;3, and AtTIP2;1 remain within syntenic blocks (Fig. 2b). Considering the distinct relationship of TIP5, if TIP2 is the primitive one, it may first give rise to -5 followed by -4. During later evolution, TIP2 generated − 1 and − 3 via transposed and dispersed duplications, respectively. More recently but before monocot-eudicot divergence, TIP2 and − 1 diverged into two subgroups via a yet unknown mechanism, i.e., TIP2A, -2B, -1 A, and − 1B. Thereby, from an evolutionary perspective, it is more suitable to classify TIP2 and − 1 into four groups. By contrast, expansion of TIP3 in tigernut appears to be lineage-specific, occurred sometime after Cyperaceae and Poaceae split or more likely by the recent WGD described in Carex littledalei C. B. Clarke (formerly known as Kobresia littledalei) [46], which is similar to the α WGD-derivation of AtTIP3;1/-3;2 (Additional file 1); expansion of TIP4 via both the ρ WGD and tandem duplication observed in rice seems to be Poaceae-specific, though the counterpart of OsTIP4;2 has lost in barley (Additional file 3). Nevertheless, we are not sure about the exact time and mechanism that CeTIP1;3 was generated, which is more distinct from CeTIP1;1 and − 1;2 but doesn’t seem to have counterparts in other examined species, including seven representative Poaceae species. In fact, besides the evolutionary distance, CeTIP1;3 differs from other TIP1s in both intron phase and ar/R selectivity filter (H-I-A-A vs. H-I-A-V), implying possible functional divergence. Additionally, TIP2 has evolved into the H-I-G-R filter, whereas TIP3 and − 4 usually exhibit the H-I-A-R filter, though some variants were also found (Fig. 1d). Moreover, we also identified two motifs specific to TIP3 (Motif 12) and − 5 (Motif 9), respectively (Fig. 1c).

CeTIP genes exhibit diverse expression profiles and are subjected to development and diurnal fluctuation regulation

Uncovering the gene expression profiles is the first step to address the importance and specific biological functions of a gene in a special tissue or certain developmental stages. According to our transcriptional profiling, CeTIP1;1 and − 2;1 were shown to constitutively express and represent two dominant members in most tissues examined, whereas other genes are relatively less expressed or even tissue-specific, e.g., tuber-specific expression of CeTIP3;1 (Fig. 3a). CeTIP1;1 has one and two orthologs in rice and Arabidopsis, respectively, i.e., OsTIP1;1, AtTIP1;1, and AtTIP1;2, where AtTIP1;2 is characterized as a recent transposed repeat of AtTIP1;1 (Additional file 1). By contrast, CeTIP2;1 possesses a single ortholog in both rice and Arabidopsis, i.e., OsTIP2;2 and AtTIP2;1, where CeTIP2;1 and OsTIP2;2 are still located within syntenic blocks (Fig. 2b), implying a conserved evolution. Interestingly, OsTIP1;1, OsTIP2;2, AtTIP1;1, AtTIP1;2, and AtTIP2;1 were also shown to be highly abundant in most tissues [16, 17, 51, 52], implying their essential roles. Possible functions of CeTIP1;1 and − 2;1 could be inferred from their orthologs, which have been proven to transport water, urea, NH3, H2O2, and glycerol [10, 13, 14, 53,54,55,56,57]. Studies showed that AtTIP1;1 expression is primarily correlated with vacuolation and cell enlargement [50]. Interestingly, despite a long time of evolution, AtTIP1;1, -1;2, and − 2;1 are redundantly involved in the emergence of new lateral root primordial [58]. CeTIP1;2, a dispersed repeat of CeTIP1;1 with a high similarity of 85.83%, also exhibits a constitutive expression pattern. However, its transcript levels are considerably lower than that of CeTIP1;1 and − 2;1, and its orthologs in rice and Arabidopsis, i.e., OsTIP1;2 and AtTIP1;3, have been proven to transport water, urea, and glycerol [57, 59]. Interestingly, in Arabidopsis, AtTIP1;3 is characterized as the pollen-specific and highly abundant AQP gene [59]. CeTIP2;2 and − 2;3, two recent dispersed repeats, are preferentially expressed in roots and rhizomes. They have one or two orthologs in rice and Arabidopsis, respectively, i.e., OsTIP2;1, AtTIP2;2, and AtTIP2;3, which have been proven to transport water, NH3, and H2O2 [56, 60]. In rice, OsTIP2;1 is preferentially expressed in roots [7]. Despite recent origin and remaining within syntenic blocks, CeTIP3;1 and − 3;2 exhibit distinct expression pattern, where CeTIP3;1 is tuber-specific gradually increasing during tuber development and − 3;2 is lowly expressed in all tissues examined in this study. Their orthologs in Arabidopsis, i.e., AtTIP3;1 and − 3;2, exhibit a strict embryo and endosperm-specific expression pattern [61], in contrast to endosperm-preferential expression of RcTIP3;1 in castor bean (Ricinus communis L.) [3]. Interestingly, OsTIP3;2 was proven to transport glycerol, which together with seed-specific expression of TIP3s in oilcrops such as canola (Brassica napus L.), soybean, peanut (Arachis hypogaea L.), and flax (Linum usitatissimum L.) imply a putative role in regulating oil content [17]. CeTIP4;1, typically expressed in tubers (downregulated during tuber maturation), roots, rhizomes as well as shoot apexes, has one or three orthologs in arabidopsis and rice, respectively, i.e., AtTIP4;1, OsTIP4;1, OsTIP4;2, and OsTIP4;3, which have been proven to transport water, urea, and glycerol [14, 54].

Diurnal variation of AQP genes has been reported in several species [7, 62]. For example, the transcripts of OsTIP1;2 and − 2;1 in roots were shown to exhibit a clear diurnal change with a large amplitude, peaking in light and dropping to the basal level in dark [7]. Similar trend was also observed in this study, and transcripts of both CeTIP1;1 and − 2;1 peaked in the daytime, 8 h–4 h after the onset of light, respectively (Fig. 3c).

Distinct subcellular localizations and interaction patterns of CeTIP1;1 and − 2;1

As the name suggests, TIPs are located mostly in tonoplasts of plant cells, which function in tetramers [5, 10, 18]. Interestingly, CeTIP1;1 and − 2;1, two dominant CeTIP genes identified in tigernut, encode proteins with distinct subcellular localizations and interaction patterns. When transiently overexpressed in tobacco leaves, CeTIP1;1 is clearly located in the vacuole membrane, in contrast to CeTIP2;1 that is located in the cell membrane. Moreover, CeTIP2;1 could not interact itself, whereas CeTIP1;1 functions in homo/heteromultimer and could mediate the tonoplast-localization of CeTIP2;1. It’s worth noting that proteomic analysis suggests the presence of AtTIPs in the thylakoid membrane of chloroplast [63], and AtTIP3s were shown to localize in both the plasma membrane and tonoplast of maturing and germinating seeds [61]. Compared with PIPs, only a few studies have been performed to investigate the interaction pattern of TIPs. In Arabidopsis, AtTIP2;1 could not only interact itself but also interact with AtTIP1;2 and − 3;1 [64]. Thereby, the reason for why CeTIP2;1 could not form homomultimer needs to be further studied.

Conclusions

To our knowledge, this is the first genome-wide characterization of the TIP subfamily in tigernut, an oil-bearing tuber plant of the Cyperaceae family. Ten members representing five phylogenetic groups identified in tigernut are equal to that present in two model plants Arabidopsis and rice, however, the group composition and/or evolution pattern are different and complex orthologous relationships were observed. Expansion of the CeTIP subfamily was contributed by WGD, transposed, and dispersed duplications. Whereas CeTIP3;1/-3;2 are recent WGD repeats, TIP4 and − 5 were proven to be old WGD repeats of TIP2, appearing sometime before monocot-eudicot divergence. CeTIP genes exhibit diverse expression profiles and are subjected to developmental and diurnal fluctuation regulation. Moreover, CeTIP1;1 and − 2;1, two dominant members, exhibit distinct subcellular localizations and interaction patterns. These findings provide valuable information for further functional analysis and genetic improvement through manipulating key members such as CeTIP1;1 and − 2;1 in tigernut.

Materials and methods

Datasets and identification of TIP genes

As shown in Additional file 1, TIP genes described in Arabidopsis and rice were obtained from TAIR11 (https://www.arabidopsis.org/) and RGAP7 (http://rice.plantbiology.msu.edu/), respectively. Their deduced proteins were used for TBLASTN (E-value, 1e–10) search of tigernut genomic and transcriptome data that were accessed from CNGBdb (https://db.cngb.org/search/assembly/CNA0051961/) and NCBI (https://www.ncbi.nlm.nih.gov/). Gene prediction and exon-intron revision with available mRNAs were conducted as previously described [48]. Briefly, gene structures of candidates were predicted using GeneMark.hmm eukaryotic (http://topaz.gatech.edu/GeneMark/gmhmme.cgi) when no gene models are available, and then manual revision was performed through aligning mRNAs (including full-length transcripts obtained via the Single-Molecule Real-Time (SMRT) sequencing) [29] to the gene sequences. Open reading frames (ORFs) of candidates were predicted using ORF Finder (http://www.bioinformatics.org/sms2/orf_find.html). Presence of the conserved MIP domain in deduced peptides was confirmed by MOTIF Search (https://www.genome.jp/tools/motif/), and physiochemical parameters were calculated using ProtParam (http://web.expasy.org/protparam/). Protein subcellular localization was predicted using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/), and gene structure was displayed using GSDS 2.0 (http://gsds.gao-lab.org/).

Synteny analysis and characterization of orthologs

Synteny analysis was conducted as previously described [28, 65], where duplicate pairs were identified using the all-to-all BLASTP method and syntenic blocks were inferred using MCScanX implemented in TBtools-II [66]: E-value, 1e-10; BLAST hits, 5. Orthologs between different species were identified using the BRH method and information from synteny analysis, whereas different modes of gene duplication were identified using the DupGen_finder pipeline [43].

Sequence alignment, phylogenetic analysis, and characterization of conserved residues/motifs

Multiple sequence alignment was conducted using MUSCLE [67] (gap open: -2.9; gap extend: 0; hydrophobicity multiplier: 1.2; clustering method: UPGMB) and phylogenetic tree was constructed using MEGA6 [68] with the maximum likelihood method (bootstrap method: 1,000 replicates; substitution model: amino acid and Jones-Taylor-Thornton (JTT) model; rates among sites: uniform rates; gaps/missing data treatment: complete deletion; ML heuristic method: Nearest-Neighbor-Interchange (NNI); initial tree for ML: Make initial tree automatically (Default - NJ/BioNJ)). Subclassification of TIPs into groups was done as described before [3, 6]. TMs and conserved residues were identified from the alignment with the structure resolved SoPIP2;1 and AtTIP2;1 [18, 19]. Conserved motifs were identified using MEME (v5.4.1, https://meme-suite.org/tools/meme) with optimized parameters of any number of repetitions, maximum number of 15 motifs, and the width of 6 and 250 residues for each motif.

Plant materials

For gene cloning and expression analysis, the tigernut variety Reyan3 as described before [23, 28] was used, and plants were grown in a greenhouse under the condition of 25 ± 0.5℃, 60–70% relative humidity, 5,000 lx, and a 12 h light/12 h dark cycle. For a day, the period from 8 a.m to 8 p.m was set to light, and mature leaves with dark green in appearance were sampled every four hours from the onset of light. Three representative stages of developmental leaf, i.e., young, mature, and senescing, were also collected at 8 a.m, where the chlorophyll content of young and senescing leaves are just a half relative to mature leaves. As for representative stages of developmental tuber, fresh tubers at 1, 5, 10, 15, 20, 25, and 35 DAI were collected as described before [22]. All samples with three biological replicates were quickly freezed with liquid nitrogen and stored at -80℃. For subcellular localization and BiFC analyses, N. benthamiana L. plants were also grown in the greenhouse as described above.

Gene expression analysis

Global expression profiles of CeTIP genes were investigated using Illumina RNA-seq samples as shown in Additional file 5, which are paired reads with 150 bp. Raw reads in the FASTQ format were obtained using fastq-dump, and quality control was performed using Trimmomatic [69]. Read mapping was performed using HISAT2 (v2.2.1) [70] with default parameters and relative transcript levels were presented in FPKM (Fragments per kilobase of exon per million fragments mapped). Differential expression genes were identified using DESeq2 (v1.22.1) [71] with parameters of “log2Fold Change > = 1” and FDR < 0.05. For qRT-PCR analysis, total RNAs were extracted using the RNAprep Pure Plant Kit (Tiangen Biotech Co., Beijing, China); synthesis of the first-strand cDNA from high quality RNAs was carried out using the PrimeScript® RT reagent kit with gDNA Eraser (Takara, Dalian, China); and PCR reaction was carried out as described before [23]. Primers used in this study are shown in Additional file 6, where CeUCE2 and CeTIP41 were used as two reference genes. The relative expression level of target genes was normalized using the 2−ΔΔCt method and statistical significance was performed using SPSS Statistics 20, where difference significance was tested following Duncan’s one-way multiple-range post hoc ANOVA.

Subcellular localization and BiFC

Primers used for plasmid construction are shown in Additional file 6. Full-length transcripts of CeTIP1;1 and − 2;1 were first isolated using CeTIP1;1F/R and CeTIP2;1F/R as primers, respectively. Then, the PCR products were employed as template, and the CDS without the termination codon were cloned to pNC-Cam1304-SubC, pNC-BiFC-Ecn, and pNC-BiFC-Enn using CeTIP1;1HF/R and CeTIP2;1HF/R as primers. The resulted recombinant plasmids, i.e., pNC-Cam1304-CeTIP1;1, pNC-Cam1304-CeTIP2;1, pNC-BiFC-Ecn-CeTIP1;1, pNC-BiFC-Ecn-CeTIP2;1, pNC-BiFC-Enn-CeTIP1;1, and pNC-BiFC-Ecn-CeTIP2;1 were introduced into Agrobacterium tumefaciens GV3101 with the helper plasmid pSoup-P19 as described before [72]. A. tumefaciens-mediated transformation was carried out as previously described [28, 72], where tobacco leaves of about 4-week-old plants were used as the receptor. About 48 h after infiltration, transformed leaves were processed for confocal laser scanning microscopy imaging (Zeiss LMS880, Germany). For subcellular localization analysis, markers for tonoplast (AtTIP1;1-RFP [10]) and plasma membrane (HbPIP2;3-RFP [35]) were also co-transformed.

Data availability

The datasets analyzed during the current study are available in the NCBI SRA repository (https://www.ncbi.nlm.nih.gov/sra/) and detailed accession numbers can be found in Additional file 5.

References

  1. Maurel C, Verdoucq L, Luu DT, Santoni V. Plant aquaporins: membrane channels with multiple integrated functions. Annu Rev Plant Biol. 2008;59:595–624.

    Article  CAS  PubMed  Google Scholar 

  2. Abascal F, Irisarri I, Zardoya R. Diversity and evolution of membrane intrinsic proteins. Biochim Biophys Acta. 2014;1840(5):1468–81.

    Article  CAS  PubMed  Google Scholar 

  3. Zou Z, Gong J, Huang QX, Mo YY, Yang LF, Xie G. Gene structures, evolution, classification and expression profiles of the aquaporin gene family in castor bean (Ricinus communis L). PLoS ONE. 2015;10(10):e0141022.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Zou Z, Yang J. Genome-wide comparison reveals divergence of cassava and rubber aquaporin family genes after the recent whole-genome duplication. BMC Genomics. 2019;20(1):380.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Wudick MM, Luu DT, Maurel C. A look inside: localization patterns and functions of intracellular plant aquaporins. New Phytol. 2009;184(2):289–302.

    Article  CAS  PubMed  Google Scholar 

  6. Johanson U, Karlsson M, Johansson I, Gustavsson S, Sjövall S, Fraysse L, et al. The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Plant Physiol. 2001;126(4):1358–69.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sakurai J, Ishikawa F, Yamaguchi T, Uemura M, Maeshima M. Identification of 33 rice aquaporin genes and analysis of their expression and function. Plant Cell Physiol. 2005;46(9):1568–77.

    Article  CAS  PubMed  Google Scholar 

  8. Jiao Y, Leebens-Mack J, Ayyampalayam S, Bowers JE, McKain MR, McNeal J, et al. A genome triplication associated with early diversification of the core eudicots. Genome Biol. 2012;13(1):R3.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Jiao Y, Li J, Tang H, Paterson AH. Integrated syntenic and phylogenomic analyses reveal an ancient genome duplication in monocots. Plant Cell. 2014;26(7):2792–802.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Maurel C, Reizer J, Schroeder JI, Chrispeels MJ. The vacuolar membrane proteinγ-TIP creates water specific channels in Xenopus oocytes. EMBO J. 1993;12(6):2241–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gerbeau P, Güçlü J, Ripoche P, Maurel C. Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes. Plant J. 1999;18(6):57–87.

    Article  Google Scholar 

  12. Bertl A, Kaldenhoff R. Function of a separate NH3-pore in aquaporin TIP2;2 from wheat. FEBS Lett. 2007;581(28):5413–7.

    Article  CAS  PubMed  Google Scholar 

  13. Dynowski M, Schaaf G, Loqué D, Moran O, Ludewig U. Plant plasma membrane water channels conduct the signalling molecule H2O2. Biochem J. 2008;414(1):53–61.

  14. Li GW, Peng YH, Yu X, Zhang MH, Cai WM, Sun WN, et al. Transport functions and expression analysis of vacuolar membrane aquaporins in response to various stresses in rice. J Plant Physiol. 2008;165(18):1879–88.

    Article  CAS  PubMed  Google Scholar 

  15. Bárzana G, Aroca R, Bienert GP, Chaumont F, Ruiz-Lozano JM. New insights into the regulation of aquaporins by the arbuscular mycorrhizal symbiosis in maize plants under drought stress and possible implications for plant performance. Mol Plant-Microbe Interact. 2014;27(4):349–63.

    Article  PubMed  Google Scholar 

  16. Regon P, Panda P, Kshetrimayum E, Panda SK. Genome-wide comparative analysis of tonoplast intrinsic protein (TIP) genes in plants. Funct Integr Genomics. 2014;14(4):617–29.

    Article  CAS  PubMed  Google Scholar 

  17. Sudhakaran S, Thakral V, Padalkar G, Rajora N, Dhiman P, Raturi G, et al. Significance of solute specificity, expression, and gating mechanism of tonoplast intrinsic protein during development and stress response in plants. Physiol Plant. 2021;172(1):258–74.

    Article  CAS  PubMed  Google Scholar 

  18. Kirscht A, Kaptan SS, Bienert GP, Chaumont F, Nissen P, de Groot BL, et al. Crystal structure of an ammonia-permeable aquaporin. PLoS Biol. 2016;14(3):e1002411.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Törnroth-Horsefield S, Wang Y, Hedfalk K, Johanson U, Karlsson M, Tajkhorshid E, et al. Structural mechanism of plant aquaporin gating. Nature. 2006;439(7077):688–94.

    Article  PubMed  Google Scholar 

  20. Larridon I, Reynders M, Huygh W, Bauters K, Van de Putte K, Muasya AM, et al. Affinities in C3Cyperus lineages (Cyperaceae) revealed using molecular phylogenetic data andcarbon isotope analysis. Bot J Linn Soc. 2011;167:19–46.

    Article  Google Scholar 

  21. De Castro O, Gargiulo R, Del Guacchio E, Caputo P, De Luca P. A molecular survey concerning the origin of Cyperus esculentus (Cyperaceae, Poales): two sides of the same coin (weed vs. crop). Ann Bot. 2015;115(5):733–45.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Zou Z, Zhao YG, Zhang L, Xiao YH, Guo AP. Analysis of Cyperus esculentus SMP family genes reveals lineage-specific evolution and seed desiccation-like transcript accumulation during tuber maturation. Ind Crop Prod. 2022;187:115382.

    Article  CAS  Google Scholar 

  23. Zou Z, Xiao YH, Zhang L, Zhao YG. Analysis of Lhc family genes reveals development regulation and diurnal fluctuation expression patterns in Cyperus esculentus, a Cyperaceae plant. Planta. 2023;257(3):59.

    Article  CAS  PubMed  Google Scholar 

  24. Stoller EW, Weber EJ. Differential cold tolerance, starch, sugar, protein, and lipid of yellow and purple nutsedge tubers. Plant Physiol. 1975;55(5):859–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Follak S, Belz R, Bohren C, De Castro O, Guacchio ED, Pascual-Sevae N, et al. Biological flora of Central Europe: Cyperus esculentus L. Perspect Plant Ecol. 2016;23:33–51.

    Article  Google Scholar 

  26. Turesson H, Marttila S, Gustavsson KE, Hofvander P, Olsson ME, Bülow L, et al. Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): a novel model system to study oil reserves in nonseed tissues. Am J Bot. 2010;97(11):1884–93.

    Article  PubMed  Google Scholar 

  27. Codina-Torrella I, Guamis B, Trujillo AJ. Characterization and comparison of tiger nuts (Cyperus esculentus L.) from different geographical origin. Ind Crop Prod. 2015;65:406–14.

    Article  CAS  Google Scholar 

  28. Zou Z, Zheng Y, Zhang Z, Xiao Y, Xie Z, Chang L, et al. Molecular characterization of oleosin genes in Cyperus esculentus, a Cyperaceae plant producing oil in underground tubers. Plant Cell Rep. 2023;42(11):1791–808.

    Article  CAS  PubMed  Google Scholar 

  29. Zou Z, Zhao YG, Zhang L, Kong H, Guo YL, Guo AP. Single-molecule real-time (SMRT)-based full-length transcriptome analysis of tigernut (Cyperus esculentus L). Chin J Oil Crop Sci. 2021;43:229–35.

    Google Scholar 

  30. Zhao X, Yi L, Ren Y, Li J, Ren W, Hou Z, et al. Chromosome-scale genome assembly of the yellow nutsedge (Cyperus esculentus). Genome Biol Evol. 2023;15(3):evad027.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Bowers JE, Chapman BA, Rong J, Paterson AH. Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. Nature. 2003;422(6930):433–8.

    Article  CAS  PubMed  Google Scholar 

  32. Wang X, Shi X, Hao B, Ge S, Luo J. Duplication and DNA segmental loss in the rice genome: implications for diploidization. New Phytol. 2005;165(3):937–46.

    Article  CAS  PubMed  Google Scholar 

  33. Tournaire-Roux C, Sutka M, Javot H, Gout E, Gerbeau P, Luu DT, et al. Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins. Nature. 2003;425(6956):393–7.

    Article  CAS  PubMed  Google Scholar 

  34. Leitão L, Prista C, Moura TF, Loureiro-Dias MC, Soveral G. Grapevine aquaporins: gating of a tonoplast intrinsic protein (TIP2;1) by cytosolic pH. PLoS ONE. 2012;7(3):e33219.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Zou Z, Zheng YJ, Qiao XY, Yang JH. Subcellular localization and multimerization analyses of HbPIP2;3, an efficient water transporter from Hevea brasiliensis. Chin J Trop Crop. 2023, https://kns.cnki.net/kcms/detail//46.1019.S.20230221.1645.004.html.

  36. Zou Z, Yang LF, Gong J, Mo YY, Wang JK, Cao JH, et al. Genome-wide identification of Jatropha curcas aquaporin genes and the comparative analysis provides insights into the gene family expansion and evolution in Hevea brasiliensis. Front Plant Sci. 2016;7:395.

  37. Shivaraj SM, Deshmukh R, Bhat JA, Sonah H, Bélanger RR. Understanding aquaporin transport system in eelgrass (Zostera marina L.), an aquatic plant species. Front Plant Sci. 2017;8:1334.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Chaumont F, Barrieu F, Wojcik E, Chrispeels MJ, Jung R. Aquaporins constitute a large and highly divergent protein family in maize. Plant Physiol. 2001;125(3):1206–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hove RM, Ziemann M, Bhave M. Identification and expression analysis of the barley (Hordeum vulgare L.) aquaporin gene family. PLoS ONE. 2015;10(6):e0128025.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Azad AK, Ahmed J, Alum MA, Hasan MM, Ishikawa T, Sawa Y, et al. Genome-wide characterization of major intrinsic proteins in four grass plants and their non-aqua transport selectivity profiles with comparative perspective. PLoS ONE. 2016;11(6):e0157735.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Wang Y, Wang X, Paterson AH. Genome and gene duplications and gene expression divergence: a view from plants. Ann N Y Acad Sci. 2012;1256:1–14.

    Article  PubMed  Google Scholar 

  42. Alix K, Gérard PR, Schwarzacher T, Heslop-Harrison JSP. Polyploidy and interspecific hybridization: partners for adaptation, speciation and evolution in plants. Ann Bot. 2017;120(2):183–94.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Qiao X, Li Q, Yin H, Qi K, Li L, Wang R, et al. Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants. Genome Biol. 2019;20(1):38.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Wang W, Haberer G, Gundlach H, Gläßer C, Nussbaumer T, Luo MC, et al. The Spirodela polyrhiza genome reveals insights into its neotenous reduction fast growth and aquatic lifestyle. Nat Commun. 2014;5:3311.

    Article  CAS  PubMed  Google Scholar 

  45. Ming R, VanBuren R, Wai CM, Tang H, Schatz MC, Bowers JE, et al. The pineapple genome and the evolution of CAM photosynthesis. Nat Genet. 2015;47(12):1435–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Can M, Wei W, Zi H, Bai M, Liu Y, Gao D, et al. Genome sequence of Kobresia Littledalei, the first chromosome-level genome in the family Cyperaceae. Sci Data. 2020;7(1):175.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hu W, Hou X, Huang C, Yan Y, Tie W, Ding Z, et al. Genome-wide identification and expression analyses of aquaporin gene family during development and abiotic stress in banana. Int J Mol Sci. 2015;16(8):19728–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Zou Z, Gong J, An F, Xie GS, Wang JK, Mo YY, et al. Genome-wide identification of rubber tree (Hevea brasiliensis Muell. Arg.) Aquaporin genes and their response to ethephon stimulation in the laticifer, a rubber-producing tissue. BMC Genomics. 2015;16:1001.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Zou Z, Zheng Y, Xie Z. Analysis of Carica papaya informs lineage-specific evolution of the aquaporin (AQP) family in Brassicales. Plants (Basel). 2023;12(22):3847.

    Article  CAS  PubMed  Google Scholar 

  50. Ludevid D, Höfte H, Himelblau E, Chrispeels MJ. The expression pattern of the tonoplast intrinsic protein gamma-TIP in Arabidopsis thaliana is correlated with cell enlargement. Plant Physiol. 1992;100(4):1633–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Beebo A, Thomas D, Der C, Sanchez L, Leborgne-Castel N, Marty F, et al. Life with and without AtTIP1;1, an Arabidopsis aquaporin preferentially localized in the apposing tonoplasts of adjacent vacuoles. Plant Mol Biol. 2009;70(1–2):193–209.

    Article  CAS  PubMed  Google Scholar 

  52. Balasaheb Karle S, Kumar K, Srivastava S, Suprasanna P. Cloning, in silico characterization and expression analysis of TIP subfamily from rice (Oryza sativa L). Gene. 2020;761:145043.

    Article  CAS  PubMed  Google Scholar 

  53. Klebl F, Wolf M, Sauer N. A defect in the yeast plasma membrane urea transporter Dur3p is complemented by CpNIP1, a Nod26-like protein from zucchini (Cucurbita pepo L.), and by Arabidopsis thaliana δ-TIP or γ-TIP. FEBS Lett. 2003;547(1–3):69–74.

    Article  CAS  PubMed  Google Scholar 

  54. Liu LH, Ludewig U, Gassert B, Frommer WB. Von Wirén N. Urea transport by nitrogen-regulated tonoplast intrinsic proteins in Arabidopsis. Plant Physiol. 2003;133(3):1220–8.

  55. Jahn TP, Møller AL, Zeuthen T, Holm LM, Klaerke DA, Mohsin B, et al. Aquaporin homologues in plants and mammals transport ammonia. FEBS Lett. 2004;574(1–3):31–6.

    Article  CAS  PubMed  Google Scholar 

  56. Loqué D, Ludewig U, Yuan L, von Wirén N. Tonoplast intrinsic proteins AtTIP2;1 and AtTIP2;3 facilitate NH3 transport into the vacuole. Plant Physiol. 2005;137(2):671–80.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Bienert GP, Møller AL, Kristiansen KA, Schulz A, Møller IM, Schjoerring JK, et al. Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. J Biol Chem. 2007;282(2):1183–92.

    Article  CAS  PubMed  Google Scholar 

  58. Reinhardt H, Hachez C, Bienert MD, Beebo A, Swarup K, Voß U, et al. Tonoplast aquaporins facilitate lateral root emergence. Plant Physiol. 2016;170(3):1640–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Soto G, Alleva K, Mazzella MA, Amodeo G, Muschietti JP. AtTIP1;3 and AtTIP5;1, the only highly expressed Arabidopsis pollen-specific aquaporins, transport water and urea. FEBS Lett. 2008;582(29):4077–82.

    Article  CAS  PubMed  Google Scholar 

  60. Dynowski M, Schaaf G, Loque D, Moran O, Ludewig U. Plant plasma membrane water channels conduct the signalling molecule H2O2. Biochem J. 2008;414(1):53–61.

    Article  CAS  PubMed  Google Scholar 

  61. Gattolin S, Sorieul M, Frigerio L. Mapping of tonoplast intrinsic proteins in maturing and germinating Arabidopsis seeds reveals dual localization of embryonic TIPs to the tonoplast and plasma membrane. Mol Plant. 2011;4(1):180–9.

    Article  CAS  PubMed  Google Scholar 

  62. An F, Zou Z, Cai XQ, Wang J, Rookes J, Lin W, Cahill D, Kong L. Regulation of HbPIP2;3, a latex-abundant water transporter, is associated to the latex dilution and latex yield in rubber trees (Hevea brasiliensis Muell. Arg) PLoS One. 2015;10(4):e0125595.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Ferro M, Brugière S, Salvi D, Seigneurin-Berny D, Court M, Moyet L, et al. AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins. Mol Cell Proteom. 2010;9(6):1063–84.

    Article  CAS  Google Scholar 

  64. Murozuka E, Hanisch S, Pomorski TG, Jahn TP, Schjoerring JK. Bimolecular fluorescence complementation and interaction of various Arabidopsis major intrinsic proteins expressed in yeast. Physiol Plant. 2013;148(3):422–31.

    Article  CAS  PubMed  Google Scholar 

  65. Zou Z, Yang JH, Zhang XC. Insights into genes encoding respiratory burst oxidase homologs (RBOHs) in rubber tree (Hevea brasiliensis Muell. Arg) Ind Crop Prod. 2019;128:126–39.

    Article  CAS  Google Scholar 

  66. Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, et al. TBtools-II: a one for all, all for one bioinformatics platform for biological big-data mining. Mol Plant. 2023;16(11):1733–42.

    Article  CAS  PubMed  Google Scholar 

  67. Edgar RC. Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015;12(4):357–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Love MI, Huber W, Anders S. Moderated estimation of Fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.

    Article  PubMed  PubMed Central  Google Scholar 

  72. Qiao XY, Zheng YJ, Yang JH, Zeng CY, Zou Z. Gene cloning, subcellular localization and multimerization analysis of HbPIP1;1 from Hevea brasiliensis. Chin J Trop Crop. 2022;43(12):2405–12.

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Acknowledgements

The authors appreciate those contributors who make the related genome and transcriptome data accessible in public databases.

Funding

This work was supported by the key research and development project of Hainan province (ZDYF2024XDNY171), China; the Project of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2022-66), China; the Central Public-interest Scientific Institution Basal Research Fund (1630052022001), China; and the National Natural Science Foundation of China (31971688 and 31700580), China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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  1. Zhi Zou and Yujiao Zheng contributed equally to this work.

Authors and Affiliations

  1. National Key Laboratory for Tropical Crop Breeding/Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions, Institute of Tropical Biosciences and Biotechnology/Sanya Research Institute of Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, 571101, P. R. China

    Zhi Zou, Yujiao Zheng, Lili Chang, Liangping Zou & Yongguo Zhao

  2. Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, College of Life Science, South-Central Minzu University, Wuhan, Hubei, 430074, P. R. China

    Li Zhang

  3. College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming, Guangdong, 525000, P. R. China

    Yongguo Zhao

  4. Hainan University, Haikou, Hainan, 570228, P. R. China

    Yi Min

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  1. Zhi Zou
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Contributions

The study was conceived and directed by ZZ. All the experiments and analyses were directed by ZZ and carried out by ZZ, ZY, CL, ZL, ZL, and ZY. ZZ, MY, and ZY wrote the paper. All the authors read and approved the final manuscript.

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Correspondence to Zhi Zou, Yi Min or Yongguo Zhao.

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Zou, Z., Zheng, Y., Chang, L. et al. TIP aquaporins in Cyperus esculentus: genome-wide identification, expression profiles, subcellular localizations, and interaction patterns. BMC Plant Biol 24, 298 (2024). https://doi.org/10.1186/s12870-024-04969-x

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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