Genome-Wide Identification and Function of Aquaporin Genes During Dormancy and Sprouting Periods of Kernel-Using Apricot (Prunus armeniaca L.)

Abstract

Aquaporins (AQPs) are essential channel proteins that play a major role in plant growth and development, regulate plant water homeostasis, and transport uncharged solutes across biological membranes. In this study, 33 AQP genes were systematically identified from the kernel-using apricot (Prunus armeniaca L.) genome and divided into five subfamilies based on phylogenetic analyses. A total of 14 collinear blocks containing AQP genes between P. armeniaca and Arabidopsis thaliana were identified by synteny analysis, and 30 collinear blocks were identified between P. armeniaca and P. persica. Gene structure and conserved functional motif analyses indicated that the PaAQPs exhibit a conserved exon-intron pattern and that conserved motifs are present within members of each subfamily. Physiological mechanism prediction based on the aromatic/arginine selectivity filter, Froger's positions, and three-dimensional (3D) protein model construction revealed marked differences in substrate specificity between the members of the five subfamilies of PaAQPs. Promoter analysis of the PaAQP genes for conserved regulatory elements suggested a greater abundance of cis-elements involved in light, hormone, and stress responses, which may reflect the differences in expression patterns of PaAQPs and their various functions associated with plant development and abiotic stress responses. Gene expression patterns of PaAQPs showed that PaPIP1-3, PaPIP2-1, and PaTIP1-1 were highly expressed in flower buds during the dormancy and sprouting stages of P. armeniaca. A PaAQP coexpression network showed that PaAQPs were coexpressed with 14 cold resistance genes and with 16 cold stress-associated genes. The expression pattern of 70% of the PaAQPs coexpressed with cold stress resistance genes was consistent with the four periods [Physiological dormancy (PD), ecological dormancy (ED), sprouting period (SP), and germination stage (GS)] of flower buds of P. armeniaca. Detection of the transient expression of GFP-tagged PaPIP1-1, PaPIP2-3, PaSIP1-3, PaXIP1-2, PaNIP6-1, and PaTIP1-1 revealed that the fusion proteins localized to the plasma membrane. Predictions of an A. thaliana ortholog-based protein-protein interaction network indicated that PaAQP proteins had complex relationships with the cold tolerance pathway, PaNIP6-1 could interact with WRKY6, PaTIP1-1 could interact with TSPO, and PaPIP2-1 could interact with ATHATPLC1G. Interestingly, overexpression of PaPIP1-3 and PaTIP1-1 increased the cold tolerance of and protein accumulation in yeast. Compared with wild-type plants, PaPIP1-3- and PaTIP1-1-overexpressing (OE) Arabidopsis plants exhibited greater tolerance to cold stress, as evidenced by better growth and greater antioxidative enzyme activities. Overall, our study provides insights into the interaction networks, expression patterns, and functional analysis of PaAQP genes in P. armeniaca L. and contributes to the further functional characterization of PaAQPs.

Keywords: aquaporin gene; cold resistance; functional analysis; genome-wide analysis; kernel-using apricot (P. armeniaca L.).

FIGURE 1

Phylogenetic relationships and microsynteny analyses of AQP genes among the P. armeniaca, A. thaliana, and P. persica genomes. (A) Deduced amino acid sequences were aligned using default parameters in ClustalW, and the phylogenetic tree was constructed via the maximum likely hood method with 1,000 bootstrap replicates with MEGA 7.0 software. Red, green, and blue circles represent the A. thaliana, P. armeniaca, and P. persica AQP gene family members. Yellow, blue, red, orange, and green arches represent the SIP, XIP, NIP, PIP, and TIP AQP gene family subgroups of A. thaliana, P. armeniaca, and P. persica. The scale bar indicates the distance calculated by way of multiple alignment. (B) The chromosome numbers of all three species are specified by different colors: blue, green, and yellow represent the P. armeniaca, A. thaliana, and P. persica chromosomes, respectively. The chromosome number is indicated on the inside with the chromosome sequence lengths in megabases. Gene pairs with syntenic relationships are linked by blue and green lines representing the microsyntenic regions between the P. armeniaca and A. thaliana chromosomes and the P. armeniaca and P. persica chromosomes, respectively.

FIGURE 2

Multiple sequence alignment of the deduced amino acid sequences of PaPIPs. Amino acids at NPA domains, ar/R selectivity filters, and Froger’s residues were identified in seven PIP family members (3 PIP1s and 4 PIP2s) in P. armeniaca.

FIGURE 3

Expression profiles of PaAQPs at different developmental stages based on RNA-seq data. ED, SP, and GS indicate that the tested materials were collected during ecological dormancy, the sprouting period, and germination stage of P. armeniaca, respectively. The number in the middle of each box represents the FPKM.

FIGURE 4

Prediction of the interaction network of PaAQP proteins based on the interactions of their orthologs in A. thaliana. (A) The interaction network of TIP. (B) The interaction network of PIP. (C) The interaction network of SIP. (D) The interaction network of NIP. (E) Y2H assays showing the protein interaction between PaNIP6-1 and WRKY6, PaTIP1-1 and TSPO, and PaPIP2-1 and ATHATPLC1G. The networks were generated from the STRING database. The red circles represent the queried protein, while the other circles are the interacting proteins. The annotation of the predicted interacting protein was derived from UniProtKB (https://www.uniprot.org/). The line thickness indicates the strength of the data support.

FIGURE 5

Changes in the transcript levels of 20 selected genes in different tissues during the dormancy and sprouting stages of P. armeniaca. The stem and flower buds were collected during the PD stage, ED stage, SP stage, and GS stage of P. armeniaca plants. All expression levels of PaAQP genes were normalized to the expression levels of PaElf (elongation factor-1α). The 2^–ΔΔCT method was used to analyze the relative gene expression. The data are means of three replicates ± SDs. The lowercase letters indicate statistical significance based on one-way ANOVA with Tukey’s HSD post hoc analysis.

FIGURE 6

PaAQP coexpression network. (A) Among the 1,500 genes coexpressed with PaAQPs, 14 (green nodes) are involved in cold resistance based on previous research. In addition, the anticold ability of 16 genes (pink nodes) is not very clear. The red node is the PaAQP genes. Cyan lines represent PaAQPs coexpressed with 14 cold resistance genes, and red lines represent PaAQPs coexpressed with 16 cold stress-associated genes. (B) Transcript levels of 14 genes involved in cold resistance in stem and flower buds during the PD stage, ED stage, SP stage, and GS stage of P. armeniaca. All expression levels of the PaAQP genes were normalized to those of PaElf. Each bar represents the mean ± SD of three technical replicates. The different letters represent significant differences at P < 0.05 (one-way ANOVA).

FIGURE 7

Localization of PaPIP1-1, PaPIP2-3, PaSIP1-3, PaXIP1-2, PaNIP6-1, and PaTIP1-1 in the cell plasma membrane. Bright, Bright-field images. DAPI, nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI), a nuclear marker; FM4-64FX, FM4-64FX dye images, a plasma membrane-specific vital dye. Merge, overlap of GFP (green) and FM4-64FX (red) fluorescence. Row 1 shows the protoplasts expressing GFP alone, which was used as a control. Row 2 shows protoplasts expressing the PaPIP1-1:GFP fusion protein with FM4-64 fluorescence. Row 3 shows the protoplasts expressing the PaPIP2-3:GFP fusion protein with FM4-64 dye. Row 4 shows protoplasts expressing the PaSIP1-3:GFP fusion protein with FM4-64 fluorescence. Row 5 shows the protoplasts expressing the PaXIP1-2:GFP fusion protein with FM4-64 dye. Row 6 shows the protoplasts expressing the PaNIP6-1:GFP fusion protein with FM4-64 dye. Row 7 shows protoplasts expressing the PaTIP1-1:GFP fusion protein with FM4-64 dye. FM4-64FX shows a plasma membrane-specific dye. Bars = 5 μm.

FIGURE 8

Overexpression of PaPIP1-3 and PaTIP1-1 increases cold tolerance and accumulation in yeast. (A) Growth of the GS115 yeast strain transformed with the empty vector pGAPZA or with pGAPZA harboring PaPIP1-1, PaPIP1-3, PaPIP2-3, or PaTIP1-1. (B) OD600 value of yeast transformants in response to low-temperature stress. The data are the means ± SDs of three replications. n.s. means not significant. The different lowercase letters indicate significant differences at P < 0.05 by ANOVA.

FIGURE 9

Cold tolerance analysis of wild-type and PaPIP1-3- or PaTIP1-1-OE plants. (A) Image of the wild-type and transgenic plants in growth chambers at 22, 16, and 16°C for 16 h/4°C 8 h. Two independent PaPIP1-3 transgenic lines (PIPOE-1and PIPOE-2) and two independent PaTIP1-1 transgenic lines (TIPOE-1and TIPOE-2), were identified by qRT-PCR. (B). After 10 days of treatment, the SOD activity (C), proline content (D), and MDA content (E) of PaPIP1-3 or PaTIP1-1 transgenic Arabidopsis and wild-type Arabidopsis were determined. PIPOE- 1-, PIPOE- 2-, and PaPIP1-3-OE transgenic lines; TIPOE- 1-, TIPOE- 2-, and PaTIP1-1-OE transgenic lines. Actin was used as a control gene. The error bars represent the SEs of three replicates. n.s. means not significant. The different letters represent significant differences at P < 0.05 (one-way ANOVA).