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Figure 1. 

Phylogenetic analysis of the identified AQPs from pea genome. (a) The pea AQPs proteins aligned with those from the cool-season legume Medicago truncatual. (b) The four un-assigned pea AQPs in (a) (denoted as NA) were further aligned with the AQPs of rice, soybean, and Arabidopsis by using the Clustal W program implemented in MEGA 7 software. The nomenclature of PsAQPs was based on homology with the identified aquaporins that were clustered together.

Figure 2. 

Chromosomal localization of the 41 PsAQPs on the seven chromosomes of pea. Chr1-7 represents the chromosomes 1 to 7. The numbers on the right of each chromosome show the physical map positions of the AQP genes (Mbp). Blue, green, orange, brown, and black colors represent TIPs, NIPs, PIPs, SIPs, and XIP, respectively.

Figure 3. 

The exon-intron structures of the AQP genes in pea. Upstream/downstream region, exon, and intron are represented by a blue box, yellow box, and grey line, respectively.

Figure 4. 

Heatmap analysis of the expression of pea AQP gene expressions in different tissues using RNA-seq data (PRJNA267198). Normalized expression of aquaporins in terms of reads per kilobase of transcript per million mapped reads (RPKM) showing higher levels of PIPs, NIPs, TIPs SIPs, and XIP expression across the different tissues analyzed. (Stage A represents 7-8 nodes; stage B represents the start of flowering; stage D represents germination, 5 d after imbibition; stage E represents 12 d after pollination; stage F represents 8 d after sowing; stage G represents 18 d after sowing, LN: Low-nitrate; HN: High-nitrate.

Figure 5. 

The visual phenotype and radicle length of pea seeds treated with water (W), 0.3 M mannitol (M), and fullerol of different concentrations dissolved in 0.3 M mannitol (MF). MF1, MF2, MF3, and MF4 indicated fullerol dissolved in 0.3 M mannitol at the concentration of 10, 50, 100, and 500 mg/L, respectively. (a) One hundred and fifty grains of pea seeds each were used for phenotype analysis at 72 h after treatment. Radicle lengths were measured using a ruler in three replicates R1, R2, and R3 in all the treatments. (b) Multiple comparison results determined using the SSR-Test method were shown with lowercase letters to indicate statistical significance (P < 0.05).

Figure 6. 

Venn diagram showing the shared and unique differentially expressed PsAQP genes in imbibing seeds under control (W), Mannitol (M) and Mannitol + Fullerol (MF3) treatments. Up-regulation (UG): PsPIP2-5, PsNIP1-1, PsNIP2-1-2, PsNIP4-2, PsNIP6-3, PsNIP1-5, PsTIP2-2, PsTIP4-1, PsSIP1-3, PsXIP2-1; Down-regulation (DG): PsTIP2-3, PsTIP3-2, PsNIP1-7, PsNIP5-1, PsXIP2-1.

Figure 7. 

The expression patterns of seven PsAQPs in imbibing seeds as revealed by RNA-Seq and qRT-PCR. The seeds were sampled after 12 h soaking in three different solutions, namely water (W), 0.3 M mannitol (M), and 100 mg/L fullerol dissolved in 0.3 M mannitol (MF3) solution. Error bars are standard errors calculated from three replicates.