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As shown in Table 1, a total of 14 PIP genes were identified from eight tigernut scaffolds (Scfs). The CDS length varies from 831 to 882 bp, putatively encoding 276–293 amino acids (AA) with a molecular weight (MW) of 29.16–31.59 kilodalton (kDa). The theoretical isoelectric point (pI) varies from 7.04 to 9.46, implying that they are all alkaline. The grand average of hydropathicity (GRAVY) is between 0.344 and 0.577, and the aliphatic index (II) ranges from 94.57 to 106.90, which are consistent with the hydrophobic characteristic of AQPs[47]. As expected, like SoPIP2;1, all CePIPs include six TMs, two typical NPA motifs, the invariable ar/R filter F-H-T-R, five conserved Froger's positions Q/M-S-A-F-W, and two highly conserved residues corresponding to H193 and L197 in SoPIP2;1 that were proven to be involved in gating[5,48], though the H→F variation was found in CePIP2;9, -2;10, and -2;11 (Supplemental Fig. S1). Moreover, two S residues, corresponding to S115 and S274 in SoPIP2;1[5], respectively, were also found in the majority of CePIPs (Supplemental Fig. S1), implying their posttranslational regulation by phosphorylation.
Table 1. Fourteen PIP genes identified in C. esculentus.
Gene name Locus Position Intron no. AA MW (kDa) pI GRAVY AI TM MIP CePIP1;1 CESC_15147 Scf9:2757378..2759502(–) 3 288 30.76 8.82 0.384 95.28 6 47..276 CePIP1;2 CESC_04128 Scf4:3806361..3807726(–) 3 291 31.11 8.81 0.344 95.95 6 46..274 CePIP1;3 CESC_15950 Scf54:5022493..5023820(+) 3 289 31.06 8.80 0.363 94.57 6 49..278 CePIP2;1 CESC_15350 Scf9:879960..884243(+) 3 288 30.34 8.60 0.529 103.02 6 33..269 CePIP2;2 CESC_00011 Scf30:4234620..4236549(+) 3 293 31.59 9.27 0.394 101.57 6 35..268 CePIP2;3 CESC_00010 Scf30:4239406..4241658(+) 3 291 30.88 9.44 0.432 98.97 6 31..266 CePIP2;4 CESC_05080 Scf46:307799..309544(+) 3 285 30.44 7.04 0.453 100.32 6 28..265 CePIP2;5 CESC_05079 Scf46:312254..314388(+) 3 286 30.49 7.04 0.512 101.68 6 31..268 CePIP2;6 CESC_05078 Scf46:316024..317780(+) 3 288 30.65 7.68 0.475 103.06 6 31..268 CePIP2;7 CESC_05077 Scf46:320439..322184(+) 3 284 30.12 8.55 0.500 100.00 6 29..266 CePIP2;8 CESC_14470 Scf2:4446409..4448999(+) 3 284 30.37 8.30 0.490 106.90 6 33..263 CePIP2;9 CESC_02223 Scf1:2543928..2545778(–) 3 283 30.09 9.46 0.533 106.47 6 31..262 CePIP2;10 CESC_10007 Scf27:1686032..1688010(–) 3 276 29.16 9.23 0.560 106.05 6 26..256 CePIP2;11 CESC_10009 Scf27:1694196..1696175(–) 3 284 29.71 9.10 0.577 105.49 6 33..263 AA: amino acid; AI: aliphatic index; GRAVY: grand average of hydropathicity; kDa: kilodalton; MIP: major intrinsic protein; MW: molecular weight; pI: isoelectric point; PIP: plasma membrane intrinsic protein; Scf: scaffold; TM: transmembrane helix. To uncover the evolutionary relationships, an unrooted phylogenetic tree was constructed using the full-length protein sequences of CePIPs together with 11 OsPIPs and 13 AtPIPs. As shown in Fig. 1a, these proteins were clustered into two main groups, corresponding to PIP1 and PIP2 as previously defined[10,49], and each appears to have evolved into several subgroups. Compared with PIP1s, PIP2s possess a relatively shorter N-terminal but an extended C-terminal with one conserved S residue (Supplemental Fig. S1). Interestingly, a high number of gene repeats were detected, most of which seem to be species-specific, i.e., AtPIP1;1/-1;2/-1;3/-1;4/-1;5, AtPIP2;1/-2;2/-2;3/-2;4/-2;5/-2;6, AtPIP2;7/-2;8, OsPIP1;1/-1;2/-1;3, OsPIP2;1/-2;4/-2;5, OsPIP2;2/-2;3, CePIP1;1/-1;2, CePIP2;2/-2;3, CePIP2;4/-2;5/-2;6/-2;7, and CePIP2;9/-2;10/-2;11, reflecting the occurrence of more than one lineage-specific whole-genome duplications (WGDs) after their divergence[50,51]. In Arabidopsis that experienced three WGDs (i.e. γ, β, and α) after the split with the monocot clade[52], AtPIP1;5 in the PIP1 group first gave rise to AtPIP1;1 via the γ WGD shared by all core eudicots[50], which latter resulted in AtPIP1;3, -1;4, and -1;2 via β and α WGDs; AtPIP2;1 in the PIP2 group first gave rise to AtPIP2;6 via the γ WGD, and they latter generated AtPIP2;2, and -2;5 via the α WGD (Supplemental Table S1). In rice, which also experienced three WGDs (i.e. τ, σ, and ρ) after the split with the eudicot clade[51], OsPIP1;2 and -2;3 generated OsPIP1;1 and -2;2 via the Poaceae-specific ρ WGD, respectively. Additionally, tandem, proximal, transposed and dispersed duplications also played a role on the gene expansion in these two species (Supplemental Table S1).
Figure 1.
Structural and phylogenetic analysis of PIPs in C. esculentus, O. sativa, and A. thaliana. (a) Shown is an unrooted phylogenetic tree resulting from full-length PIPs with MEGA6 (maximum likelihood method and bootstrap of 1,000 replicates), where the distance scale denotes the number of amino acid substitutions per site. (b) Shown are the exon-intron structures. (c) Shown is the distribution of conserved motifs among PIPs, where different motifs are represented by different color blocks as indicated and the same color block in different proteins indicates a certain motif. (At: A. thaliana; Ce: C. esculentus; PIP: plasma membrane intrinsic protein; Os: O. sativa).
Analysis of gene structures revealed that all CePIP and AtPIP genes possess three introns and four exons in the CDS, in contrast to the frequent loss of certain introns in rice, including OsPIP1;2, -1;3, -2;1, -2;3, -2;4, -2;5, -2;6, -2;7, and -2;8 (Fig. 1b). The positions of three introns are highly conserved, which are located in sequences encoding LB (three residues before the first NPA), LD (one residue before the conserved L involved in gating), and LE (18 residues after the second NPA), respectively (Supplemental Fig. S1). The intron length of CePIP genes is highly variable, i.e., 109–993 bp, 115–1745 bp, and 95–866 bp for three introns, respectively. By contrast, the exon length is relatively less variable: Exons 2 and 3 are invariable with 296 bp and 141 bp, respectively, whereas Exons 1 and 4 are of 277–343 bp and 93–132 bp, determining the length of N- and C-terminus of PIP1 and PIP2, respectively (Fig. 1b). Correspondingly, their protein structures were shown to be highly conserved, and six (i.e., Motifs 1–6) out of 15 motifs identified are broadly present. Among them, Motif 3, -2, -6, -1, and -4 constitute the conserved MIP domain. In contrast to a single Motif 5 present in most PIP2s, all PIP1s possess two sequential copies of Motif 5, where the first one is located at the extended N-terminal. In CePIP2;3 and OsPIP2;7, Motif 5 is replaced by Motif 13; in CePIP2;2, it is replaced by two copies of Motif 15; and no significant motif was detected in this region of CePIP2;10. PIP1s and PIP2s usually feature Motif 9 and -7 at the C-terminal, respectively, though it is replaced by Motif 12 in CePIP2;6 and OsPIP2;8. PIP2s usually feature Motif 8 at the N-terminal, though it is replaced by Motif 14 in CePIP2;2 and -2;3 or replaced by Motif 11 in CePIP2;10 and -2;11 (Fig. 1c).
Gene localization and synteny analyses reveal complex evolution patterns of CePIP genes
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As shown in Fig. 2a, gene localization of CePIPs revealed three gene clusters, i.e., CePIP2;2/-2;3 on Scf30, CePIP2;4/-2;5/-2;6/-2;7 on Scf46, and CePIP2;10/-2;11 on Scf27, which were defined as tandem repeats for their high sequence similarities and neighboring locations. The nucleotide identities of these duplicate pairs vary from 70.5% to 91.2%, and the Ks values range from 0.0971 to 1.2778 (Table 2), implying different time of their birth. According to intra-species synteny analysis, two duplicate pairs, i.e., CePIP1;1/-1;2 and CePIP2;2/-2;4, were shown to be located within syntenic blocks (Fig. 2b) and thus were defined as WGD repeats. Among them, CePIP1;1/-1;2 possess a comparable Ks value to CePIP2;2/-2;3, CePIP1;1/-1;3, and CePIP2;4/-2;8 (1.2522 vs 1.2287–1.2778), whereas CePIP2;2/-2;4 harbor a relatively higher Ks value of 1.5474 (Table 2), implying early origin or fast evolution of the latter. While CePIP1;1/-1;3 and CePIP2;1/-2;8 were characterized as transposed repeats, CePIP2;1/-2;2, CePIP2;9/-2;10, and CePIP2;8/-2;10 were characterized as dispersed repeats (Fig. 2a). The Ks values of three dispersed repeats vary from 0.8591 to 3.0117 (Table 2), implying distinct times of origin.
Figure 2.
Duplication events of CePIP genes and synteny analysis within and between C. esculentus, O. sativa, and A. thaliana. (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 C. esculentus, O. sativa, and A. thaliana. (c) Synteny analysis within and between C. esculentus, C. cristatella, R. breviuscula, and J. effusus. Shown are PIP-encoding chromosomes/scaffolds and only syntenic blocks that contain PIP genes are marked, i.e., red and purple for intra- and inter-species, respectively. (At: A. thaliana; Cc: C. cristatella; Ce: C. esculentus; Je: J. effusus; Mb: megabase; PIP: plasma membrane intrinsic protein; Os: O. sativa; Rb: R. breviuscula; Scf: scaffold; WGD: whole-genome duplication).
Table 2. Sequence identity and evolutionary rate of homologous PIP gene pairs identified in C. esculentus. Ks and Ka were calculated using PAML.
Duplicate 1 Duplicate 2 Identity (%) Ka Ks Ka/Ks CePIP1;1 CePIP1;3 78.70 0.0750 1.2287 0.0610 CePIP1;2 CePIP1;1 77.20 0.0894 1.2522 0.0714 CePIP2;1 CePIP2;4 74.90 0.0965 1.7009 0.0567 CePIP2;3 CePIP2;2 70.50 0.1819 1.2778 0.1424 CePIP2;4 CePIP2;2 66.50 0.2094 1.5474 0.1353 CePIP2;5 CePIP2;4 87.30 0.0225 0.4948 0.0455 CePIP2;6 CePIP2;5 84.90 0.0545 0.5820 0.0937 CePIP2;7 CePIP2;6 78.70 0.0894 1.0269 0.0871 CePIP2;8 CePIP2;4 72.90 0.1401 1.2641 0.1109 CePIP2;9 CePIP2;10 76.40 0.1290 0.8591 0.1502 CePIP2;10 CePIP2;8 64.90 0.2432 3.0117 0.0807 CePIP2;11 CePIP2;10 91.20 0.0562 0.0971 0.5783 Ce: C. esculentus; Ka: nonsynonymous substitution rate; Ks: synonymous substitution rate; PIP: plasma membrane intrinsic protein. According to inter-species syntenic analysis, six out of 14 CePIP genes were shown to have syntelogs in rice, including 1:1, 1:2, and 2:2 (i.e. CePIP1;1 vs OsPIP1;3, CePIP1;3 vs OsPIP1;2/-1;1, CePIP2;1 vs OsPIP2;4, CePIP2;2/-2;4 vs OsPIP2;3/-2;2, and CePIP2;8 vs OsPIP2;6), in striking contrast to a single one found in Arabidopsis (i.e. CePIP1;2 vs AtPIP1;2). Correspondingly, only OsPIP1;2 in rice was shown to have syntelogs in Arabidopsis, i.e., AtPIP1;3 and -1;4 (Fig. 2b). These results are consistent with their taxonomic relationships that tigernut and rice are closely related[50,51], and also imply lineage-specific evolution after their divergence.
Comparative genomics analyses reveals lineage-specific evolution of PIP genes in Cyperaceae
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As described above, phylogenetic and syntenic analyses showed that the last common ancestor of tigernut and rice is more likely to possess only two PIP1s and three PIP2s. However, it is not clear whether the gene expansion observed in tigernut is species-specific or Cyperaceae-specific. To address this issue, recently available genomes were used to identify PIP subfamily genes from C. cristatella, R. breviuscula, and J. effuses, resulting in 15, 13, and nine members, respectively. Interestingly, in contrast to a high number of tandem repeats found in Cyperaceae species, only one pair of tandem repeats (i.e., JePIP2;3 and -2;4) were identified in J. effusus, a close outgroup species to Cyperaceae in the Juncaceae family[36,37]. According to homologous analysis, a total of 12 orthogroups were identified, where JePIP genes belong to PIP1A (JePIP1;1), PIP1B (JePIP1;2), PIP1C (JePIP1;3), PIP2A (JePIP2;1), PIP2B (JePIP2;2), PIP2F (JePIP2;3 and -2;4), PIP2G (JePIP2;5), and PIP2H (JePIP2;6) (Table 3). Further intra-species syntenic analysis revealed that JePIP1;1/-1;2 and JePIP2;2/-2;3 are located within syntenic blocks, which is consistent with CePIP1;1/-1;2, CePIP2;2/-2;4, CcPIP1;1/-1;2, CcPIP2;3/-2;4, RbPIP1;1/-1;2, and RbPIP2;2/-2;5 (Fig. 2c), implying that PIP1A/PIP1B and PIP2B/PIP2D were derived from WGDs occurred sometime before Cyperaceae-Juncaceae divergence. After the split with Juncaceae, tandem duplications frequently occurred in Cyperaceae, where PIP2B/PIP2C and PIP2D/PIP2E/PIP2F retain in most Cyperaceae plants examined in this study. By contrast, species-specific expansion was also observed, i.e., CePIP2;4/-2;5, CePIP2;10/-2;11, CcPIP1;2/-1;3, CcPIP2;4/-2;5, CcPIP2;8/-2;9, CcPIP2;10/-2;11, RbPIP2;3/-2;4, and RbPIP2;9/-2;10 (Table 3 & Fig. 2c).
Table 3. Twelve proposed orthogroups based on comparison of representative plant species.
Orthogroup C. esculentus C. cristatella R. breviuscula J. effusus O. sativa A. thaliana PIP1A CePIP1;1 CcPIP1;1 RbPIP1;1 JePIP1;1 OsPIP1;3 AtPIP1;1, AtPIP1;2,
AtPIP1;3, AtPIP1;4,
AtPIP1;5PIP1B CePIP1;2 CcPIP1;2, CcPIP1;3 RbPIP1;2 JePIP1;2 − − PIP1C CePIP1;3 CcPIP1;4 RbPIP1;3 JePIP1;3 OsPIP1;1, OsPIP1;2 − PIP2A CePIP2;1 CcPIP2;1 RbPIP2;1 JePIP2;1 OsPIP2;1, OsPIP2;4,
OsPIP2;5AtPIP2;1, AtPIP2;2,
AtPIP2;3, AtPIP2;4,
AtPIP2;5, AtPIP2;6PIP2B CePIP2;2 CcPIP2;2 RbPIP2;2 JePIP2;2 OsPIP2;2, OsPIP2;3 − PIP2C CePIP2;3 CcPIP2;3 RbPIP2;3, RbPIP2;4 − − − PIP2D CePIP2;4, CePIP2;5 CcPIP2;4, CcPIP2;5 RbPIP2;5 − − − PIP2E CePIP2;5 CcPIP2;5 RbPIP2;6 − − − PIP2F CePIP2;6 CcPIP2;6 − − − − PIP2G CePIP2;7 CcPIP2;7 RbPIP2;7 JePIP2;3, JePIP2;4 − − PIP2H CePIP2;8 CcPIP2;8, CcPIP2;9 RbPIP2;8 JePIP2;5 OsPIP2;6 AtPIP2;7, AtPIP2;8 PIP2I CePIP2;9, CePIP2;10,
CePIP2;11CcPIP2;10, CcPIP2;11 RbPIP2;9, RbPIP2;10 JePIP2;6 OsPIP2;7, OsPIP2;8 − At: A. thaliana; Cc: C. cristatella; Ce: C. esculentus; Je: J. effuses; Os: O. sativa; Rb: R. breviuscula; PIP: plasma membrane intrinsic protein. Tissue-specific expression analysis of CePIP genes identifies three dominant members
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Tissue-specific expression profiles of CePIP genes were investigated using transcriptome data available for young leaf, mature leaf, sheath, root, rhizome, shoot apex, and tuber. As shown in Fig. 3a, CePIP genes were mostly expressed in roots, followed by sheaths, moderately in tubers, young leaves, rhizomes, and mature leaves, and lowly in shoot apexes. In most tissues, CePIP1;1, -2;1, and -2;8 represent three dominant members that contributed more than 90% of total transcripts. By contrast, in rhizome, these three members occupied about 80% of total transcripts, which together with CePIP1;3 and -2;4 contributed up to 96%; in root, CePIP1;1, -1;3, -2;4, and -2;7 occupied about 84% of total transcripts, which together with CePIP2;1 and -2;8 contributed up to 94%. According to their expression patterns, CePIP genes could be divided into five main clusters: Cluster I includes CePIP1;1, -2;1, and -2;8 that were constitutively and highly expressed in all tissues examined; Cluster II includes CePIP2;2, -2;9, and -2;10 that were lowly expressed in all tested tissues; Cluster III includes CePIP1;2 and -2;11 that were preferentially expressed in young leaf and sheath; Cluster IV includes CePIP1;3 and -2;4 that were predominantly expressed in root and rhizome; and Cluster V includes remains that were typically expressed in root (Fig. 3a). Collectively, these results imply expression divergence of most duplicate pairs and three members (i.e. CePIP1;1, -2;1, and -2;8) have evolved to be constitutively co-expressed in most tissues.
Figure 3.
Expression profiles of CePIP genes in various tissues, different stages of leaf development, and mature leaves of diurnal fluctuation. (a) Tissue-specific expression profiles of 14 CePIP genes. The heatmap was generated using the R package implemented with a row-based standardization. Color scale represents FPKM normalized log2 transformed counts, where blue indicates low expression and red indicates high expression. (b) Expression profiles of CePIP1;1, -2;1, and -2;8 at different stages of leaf development. (c) Expression profiles of CePIP1;1, -2;1, and -2;8 in mature leaves of diurnal fluctuation. Bars indicate SD (N = 3) and uppercase letters indicate difference significance tested following Duncan's one-way multiple-range post hoc ANOVA (p< 0.01). (Ce: C. esculentus; FPKM: Fragments per kilobase of exon per million fragments mapped; PIP: plasma membrane intrinsic protein)
Expression analysis reveals that CePIP transcripts in leaves are subjected to development regulation
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As shown in Fig. 3a, compared with young leaves, transcriptome profiling showed that CePIP1;2, -2;3, -2;7, -2;8, and -2;11 were significantly down-regulated in mature leaves, whereas CePIP1;3 and -2;1 were up-regulated. To confirm the results, three dominant members, i.e., CePIP1;1, -2;1, and -2;8, were selected for qRT-PCR analysis, which includes three representative stages, i.e., young, mature, and senescing leaves. As shown in Fig. 3b, in contrast to CePIP2;1 that exhibited a bell-like expression pattern peaking in mature leaves, transcripts of both CePIP1;1 and -2;8 gradually decreased during leaf development. These results were largely consistent with transcriptome profiling, and the only difference is that CePIP1;1 was significantly down-regulated in mature leaves relative to young leaves. However, this may be due to different experiment conditions used, i.e., greenhouse vs natural conditions.
qRT-PCR analysis reveals that the transcription of CePIP1;1, -2;1, and -2;8 in mature leaves is subjected to diurnal regulation
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Diurnal fluctuation expression patterns of CePIP1;1, -2;1, and -2;8 were also investigated in mature leaves and results are shown in Fig. 3c. Generally, transcripts of all three genes in the day (8, 12, 16, and 20 h) were higher than that in the night (24 and 4 h). During the day, both CePIP1;1 and -2;8 exhibited an unimodal expression pattern that peaked at 12 h, whereas CePIP2;1 possessed two peaks (8 and 16 h) and their difference was not significant. Nevertheless, transcripts of all three genes at 20 h (onset of night) were significantly lower than those at 8 h (onset of day) as well as 12 h. In the night, except for CePIP2;1, no significant difference was observed between the two stages for both CePIP1;1 and -2;8. Moreover, their transcripts were comparable to those at 20 h (Fig. 3c).
Expression profiles of CePIP genes during tuber development
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To reveal the expression patterns of CePIP genes during tuber development, three representative stages, i.e., 40 DAS (early swelling stage), 85 DAS (late swelling stage), and 120 DAS (mature stage), were first profiled using transcriptome data. As shown in Fig. 4a, except for rare expression of CePIP1;2, -2;2, -2;9, and -2;10, most genes exhibited a bell-like expression pattern peaking at 85 DAS, in contrast to a gradual decrease of CePIP2;3 and -2;8. Notably, except for CePIP2;4, other genes were expressed considerably lower at 120 DAS than that at 40 DAS. For qRT-PCR confirmation of CePIP1;1, -2;1, and -2;8, seven stages were examined, i.e., 1, 5, 10, 15, 20, 25, and 35 DAI, which represent initiation, five stages of swelling, and maturation as described before[32]. As shown in Fig. 4b, two peaks were observed for all three genes, though their patterns were different. As for CePIP1;1, compared with the initiation stage (1 DAI), significant up-regulation was observed at the early swelling stage (5 DAI), followed by a gradual decrease except for the appearance of the second peak at 20 DAI, which is something different from transcriptome profiling. As for CePIP2;1, a sudden drop of transcripts first appeared at 5 DAI, then gradually increased until 20 DAI, which was followed by a gradual decrease at two late stages. The pattern of CePIP2;8 is similar to -1;1, two peaks appeared at 5 and 20 DAI and the second peak was significantly lower than the first. The difference is that the second peak of CePIP2;8 was significantly lower than the initiation stage. By contrast, the second peak (20 DAI) of CePIP2;1 was significantly higher than that of the first one (1 DAI). Nevertheless, the expression patterns of both CePIP2;1 and -2;8 are highly consistent with transcriptome profiling.
Figure 4.
Transcript and protein abundances of CePIP genes during tuber development. (a) Transcriptome-based expression profiling of 14 CePIP genes during tuber development. The heatmap was generated using the R package implemented with a row-based standardization. Color scale represents FPKM normalized log2 transformed counts, where blue indicates low expression and red indicates high expression. (b) qRT-PCR-based expression profiling of CePIP1;1, -2;1, and -2;8 in seven representative stages of tuber development. (c) Relative protein abundance of CePIP1;1, -2;1, and -2;8 in three representative stages of tuber development. Bars indicate SD (N = 3) and uppercase letters indicate difference significance tested following Duncan's one-way multiple-range post hoc ANOVA (p < 0.01). (Ce: C. esculentus; DAI: days after tuber initiation; DAS: days after sowing; FPKM: Fragments per kilobase of exon per million fragments mapped; PIP: plasma membrane intrinsic protein).
Protein abundances of CePIP1;1, -2;1, and -2;8 in tubers are positively correlated with the moisture content during tuber development
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Since protein abundance is not always in agreement with the transcript level, protein profiles of three dominant members (i.e. CePIP1;1, -2;1, and -2;8) during tuber development were further investigated. For this purpose, we first took advantage of available proteomic data to identify CePIP proteins, i.e., leaves, roots, and four stages of tubers (freshly harvested, dried, rehydrated for 48 h, and sprouted). As shown in Supplemental Fig. S2, all three proteins were identified in both leaves and roots, whereas CePIP1;1 and -2;8 were also identified in at least one of four tested stages of tubers. Notably, all three proteins were considerably more abundant in roots, implying their key roles in root water balance.
To further uncover their profiles during tuber development, 4D-PRM-based protein quantification was conducted in three representative stages of tuber development, i.e., 1, 25, and 35 DAI. As expected, all three proteins were identified and quantified. In contrast to gradual decrease of CePIP2;8, both CePIP1;1 and -2;1 exhibited a bell-like pattern that peaked at 25 DAI, though no significant difference was observed between 1 and 25 DAI (Fig. 4c). The trends are largely in accordance with their transcription patterns, though the reverse trend was observed for CePIP2;1 at two early stages (Fig. 4b & Fig. 4c).
Subcellular localization analysis reveals that CePIP1;1, -2;1, and -2;8 function in the cell membrane
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As predicted by WoLF PSORT, CePIP1;1, -2;1, and -2;8 may function in the cell membrane. To confirm the result, subcellular localization vectors named pNC-Cam1304-CePIP1;1, pNC-Cam1304-CePIP2;1, and pNC-Cam1304-CePIP2;8 were further constructed. When transiently overexpressed in tobacco leaves, green fluorescence signals of all three constructs were confined to cell membranes, highly coinciding with red fluorescence signals of the plasma membrane marker HbPIP2;3-RFP (Fig. 5).
Figure 5.
(a) Schematic diagram of overexpressing constructs, (b) subcellular localization analysis of CePIP1;1, -2;1, and -2;8 in N. benthamiana leaves. (35S: cauliflower mosaic virus 35S RNA promoter; Ce: C. esculentus; EGFP: enhanced green fluorescent protein; kb: kilobase; NOS: terminator of the nopaline synthase gene; RFP: red fluorescent protein; PIP: plasma membrane intrinsic protein).
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All the relevant data is available within the published article.
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About this article
Cite this article
Zou Z, Zheng Y, Xiao Y, Liu H, Huang J, et al. 2024. Molecular insights into PIP aquaporins in tigernut (Cyperus esculentus L.), a Cyperaceae tuber plant . Tropical Plants 3: e027 doi: 10.48130/tp-0024-0030
Molecular insights into PIP aquaporins in tigernut (Cyperus esculentus L.), a Cyperaceae tuber plant
- Received: 01 May 2024
- Accepted: 15 July 2024
- Published online: 22 August 2024
Abstract: Plasma membrane intrinsic proteins (PIPs) constitute a subfamily of aquaporin that mediate the efficient water transport across the cell membrane. In the present study, the first genome-wide characterization of PIP genes in tigernut (Cyperus esculentus L.), a Cyperaceae plant producing oil in underground tubers, is presented. A high number of 14 PIP genes representing two phylogenetic groups or 12 orthogroups were identified from the tigernut genome. Comparative genomics analyses revealed that the expansion of CePIP genes was mainly contributed by whole-genome duplication and transposed/tandem duplications, and some of them are lineage or even species-specific. Expression analyses indicated that CePIP1;1, -2;1, and -2;8 are three dominant members, which are constitutively expressed in most tissues including leaf and tuber. Whereas transcription of CePIP1;1, -2;1, and -2;8 in leaves is subjected to development and diurnal regulation, their mRNA/protein abundances in tubers are positively correlated with the moisture content during tuber development. Moreover, when transiently overexpressed in Nicotiana benthamiana leaves, all three proteins were shown to be located in the cell membrane. These findings provide valuable information for further functional analysis and genetic improvement in tigernut.