Genome-wide analysis of the aquaporin genes in melon (Cucumis melo L.).

Melon (Cucumis melo L.) is a very important crop throughout the world and has great economic importance, in part due to its nutritional properties. It prefers well-drained soil with low acidity and has a strong demand for water during fruit set. Therefore, a correct water balance-involving aquaporins-is necessary to maintain the plants in optimal condition. This manuscript describes the identification and comparative analysis of the complete set of aquaporins in melon. 31 aquaporin genes were identified, classified and analysed according to the evolutionary relationship of melon with related plant species. The individual role of each aquaporin in the transport of water, ions and small molecules was discussed. Finally, qPCR revealed that almost all melon aquaporins in roots and leaves were constitutively expressed. However, the high variations in expression among them point to different roles in water and solute transport, providing important features as that CmPIP1;1 is the predominant isoform and CmTIP1;1 is revealed as the most important osmoregulator in the tonoplast under optimal conditions. The results of this work pointing to the physiological importance of each individual aquaporin of melon opening a field of knowledge that deserves to be investigated.

Introduction

Aquaporins are highly conserved transmembrane proteins, present in all domains of life, whose main function is the selective, bidirectional and passive transport of water and some small neutral solutes and ions[1]. Plant aquaporins are classified within the superfamily of Major Intrinsic Proteins (MIPs). The classification of the aquaporins into five subfamilies provide the PIPs, (plasma membrane intrinsic Proteins), the TIPs (tonoplast intrinsic proteins), the NIPs (nodulin26-like intrinsic proteins), SIPs (small basic intrinsic proteins)[2], XIPs (X-intrinsic proteins), depending on their sequence homology and subcellular localisation. Aquaporins are formed by four monomers, each monomer constituting a functional pore, that together constitute stable tetramers formed by interaction of the monomers with each other through neighbouring membrane-spanning α-helices, via hydrophobic interactions and hydrogen bonds[3]. This close association forms an additional fifth, central pore with suggested functions in gases and ion transport[4,5]. Each monomer is configured by six membrane-spanning α-helices (H1–H3 and H4–H6) and two re-entrant short α-helices (HB and HE) with five interconnecting loops (LA-LE) that collectively form a right-handed α-helical bundle, configuring the typical hourglass morphology with two opposite vestibules. The N- and C-termini are both located on the cytoplasmic side of the membrane[6].

The specificity of the solute transport through the different aquaporin homologues is mainly controlled by steric occlusion provided by the specific residues that constitute each monomer; among these, the two main constrictions of the pore channel appear to have a determining role. The first constriction is formed at the center of the “hourglass” pore by the opposite juxtaposition of two Asn-Pro-Ala (NPA) motifs situated at the end of the two re-entrant helices, in loops B and E[6]. NPA motifs have a critical function in proton exclusion and as a size barrier. The second constriction, known as the aromatic/arginine (ar/R) selectivity filter, is formed at the extracellular vestibule by four residues, one each in helices 2 and 5 (H2, H5) and two in Loop E (LE1 and LE2), and is the narrowest part of the pore in most of the aquaporin homologues[7] that give rise to the possibility of transporting different specific molecules while serving as an exclusion barrier for others[8] (for a review, see Luang and Hrmova, 2017)[3]. However, in addition to these two well-known filters, other amino acid residues have been shown to be important in the discrimination between the transport of molecules, the Froger’s positions (FPs)[9] located in Loop C (P1), Loop E (P2–P3) and transmembrane helix 6 (P4–P5). These residues were described in the first years of aquaporins discovery, but they have been little studied[10-12].

In this way, in addition to water, plant aquaporins have been investigated for their capacity to transport a wide range of solutes such as carbon dioxide (CO2), hydrogen peroxide (H2O2), short polyols like glycerol (Gly), NH3, urea, boric acid (boron, B), silicic acid (silicon, Si), arsenic (As), antimonite (Sb) and some other compounds like germanic acid, selenic acid, lactic acid, formamide and acetamide (for a detailed bibliography see Luang and Hrmova, 2017)[3]. This reveals the great influence that aquaporins can have on the control of growth, nutrition, osmoregulation, signaling, ion homeostasis and defense against different stresses[13-15].

Melon (Cucumis melo L.) is a eudicot diploid species (2n = 24) and one of the most important cucurbits. The great importance of melon is due not only to its great economic importance but also to its nutritional properties as the presence of health-promoters including β-carotenes, folic acid, phenolic acids, vitamins A and C and minerals[16,17], being of special interest in nutrition and human healthcare[18]. The crop highly demand an appropriate water balance[19] related with its capacity to adapt quickly under abiotic stress conditions. This suggests strong control of water and nutrients transport in membranes, pointing to aquaporins as one of the most interesting targets in these plants.

Therefore, as each aquaporin is structurally unique and that simple variations of specific residues can produce an altered solute selectivity, the aim of this study was to identify, compare and analyse the complete set of aquaporins in melon. The comparative analysis of aquaporins was performed to understand the evolutionary relationship of melon with related plant species, to predict their possible roles in the transport of other compounds apart from water and to determine their constitutive expression (determining structural importance of NPA motifs, the ar/R region and FPs). Analysis by qPCR was carried out in roots and leaves after the design of primers and a comparison with RNA-seq results was done with existing databases.

Material and methods

Identification of putative C. melo aquaporins (CmAQPs)

The complete set of aquaporin protein sequences of watermelon (Citrullus lanatus L.)[20], identified by Zhou et al., 2019, was used as a template in the PSI-Blast tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with default parameters and the three-iterations method. This was performed against the C. melo database and using the non-redundant protein sequences (nr) database.

All the sequence-related information was retrieved from the National Centre of Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA246165)[21] and compared with the corresponding sequences found in the Cucurbit database (CuGenDB) (http://cucurbitgenomics.org/organism/18) collection. Exon–intron analysis of sequences were performed using Microsoft Office package (2016) software (Supplementary Figure S1).

Protein properties, sequence analysis and phylogenetic studies

Protein features such as the isoelectric point (Pi) and molecular weight (Mw) were calculated with Expasy’s ProtParam tool (https://web.expasy.org/protparam/). The transmembrane domains and number of transmembrane helices were elucidated using the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/)[22]. Motifs locations was performed by MEME web server (http://meme-suite.org/tools/meme). The subcellular location was predicted with two different prediction softwares: Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) and WoLF PSORT (https://www.genscript.com/wolf-psort.html), using the default parameters. Protein tertiary structures were predicted with the PSIPRED server[23].

Furthermore, the important residues involved in the specific transport functions of the aquaporins were identified, first, with alignments of each aquaporin family (PIPs, TIPs, NIPs, SIPs and XIP) performed in the Mega X software with the MUSCLE algorithm[24]. Second, we used the already-described Spinacia oleracea PIP1 protein (accession number AAA99274.2) as a pattern to locate all the functional residues—like the NPA motifs (loop B and loop E, respectively), the ar/R filters (H2, H5, LE1, LE2)[6,25,26] and the FPs (P1-P5) described by Froger et al. (1998)[9]. The prediction of the transport of substances other than water was obtained by comparison with orthologues and their known functions in Arabidopsis thaliana L., rice (Oryza sativa L.) and maize (Zea mays L.). Their functions as transporters are related to the same structural positions[11,12]. In addition to this, comparisons of homologues[10,11], mutational studies[12,26-28], pore structure analyses[29,30] and experimental studies[12,13,26-28,31-46] have been taken into account, leading us to suggest the transport possibilities for each aquaporin of C. melo.

For the phylogenetic analysis and tree construction, C. melo aquaporins protein sequences were aligned with all the characterised aquaporins from C. lanatus[20], A. thaliana[2] and Cucumis sativus L.[47] (Fig. 1). Mega X was employed to build the phylogenetic tree. We aligned all the sequences with the MUSCLE method and then used a Neighbour Joining (NJ) algorithm employing 1000 bootstrap replicates, a Poisson model and pairwise deletion. The same analysis and tree construction were performed for the C. melo, A. thaliana, Z. mays[48] and O. sativa[49] phylogenetic tree (Supplementary Figure S2).

Figure 1

Phylogenetic analysis of aquaporin family proteins of C. melo (filled circles), A. thaliana (triangles), C. lanatus (stars) and C. sativus (squares). We used MUSLE to align the protein sequences and the NJ method (with 1000 bootstrap replications) to build the tree, all with MEGA X. Phylogenetic tree design has been done with the online tool “Interactive Tree Of Life” (iTOL; https://itol.embl.de/). The different MIP sub-families are highlighted as: PIPs (green; PIP1s light green and PIP2s dark green), TIPs (sky blue), NIPs (orange), SIPs (pink) and XIPs (purple). The abbreviation of the species is as follows: Cm (C. melo), At (A. thaliana), Cl (C. lanatus), Cs (C. sativus). The meanings of the suffixes in the C. sativus names are: lk: like, pb: probable, pd: predicted.

Plant material and growth conditions

Seeds of melon, C. melo var. Grand Riado, supplied by SAKATA SEED IBERICA S.L.U, were hydrated with deionised water and aerated for 16 h. After this, they were germinated in vermiculite, in the dark at 28 °C, for 2 d. They were then transferred to a controlled-environment chamber having a 16-h light and 8-h dark cycle with temperatures of 25 and 20 °C and relative humidity of 60 and 80%, respectively. Photosynthetically active radiation (PAR) of 400 µmol m−2 s−1 was provided by a combination of fluorescent tubes (PHILIPS TLD 36 W/83, Jena, Germany and Sylvania F36 W/GRO, Manchester, NH, USA) and metal halide lamps (OSRAM HQI, T 400 W, Berlin, Germany). After 3 d, once the seeds germinated, 16 seedlings were placed in 15-L containers, 4 seedlings per container, with continuously-aerated Hoagland nutrient solution[50]. The solution was replaced completely every week. After a month of growth, 6 plants were harvested separating roots and leaves from the rest of the plant, instantly frozen in liquid nitrogen and stored at − 80 ºC. The experimental design was completed randomized design (CRD).

RNA extraction and retro transcription (RT)

Total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s protocol. The quantity and purity of RNA was measured with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). Integrity of the RNA was measured by electrophoresis in agarose gel. Contaminating DNA was removed using RNase-free DNase solution, (Thermo Fisher Scientific) according to the manufacturer’s instructions. The RNA extracted was stored at − 80 °C until use. The absence of DNA contamination was checked in all samples by polymerase chain reaction (PCR) using recombinant Taq DNA Polymerase (Thermo Fisher Scientific) with aliquots of the same RNA that had been subjected to the DNase treatment but not to the reverse-transcription step according to the manufacturer’s protocol. The High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used to synthesise cDNA from 2 µg of total RNA, according to the manufacturer’s protocol.

Primers design

The primer sets used to amplify each aquaporin gene were specifically designed in the 3′ or 5′ non-coding region of each gene, in order to avoid the non-specific amplification of other aquaporin genes, since in the coding region the homology between their sequences is very high[51]. For this same reason, the primer design was carried out manually, meeting the specific requirements imposed by the high homology of the sequences used and the technique used for the analysis. Virtual analysis of melting temperature, primer hairpins, self-dimers, hetero-dimers and individual and total ΔG was performed with PCR Primer Stats (https://www.bioinformatics.org/sms2/pcr_primer_st) and IDT Oligo Analyzer Tools (https://eu.idtdna.com/calc/analyzer/). The ΔG accepted for dimer analysis was always less than − 6.5 kcal/mol (Table 1). The specificity of the amplicons was checked using the virtual nucleotide basic local alignment search tool (NCBI nucleotide BLAST: https://blast.ncbi.nlm.nih.gov/Blast.cgi) and then physically, with the total DNA extracted from 50 mg of frozen sample (DNeasy Plant Pro Kit. QIAGEN, Hilden, Germany) according to the manufacturer’s protocol, by standard PCR using Taq DNA Polymerase, recombinant (Thermo Scientific). The efficiency of the primer sets was evaluated with the software QuantStudio 5 (QuantStudio Design and Analysis Software version 1.4.0.0), by analysing the threshold cycle (Ct)/fluorescence ratio at six independent points of PCR curves[52], giving values between 95 and 100% (Table 1). Five housekeeping primers—CmACT (gi: MU51303), CmADP (gi: MU47713), CmGAPC2 (gi: MU54550), CmRAM (gi: MU45556) and CmRLP (gi: MU45916)—for C. melo, selected according to Kong et al. (2014)[53], were checked in each cDNA used in the quantitative PCR quantification (qPCR) and were measured using a Visual basic application for Excel (GeNorm) that automatically calculates the gene stability[54]. CmRAN (encoding the GTP-binding nuclear protein) was then selected as the reference gene for the standardisation of each sample. The sequences and features of the primers used for the 31 melon aquaporin genes and one constitutively-expressed gene are shown in Table 1.

Table 1

Primer used for measurement of C. melo (Cm) aquaporins expression by RT-qPCR.

Gene name	Primer sequence 5′– > 3’forward/reverse primer)	Product size (bp)	ΔG Primer	ΔG Self-dimer	ΔG hetero-dimer	E (%)	Tª melting	Tª annealing	DNA dilution
CmRAN #	F:TGCACCCTTTGGACTTCTTC R:GATGTAGTAGCCATCCCGTAAAC	104	–	–	–	98.7	–	–	–
CmPIP1;1	F: GCTTCTTCAATCAATCACAGC R: ACCATTACATAGCTTCATAGCC	181	− 37.26 − 38.53	− 4.74 − 6.34	− 4.74	99.7	75.81	60 °C	1:10
CmPIP1;2	F: CATTCTTCCCCAAAAGCAAA R: AGTTTTCAGAAAGGCAGCCA	179	− 39.64 − 38.89	− 3.54 − 6.21	− 5.49	98.81	76.17	60 °C	1:10
CmPIP2;1	F: TTAAGCCTAATAGTTGTGTGC R: AAAAGAGAGCAAAAACCACG	147	− 36.17 − 39.91	− 4.85 − 3.61	− 5.09	100.36	75.16	60 °C	1:2
CmPIP2;2	F: GGATCTAAAATGTGTGATTAGG R: ATAAAACCTTCAAAATATGTGC	158	− 36.73 − 37.88	− 4.62 − 3.91	− 4.67	100.15	73.91	57 °C	1:10
CmPIP2;3	F: ATCTCTAACATCCATCACTCC R: TAGTTAGCTTGTGGTGGTCG	94	− 34.68 − 36.3	− 1.47 − 6.34	− 5.85	99.98	76.96	60 °C	1:5
CmPIP2;4	F: CTCTCGTCTGCTTTGGTCC R: ATACATAGGATGAGAATGAGC	158	− 36.06 − 34.27	− 3.61 − 3.43	− 4.75	99.13	75.14	60 °C	1:5
CmPIP2;5	F: TTCCTTTTGCTATTGGAATGG R: TTGCCTGTTACATTACTAGG	89	− 42.08 − 36.1	− 6.59 − 4.67	− 5.37	100.32	71.69	57 °C	1:5
CmPIP2;6	F: TCTGCTCTATAAATCTTATCCC R: TAACACACTTCATTAGTTCAGC	144	− 37.07 − 35.44	− 4.38 − 3.29	− 4.74	99.43	74.25	57 °C	1:2
CmPIP2;7	F: CCATTCCATAAGCAAAAGACT R: GAACATTAGTAAGCCAAGTGG	177	− 38.06 − 36.71	− 3.14 − 5.02	− 5.02	96.27	72.00	60 °C	1:2
CmPIP2;8	F: ACAAAACCAAAGAAGTGTTCG R: GCAGGATCTCAGTGAATGTG	158	− 37.87 − 34.65	− 3.61 − 4.62	− 3.52	99.72	76.51	60 °C	1:5
CmPIP2;9	F: CTTCTTCCTTACACTTCATGC R: TTACCCAATTACAAAAGATTGC	119	− 36.09 − 39.48	− 5.38 − 5.37	− 5.12	97.43	73.48	60 °C	1:2
CmPIP2;10	F: AAGAAGATGATGGTAGAAGTGG R: CATTCAAAGACAATCCCTTCC	120	− 37.12 − 38.58	− 1.47 − 3.54	− 5.12	99.95	73.21	60 °C	1:10
CmTIP1;1	F: CGTCAACTTCTTTGTTCTACGT R: CAATTTAATACGACATCAAAATGG	90	− 38.04 − 41.76	− 6.30 − 5.36	− 5.84	95.41	73.29	60 °C	1:10
CmTIP1;2	F: TCAACCACCACCACCACC R: GACACGACCAAACCCATCC	129	− 34.34 − 37.21	Non − 3.61	− 1.57	99.44	75.19	60 °C	1:5
CmTIP1;3	F: TCTTGACTTTATTCAGAGACC R: ATTCTCTTCCTGATTCTTAGC	105	− 34.14 − 35.72	− 3.53 − 3.14	− 6.35	99.90	72.66	57 °C	1:2
CmTIP2;1	F: TCCCTTTGTAATAAGAGGAGG R: AAGAAGAGAATCCAATGAACC	133	− 37.87 − 36.73	− 4.67 − 1.95	− 4.64	99.64	73.77	60 °C	1:10
CmTIP2;2	F: GTGTAAAAAATGAAACCAAAACG R: TTGAGGGAAAACCGAAGAAGG	150	− 41.36 − 41.95	− 1.95 − 3.61	− 3.90	99.82	77.43	60 °C	1:2
CmTIP3;1	F: TTTCTGCTCTATATGTTGTAGG R: CTGTATGACATTTATTACCTTC	144	− 35.96 − 34.39	− 3.91 − 3.43	− 4.77	100.3	75.69	57 °C	1:10
CmTIP4;1	F: GTCATCATACTTACCATTTGC R: ACTACAAGAAACTGGAAAGG	104	− 34.98 − 34.30	− 3.14 − 1.95	− 5.02	98.37	74.58	60 °C	1:2
CmTIP5;1	F: TTTAAGCGTTGGTTTTGTGC R: GATAAAAATTCATGTTAGATACAC	97	− 39.02 − 36.66	− 4.85 − 5.38	− 5.83	99.10	72.01	60 °C	1:2
CmNIP1;1	F: CCTTACTTCACATGAAACTAGG R: CAGCCATCAAGAAGTTTGG	99	− 36.74 − 35.63	− 5.47 − 5.02	− 6.83	100.3	73.11	60 °C	1:5
CmNIP2;1	F: ATAGTTTGAGTGTTTTAATGAGC R: GGCTACTTCTGATACATTGC	124	− 37.51 − 34.6	− 4.85 − 3.14	− 5.37	100.3	71.99	60 °C	1:10
CmNIP2;2	F: GAGAAGAATGAATCTGAAATAGG R: GAAAAGAAGAACCAATTTTATGG	115	− 37.93 − 40.72	− 3.17 − 5.83	− 5.36	99.05	70.03	60 °C	1:5
CmNIP4;1	F: AAAGGAAGAACATAAACGATAAC R: ATTGAGTCTCAGAAAGAAAGG	86	− 39.06 − 35.38	− 3.61 − 5.13	− 3.9	99.21	72.27	60 °C	1:5
CmNIP5;1	F: AGAATAAAGTTGAGAAGAAAAGG R: GGCAAGTAGAAACAATATAGCA	95	− 38.65 − 37.87	− 1.94 − 3.91	− 3.9	98.87	73.47	60 °C	1:10
CmNIP5;2	F: TGATAATGATAGTGGTCGTTG R: TTGACATGAAAGTAAAAGGTCG	75	− 35.08 − 38.59	− 3.61 − 5.38	− 3.61	100.2	72.54	60 °C	1:5
CmNIP6;1	F: CCCAGAGAACACTTTGAACC R: ATACACAATGACCAATACTTGC	137	− 36.39 − 36.7	− 1.95 − 3.90	− 3.9	100.2	75.56	60 °C	1:5
CmNIP7;1	F: CCCTCTATATTTCCAGTTGC R: AAGTAAGGTTTAATTTGATTACCG	132	− 36.25 − 41.89	− 3.91 − 5.36	− 4.67	100.7	72.46	60 °C	1:2
CmSIP1;1	F: GCAGTTATGTTTAGTTTGATTC R: CTAATGTCCAAAGTCTATAAGC	92	− 35.91 − 35.95	− 3.14 − 3.40	− 5.84	100.5	71.54	60 °C	1:2
CmSIP2;1	F: CTCTTAATGATTCCAATGTAGTG R: TGACGATGTTGTCGGATTCC	101	− 37.21 − 37.96	− 4.85 − 5.19	− 4.64	98.77	76.71	60 °C	1:2
CmXIP1;1	F: TCTTCCTCTTTTCTCTCAAGG R: GCAGTCGCTACTAATTCTGTC	101	− 36.86 − 36.23	− 4.67 − 5.36	− 1.95	NM	71.82	60 °C	1:2

Columns: gene name, primer sequence: up the forward (F) and below the reverse (R), product size (bp), ΔG primer, ΔG self-dimer, ΔG hetero-dimer between primers, % efficiency (E), Tª melting and qPCR conditions (Tª annealing and DNA dilution).

Shortening code: NM, No measurement.

#CmRAN primers were obtained from Kong et al.[53].

Quantitative real-time PCR (RT-qPCR) analyses

To compare the expression of all aquaporin genes, RT-qPCR was carried out. It was performed using 2 μL of 1:2, 1:5 or 1:10 diluted cDNA samples, depending on the gene being analyzed (see Table 1), in 10 μL of reaction medium containing 500 nM gene-specific primers and 5 μL of Power SYBR Green PCR Master Mix (Applied Biosystems by Thermo Fisher Scientific), in a QuantStudio 5 Flex, a Real-Time qPCR system (Applied Biosystems by Thermo Fisher Scientific) following the manufacturer’s instructions. The qPCR program consisted of a 10 min initial denaturation at 95 °C and then amplification in a two-step procedure: 15 s of denaturation at 95 °C and 60 s of annealing and extension at a primer-specific temperature for 40 cycles, followed by a dissociation stage. Data collection was carried out at the end of each round in step 2. These conditions were used for both target and reference genes, and the absence of primer-dimers was checked in controls lacking templates. Real-time PCR analysis was performed on 3–6 independent samples for each treatment (biological replicates) and each sample reaction was carried out in triplicate (technical replicates) in 96-well plates. The transcript levels were calculated using the 2−ΔΔCt method[55]. Negative controls without cDNA were used in all PCR reactions. Finally, the normalised expression levels were rescaled and presented as relative units (ru) with respect to the most highly-expressed aquaporin, CmTIP1;1, which was assigned a value of 100. The extremely high expression of CmTIP1;1 masks the presence of other aquaporins, which is why we separated them into three different groups depending on their relative levels of expression.

Comparison with previous RNA-seq

Our qPCR analyses were compared with the RNA-seq analyses in databases, namely, the work of Latrasse et al. (2017)[56]. For this, the levels of all RT-qPCRs were relativized to that of the gene that had the highest expression levels, as previously described. The same procedure was followed with all the genes analysed by RNA-seq. In this case, the maximum value was for PIP1;1 in roots, which received the same fixed value of 100, and the rest were relativized to it. Finally, the values of each aquaporin in the RNA-seq were compared with those obtained from our RT-qPCR. After being relativized, all aquaporins data were separated in groups of similar relative expression, as in the previous case of the qPCR data.

Data analysis

Statistical analyses were performed using the SPSS 25.0.0.1 software package. For the qPCR analyses, the Student’s t-test was performed. The qPCR and RNA-seq comparisons were analysed using one-way ANOVA, followed by the post hoc Tukey multiple comparison test. Significant differences between the values of all parameters were determined at P ≤ 0.05, according to Tukey’s test. The values presented are the means ± SE. To detect outliers in the qPCRs performed, the SPSS 25.0.0.1 software package was used.

Ethical approval

This article does not contain any studies with human or animal subjects.

Consent to participate

All the authors referred in this study acknowledge the authorship and agreed with the content.

Consent of publication

All the authors referred in this study give explicit consent to submit this article and consent to the publication of the data presented here.

Results

Genome-wide identification of CmAQP genes

A search of the whole genome for aquaporins proteins revealed 57 matches in the NCBI protein database, 34 corresponding to C. melo, while the rest of the non-selected sequences were specific to concrete varieties.

First, we analysed each matched sequence and searched for its accession in the Cucurbit genomics database. During this procedure we found three sequences (MELO3C025166.2, MELO3C000776.2 and MELO3C025165.2) that were not previously located in our first search of the NCBI protein database. After this, we analysed all the sequences, including these new finds, which resulted in the elimination of some sequences based on the following.

The CmTIP1;3 gene had three transcript variants in the nucleotide database and only one of them had the characteristic NPA motifs when transcribed to protein. So, the other two transcript variants were omitted.

Also, we found a high nucleotide sequence similarity among five sequences, the three new sequences found in the Cucurbit genomics database and two sequences from the 34 found in the NCBI database (XP_008466925, XP_008463222.1). Only XP_008463222.1 (from now on named CmPIP1;1) was a complete protein sequence including the two characteristic NPA motifs. Sequence alignments showed that XP_008466925 was, indeed, a partial sequence of CmPIP1;1 with 100% sequence identity. In addition, the other three sequences also had a high similarity to CmPIP1;1 but the alignment seemed to be shifted, so we thought these sequences were from the upstream region of the CmPIP1;1 gene, integrating only the second NPA motif. To prove this theory, we extracted the sequence downstream and upstream of the CmPIP1;1 gene in chromosome 10 and then aligned it against these three unknown sequences. As a result, all the sequences had at least 98–99% identity with the sequence including the CmPIP1;1 gene and the alignments were located upstream of CmPIP1;1, containing only the second NPA motif in their sequences, as we thought at the beginning. With these results and due to the lack of the two NPA motifs, we decided to omit the sequences from further analysis. The presence of this type of sequence in the database could be due to a non-curated identification method without experimental evidence.

In all the analyses carried out, a total of 31 putative aquaporin genes were identified (Table 2).

Table 2

List of the 31 aquaporins found in C. melo (Cm).

Identifiers			Gene features		Protein features					Subcellular location
Gene name	CCDBa	Geneb	Chromosome location	Exon no	mRNA length	Protein length	Mwc (kDa)	Isoelectric point	TMHc	Plant-mPLocd	WolF-PSORTd
CmPIP1;1	MELO3C025164.2	103501427	chr10: 9455124 .. 9456876 (−)	3	1202 bp	292 aa	31.47	7.67	5	plas	plas
CmPIP1;2	MELO3C005685.2	103482758	chr09: 23466873 .. 23469204 (+)	4	1275 bp	286 aa	30.71	9.13	6	plas	plas
CmPIP2;1	MELO3C014240.2	103491188	chr05: 4877174 .. 4878743 (+)	4	1172 bp	284 aa	29.86	7.71	7	plas	plas
CmPIP2;2	MELO3C014241.2	103491189	chr05: 4867151 .. 4869395 (−)	4	1239 bp	284 aa	30.21	8.78	6	plas	plas
CmPIP2;3	MELO3C014239.2	107990277	chr05: 4880993 .. 4882676 (+)	4	1245 bp	284 aa	29.89	8.22	6	plas	plas
CmPIP2;4	MELO3C025772.2	103501948	chr11: 28209027 .. 28210647 (−)	3	1381 bp	283 aa	30.36	7.63	6	plas	plas
CmPIP2;5	MELO3C019794.2	103496419	chr03: 23650515 .. 23655241 (+)	4	1390 bp	276 aa	29.37	9.56	6	plas	plas
CmPIP2;6	MELO3C014238.2	103491187	chr05: 4886375 .. 4887984 (+)	4	1178 bp	279 aa	30.02	8.58	6	plas	plas
CmPIP2;7	MELO3C012429.2	103489467	chr10: 537492 .. 541727 (−)	4	1116 bp	287 aa	31.24	9.41	6	plas	plas
CmPIP2;8	MELO3C009337.2	103486477	chr04: 32797579 .. 32799526 (+)	3	1490 bp	289 aa	31.03	9.10	6	plas	plas
CmPIP2;9	MELO3C014244.2	103491191	chr05: 4857028 .. 4859643 (−)	4	1308 bp	278 aa	29.40	8.58	6	plas	plas
CmPIP2;10	MELO3C013347.2	103490279	chr01: 16804141 .. 16806721 (−)	4	1199 bp	280 aa	29.91	9.24	6	plas	plas
CmTIP1;1	MELO3C024483.2	103500838	chr08: 10141935 .. 10143868 (+)	2	975 bp	250 aa	25.67	5.64	7	tono	plas
CmTIP1;2	MELO3C009377.2	103486517	chr04: 32517961 .. 32520018 (+)	2	988 bp	253 aa	26.34	6.03	7	tono	cyto, tono
CmTIP1;3	MELO3C025466.2	103501648	chr09: 5838747 .. 5839429 (−)	3	1145 bp	253 aa	26.55	5.53	6	tono	cyto, plas, tono
CmTIP2;1	MELO3C024263.2	103500601	chr01: 35574300 .. 35575883 (+)	3	1232 bp	248 aa	25.43	5.66	6	tono	tono
CmTIP2;2	MELO3C005526.2	103482603	chr09: 22169680 .. 22172778 (−)	3	1150 bp	250 aa	25.09	5.39	6	tono	tono
CmTIP3;1	MELO3C002183.2	103482730	chr12: 25767948 .. 25770390 (−)	3	1137 bp	284 aa	30.08	7.17	5	tono	mito
CmTIP4;1	MELO3C011146.2	103488186	chr03: 28321088 .. 28322961 (+)	3	1087 bp	247 aa	25.70	5.91	7	tono	tono
CmTIP5;1	MELO3C005441.2	103504693	chr09: 21620302 .. 21621635 (−)	3	957 bp	260 aa	26.86	8.31	6	Plas	chlo
CmNIP1;1	MELO3C007188.2	103484424	chr08: 1361212 .. 1363293 (−)	5	1326 bp	276 aa	29.54	9.48	6	plas	plas
CmNIP2;1	MELO3C009870.2	103487002	chr04: 28593871 .. 28598610 (−)	5	1360 bp	287 aa	30.41	9.15	6	plas	plas, tono
CmNIP2;2	MELO3C009871.2	103487003	chr04: 28560873 .. 28564531 (−)	5	1232 bp	261 aa	27.52	6.29	6	plas	plas
CmNIP4;1	MELO3C020281.2	103496839	chr06: 14916752 .. 14920413 (−)	5	1021 bp	269 aa	28.81	7.64	5	plas	tono
CmNIP5;1	MELO3C005818.2	103482897	chr09: 24541790 .. 24547172 (−)	4	1794 bp	298 aa	30.83	8.64	5	plas	plas
CmNIP5;2	MELO3C005817.2	103482896	chr09: 24536633 .. 24540050 (−)	5	1044 bp	250 aa	26.19	8.62	6	plas	tono
CmNIP6;1	MELO3C017831.2	103494651	chr07: 27500120 .. 27503882 (+)	5	2218 bp	304 aa	31.66	7.64	6	plas	tono
CmNIP7;1	MELO3C006559.2	103483738	chr06: 4173677 .. 4176268 (−)	5	1123 bp	268 aa	28.52	6.38	6	plas	plas
CmSIP1;1	MELO3C008793.2	103485971	chr08: 26514604 .. 26519163 (+)	3	1287 bp	243 aa	25.58	9.55	6	plas	tono
CmSIP2;1	MELO3C009719.2	103486855	chr04: 29912868 .. 29916655 (−)	3	1275 bp	316 aa	34.88	9.72	6	plas	plas
CmXIP1;1	MELO3C020774.2	103497290	chr11: 4268119 .. 4268934 (+)	2	1148 bp	316 aa	34.21	6.88	7	plas	plas

Columns: Identifiers (Gene name, ID from Cucurbit genomics database and NCBI accession), Gene features (chromosome location and exons number), protein features (mRNA length, protein length, molecular weight, isoelectric point and predicted number of transmembrane domains) and subcellular location prediction by Plant-mPLoc and Wolf-PSORT programs.

aID from Cucurbit genomics database.

bNCBI accessions.

cMw: molecular weight in kDa and TMHC: predicted number of transmembrane domains.

dShortening codes from subcellular location in Plant-mPLoc and Wolf-PSORT. Plas: plasma membrane; cyto: cytoplasm; tono: tonoplast; mito: mitochondria; chlo: chloroplast.

Nomenclature and classification

For the 31 putative aquaporin genes finally selected, their amino acid sequences were aligned for a phylogenetic analysis that divided them into sub-families, to help us to name them correctly. The C. melo aquaporins were named relative to their homology and phylogenetic relationships with those of C. sativus, C. lanatus and A. thaliana (Fig. 1).

The proteins were grouped into five sub-families consisting of twelve PIPs, nine TIPs, eight NIPs, two SIPs and one XIP (Fig. 1). The PIP sub-family was divided into two groups (PIP1 and PIP2), with two members in the PIP1 group and ten in PIP2. The TIP sub-family was divided into five groups: TIP1, with three members, TIP2, with two members and one member each in groups TIP3, TIP4 and TIP5. The NIPs were divided into NIP1, NIP4 and NIP7, with one member each, and groups NIP2 and NIP5, with two members each. The SIP sub-family was formed by SIP1 and SIP2, with one member each. Lastly, only one sequence represented the XIP group.

Chromosomal location, protein features and subcellular localisation prediction

These 31 putative aquaporin-encoding genes were located on all chromosomes except chromosome 2, in a non-uniform manner: chromosome 9 incorporated six aquaporins genes, while chromosomes 4 and 5 had five genes each, chromosome 8 had three genes, two genes were identified on each of chromosomes 1, 3, 6, 10 and 11 and chromosomes 7 and 12 possessed only one putative aquaporin gene (Fig. 2). Exon–intron analysis showed a similar distribution on exons structures among each aquaporin family displaying 3–5 exons per gene (Supplementary Figure S1).

Figure 2

Chromosomal distribution of aquaporin genes in C. melo. The different MIP sub-families are distinguished using different-coloured boxes: PIPs (red), TIPs (yellow), NIPs (green), SIPs (pink) and XIPs (purple). The abbreviations are as follows: Cm (C. melo), Chr (chromosome). This figure was made with Microsoft Office 2016 package.

The list of identified aquaporins, together with basic statistics on the primary protein sequences, is reported in Table 2. The PIPs protein length ranged from 276 (CmPIP2;5) to 292 residues (CmPIP1;1), with a median of 283 amino acids (aa) per protein, while that of the TIP sub-family ranged from 247 to 284 residues (CmTIP4;1 and CmTIP3;1, respectively), with a median of 255 aa. In the case of the NIPs, the length varied between 250 (CmNIP5;2) and 305 residues (CmNIP6;1), with an average of 276 aa. Finally, the SIP sub-family (with only two members) had a mean length of 275 aa. Motifs structure of each AQP of the same subfamily showed similar motifs structures (PIPs, TIPs and NIPs), NPA motif, and a large motif (YRALIAEFIATLLFLFVGVLTVIGYSKQTDTASLGGIG) conserved in all the sequences. However, SIPs and XIP1.1 showed different motif distribution, with some similarities between themselves. (Supplementary Figure S3).

Table 2 also displays the Pi and Mw data. The Pi varied among the families but was more or less constant among the sub-family members, with some exceptions. The PIPs sub-family had an average Pi of 8.58. In contrast, the TIPs group showed a mean Pi of 6.08, the lowest of all the sub-families, but CmTIP5.1 stood out with a Pi of 8.31, quite high for this group. The median Pi was 7.81 in the NIPs and, lastly, the SIPs displayed the highest Pi of all the sub-families, with an average of 9.63. The Mw did not vary much among the sub-families, unlike the Pi. The PIPs, NIPs and SIPs had mean values of 30.27 kDa, 29.09 kDa and 29.32 kDa, respectively; only the TIPs presented a notable difference, with an average of 26.39 kDa.

One of the principal features that define not only the aquaporins but also Integral Membrane Proteins (IMPs) is the presence of transmembrane helices. In the case of aquaporins, they must number at least six. We present, in Table 2, a prediction of the possible number of transmembrane motifs of each protein. For four of them, only five transmembrane helices were predicted and in order to confirm these results, we analysed the 3D structures of these proteins (Fig. 3); it can be seen that these four proteins displayed six transmembrane helices.

Figure 3

3D protein structure prediction for C. melo aquaporins: CmPIP1.1, CmTIP1.3, CmNIP4.1 and CmNIP5.1. Identification of the six transmembrane helices (H1–H6) and the additional two re-entrant helices are highlighted with different colours (H1 fuchsia, H2 green, H3 orange, H4 yellow, H5 sky blue, H6 dark blue, Re-entrant helices salmon). Protein structures were performed using Jmol software.

Prediction of the subcellular localisation, based on bioinformatics tools (the Plant-mPLoc and Wolf-PSORT programs), predicted all the PIP, SIP and XIP aquaporins situated on the plasma membrane. Most of the TIPs were predicted in the tonoplast, with certain exceptions: The Wolf-PSORT program predicts CmTIP1;1 in the plasma membrane, CmTIP3;1 in the mitochondrial membrane and CmTIP5.1 in the chloroplast membrane. As for the NIPs, almost all were predicted to have a plasma membrane localisation—except CmNIP2;1, CmNIP4;1, CmNIP5;2 and CmNIP6;1, which Wolf-PSORT placed in the tonoplast.

Study of functional residues in aquaporins and possible solute transport

The functions of aquaporins are highly delimited by a few aa residues, mostly the NPA motifs, ar/R filter and FPs. These functional residues are listed in Table 3. The AQP phylogenetic framework can be used to predict the putative function of individual AQPs on the basis of orthologous genes from A. thaliana and other species[11]. Thus, the identification of conserved motifs in each subfamily and in each cluster of orthologous genes offers a framework for studying their possible functional implication[57]. Based on this principle, a comparative analysis of the main important residues of orthologous genes in C. melo and A. thaliana and their homologues in other species such as rice and maize was performed (Supplementary Table S1), as the transport of different solutes by their aquaporins has been studied previously, both functionally and experimentally. In addition to this comparison of homologues, mutational studies, pore structure analyses and experimental studies were considered, to propose the transport possibilities of each C. melo aquaporin, specified in Table 3.

Table 3

Identification of important residues and transport prediction for C. melo (Cm) aquaporins.

Gene name	NPA motif	ar/R selectivity filter				Froger’s positions					Transport prediction
	LB/LE	H2	H5	LE1	LE2	P1	P2	P3	P4	P5
CmPIP1;1	NPA/NPA	F	H	T	R	Q	S	A	F	W	CO2abc, H2O2ab, B*1
CmPIP1;2	NPA/NPA	F	H	T	R	E	S	A	F	W	CO2b*2, H2O2b, Urea *1
CmPIP2;1	NPA/NPA	F	H	T	R	Q	S	A	F	W	CO2ab, H2O2ac
CmPIP2;2	NPA/NPA	F	H	T	R	Q	S	A	F	W	CO2ab, H2O2ac
CmPIP2;3	NPA/NPA	F	H	T	R	Q	S	A	F	W	CO2ab, H2O2ac
CmPIP2;4	NPA/NPA	F	H	T	R	Q	S	A	F	W	CO2ab, H2O2ac
CmPIP2;5	NPA/NPA	F	H	T	R	Q	S	A	F	W	CO2ab, H2O2a
CmPIP2;6	NPA/NPA	F	H	T	R	Q	S	A	F	W	CO2ab, H2O2a
CmPIP2;7	NPA/NPA	F	H	T	R	Q	S	A	F	W	CO2ab, H2O2a
CmPIP2;8	NPA/NPA	F	H	T	R	Q	S	A	F	W	CO2ab, H2O2a
CmPIP2;9	NPA/NPA	F	N	A	R	K	S	A	F	W	–
CmPIP2;10	NPA/NPA	F	H	T	R	M	S	A	F	W	CO2abc, H2O2*2, Asc*3
CmTIP1;1	NPA/NPA	H	I	A	V	T	A	S	Y	W	H2O2*2, NH3j*2, Ureab*2, B*1
CmTIP1;2	NPA/NPA	H	I	A	V	I	A	A	Y	W	H2O2*2, NH3j*2, Ureab*2, B*1
CmTIP1;3	NPA/NPA	H	I	A	V	T	T	A	Y	W	H2O2*2, NH3j*2, Ureab*2, B*1 Gly*3
CmTIP2;1	NPA/NPA	H	I	G	R	T	S	A	Y	W	H2O2ac, NH3abcghj, Ureaabcdg
CmTIP2;2	NPA/NPA	H	I	G	R	T	S	A	Y	W	H2O2ac, NH3abcghj, Ureaabcdg
CmTIP3;1	NPA/NPA	H	I	A	R	T	A	S	Y	W	NH3gj, Ureabd
CmTIP4;1	NPA/NPA	H	I	A	R	T	S	A	Y	W	H2O2a, NH3ahj, Ureaabd*2
CmTIP5;1	NPA/NPA	N	V	G	C	I	A	A	Y	W	Uread
CmNIP1;1	NPA/NPA	W	V	A	R	F	S	A	Y	I	H2O2a*1*2, Asc*2*3, Sb*2, B*1, Glyce*1*2
CmNIP2;1	NPA/NPV	G	S	G	R	L	T	A	Y	F	Siabik*1*3, H2O2*1, Asac*1*3, Sbak*1*3, Ureaabc*3, Bbck*1*3, Glyc*1
CmNIP2;2	NPA/NPA	C	S	G	R	L	S	A	Y	M	Sik, Ask, Bk
CmNIP4;1	NPA/NPA	W	V	A	R	L	T	A	Y	I	Glye
CmNIP5;1	NPS/NPV	A	I	G	R	F	T	A	Y	L	Asack*2, Sba*2, Back*2, Ureaf, Glycf
CmNIP5;2	NPS/NPV	S	I	G	R	F	T	A	Y	L	–
CmNIP6;1	NPA/NPV	T	V	A	R	F	T	A	Y	L	Sba
CmNIP7;1	NPA/NPA	A	V	A	R	F	S	A	Y	I	Ureaf, Glyf
CmSIP1;1	NPT/NPA	F	I	P	N	M	A	A	Y	W	–
CmSIP2;1	NPL/NPA	I	H	G	S	F	V	A	Y	W	–
CmXIP1;1	SPI/SPA	I	I	V	R	M	C	A	F	W	–

Columns: gene name; NPA motif (LB and LE positions); ar/R selectivity filter (H2, H5, LE1, LE2), Froger’s positions (P1–P5) and transport prediction.

Shortening codes: LB, loop B; LE, loop E; H, hélix; Si, silicon; B, boron; As, arsenic; Sb, antimonite; Gly, glycerol. Aminoacide residues named with letters according the international code.

aTransport prediction according to Azad et al.[12].

bTransport prediction according to Hove and Bhave[10].

cTransport prediction according to Perez Di Giorgio et al.[11].

dTransport prediction according to Dynowski et al.[28].

eTransport prediction according to Wallace et al.[29].

fTransport prediction according to Wallace and Roberts[27].

gTransport prediction according to Azad et al. (2011).

hTransport prediction according to Kirscht et al.[74].

iTransport prediction according to Deshmukh et al.[85].

jTransport prediction according to Jahn et al.[30].

kTransport prediction according to Mitani-Ueno et al.[26].

*1 prediction based on Zea mays L. aquaporins orthologues and homologues.

*2 prediction based on Arabidopsis thaliana L. aquaporins orthologues and homologues.

*3 prediction based on Oryza sativa L. aquaporins orthologues and homologues.

Expression analysis

After primer verification, the expression of 31 aquaporins of C. melo was analysed in root and leaf tissue by RT-qPCR. All aquaporins were detected in both tissues, except PIP2;9, which was only detected in roots, and XIP1;1, that could not be detected in any sample.

Figure 4 shows the aquaporins grouped according to their levels of expression (see Material and Methods). Group 1 contains the aquaporins that showed higher expression (10–100 ru) in both tissues: TIP1;1, PIP1;2 and PIP1;1 (Fig. 4a). Group 2 contains those with medium levels of expression (1–10 ru): PIP2;2, PIP2;3, PIP2;6, PIP2;10, TIP3;1, NIP2;1, NIP2;2, NIP5;1 and NIP5;2 (Fig. 4b). Finally, group 3 contains the aquaporins PIP2;1, PIP2;4, PIP2;5, PIP2;7, PIP2;8, PIP2;9, TIP1;2, TIP1;3, TIP2;1, TIP2;2, TIP4;1, TIP5;1 NIP1;1, NIP4;1, NIP6;1, NIP7;1, SIP1;1, SIP1;2 and XIP1;1, which showed low levels of expression (0–1 ru) (Fig. 4c).

Figure 4

Expression levels of aquaporins genes. All the analyses were performed using qPCR, for leaves and roots. Statistical analysis was performed using SPSS 25.0.0.1. The values are the means ± s.e.m. of 3 to 6 biological replicates. Columns with * differ significantly according to Tukey’s test (p = 0.05). Each group is formed by aquaporins genes with similar expression levels represented as relative units (ru) (a) Group 1 (10–100 ru): PIP1;1, PIP1;2 and TIP1;1 (b) Group 2 (1–10 ru): PIP2;2, PIP2;3, PIP2;6, PIP2;10, TIP3;1, NIP2;1, NIP2;2, NIP5;1 and NIP5;2 (c) Group 3 (0–1 ru): PIP2;1, PIP2;4, PIP2;5, PIP2;7, PIP2;8, PIP2;9, TIP1;2, TIP1;3, TIP2;1, TIP2;2, TIP4;1, TIP5;1 NIP1;1, NIP4;1, NIP6;1, NIP7;1, SIP1;1, SIP1;2 and XIP1;1. This figure was made using Microsoft Office 2016 package.

The aquaporin that showed the highest gene expression in the analysed tissues was TIP1;1. Among the PIP subgroups, the PIP1s clearly showed higher expression levels, PIP1;1 being stronger in the leaves and both PIP1;1 and PIP1;2 in the roots, while of the PIP2s the most-highly expressed was PIP2;10, in both roots and leaves, this latter tissue also showing high expression of PIP2;6. Nor should the presence of PIP2;2 and PIP2;3, in both tissues, and of PIP2;4, in roots, be neglected. Within the TIPs subfamily, TIP1;1 exhibited the highest expression in both roots and (especially) leaves, followed by TIP3;1, in both tissues. Attending to the NIPs groups, the most expressed were NIP2;1, NIP2;2, NIP5;1 and NIP5;2; the expression of NIP2;1 was more important in leaves while both NIPs5 and NIP2;2 were expressed in roots and leaves at similar levels. Regarding the rest of the aquaporins, it should be noted that most of them appeared to a greater extent in the roots, being either lower, or practically nil, their presence in leaves, with no significant differences between them. As exceptions, PIP2;5, TIP2;2 and TIP1;3 had practically the same level of expression in both tissues, although slightly higher in leaves.

Comparison with RNA-seq expression

After our expression analysis, we decided to compare the expression of 31 aquaporins of C. melo var. Grand Riado with the RNA-seq of another variety, in this case Cantalupo (cantaloupe)[56]. The comparative analysis of the two techniques highlighted three different behaviour patterns (Fig. 5). Some aquaporins had very similar levels in both root and leaf tissues, such as PIP2;6, TIP1;1, TIP2;1, TIP1;3, TIP5;1 and XIP1;1. Other aquaporins had similar levels in one tissue: like PIP1;2, PIP2;9 and PIP2;10 in roots, and PIP2;1, PIP2;3, TIP3;1, TIP4;1, NIP1;1, NIP2;1, NIP5;1, NIP5;2 and NIP7;1 in leaves. By contrast, we obtained completely different patterns for other aquaporins: namely, PIP1;1, PIP2;2, PIP2;4, PIP2;7, TIP1;2, TIP2;2, NIP2;2, SIP1;1 and SIP2;1, which all had lower expression in our analysis than in the RNA-seq analysis, and PIP2;5, PIP2;8, NIP4;1 and NIP6;1, which all had higher expression in our analysis.

Figure 5

Comparison of the expression levels of aquaporins genes determined by qPCR with RNA-seq analysis in leaves and roots. Statistical analysis was performed using SPSS 25.0.0.1. Each bar represents the mean of 2 biological replicates for the RNA-seq data, and of 3 to 6 biological replicates for RT-qPCR. Columns with * differ significantly according to Tukey’s test (p = 0.05). Each group is formed by aquaporins genes with similar expression levels represented as relative units (ru). (a) Group 1 (20–120 ru): PIP1;1, PIP1;2 PIP2;4, TIP1;1 and TIP2;2. (b) Group 2 (2–20 ru): PIP2;1, PIP2;2, PIP2;6, PIP2;10, TIP1;2, TIP4;1, NIP2;1, NIP2;2, NIP5;1, SIP1;1 and SIP1;2. (c) Group 3 (0–2 ru): PIP2;3, PIP2;5, PIP2;7, PIP2;8, PIP2;9, TIP1;3, TIP2;1, TIP3;1, TIP5;1, NIP1;1, NIP4;1, NIP5;2, NIP6;1, NIP7;1 and XIP1;1. This figure was made using Microsoft Office 2016 package.

Based on the RNA-seq analysis, TIP1;1 and PIP1;1 were the most expressed aquaporin isoforms in melon plants (Fig. 5a). Both PIP1s were among the aquaporins with more presence in both tissues (roots and leaves). Of the PIP2 isoforms, PIP2;4 was highly expressed, especially in root tissues, closely followed by PIP2;10, with a strong presence in leaves. Other prominent isoforms within this subgroup were PIP2;1, PIP2;2 and PIP2;6 (Fig. 5b). Within the TIPs subfamily, TIP2;2 seemed to be important in both tissues (Fig. 5a). Attending to the NIPs subfamily, NIP5.1, NIP2.1 and NIP2.2 were the most expressed, being very striking the high level of NIP5.1 in roots (Fig. 5a). Comparing the NIP2s, NIP2.1 was expressed more in roots while NIP2.2 was higher in leaf tissue (Fig. 5b). Of the SIPs, SIP1.1 was the most expressed, its presence being more important in roots (Fig. 5b).

Discussion

The availability of the whole genome sequence of melon would facilitate genome-wide analysis to identify the complete set of aquaporins. Also, some analyses of the expression of diverse aquaporins with RNA-seq techniques have been performed; thanks to these it is possible to find in the various databases the nucleotide sequences of a large number of melon aquaporins genes, although many of them come from diverse origins and have not been ordered, compared and classified. In this way, the number of aquaporins genes identified in melon (31) was comparable with the number found in other plant species, such as rice (33)[49], A. thaliana (35)[2] and watermelon (35)[20]. It seems that some aquaporin genes physically cluster very close to each other in certain regions, suggesting the occurrence of more than one gene tandem duplication event in the evolutionary history of melon, as was previously studied in tobacco plants[58]. There is an accumulation of the PIP2 genes in chromosome 5. In the same way, the co-location of CmNIP2;1 and CmNIP2;2 in chromosome 4 and of CmNIP5;1 and CmNIP5;2 in chromosome 9 could allude also to genomic duplication events. Despite this, further investigations are needed to clarify the evolutionary events that have occurred across the aquaporin family in C. melo.

The protein features, such as the mean Mw, of each subfamily also showed similarities to those of A. thaliana[59]. However, a notable difference is shown in the SIPs subfamily, the Mw being greater in melon than in A. thaliana; this could be due to the larger sequence found in CmSIP2;1, compared with AtSIP2;1. Furthermore, when the protein sequence of CmSIP2;1 was compared with those of homologous SIP2;1 proteins in other species (watermelon, arabidopsis and cucumber)[2,20,47], all the sequences aligned from the second methionine residue of CmSIP2;1. This suggests that this site is the starting point of translation, resulting in a protein of 238 aa and 25.99 kDa, thus equating the results to those obtained in arabidopsis for this subgroup. However, this hypothesis needs to be studied in order to clarify all the protein features previously predicted. In addition, TIPs were not found to be smaller than PIPs and NIPs, but most of them were more acidic (with lower Pi) than PIPs, NIPs, SIPs and XIP. The Pi could reflect a functional constraint imposed on MIPs[60]. Indeed, sequence analysis in arabidopsis revealed that the cause of this difference in Pi lies in the C-terminal regions, which are more basic in PIPs and NIPs than in TIPs. Therefore, it is possible that phosphorylation sites or sorting signals in the C-terminal regions form part of the hypothesised functional constraint on the sequences[60].

The aquaporins monomers found a highly conserved structure, with six transmembrane helices in all CmAQPs (Table 1, Fig. 3). These strongly conserved regions has been reported to be likely constrained to maintain the structural integrity of the aquaporin monomer and the conservation of critical residues which was essential for tetramer formation[61]. In this way the subcellular location of aquaporins, PIPs, NIPs and XIPs are usually located in the plasma membrane, while TIPs and SIPs are normally localised to the tonoplast and endoplasmic reticulum (ER), respectively[62,63]. However, our results showed that the PIPs, NIPs, SIPs and XIP were located principally in the plasma membrane (Table 2). This was expected, with the exception of the SIPs—that were not located in the ER by any of the programs used, although this is their most probable location. Nevertheless, a few of NIP proteins were located not only in the plasma membrane but also in the tonoplast and the TIPs showed a diverse range of possible subcellular locations depending on the chosen prediction software (chloroplast, mitochondria, plasma membrane and cytoplasm). Significantly, CmTIP5;1 was the only aquaporin assigned to chloroplasts, while CmTIP3;1 was localized in mitochondria (Table 2). As cell compartmentalization has represented the main driving force in the diversification of aquaporins in plants, no location should be excluded without specific analysis and more studies must be made to confirm these preliminary data. Nevertheless, NIPs isoforms have not yet been found in the tonoplast, so this does not appear to be their most likely location. A role for AtTIP5;1 as a nitrogen transporter in mitochondria has been proposed[64] and different isoforms of PIPs and TIPs have been detected in chloroplasts[65,66]; a role in the transport of CO2, water and H2O2 in chloroplast membranes could be beneficial to the plant dynamics[62]. So, the predicted localization of CmTIP3;1 in mitochondria and of CmTIP5;1 in chloroplasts cannot be rejected, even if this location for CmTIP5;1 does not seem to coincide with the gene expression data (Fig. 4c), which place it mostly in roots.

Analysis of possible functions in solutes transport (Table 3)

The function of PIPs in water transport is very specialised and their structure is highly preserved[67]. All PIPs have the same NPA motifs and ar/R selectivity filters residues (F, H, T and R in H2, H5, LE1 and LE2, respectively) (Table 3), directly related to their main role in water transport[7], while phylogenetic and functional studies suggest that they are equally capable of transporting CO2[10,12]. The FPs are also very similar in most PIP aquaporins from both subgroups (PIP1s and PIP2s). The S-A-F-W residues are well conserved in all cases in positions P2 to P5 respectively. In nine of the 12 PIPs, P1 was a Q (Gln) residue; the exceptions were CmPIP1;2 with E (Glu), CmPIP2;9 with K (Lys) and CmPIP2;10 with M (Met) (Table 3). The comparison of melon FPs with homologues in other species predicted the possible transport of H2O2 by almost all melon PIPs[12]. The only exception was the unusual CmPIP2;9, whose aa residues in the ar/R filter (F-N-A-R) and in the P1-FPs (K) differed from those of the PIP2s characterised to date and whose particular sequence has not been previously described. The sequence homology analysis (Supplementary Table S1) of PIPs that have been shown to transport B, urea and As resulted in the prediction of matching transport by orthologues in C. melo[11]. According to this, CmPIP1s could be able to transport boric acid and/or urea, like their orthologues ZmPIP1;1 and ZmPIP1;5[31,32], while CmPIP2;10 could transport As, as OsPIP2;6 does[45] (Supplementary Figure S2).

Based on the ar/R filter[68], the CmTIPs were classified in four groups attending to their homology. Group I is formed by CmTIP1;1, CmTIP1;2 and CmTIP1;3, Group IIa is formed by CmTIP2;1 and CmTIP2;2, Group IIb is constituted by CmTIP3;1 and CmTIP4;1 and Group III has only one member, CmTIP5;1. In general, TIPs seem to have developed the capacity to transport nitrogenous compounds[69]. The great importance of the H2 and H5 positions (H–I) with a non-polar LE1 (A/G) in the groups I, IIa and IIb has been related with NH3 transport[30]. Many TIPs have been characterised as urea transporters[32,33,46,70]. In the TIPs group I, the substitution of the typical R by V in the LE2 position has been proved to be involved in the transport of not only NH3 and but also H2O2, in mutagenic studies[30,71,72]. Regarding the FPs, the CmTIP1s presented slight differences, and a direct homology with residues of the other plants studied was not found (Supplementary Table S1). However, the phylogenetic analysis clearly related the CmTIP1s with the TIP1s of A. thaliana (Fig. 1). All the AtTIP1s were predicted to transport urea and H2O2[10-12] and this has been proven experimentally in heterologous systems[33,36,70], while NH3 transport has only been tested in mutagenic studies[72]. All this suggests that C. melo TIP1s are able to transport these solutes. The clade including CmTIP1;3, AtTIP1;3, ZmTIP1;2 and OsTIP1;2 diverged earlier, followed by the separation of CmTIP1;2 that presents a greater evolutionary divergence from AtTIP1s but is closer to OsTIP1;1 and ZmTIP1:1 (Supplementary Figure S2). Interestingly, it has been shown that OsTIP1;2 can transport glycerol[73] and that ZmTIP1;1 and ZmTIP1;2 can transport boric acid, in addition to NH3, urea and H2O2[46]. So, CmTIPs1 might be able to transport B and CmTIP1;3 might be able to transport Gly also (Table 3). Regarding Group IIa (TIP2s), their ar/R region has been directly related to the transport of NH3 and urea[28,33,72,74]. A specific amino acid, histidine (H), in loop C seems to be key for the de-protonation of NH4+, which would allow the transport of NH3 independent of pH[74]. This residue is present in CmTIP2s and CmTIP4.1, but not in the other TIPs subgroups, which lack the ability to extract a proton from an NH4+ ion; thus, the passage of ammonia relies on the pH-dependent concentration of uncharged NH3 in the medium[71]. The transport predictions for FPs indicate that they can transport not only NH3 and urea but also H2O2[10-12]; their relationship with AtTIP2s (Fig. 1) also suggests this capability, while the ability of orthologues to transport H2O2 has only been tested in assays with mutants[75]. Phylogenetic analysis of the TIPs group IIb showed that CmTIP3;1 is closely related to AtTIP3s (Fig. 1), although there is a substitution in FPs P3 (A is replaced by S) (Table 3). The ar/R analysis supports the NH3 and urea transport capacity[28,30,72]. As stated previously, CmTIP4;1 and CmTIP2s share all FPs, the His in loop C[74] and the same ar/R region (with the only exception of G instead of A in LE1) (Table 3), pointing to selective NH3 transport. Urea transport by CmTIP4;1 is supported by mutant studies[28] and has been shown to be transported by the orthologous AtTIP4.1[33]. The prediction based on the phylogenetic framework, and including FPs, also indicated the ability to transport H2O2[12]. CmTIP5;1 is the only aquaporin in group III and its orthologue AtTIP5;1 has been shown to transport urea[70]. Their FPs differ only in the P1 position (Table 3), which in C. melo is a residue of I instead of V (both non-polar); therefore, the transport of urea by CmTIP5;1 seems to be possible and is supported by mutagenic analysis of the ar/R region[28].

The NIPs form a monophyletic group[76] and overwhelming evidence supports a main role for NIPs in metalloids transport[77] beside glycerol—including B, Si, As and Sb [reviewed in Pommerrenig et al. (2015)]. Interestingly, all these compounds, in their uncharged states, have a geometry that is substantially similar to a conformation of glycerol in a retracted state; this would readily allow adaptation of the pore of the original aquaglyceroporin NIPs to the transport of these compounds[78]. Eight NIPs have been found in C. melo and have been classified into the three subgroups of NIPs based on their ar/R filter[79]. CmNIP1;1 and CmNIP4;1 belong to Group I. CmNIP5;1, CmNIP5;2, CmNIP6;1 and CmNIP7;1 belong to group II, although some of their compositions vary from the typical ar/R residues (discussed below), and CmNIP2;1 and CmNIP2;2 belong to Group III. The typical W-V-A-R in H2, H5, LE1 and LE2, respectively, are the characteristic ar/R residues in Group I and have been directly related to water and glycerol transport[29]. Both CmNIP1;1 and CmNIP4;1 have these characteristic motifs (Table 3). The phylogenetic tree places both in the same clade along with AtNIP1;1, AtNIP1;2, AtNIP2;1 and AtNIP3;1 (all in a branch with CmNIP1;1), AtNIP4;1 and AtNIP4;2 (both in the same branch as CmNIP4.1) (Fig. 1). Experimental studies showed that AtNIP1s were able to transport As, Sb, H2O2 and Gly[37,42,75,80]. CmNIP1;1 appears to have separated from AtNIP1s earlier, in an evolutionary branch that it shares with maize and rice NIP1s (Supplementary Figure S2). OsNIP1;1 can transport As[38] while ZmNIP1;1 has been shown, in heterologous systems, to transport H2O2 and glycerol; unexpectedly, it is also able to transport boric acid efficiently[46]. Thus, CmNIP1;1 might transport As, Sb, H2O2 and Gly, while B cannot be ruled out. Interestingly, while CmNIP4;1 belongs to group I and has the same ar/R motifs, its FPs are more similar to those of NIPs group II. This increases the possibility that it transports other interesting solutes, such as those that can be transported by NIP2s (discussed below). Despite this, the homology-based prediction did not allow a specific transport role to be assigned to CmNIP4;1, beyond glycerol transport compatibility[29], due to its clear differences in FPs from its orthologues (Supplementary Figure S2 & Supplementary Table S1). Strong evidence supports the role of the NIPs II group in boric acid transport[34,41,81]. Among the C. melo NIPs belonging to this group, the most important variation is in H2; in CmNIP5;2 it is an S residue and in CmNIP6;1 a T residue (Table 3). However, both CmNIP5;2 and CmNIP6;1 conserve FPs identical to those of CmNIP5;1 and homologous to those of other aquaporins of group II found in arabidopsis, rice and maize (Supplementary Table S1), all of them belonging to the same clade in the phylogenetic tree (Supplementary Figure S2). CmNIP5;1 and CmNIP5;2 are very close to AtNIP5;1, which has been proved to transport As, Sb and B in experimental assays[26,34,82]. CmNIP6;1 is related directly to AtNIP6;1, the latter being able to transport not only As, Sb and B but also urea and Gly[27,41,82] (Fig. 1). The prediction based on the phylogenetic framework, and including FPs, assigned to CmNIP5;1 the ability to transport As, Sb and B, as AtNIP5;1 does. This As and B transport is supported by mutagenic analysis[26] and its ability to transport Gly and/or urea must also be considered[27]. The variation in H2 of CmNIP5;2 (Table 3) prevented the prediction of any transport ability, although it should not be ruled out that it fulfils functions similar to those of CmNIP5;1. The same applies to CmNIP6;1, for which the prediction points only to Sb transport, although this is most likely not its only function. Regarding CmNIP7;1, its phylogenetic divergence does not separate it from group II of the NIPs and its proximity to AtNIP7;1 suggests that it can perform similar functions (Fig. 1). AtNIP7;1 can transport As, Sb, B, urea and Gly[73,82]. The possible transport of Gly and/or urea is also supported by studies with point mutations[27] and by the presence of a specific Y64 residue (Y81 in AtNIP7;1) which has been directly related to gating and regulation of urea and Gly transport[83]. The aquaporins CmNIP2;1 and CmNIP2;2 are included in the NIP III group[79]. They possess a unique ar/R selectivity filter due to the small size of the aa in the H2 and H5 positions, giving the largest pore diameter described in aquaporins, that may allow the passage of very large solutes such as Si (4.38 Å)[84]. Indeed, the transport of Si has been specifically associated with the motifs that are present in CmNIP2;1[85]. Si transporters are absent from the Brassicaceae and, therefore, NIP III aquaporins have not been found in A. thaliana, but they are present in all silicon-accumulator plants. Phylogenetic analysis assigned CmNIP2;1 and CmNIP2;2 to the same clade as the Z. mays and O. sativa NIP2s (Supplementary Figure S2), strongly suggesting that all members of the C. melo NIPs III group are Si transporters. In addition to silicon, members of the NIPs group III from Z. mays and O. sativa have been proved experimentally to transport As, Sb, B and urea[39,46,79,86-88]; also, ZmNIP2:1 was able to transport small amounts of H2O2 and Gly in heterologous expression experiments[46]. Thus, according to the analysis of orthologues, the transport of most of these solutes could occur via C. melo NIP2s and the prediction based on homologies also points to such transport by CmNIP2;1[10-12]. The case of CmNIP2;2 is peculiar since it clearly belongs to the same protein family but has a variation in the H2 position of the ar/R selectivity filter (C instead of G), and also its FPs are slightly different (S in P2, instead of T) from those of the NIPs III group (Table 3); hence, a prediction could not be performed based on homologous genes. The substitution of G in H2 by another non-polar small residue (A) did not affect the ability of OsNIP2;1 to transport Si, As or B[26], so it is reasonable to propose that CmNIP2;2 can also transport some of these solutes.

Very little is known about the function of SIPs in planta. They are localised in the ER and facilitate water transport[89] but no data on transport of non-aqueous substrates are available. Despite this, homologues- and structure-based analyses predict that the SIP1s of A. thaliana, Z. mays and O. sativa are capable of transporting urea, while the SIP2s could transport H2O2 and As[12]. Phylogenetic analysis showed that the SIPs of C. melo are clearly related to their orthologues in A. thaliana (Fig. 1), although the variation that both CmSIP1;1 and CmSIP2;1 show in H2 impairs the direct assignation of solutes transport to these proteins (Table 3).

The absence of XIPs in the Brassicaceae and monocots makes it impossible to compare the sequence found in C. melo with those of A. thaliana, Z. mays and O. sativa. For this reason, we compared the important residues with those of XIPs in other species[90]. CmXIP1;1 belongs clearly to the XIP-A subfamily, which does not yet have known transport functions. The main particularity of Cucumis (both sativus and melo) XIPs is that they present an SPI/SPA motif rather than a NPA/NPA motif, while conserving other MIP-specific sequence features. It is usual to find variations in the first NPA motif of XIP1s—as they have been observed in other species, such as Ricinus communis L., Lotus japonicus L., Prunus persica L. and Glycine max L.[90]—but it is quite odd to find a mutation in the second NPA motif. Also, in the first NPA motif, the A is substituted by I, which is also quite rare and has been observed only in Cucumis to date. It is still unknown what kind of functional effect this residue change might have on the protein.

Constitutive expression of CmAQPs and possible implication in melon physiology

Our results clearly show that the main aquaporin expressed in C. melo was CmTIP1;1, by a wide margin with respect to the other isoforms (Fig. 4a). The water transport capacity has been maintained in TIPs, especially in the TIP1s, for which the magnitude stands out, being mostly responsible for the high permeability of the tonoplast[91]. CmTIP1;1, therefore, should be mainly responsible for the water transport between the cytoplasm and vacuole in melon plants. The other TIP1s in C. melo showed very slight expression (Fig. 4c). If this is due to a redundancy of functions because of the importance of CmTIP1;1, or to their specialisation in other tissues or environmental conditions, remains to be investigated. Just below CmTIP1;1, the PIP1s isoforms were the aquaporin genes most expressed in our studies, in both roots and leaves (Fig. 4a). Other studies showed patterns similar to ours. For example, in maize, in the central zone of the leaf, the aquaporin gene most expressed was shown to be ZmPIP1;1, with PIP1s contributing more than 70% of the expression of PIPs in leaves[92]. The characteristic structure of PIPs is also clearly compatible with CO2 and H2O2 transport besides that of water. So, certain PIP1 isoforms might specialise in such functions, depending on their location, and some PIP1s were shown to be directly related with CO2 transport in mesophyll cells and chloroplasts[66,92,93]. This allows us to propose a function of CmPIP1s in the transport of both water and CO2 in melon, which could explain their extremely high expression in leaves. The PIP2s isoforms, discounting their specific function as water transporters, have been related with ROS signalling through their regulation by and transport of H2O2[36,75,94]. Attending to the expression profile, CmPIP2;6 seems to have also a fundamental role in leaves (Fig. 4b), as occurs in other species such as Arabidopsis, in which one PIP2 (AtPIP2;6) is predominant in leaves[95], pointing to a possible implication in H2O2 or even CO2 transport besides that of water. Indeed, it has been shown that, despite the high homology among the different PIPs, some isoforms of plants possess the ability to transport water but not CO2 (NtPIP2;1) or vice versa (PIP1, NtAQP1), the proportion of each of them in the tetramer allowing the passage of water or CO2 to be regulated. This opens the door to the involvement of the fifth, central pore in the transport of gases and to possible regulation of the transport by competition between subunits in the formation of the tetramer[5]. How tetramers can be established preferentially with one concrete isoform, to regulate such transport, could be of interest in future studies to see if CmPIP2;6 is preferentially selected in tetramers with PIP1s involved in CO2 conduction.

In addition, PIP1s also had high expression in roots along with CmPIP2;10, which is probably the main water transporter in C. melo, especially in roots, followed by CmPIP2;2, CmPIP2;3 and CmPIP2;4 (Fig. 4b,c). As we show in this study, and in general under optimal conditions, there is greater expression of aquaporins in the roots[51,96,97], since these are the point of entry of water and nutrients into the plant, and the predominance of a specific PIP2 isoform also seems to be habitual. As an example, ZmPIP2;5 is the main PIP gene expressed in the roots of maize and has been shown to be essential for water to cross the Casparian barrier in the exodermis[51]. The heterotetrameric conformation that includes PIP2 and PIP1 subunits has been shown to improve water transport, versus heterotetramers consisting exclusively of PIP2s[98,99]. This could be a valuable source of water transport regulation in the roots of melon plants as the interaction of the two isoforms is essential for PIP1s to access their active location in the plasma membrane[99]. Interestingly, the orthologues of CmPIP1;1, CmPIP1;2 and CmPIP2;10, the most expressed PIP isoforms in melon roots, have been found to transport other solutes such as urea, B and As[31,32,45]. The occurrence of this transport through these isoforms in melon could be of great interest to understand their expression patterns. Apart from CmTIP1;1, within the TIPs, only CmTIP3;1 seems to have a significant presence under optimal conditions, in both roots and leaves (Fig. 4b). Given that the prediction of the subcellular location (Table 2) places CmTIP3;1 in the mitochondria and the well-known ability of TIPs to transport nitrogen compounds then, if this location is confirmed, they could also play a role in the transport of NH3 produced by photorespiration[63]. Attending to the CmNIPs, the expression of CmNIP5;1 and CmNIP5;2 was similar, being low but significant in both roots and leaves (Fig. 4b). Strong evidence supports the role of the NIPs II group in boric acid transport[34,41,81] and this is probably the main function of the CmNIP5s. The members of the NIPs III group have been defined clearly as Si transporters[100] and the CmNIP2s were the most expressed NIPs genes in melon plants, especially CmNIP2;1, whose expression was higher in leaf tissues, while CmNIP2;2 was expressed mostly in roots (Fig. 4b). In rice it has been shown that OsNIP2;1 is the main Si transporter involved in Si uptake, while OsNIP2;2 is mostly implicated in the unloading of Si from xylem vessels in leaves[35,43]. Similarly, the stronger presence of CmNIP2;1 in leaves suggests a Si-unloading function in leaves, while the two isoforms had the same expression in roots, implicating both in the uptake and translocation of Si in roots. However, although neither B transport by CmNIP5;2 nor Si transport by CmNIP2;2 could be directly predicted based on previous analyses (Table 3), these are probably their main functions. This idea is supported by the tight tandem that each of them forms with its respective paralog in chromosomes 9 and 5, respectively. These probably represent the result of gene duplication events that yielded differences in discrete functions or sub-localisations within the same tissue, or even between different tissues, as seems to have been the case in the NIP2s.

Finally, it is worth noting the expression of the aquaporin CmSIP1;1 in roots, which was at the level of many PIPs and more than half of the isoforms analysed. Thus, although specific functions are not known, it seems to have some importance in the physiology of melon roots.

Comparison of expression profiles in RNA-seq versus qPCR

Quantitative reverse transcription PCR (RT-qPCR) was the tool used most widely for the quantification of gene expression due to its sensitivity and precision[101], being considered the most important medium-performance gene-expression analysis technology[102], and it should be used to validate RNA sequencing, which is the most widely used tool today. Based on this, we decided to compare the expression of the 31 aquaporins genes of C. melo var. Grand Riado with the RNA-seq data available in the databases, which refer to another variety, in this case Cantaloupe (melon)[56].

We found numerous similarities between our qPCR analyses and the RNA-seq data. Among them, the aquaporins with higher levels of expression showed better correlations for one or both tissues; this was the case for PIP1;2, PIP2;1, PIP2;6, PIP2;10, TIP1;1, TIP4;1, NIP2;1 and NIP5;1. The differences in expression between the qPCRs and RNA-seq can be explained, since the sensitivity of the qPCRs decreases from cycle 34 onwards and most of the differences were found in aquaporins that appeared from this cycle onwards due to their low expression. Other mismatches between the two analyses can be explained by the use of different melon varieties. This could have led to changes in the expression pattern of some aquaporins—as seen in other plant varieties, where differences have been found, even between different cultivars[103]. Lastly, the growth conditions were different: Latrasse et al. (2017) used constant humidity (60%) and a temperature of 21–27 ºC in a glasshouse (for melon plants grown on rock compost) and also grew plants in the field (in soil), while we used hydroponic culture, at 60–80% humidity and a temperature of 20–25 ºC[56].

Despite the discrepancies, a clear relationship is seen regarding the importance of certain isoforms in C. melo. Clearly, CmTIP1;1 is revealed as the most important osmoregulator in the tonoplast under optimal conditions. The PIP1s are of greater importance, in both roots and leaves, CmPIP1;1 being the predominant isoform. CmPIP2;6 is very important in leaf tissues, while in the case of the variety Cantaloupe its function seems to be shared with CmPIP2;10, which had a strong presence in leaves. In roots, the Cantaloupe aquaporins isoforms that seem to have the major role in water uptake are CmPIP2;4, followed by PIP2;10, PIP2;2 and, finally, PIP2;1. Of the NIPs, NIP5;1 and the NIP2s are undoubtedly important in melon plants. In the variety Cantaloupe, the presence of NIP5;1 is extremely striking in roots, being one of the most expressed isoforms, while in Grand Riado both NIP5 isoforms may share redundant functions. The genetic duplication of the NIP2s in a narrow chromosomal tandem and their matching transport abilities, together with their differentiation with respect to the expression in roots and leaves, suggest that they have acquired tissue-specific roles, these possibly being different in each of the two varieties. Finally, both analyses indicated that SIP1;1 plays a constitutive and important, but still unknown, role. It seems that, in each variety, slight changes in the weight of different paralogs have developed and these changes could be related to the transport function and/or localisation. The abundant solute transport possibilities for each aquaporin isoform could have played an important role in the specialisation within each variety, depending on the needs of each of these two varieties throughout their evolution and development in different environments.

Concluding remarks

In this work, the sequences of aquaporins genes have been assessed in melon. The comparative analysis with related plant species was very useful to predict the possible roles of some of the aquaporins in the transport of certain solutes. This analysis together with qPCR carried out in roots and leaves revealed the constitutive expression of almost all aquaporins genes in both organs. However, the role of each aquaporin must be elucidated in future work. Therefore, the exhaustive design of all the primers presented here is intended to serve as the basis for future research into all aquaporins in melon, regarding their roles in plant development and in the response to stress conditions. Finally, this work will serve as a basis for other researchers who wish to carry out a comprehensive analysis of the genome, explaining and clarifying a simple way to carry out this type of research.

Supplementary Information.