Figures (5)  Tables (3)

    • 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).

    • 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).

    • 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)

    • 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).

    • 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).

    • 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.

      Table 1. 

      Fourteen PIP genes identified in C. esculentus.

    • 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.

      Table 2. 

      Sequence identity and evolutionary rate of homologous PIP gene pairs identified in C. esculentus. Ks and Ka were calculated using PAML.

    • 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;5
      PIP1B 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;5      		 AtPIP2;1, AtPIP2;2,
      AtPIP2;3, AtPIP2;4,
      AtPIP2;5, AtPIP2;6
      PIP2B 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;11      		 CcPIP2;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.

      Table 3. 

      Twelve proposed orthogroups based on comparison of representative plant species.