1. Introduction
Water is indispensable at every stage of plant growth and development, and is an essential component for the maintenance of normal physiological functions in cells. Plants constantly undergo water absorption, transmembrane transport, and inter-tissue transfer. Aquaporins (AQPs) are membrane proteins embedded in the biological membrane that efficiently transport water molecules and other small molecular compounds. AQPs can enhance the transmembrane transport efficiency of water molecules, increasing the permeability by more than tenfold [
1]. They also regulate the flow of water within cells, with approximately 70%–90% of intracellular water movement facilitated through AQPs [
2]. Additionally, AQPs are involved in various physiological and metabolic processes [
3,
4,
5].
Aquaporin primary structures exhibit a high degree of homology between the amino (N) terminus and carboxyl (C) terminus sequences within the protein [
6]. Aquaporins have small NPA motifs at both ends composed of highly conserved amino acid residues (Asn–Pro–Ala sequence). In higher plants, AQPs are divided into five subfamilies based on sequence homology, similarity, and subcellular localization: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs), and uncharacterized X intrinsic proteins (XIPs). Two additional subfamilies, GlpF-like intrinsic proteins (GIPs) and hybrid intrinsic proteins (HIPs) are found only in mosses. Studies have demonstrated that aquaporins play a crucial role not only as indispensable mediators in plant water transport but also in maintaining intracellular homeostasis and reducing external damage under abiotic stress [
7,
8,
9].
Low-temperature stress is a common abiotic stress factor that severely limits plant growth and development. Numerous studies have demonstrated a close relationship between aquaporins and plant cold resistance. Low temperatures reduce the water absorption rate of plant roots and water transport within the plant body. Cold-tolerant plants can recover growth after exposure to low temperatures, while cold-sensitive plants may lose their ability to regulate water under cold stress and subsequently die from dehydration during recovery [
10]. As one of the main pathways for transmembrane water transport, aquaporins play a crucial role when plants face low-temperature stress [
11]. Research has shown that plant aquaporins exhibit a cold-stress response at the beginning of cold stress. For example, transgenic
Musa nanas overexpressing
MusaPIP1;2 and
MusaPIP1;2 exhibits enhanced tolerance to low-temperature stress [
12], and transgenic
Nicotiana tabacum overexpresses the
Triticum aestivum aquaporin gene TaAQP7 (PIP2), showing increased cold and drought tolerance [
13,
14]. When
Oryza sativa is exposed to low temperatures over a long period, it increases the expression of
OsPIP2;5 to enhance root hydraulic conductivity (
Lpr), thereby mitigating the impact of the cold stress on roots [
15]. Studies indicate that plants such as
O. sativa [
16],
M. nana [
12,
17,
18],
T. aestivum [
13,
19], and
Sorghum bicolor [
20] enhance their cold tolerance by increasing or suppressing the expression of related aquaporin genes under low-temperature stress, with many of the cold-stress-related aquaporin genes being PIP genes.
Catalpa bungei, a large deciduous tree in the Bignoniaceae family, is a traditional and precious native tree species unique to China, mainly used for wood processing and landscape greening, historically referred to as the “King of Woods” [
21]. Due to its poor cold tolerance, low temperatures significantly limit its cultivation scope in China. This study aims to identify the aquaporin gene family of
C. bungei (CbAQP), analyze their expression patterns, and investigate the changes in the expression of the CbAQP gene family during different cold stress periods, selecting candidate aquaporin genes responsive to low-temperature stress. The results of this study lay the foundation for further research into the biological functions of candidate aquaporin genes related to the cold resistance of
C. bungei, provide a theoretical basis for improving the quality of
C. bungei seedlings and cold-resistant breeding, and expanding its distribution range to the south and north.
4. Discussion
Leaves are highly sensitive and adaptable to environmental changes during plant evolution [
34]. Their external morphology can intuitively and rapidly reflect the growth status of the plant and its sensitivity to adversity [
35]. Low temperatures disrupt the normal physiological metabolism of plants, affecting the transport of water between cells, resulting in symptoms such as leaf curling and wilting due to dehydration. In this study, under cold stress, the external morphology of the leaves of the less cold-tolerant “Qiuza 1” exhibited damage symptoms later than the more cold-tolerant “Qiuza 2”. However, as the duration of cold stress increased, “Qiuza 1” showed severe damage symptoms earlier than “Qiuza 2”. This suggests that “Qiuza 1” has weaker cold tolerance compared to “Qiuza 2” under prolonged cold conditions. This characteristic of delayed initial damage but earlier severe damage under prolonged cold treatment is inconsistent with the weak cold tolerance observed by Huang et al. [
36] and Wei [
37]. This may indicate that
C. bungei has a unique mechanism in response to cold tolerance.
In vertebrates, there are approximately 11 to 13 AQPs, and the number of AQPs in most plants ranges from 30 to 50 [
38]. Although the number of genes encoding aquaporin proteins in
C. bungei is relatively small, the overall distribution ratio of aquaporin protein genes within subfamilies is consistent with
A. thaliana. Through transcriptome sequencing, this study identified 15 aquaporin protein genes in
C. bungei, which can be classified into 5 subfamilies: PIPs, TIPs, NIPs, SIPs, and XIPs. The specific number of CbAQP gene family members awaits further validation through genome sequencing.
In the evolution of plants, XIP genes are prone to events such as substitution of Ar/R selective filtering sites, insertion and loss of the C loop, and loss of introns [
30]. Currently, XIP subfamilies are absent in monocotyledonous plants such as
O. sativa,
Zea mays, and
S. bicolor, as well as in some dicotyledonous plants like
A. thaliana. However, XIP subfamilies are present in dicotyledonous plants such as
P. trichocarpa,
G. hirsutum, and
C. sativus (
Table 3). This study found that a XIP gene also exists in dicotyledonous
C. bungei, suggesting that XIP subfamilies may only exist in dicotyledonous plants in the plant kingdom. Evolutionary analysis indicates that CbAQPs have a closer relationship with AQPs in
A. thaliana than with those in
O. sativa, which is consistent with the current view of the differentiation between monocotyledons and dicotyledons in plant evolution [
39].
The study found that PIPs exhibit high selectivity in transporting substrates and play an important role in maintaining the water balance in plant cells. Whether plant plasma membrane aquaporin proteins can accurately locate to the plasma membrane determines their ability to function as water channel proteins. Among the 12 cold-related genes screened in this study, 5 belong to the PIPs subfamily, indicating that the water balance in
C. bungei under low-temperature stress mainly relies on the PIPs subfamily of aquaporin proteins. This result is consistent with previous research findings on aquaporin proteins responding to low-temperature stress [
40]. What sets this study apart from other research is that genes in the NIPs and TIPs subfamilies of the CbAQP gene family also respond to low-temperature stress.
When plants are subjected to low-temperature stress, the water balance within the plant is disrupted. Aquaporin protein, as a key factor in transmembrane water transport, actively responds to low-temperature stress. However, the response pattern of plant aquaporin proteins may vary depending on the species, organ, and subfamily, indicating that aquaporin proteins may have different functions within plants. Seong et al. [
11] found that overexpression of
PIP2;5 in
A. thaliana resulted in increased tolerance to low temperatures in stems, leaves, and roots compared to wild-type plants. Matsumoto et al. [
32] chemically treated
O. sativa to abolish its cold resistance and found that PIP1 was closely associated with the plant’s cold resistance.
Under 4 °C low-temperature treatment conditions, overexpression of Os
PIP1;3 can enhance the cold resistance of
O. sativa [
41]. Researchers have also found that although the water permeability of
PIP1;3 is lower than that of
OsPIP2;2 and
OsPIP2;4, co-expression of
PIP1;3 with either
OsPIP2;2 or
OsPIP2;4 significantly enhances the water permeability of
OsPIP2;2 or
OsPIP2;4. Interaction between PIP1 and PIP2 in
O. sativa significantly enhances the plant’s cold resistance. After low-temperature stress treatment, overexpression of the
MusaPIP1;2 gene in
M. nanas improves resistance to various stresses, including low-temperature stress [
17]. Overexpression of
MaPIP2;7 reduces the levels of malondialdehyde (MDA) and ion leakage in plants while increasing the levels of chlorophyll, proline, soluble sugars, and abscisic acid (ABA), thereby enhancing tolerance to various stresses such as cold [
18]. Overexpression of genes such as TaAQP7 (PIP2),
MaSIP2;1, and
OsPIP2;7 regulates osmotic balance in plants, reduces membrane damage and oxidation, and enhances cold tolerance by regulating levels of hormones such as ABA and GA.
Based on the CbAQP genes and cold-stress-related aquaporin genes (
Figure 4), the CbAQP gene
CbPIP2;5 shows the highest similarity to the
A. thaliana aquaporin
AtPIP2;5. Jang et al. found that
AtPIP2;5 is the main aquaporin responding to low-temperature stress when overexpressed in
A. thaliana and
N. tabacum [
42]. In both
A. thaliana and
N. tabacum subjected to low-temperature stress, the expression of
PIP2;5 was highly induced on the first day of low-temperature stress (compared to days 1, 7, and 14), followed by a gradual decrease in expression during continued low-temperature stress. The expression pattern of the CbAQP gene
CbPIP2;5 is highly consistent with that of
AtPIP2;5, suggesting that
CbPIP2;5 plays a crucial role in response to low-temperature stress in CbAQP. Comparing the expression levels of
CbPIP2;5 in the two CbAQP varieties at the same stage, it was found that the expression level of
CbPIP2;5 in the less cold-resistant variety “Qiuza 1” was significantly higher than that in the more cold-resistant variety “Qiuza 2” when facing low-temperature stress. This indicates that to maintain water homeostasis within the plant, CbAQP upregulates
PIP2;5 expression to maintain root water permeability and water transport within the plant, enabling rapid response to low-temperature stress. However, with prolonged exposure to low temperatures, overexpression of
PIP2;5 reduces the sensitivity of plant roots to low temperatures, which is not conducive to long-term adaptation of CbAQP to low-temperature environments. In this study, under cold stress, the time at which “Qiuza 1” exhibited upward curling at the leaf edges was later than “Qiuza 2”. However, the time at which “Qiuza 1” showed slight downward leaf curling was earlier than “Qiuza 2”. These changes in external morphology were consistent with the expression patterns and functions of
CbPIP2;5. Overexpression of the
T. aestivum aquaporin gene
TdPIP2;1 effectively enhances
T. aestivum’s stress resistance [
43], while increased expression of
OsPIP2;5,
OsPIP2;8,
OsPIP2;3, and
OsPIP2;7 in
O. sativa effectively enhances its cold resistance [
15], which is consistent with the short-term cold stress response observed in CbAQP.
The CbAQP gene
CbPIP1;2 belongs to the PIPs subfamily and shows the highest similarity to the
A. thaliana AQPs
AtPIP1;4 and
AtPIP1;5. It is reported that
AtPIP1;4 exhibits a certain functional synergy with
AtPIP2;5 and affects root water permeability by upregulating expression [
11].
AtPIP1;4 also demonstrates higher sensitivity to low temperatures, and its overexpression can maintain the high water permeability of cells.
CbPIP1;2 exhibits peak expression at 18 h of low-temperature stress in the less cold-resistant “Qiuza 1”, while in the more cold-resistant “Qiuza 2”, peak expression occurs at 6 h of low-temperature stress. The delayed peak expression in “Qiuza 1” suggests that “Qiuza 2” responds more rapidly to low temperatures. Additionally, the expression level of
CbPIP1;2 in “Qiuza 1” shows a fluctuating upward trend, indicating that prolonged periods of high water permeability may lead to increased vulnerability to damage during the later stages of low-temperature stress.
The CbAQP gene
CbTIP4;1 belongs to the TIPs subfamily, which is an important subfamily of plant aquaporins. Overexpression of the ginseng PgTIP gene in
A. thaliana significantly alters nutrient growth and reproductive development and reduces resistance to low-temperature stress. When facing low-temperature stress, the expression level of
CbTIP4;1 in “Qiuza 1” significantly increases, far exceeding that in “Qiuza 2”. Therefore, we believe that the high expression of
CbTIP4;1 may reduce the cold resistance of
C. bungei. In low-temperature environments, plant tissues usually freeze due to heterogeneous ice nucleation occurring extracellularly [
44]. Because the water potential of ice is lower than that of water, the cell sap moves out of the cell along the gradient, leading to cell dehydration. It can be speculated that plant cold resistance should include mechanisms to resist cell dehydration induced by freezing. The downregulation of TIPs may be part of this strategy. Therefore, the overexpression of TIPs in plant cells may reduce the cold resistance of plants.
The CbAQP
CbNIP2;1 belongs to the NIPs subfamily, and there are few reports on the response of the NIPs subfamily to low-temperature environments in plants. Based on the family properties of Nodulin26 intrinsic membrane protein of NIPs, we speculate that the
CbNIP2;1 gene may affect the absorption and release of metal ions by plant cell ion channels, thereby affecting the concentration of solutes in plant cells and changing the ion concentration of plant cells in low-temperature environments.
CbNIP2;1 peaks in expression at 6 h and 12 h in “Qiuza 1” and “Qiuza 2”, respectively, with overall expression levels in “Qiuza 2” significantly higher than in “Qiuza 1” (
p < 0.05). This result is consistent with Verma’s study in
O. sativa [
45], indicating that the high expression level of
CbNIP2;1 helps to improve plant cold resistance. However, further research is needed to determine how
CbNIP2;1 specifically affects plant ion channels.
In this study, by comparing the CbAQP genes with other reported cold-related aquaporin genes and analyzing the changes in CbAQP during four periods of low-temperature stress, we identified the specific expression patterns of individual members of this gene family during low-temperature stress. Among the 15 CbAQP genes, we found 12 CbAQP genes responsive to low-temperature stress, including 5 in the CbPIPs subfamily, 2 in the CbTIPs subfamily, 3 in the CbNIPs subfamily, 1 in the CbSIPs subfamily, and 1 in the CbXIPs subfamily. Based on reported sequences related to cold stress, we found that the sequences CIAW and GGMI in motif 6 are unique to the CbAQP PIPs subfamily, suggesting that these two gene sequences may not be key sequences in the response of CbAQP to low-temperature stress. The motifs IAFEXXT, SGGHINPAVT, and GTFVLVYTVF are distributed in motifs 1 and 2, and motifs 1 and 2 are simultaneously present in the PIPs, TIPs, and NIPs subfamilies of CbAQP. We speculate that these gene sequences may play a role when C. bungei faces low-temperature stress. Upon analyzing the gene sequences of CbTIP4;1, CbNIP2;1, CbPIP1;2, and CbPIP2;5, we found that the sequences IXEXIAT and EIXXTF are highly conserved among AQPs in different subfamilies. Therefore, we speculate that these two gene sequences may play a key role when C. bungei faces low-temperature stress. The absorption and transportation of water by plant roots directly depend on the transcriptional regulation of aquaporins and other factors that change the permeability of cell membranes to water. Facing low-temperature stress, C. bungei regulates the expression levels of AQP genes and the corresponding protein activities to adjust the water permeability of roots, thereby maintaining water balance within the plant and ensuring normal physiological activities.