Genome-Wide Identification of Aquaporin Gene Family in Pitaya Reveals an HuNIP6;1 Involved in Flowering Process

Aquaporins (AQPs) are essential membrane proteins involved in seed maturation and germination, stomata movement, photosynthesis, and regulation of plant flowering processes. Pitaya flowers are open at night and wither at daybreak, which shows an obvious circadian rhythm. In this study, a comprehensive genome-wide analysis of AQPs in Hylocereus undantus was conducted to screen key genes associated with flowering processes. A total of 33 HuAQP genes were identified from the H. undantus genome. The 33 HuAQPs were grouped into four subfamilies: 10 PIPs, 13 TIPs, 8 NIPs, and 2 SIPs, which were distributed on 9 out of 11 pitaya chromosomes (Chr) (except for Chr7 and Chr10). Results from expression profiles showed that HuNIP6;1 may be involved in pitaya’s floral opening. HuNIP6;1 was localized exclusively in the cell membrane. Overexpression of HuNIP6;1 in Arabidopsis thaliana significantly promoted early flowering through regulating negative flowering regulators of MJM30, COL9, and PRR5, suggesting that HuNIP6;1 plays key roles in regulating flowering time. The present study provides the first genome-wide analysis of the AQP gene family in pitaya and valuable information for utilization of HuAQPs.


Introduction
Aquaporins (AQPs) belonging to the major intrinsic protein (MIP) superfamily have been found in all types of organisms, including bacteria, fungi, animals, and plants. In 1988, a new intact membrane protein named CHIP28 was purified from erythrocyte and renal cell membrane [1]. Then, CHIP28 was termed as Aquaporin-1 (AQP1), since it showed the function of water transportation [2]. With the rapid increase in available whole genomic sequences in recent years, AQPs have been identified in various plant species, including 35 from Arabidopsis thaliana [3], 35 from Zea mays [4], 28 from Beta vulgaris [5], and 35 from Citrullus lanatus [6]. According to the homology of amino acids and subcellular location, plant AQPs are classified into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs), uncategorized (X) intrinsic proteins (XIPs), and small basic intrinsic proteins (SIPs) [7].
Each AQP contains six transmembrane (TM) domains (TM1-TM6) connected by five loops (Loops A-E), as well as the two signature asparagine-proline-alanine (NPA) motifs located at the N-terminus of the two half-helices (HB and HE) in LB and LE [8]. The NPA motifs (signature motifs) are located inside the pore responsible for electrostatic repulsion

Identification, Classification, Nomenclature, and Properties of HuAQPs
A total of 43 gene sequences were obtained in the pitaya genome database (http: //www.pitayagenomic.com/) using the keyword 'aquaporin', and 33 AQP genes (HuAQPs) had one complete MIP domain and two NPA motifs (Table 1). Based on sequence cluster analyses of H. undantus, A. thaliana, D. caryophyllus, and B. vulgaris, those HuAQPs were classified into four subfamilies: 10 PIPs, 13 TIPs, 8 NIPs, and 2 SIPs (Figure 1). The PIPs subfamily included PIP1s and PIP2s subgroups, with five PIP1s and five PIP2s. The TIPs subfamily was divided into five subgroups: four TIP1s, five TIP2s, one TIP3s, two TIP4s and one TIP5. The NIPs subfamily contained four subgroups; i.e., four NIP4s, one NIP5, two NIP6, and one NIP7. HuAQPs were named according to their percentage of identity to the 35 AQP proteins from A. thaliana [3]. Among the 33 predicted HuAQP proteins, the largest one was HuNIP5;1, with 310 amino acids (AAs), while the smallest protein was HuNIP6;2 with 217 AAs (Table 1).

Gene Structure and Conserved Motif Analyses of HuAQPs
The exon-intron structures play vital roles in plant evolution. The exon-intron structures and conserved motifs of HuAQPs were analyzed. Most PIP subfamilies had four exons, except for HuPIP2;1 and HuPIP2;2 containing three exons. TIP subfamilies had three exons, except for HuTIP1;1 with two exons. NIP subfamilies contained five exons, while HuNIP5;1 and HuNIP6;2 had four exons and three exons, respectively. All SIP subfamilies contained three exons ( Figure 2B). As shown in Figure 2C, Motif 1 and Motif 3 were found in all HuAQPs, except HuTIP1;3 without Motif 3. Both PIP and TIP subfamilies contained seven motifs, suggesting that they were highly conserved in pitaya. Motif 5 and Motif 9 were unique to the PIP subfamilies, while Motif 10 specifically existed in the TIP subfamilies. NIP subfamilies had Motif 1, Motif 2, Motif 5, Motif 6, and Motif 7, except for NIP6;2.

Gene Structure and Conserved Motif Analyses of HuAQPs
The exon-intron structures play vital roles in plant evolution. The exon-intron structures and conserved motifs of HuAQPs were analyzed. Most PIP subfamilies had four exons, except for HuPIP2;1 and HuPIP2;2 containing three exons. TIP subfamilies had three exons, except for HuTIP1;1 with two exons. NIP subfamilies contained five exons, while HuNIP5;1 and HuNIP6;2 had four exons and three exons, respectively. All SIP subfamilies contained three exons ( Figure 2B). As shown in Figure 2C, Motif 1 and Motif 3 were found in all HuAQPs, except HuTIP1;3 without Motif 3. Both PIP and TIP subfamilies

Gene Mapping on the Pitaya Chromosomes
According to the gene loci information, the 33 HuAQP genes were unevenly distributed in nine chromosomes. HuAQPs were mainly distributed at both ends of chromosomes, and density of HuAQPs varied on individual chromosomes. Most of the HuAQPs were located on Chr 5 (seven genes; 21.2%) and Chr 6 (seven genes; 21.2%). However, there was only one HuAQP on Chr 11 ( Figure 3).

Gene Mapping on the Pitaya Chromosomes
According to the gene loci information, the 33 HuAQP genes were unevenly distributed in nine chromosomes. HuAQPs were mainly distributed at both ends of chromosomes, and density of HuAQPs varied on individual chromosomes. Most of the HuAQPs were located on Chr 5 (seven genes; 21.2%) and Chr 6 (seven genes; 21.2%). However, there was only one HuAQP on Chr 11 ( Figure 3).

Expression Patterns of HuAQPs during the Flower-Opening Process in Pitaya
The expression differences of HuAQP genes were analyzed in petals at six floweropening stages under 14 h/10 h day/night and 24 h/0 h day/night (Figure 4)

Expression Patterns of HuNIP6;1 during Flower Opening in Pitaya
The expression patterns of HuNIP6;1 were further analyzed in various tissues (ovaries, petals sepals, and the first and second segments of calyx) by RT-qPCR ( Figure 5).

Expression Patterns of HuNIP6;1 during Flower Opening in Pitaya
The expression patterns of HuNIP6;1 were further analyzed in various tissues (ovaries, petals sepals, and the first and second segments of calyx) by RT-qPCR ( Figure 5). Higher expression levels of HuNIP6;1 were detected at Os 3 in the petals and sepals, Os 5 in ovary, and the first segment of calyx under 14 h/10 h day/night. However, HuNIP6;1 showed higher expression levels at Os 2 in the second segment of calyx, petals, and sepals under 24 h/0 h day/night. HuNIP6;1 was upregulated from Os 3 to Os 4 in all tissues except for the sepals under 14 h/10 h day/night, compared to down-regulation from Os 5 to Os 6 in all tissues at the two conditions.
Higher expression levels of HuNIP6;1 were detected at Os 3 in the petals and sepals, Os 5 in ovary, and the first segment of calyx under 14 h/10 h day/night. However, HuNIP6;1 showed higher expression levels at Os 2 in the second segment of calyx, petals, and sepals under 24 h/0 h day/night. HuNIP6;1 was upregulated from Os 3 to Os 4 in all tissues except for the sepals under 14 h/10 h day/night, compared to down-regulation from Os 5 to Os 6 in all tissues at the two conditions.

Expression Pattern of HuNIP6;1 in Diurnal Cycle in Pitaya under 14 h/10 h Day/Night
Circadian rhythm is synchronized with the circadian cycle, and plants contain a circadian clock that can coordinate internal and external cues to adapt to the environment. The expression patterns of HuNIP6;1 in the diurnal cycle were analyzed in petals by RT-qPCR ( Figure 6). During the flower opening of pitaya, the expression levels of HuNIP6;1 increased from 0 h and reached its maximum at 12 h, and decreased thereafter. The results were consistent with the process of pitaya flower opening.

Subcellular Localization of HuNIP6;1
Subcellular localization of HuNIP6;1 was analyzed using transient expression of constructs in epidermal cells of N. benthamiana. The fluorescence for HuNIP6;1 was apparently localized in the cell membrane, while the GFP signal for the positive control was observed in both the cell membrane and nucleus (Figure 7).

Over-expression of HuNIP6;1 in Arabidopsis thaliana
To further confirm the role of HuNIP6;1, we generated transgenic A. thaliana plants constitutively expressing HuNIP6;1 driven by the 35S promoter (Figures 8 and 9). Six different transgenic lines were obtained. Over-expression of HuNIP6;1 in A. thaliana plants resulted in early flowering. The flowering time of 35S::HuNIP6;1 transgenic plants was significantly earlier than that of wild-type. The transgenic lines had higher bolting height, as well as more flower buds, fruit pods, rosette leaves, and bolting ( Table 2). The expression levels of HuNIP6;1, MJM30, COL9, and PRR5 in the transgenic lines were significantly

Over-Expression of HuNIP6;1 in Arabidopsis thaliana
To further confirm the role of HuNIP6;1, we generated transgenic A. thaliana plants constitutively expressing HuNIP6;1 driven by the 35S promoter (Figures 8 and 9). Six different transgenic lines were obtained. Over-expression of HuNIP6;1 in A. thaliana plants resulted in early flowering. The flowering time of 35S::HuNIP6;1 transgenic plants was significantly earlier than that of wild-type. The transgenic lines had higher bolting height, as well as more flower buds, fruit pods, rosette leaves, and bolting ( Table 2). The expression levels of HuNIP6;1, MJM30, COL9, and PRR5 in the transgenic lines were significantly higher than that of wild-type. However, no significant changes of positive flowering regulators such as FT, FD, CO, LFY, and SCO1 were detected in transgenic HuNIP6;1 Arabidopsis (data not shown). Those results suggested that HuNIP6;1 has significant functions in flowering of transgenic A. thaliana through regulating the other flowering-related genes.

Discussion and Conclusions
AQPs are crucial membrane transport proteins participating in the transport of water and/or small neutral solutes in plants. AQPs family members have been identified in plant species such as A. thaliana [3], Brassica oleracea [35], Zea mays [4], B. vulgaris [5], and C. lanatus [6]. In this study, the genome-wide identification and characterization of pitaya AQPs (HuAQPs) was performed using available genomic sequence data (http: //www.pitayagenomic.com/) (Accessed on 15, July, 2021). The evolutionary relationships, gene structures, exon-intron distribution, conserved motifs, chromosomal distribution, and expression patterns of HuAQPs were analyzed. After excluding incomplete ORFs without MIP domain and NPA motifs, a total of 33 HuAQP genes (10 PIPs, 13 TIPs, 8 NIPs, and 2 SIPs) distributed in 9 chromosomes were identified. All groups contained HuAQP genes, except for XIP group. The XIP group contained one gene from D. caryophyllus and B. vulgaris [5,29]. Similar results were reported in chickpea, Medicago, and Lotus japonicus [36,37]. The number of exons in most HuAQP genes within PIP, TIP, NIP, and SIP was four, three, five, and three, respectively, suggesting that HuAQPs were highly conserved. A similar exon-intron structure is found in the other plants such as sweet orange [38], M. truncatula [39], watermelon [6], and longan [40].
Petals were the crucial to the flower opening and closure of Nymphaea caerulea [41]. The growth of carnation petals is controlled by coordinated gene expression during the progress of flower opening [42,43]. AQPs play an important role in the expansion of petal cells [17,26,42]. Rh-PIP2;1, a rose AQP gene, is involved in ethylene-regulated petal expansion. After silencing of Rh-PIP2;1 in rose flowers, petal expansion was greatly inhibited [26]. DcaPIP1;3, DcaPIP2;2, DcaPIP2;5, DcaTIP1;4, and DcaTIP2;2 are considered to be involved in flower-opening stages in carnation [29]. In this study, the similar gene structure and the conserved motif were found in the same subfamilies; however, their expression patterns were different (Figure 4), suggesting that different members of the same subfamily played different roles during pitaya flowering. The expression of HuPIP1;1, HuPIP1;2, HuPIP1;4, HuPIP1;5, HuPIP2;1, HuPIP2;2, and HuPIP2;5 showed increasing patterns, and then decreased during pitaya flower opening under 14 h/10 h day/night, indicating they may play roles in flower opening. HuTIP5;1, HuSIP1;1, HuSIP1;2, HuNIP4;3, and HuNIP6;2 had preferential expressions in the Os 6 (periods after flowering) under the two conditions, indicating that they may be involved in flower withering.
HuNIP6;1, a novel AQP gene located in Chr 3, was obtained from the genome data of pitaya. The length of the HuNIP6;1 protein was 304 AAs, which is similar to other plants, such as 306 AAs in B. vulgaris [5] and 307 AAs in D. caryophyllus [29]. Results from expression analyses suggested that HuNIP6;1 may be involved in the process of pitaya flowering. The subcellular localization showed that HuNIP6;1 is located at the cell membrane. Over-expression of HuNIP6;1 in A. thaliana significantly resulted in early flowering (Figures 8 and 9). MJM30, COL9, and PRR5 were negative flowering regulators [44][45][46]. In the present study, over-expression of HuNIP6;1 in Arabidopsis significantly prompted expression of MJM30, COL9, and PRR5. Those results suggested that the over-expression of HuNIP6;1 broke the original cycle by regulating the expression of MJM30, COL9, and PRR5 to a normal rhythm in Arabidopsis. However, the mechanism of interaction between HuNIP6;1 and the other flowering-related genes is unknown, and further study is necessary to elucidate it.
In summary, our study provides the first genome-wide analysis of the AQP family in pitaya. A total of 33 HuAQP genes were identified and divided into four subfamilies. A novel AQP gene; i.e., HuNIP6;1, belonging to a member of the Group NIP family, was obtained, and was located at the cell membrane. Over-expression of the HuNIP6;1 gene in A. thaliana resulted in a significant early flowering, suggesting that HuNIP6;1 plays a significant role in pitaya flowering. The results of the present study provide valuable information for a better understanding of AQPs during the flower-opening process of pitaya.

Gene-Structure Analysis and Identification of Conserved Motifs
TBtools software (http://www.tbtools.com/) was used for the exon-intron structure analysis, and the conserved protein motifs were analyzed using the MEME (http://memesuite.org/) website.

Chromosomal Distribution
The location information of the AQPs was obtained from the pitaya genome database (http://www.pitayagenomic.com/) (Accessed on 15 July 2021). The gene-location map was constructed using MapChart [48].

Plant Materials
The flowers of 'Hongguan No. 1 pitaya (H. monacanthus) were collected from Baiyun District, Guangzhou City, Guangdong Province, China. Flowers with 25 cm stems were picked on the 15th day after the flower bud formation in July and cultured at two different conditions: 14 h/10 h day/night and 24 h/0 h day/night at 25 • C. Samples were collected every 4 h from 20:00 on the same day (supposed as the 0 h). The flower-opening process was separated into six stages, including opening stage 1 (Os 1), 12 h after culture (periods before flowering); Os 2, 24 h after culture (periods before flowering); Os 3, 28 h after culture (periods during flowering); Os4, 32 h after culture (periods during flowering); Os 5, 36 h after culture (periods after flowering); and Os 6, 40 h after culture (periods after flowering) ( Figure 10A). Ovaries, petals, sepals, and the first and second segments of calyx were collected for gene cloning and expression analyses ( Figure 10B). To analyze the rhythmic expression of HuNIP6;1 in pitaya petal, the samples were collected every 4 h from 12:00 on the day of flowering (supposed as the 0 h). Three uniformly sized flowers from every period were sampled as three replicates. All samples were immediately frozen in liquid nitrogen and stored at -80 • C until use.

Gene-Structure Analysis and Identification of Conserved Motifs
TBtools software (http://www.tbtools.com/) was used for the exon-intron structure analysis, and the conserved protein motifs were analyzed using the MEME (http://memesuite.org/) website.

Chromosomal distribution
The location information of the AQPs was obtained from the pitaya genome database (http://www.pitayagenomic.com/) (Accessed on 15 July 2021). The gene-location map was constructed using MapChart [48].

Plant Materials
The flowers of 'Hongguan No. 1′ pitaya (H. monacanthus) were collected from Baiyun District, Guangzhou City, Guangdong Province, China. Flowers with 25 cm stems were picked on the 15th day after the flower bud formation in July and cultured at two different conditions: 14 h/10 h day/night and 24 h/0 h day/night at 25 °C. Samples were collected every 4 h from 20:00 on the same day (supposed as the 0 h). The flower-opening process was separated into six stages, including opening stage 1 (Os 1), 12 h after culture (periods before flowering); Os 2, 24 h after culture (periods before flowering); Os 3, 28 h after culture (periods during flowering); Os4, 32 h after culture (periods during flowering); Os 5, 36 h after culture (periods after flowering); and Os 6, 40 h after culture (periods after flowering) ( Figure 10A). Ovaries, petals, sepals, and the first and second segments of calyx were collected for gene cloning and expression analyses ( Figure 10B). To analyze the rhythmic expression of HuNIP6;1 in pitaya petal, the samples were collected every 4 h from 12:00 on the day of flowering (supposed as the 0 h). Three uniformly sized flowers from every period were sampled as three replicates. All samples were immediately frozen in liquid nitrogen and stored at -80 °C until use.

RNA isolation, cDNA synthesis and RT-qPCR analyses
RNA was isolated using the EASYspin Plus Plant Quick RNA isolation Kit (RN38) (Aidlab Biotechnology, Beijing, China) according to the manufacturer's instructions. The first strand cDNA was synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). The primers of RT-qPCR were designed by Batch-Primer3v1.0 (http://batchprimer3.bioinformatics.ucdavis.edu/index.html) and the Actin (1) reference gene was used as the internal control [49]. The primers used for RT-qPCR are listed in Table S1. RT-qPCR was carried out using a LightCycler 480Ⅱ real-time PCR system (Roche, Switzerland) using the RealUniversal Color PreMix (SYBR Green) (TIANGEN, Beijing, China) according to the manufacturer's instructions. Three biological replicates were performed for each sample. The relative expression levels were calculated using the comparative 2 −△△C T method [50].

Cloning of HuNIP6;1 Gene
HuNIP6;1 was cloned using specific primers of ATGGATGCTGAGGATCCCGG and TCATCTCCGGAAGCTTGGTG following the procedure of Xie et al. [51].

Arabidopsis thaliana Transformation and Phenotypic Analysis
The full-length of HuNIP6;1 was subcloned into the pPZP6k90 vector under the control of the 35S promoter and transformed into A. thaliana by the Agrobacterium tumefaciensmediated method. The 35S:HuNIP6;1 transformed lines were identified by PCR detection, and the expression levels of HuNIP6;1 were analyzed by RT-qPCR. The phenotypes of T4 homozygous transgenic plants were analyzed and photographed with a digital camera (G16, Canon).

RNA Isolation, cDNA Synthesis and RT-qPCR Analyses
RNA was isolated using the EASYspin Plus Plant Quick RNA isolation Kit (RN38) (Aidlab Biotechnology, Beijing, China) according to the manufacturer's instructions. The first strand cDNA was synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). The primers of RT-qPCR were designed by BatchPrimer3v1.0 (http://batchprimer3.bioinformatics.ucdavis.edu/index.html) and the Actin (1) reference gene was used as the internal control [49]. The primers used for RT-qPCR are listed in Table S1. RT-qPCR was carried out using a LightCycler 480II real-time PCR system (Roche, Switzerland) using the RealUniversal Color PreMix (SYBR Green) (TIANGEN, Beijing, China) according to the manufacturer's instructions. Three biological replicates were performed for each sample. The relative expression levels were calculated using the comparative 2 − C T method [50].

Cloning of HuNIP6;1 Gene
HuNIP6;1 was cloned using specific primers of ATGGATGCTGAGGATCCCGG and TCATCTCCGGAAGCTTGGTG following the procedure of Xie et al. [51].

Arabidopsis thaliana Transformation and Phenotypic Analysis
The full-length of HuNIP6;1 was subcloned into the pPZP6k90 vector under the control of the 35S promoter and transformed into A. thaliana by the Agrobacterium tumefaciensmediated method. The 35S:HuNIP6;1 transformed lines were identified by PCR detection, and the expression levels of HuNIP6;1 were analyzed by RT-qPCR. The phenotypes of T 4 homozygous transgenic plants were analyzed and photographed with a digital camera (G16, Canon).

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10.3 390/ijms22147689/s1. Author Contributions: Conception and design of the experiments, X.Y., Y.G. and Y.Q.; performance of the experiments, X.Y.; analysis of data, X.Y., Y.G., C.C., F.X. and Q.H.; contribution of reagents/materials/analysis tools, Z.Z., R.Z., J.Z. and G.H.; writing and revision of the paper, X.Y. and Y.Q. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.