Water deficit—caused by drought, high salinity/alkaline, high temperature, cold/freezing conditions or other abiotic stressors— can negatively affect plant growth and survival. However, plants have developed intricate mechanisms to cope with this type stress, including alterations to signal perception and transduction and differential expression of stress responsive genes through complex networks. Aquaporins are a class of integral membrane proteins that facilitate the diffusion of water and other small solutes. Plants often maintain large and diverse AQP families compared to animals and microorganisms. Aquaporins have been reported to play crucial roles in plant water balance and homeostasis under adverse growing conditions [5, 21] and in response to specific biotic challenges [36, 37]. In this study, we performed genome-wide identification and characterization of AQPs in C. rosea to understand the evolution of this family and its molecular role. We were particularly interested in resolving the molecular mechanisms underlying this extremophile halophyte’s adaptation to coral reef habitats and its responses to acute salt, alkaline, and drought stressors.
The AQP protein family within the C. rosea genome was characterized and 37 putatively functional CrAQP isoforms (based on Pfam domain sequences) were identified, belonging to the PIP (11 isoforms), TIP (10), NIP (11), SIP (4), and XIP (1) families (Table 1). We performed whole genome sequencing of C. rosea, and our result indicates that this species is diploid, with a 534.94 Mbp genome size (data not published). The number of AQPs was similar to other diploid plant species (Table 2) and their protein sequences were highly similar. This indicates that the number of AQPs and sequence specificity may not be directly related to the adaptation of C. rosea to extreme environments. The roles that CrAQPs play in stress tolerance needs to be further studied from other perspectives, such as transcriptional regulation, protein modification, and the regulation of AQP transmembrane transport activities.
Although numerous studied have identified AQPs in model plant species, research on this gene family has increasingly focused on plants that inhabit novel environments. This is largely because AQP genes are seen as candidates for use in genetic modification of crops to increase agricultural productivity [38, 39]. The saltbush Atriplex canescens is highly tolerant of saline-alkaline soils, drought, heavy metals, and cold, and the AQP genes AcPIP2 and AcNIP5;1 have been shown to be involved in abiotic stress tolerance in this species, and their overexpression in transgenic Arabidopsis caused altered tolerance to drought and salt [40, 41]. Compared with cultivated soybean, the wild Glycine soja is relatively salt-alkaline tolerant. Two AQP genes from G. soja, GsTIP2;1 and GsPIP2;1, minimized tolerance to salt and dehydration stress when overexpressed in Arabidopsis, implying they have negative impacts on stress tolerance by regulating water potential [42, 43]. In most functional analyses conducted in transgenic plants, the overexpression of AQP genes caused elevated tolerance to salt and drought, such as in Malus zumi (gene MzPIP2;1) [44], Sesuvium portulacastrum (SpAQP1) [45], Stipa purpurea (SpPIP1) [46], Simmondsia chinensis (ScPIP1) [47], Thellungiella salsuginea (TsPIP1;1) [48], and Phoenix dactylifera (PdPIP1;2) [49]. The elevated expression of AQP genes in plants can lead to cellular changes in water potential, which cause alterations in water uptake and transpiration, and ultimately modify tolerance to water deficit stress. In this respect, understanding the distribution, expansion, regulation, phylogenetic diversity, and evolutionary selection of AQP genes in extremophile plants like C. rosea is an important step toward potentially improving the water utilization abilities and drought adaptations of other plant species, including agricultural crops.
Plant AQPs play versatile physiological roles in combatting abiotic stress, not only by regulating water content and potential, but also by transporting certain signaling molecules and nutrients. Generally, AQPs consist of six transmembrane helices connected by five loops (A–E) and cytosolic N- and C-termini. Loops B (cytosolic) and E (non-cytosolic) both contain the highly conserved NPA (asparagine-proline-alanine) motifs that form part of the core of these proteins. The aromatic/arginine (ar/R) constriction is located at the non-cytosolic end of the pore. The substrate specificity of AQPs is closely related to several different signature sequences, including NPA motifs, the ar/R filter, and Froger’s positions (FPs) [50]. In all CrAQP NPA motifs, the first two residues were the most conserved, except for CrSIP1;3 and CrXIP1;1, in which the loop B and loop E NPA motifs degenerated into NLG and SPV. The third residue of NPA motifs was more variable, in which A was frequently replaced by either L, S, T, or V. However compared to the NPA motifs, the 10 amino acid residues at the ar/R filter and Froger’s positions were more variable in all CrAQPs (Table 3). In some subfamilies, the ar/R selectivity filter sequences were similar, such as in CrPIPs (F-H-T-R), CrTIP1s (H-I-A-V), and CrNIP1s (W-V-A-R). We also analyzed Froger’s positions (P1–P5), five conserved amino acid residues that are related to glycerol transport in water-conducting AQPs. The P2, P3, P4, and P5 Froger’s positions in CrPIPs were relatively conserved (S-A-F-W), and in CrTIPs, they were less conserved (S-A-Y/F-W). In CrNIPs and CrSIPs, the P3 and P4 positions mostly stayed A and Y. It is supposed that plant TIPs may transport various small solutes, including H2O2, NH4+, and urea, in addition to water [39]. As with other plant TIPs, CrTIPs are mainly located in vacuolar membranes and may be involved in the regulation of water flow across subcellular compartments of organelles [51]. The variation of CrTIPs in ar/R selectivity filter sequences may contribute to their multiple transport functions, and their NPA spacing varies from 79 to 127 amino acid residues, which indicates that CrTIPs might also be involved in the transmembrane transport of multiple small molecules.
Gene structure organization, gene expansion, and gene diversity are critical indicators of the evolution of gene families. The CrPIP and CrTIP subfamilies exhibit relatively stable gene structure in comparison with other subfamilies (Fig. 3). Most of them possess three (CrPIPs) or two (CrTIPs) introns, suggesting that they might share a common ancestral origin. Similar to previous reports showing very few or no intronless AQP genes in other plant species [30, 31, 52], only one intronless AQP was identified in C. rosea. The intronless gene, CrSIP1;2, might have evolved recently through a retrotransposon process. The CrAQP family has undergone a number of duplication events consistent with the highly duplicated nature of plant genomes (Fig. 2; Table 4). The duplication events concerning segmental and tandem duplications identified in this study have also been reported in other plant species [33, 34]. In the present study, some duplicated CrAQPs have distinct patterns of expression in different tissues and habitats, and under different stressors and hormone exposure (Figs. 5 and 6). It is likely that these duplicated gene pairs have similar protein functions yet function in different biological processes, probably mediated by transcriptional regulation or posttranscriptional modification.
Canavalia rosea is a salt- and alkaline-tolerant and drought-adapted halophyte, and abiotic stressors, such as saline-alkaline soil, seasonal drought, strong solar irradiance, and high temperatures, are the main limiting factors that induce osmotic stress and disturb water balance for this species and other tropical seaside plants. Aquaporin genes, especially the PIP isoforms, play major roles in maintaining plant water homeostasis and responses to abiotic stress. Gene transcript levels are dependent upon the structures of their promoters. Therefore, the cis-acting elements in promoter regions might provide the key to understanding genetic factors influencing the responses of signal molecules and environmental elicitors. We summarized the abiotic stress-related cis-acting elements in CrAQP promoters (Fig. 4) and our findings suggest diversity in CrAQP expression patterns. Furthermore, the expression profiles of CrAQPs in different tissues revealed by RNA-Seq indicate that some of the CrPIP and CrTIP subfamilies had higher expression levels than other subfamilies (Fig. 5a), and habitat-specific RNA-Seq data further indicated the most of the CrPIP members had greater expression levels in coastal C. rosea (YX) than in inland C. rosea (SCBG; Fig. 5b). Our results suggest that differential expression of CrPIPs might be associated with different water use strategies in different habitats, and the higher expression level of CrAQPs in coastal C. rosea plants might be an adaptive mechanism to deal with intracellular and extracellular water-deficit signals. Therefore, we further investigated the expression patterns of CrPIPs under salt, alkaline, and drought stress and the ABA hormone treatment using qRT-PCR (Fig. 6). The results showed that CrPIP expression was most affected under the saline-alkaline, high osmotic stress, and ABA treatments. Furthermore, some CrPIPs showed clearly different and even opposite expression patterns in roots, vines, and leaves. This can be attributed to the fact that in roots the PIP proteins mainly facilitate water absorption from external environments, while in vines and leaves, the PIPs may play a larger role in transpiration. Broadly, our results suggest a role for PIPs in regulating C. rosea hydraulics and probably adaptation to the challenging environmental conditions found on tropical coral reefs and islands.
We performed protein-protein interaction studies using yeast two-hybrid assays and found that two CrPIP members, encoded by CrPIP1;5 and CrPIP2;3, that were highly expressed in all tested tissues and almost constitutively expressed under the abiotic stress challenges and ABA treatment (Fig. 5a and Fig. 6). These two CrPIP members could bind to themselves and each other to form homodimers and heterodimers (Fig. 7). This is consistent with previous findings that some PIP1 and PIP2 members could assemble as homotetramers and heterotetramers, thereby triggering channel activities, influencing substrate specificity, and regulating PIP trafficking [53]. Here, our results on the expression patterns of CrPIP1;5 and CrPIP2;3 provide a detailed understanding of their regulatory modes and help to illuminate CrAQP functions. These data are especially helpful for characterizing AQP-interacting protein complexes involved in C. rosea’s adaptations to harsh environmental conditions such as low water availability and saline-alkaline soils.
Our results from the yeast overexpression system indicate that CrPIP1;5 is an active transmembrane H2O and H2O2 transporter (Fig. 8). We assessed the overexpression of CrPIP1;5 in transgenic Arabidopsis, and CrPIP1;5 lead to slightly reduced saline-alkaline and drought tolerance. This suggests that CrPIP1;5 could play a key role in water transport. We also found that high levels of salt, alkaline, and ABA slightly decreased the expression of CrPIP1;5 in C. rosea., This further suggests that this gene is highly important for water movement between cells and tissues, and is indeed involved in a stress response pathway that protects plants from water loss under high salinity conditions and promotes water release under high osmotic stress caused by PEG or sorbitol (Fig. 8b). The overexpression of CrPIP1;5 in transgenic Arabidopsis is in contrast to most previous findings [39], suggesting that overexpression of plant PIPs results in improved agronomic and abiotic stress tolerance. There are also few studies reporting that the overexpression of plant PIPs could increase sensitivity to drought stress. For example, transgenic tobacco (Nicotiana tabacum) plants overexpressing AtPIP1;4 and AtPIP2;5 displayed rapid water loss under dehydration stress and showed enhanced water flow under drought stress [54]. The Glycine soja gene GsPIP2;1 negatively impact salt and drought stress tolerance by regulating water potential when overexpressed in transgenic Arabidopsis [43]. In addition, Arabidopsis plants overexpressing AcPIP2 (a PIP gene from saltbush A. canescens) exhibited drought-sensitive phenotypes [40]. Together, these studies suggest that regulation of PIP genes within different plant species promotes plant responses to abiotic stressors by maintaining water homeostasis.