A banana aquaporin gene, MaPIP1;1, is involved in tolerance to drought and salt stresses
© Xu et al.; licensee BioMed Central Ltd. 2014
Received: 15 October 2013
Accepted: 18 February 2014
Published: 8 March 2014
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© Xu et al.; licensee BioMed Central Ltd. 2014
Received: 15 October 2013
Accepted: 18 February 2014
Published: 8 March 2014
Aquaporin (AQP) proteins function in transporting water and other small molecules through the biological membranes, which is crucial for plants to survive in drought or salt stress conditions. However, the precise role of AQPs in drought and salt stresses is not completely understood in plants.
In this study, we have identified a PIP1 subfamily AQP (MaPIP1;1) gene from banana and characterized it by overexpression in transgenic Arabidopsis plants. Transient expression of MaPIP1;1-GFP fusion protein indicated its localization at plasma membrane. The expression of MaPIP1;1 was induced by NaCl and water deficient treatment. Overexpression of MaPIP1;1 in Arabidopsis resulted in an increased primary root elongation, root hair numbers and survival rates compared to WT under salt or drought conditions. Physiological indices demonstrated that the increased salt tolerance conferred by MaPIP1;1 is related to reduced membrane injury and high cytosolic K+/Na+ ratio. Additionally, the improved drought tolerance conferred by MaPIP1;1 is associated with decreased membrane injury and improved osmotic adjustment. Finally, reduced expression of ABA-responsive genes in MaPIP1;1-overexpressing plants reflects their improved physiological status.
Our results demonstrated that heterologous expression of banana MaPIP1;1 in Arabidopsis confers salt and drought stress tolerances by reducing membrane injury, improving ion distribution and maintaining osmotic balance.
Plant growth depends greatly on water absorption from the soil and the movement of water from the roots to other plant parts . However, environmental stresses such as drought, salt and cold can lead to water loss in plants. Such environmental stresses severely affect plant growth and productivity worldwide. Translocation of water is an important process to maintain the ability to tolerate desiccation and high salt stresses [2–4]. In plants, water movement is controlled by both apoplastic and symplastic pathways . When plants are experiencing abiotic stress, the symplastic pathway is efficient for transporting water across membranes [5–7], and the symplastic pathway is regulated mainly by members of the aquaporin family of proteins .
Aquaporins (AQPs) transport water as well as other small molecules such as glycerol, CO2 and boron through membranes [9–11]. Biological activities associated with AQPs are diverse and include seed germination, stomatal movement, cell elongation, reproductive growth, phloem loading and unloading and stress responses in plants [12, 13]. Many genes encoding AQP proteins have been identified from different plant species, including 35 from Arabidopsis, 33 from rice  and 36 from maize . These orthologs can be subdivided into four groups characterized by highly conserved amino acid sequences and stereotypical intron positions within each group: the tonoplast intrinsic proteins (TIPs), the plasma membrane intrinsic proteins (PIPs), the nodulin-like plasma membrane intrinsic proteins (NIPs) and the small intrinsic proteins (SIPs) .
The expression and biological activities of AQPs are affected by a number of signals, including abiotic stresses, plant hormones and light [10, 14, 18–21]. The regulation and biochemical functions of AQPs in response to abiotic stresses are complex and not well understood. In a number of transgenic approaches, some AQPs have been demonstrated to confer tolerance to abiotic stresses [6, 11, 13, 22–26]. For example, overexpression of TaAQP8 results in increased root elongation under salt stress . Tobacco NtAQP1 is involved in improving water use efficiency, hydraulic conductivity, and yield production under salt stress . However, overexpression of a distinct aquaporin, HvPIP2;1, leads to an increased transpiration rate and slightly decreased intrinsic water-use efficiency . These attempts to use AQPs to improve crop tolerance to abiotic stresses have yielded contradictory results depending on the isoforms of AQPs. Therefore isoforms that are shown to confer improved physiological status under stress are of major importance in crop science.
Banana (Musa acuminata L.) is a large annual monocotyledonous herbaceous plant found in tropical and subtropical climates, and is one of the most popular fresh fruits enjoyed worldwide. Because banana has shallow roots and a permanent green canopy, it is especially sensitive to conditions that lead to water deficit [28, 29]. A better understanding of the mechanisms employed by banana plants to tolerate abiotic stresses will be helpful for increasing crop production and quality of this economically valuable fruit. In banana, only one aquaporin gene, MusaPIP1;2, has been characterized as a positive factor in abiotic stress tolerance. Transgenic plants overexpressing MusaPIP1;2 constitutively exhibited better abiotic stress survival characteristics including lower malondialdehyde content, elevated relative water content, elevated proline levels and a higher photosynthetic efficiency relative to controls under different abiotic stress conditions . In our previous study, a transcript displaying upregulated expression at the early stage of post-harvest banana ripening was identified by cDNA microarray . Sequence analysis suggested that this cDNA fragment exhibited high similarity to AQP genes from other plant species. In this study, a full-length cDNA encoding MaPIP1;1 was cloned and characterized. We investigated the function of MaPIP1;1 during drought and salt stresses, which will lead to increased understanding of the mechanisms of environmental stress tolerance employed by plants.
A cDNA fragment was identified by cDNA microarray from genes that were differentially expressed at the early stage of post-harvest banana ripening and the full-length cDNA, designated as MaPIP1;1 (GenBank: KC969669), was obtained using the rapid amplification of cDNA ends (RACE) method. The full-length MaPIP1;1 cDNA is 1123 bp in length with a 861 bp open reading frame (ORF) that encodes 286 amino acids. BLASTX analysis demonstrated that MaPIP1;1 had 94% sequence identity with HcPIP1 from Hedychium coronarium and OsPIP1;2 from Oryza sativa Japonica Group. The predicted MaPIP1;1 protein has a highly conserved amino acid sequence (‘HINPAVTFG’) characteristic of the MIP family, six putative transmembrane helices and two ‘NPA’ motifs (Additional file 1: Figure S1). Phylogenetic analysis of MaPIP1;1 with other AQPs from Arabidopsis and rice that MaPIP1;1 is close to PIP1 subfamily (Additional file 1: Figure S2). These results suggest that the MaPIP1;1 gene cloned in this study is a member of the PIP1 subfamily in banana.
Several lines of evidence have shown that AQPs are involved in abiotic stress tolerance [11, 13, 22, 25, 26, 36]. In our study, we observed that expression of MaPIP1;1 in leaves and roots was significantly induced after drought and salt treatment, implying that this gene product may play a positive role in mediating responses to drought and salt stresses. To better understand the function of MaPIP1;1 during abiotic stress, we generated a number of MaPIP1;1-overexpressing Arabidopsis transgenic lines. The transgenic seedlings and adult plants exhibited increased tolerance to drought and salt stresses compared to WT. These results are consistent with previous studies demonstrating that overexpression of AQP genes confers abiotic stress tolerance to transgenic plants [11, 13, 25, 26, 36].
Na+ is toxic to cell metabolism and has a deleterious effect on some proteins. High Na+ levels also reduce photosynthesis and lead to oxidative damage . Additionally, drought stress can induce a rapid accumulation of ROS leading to damage of the cell membrane and oxidation of proteins, lipids, and DNA [38, 39]. MDA, the product of lipid peroxidation caused by ROS, can be used to evaluate ROS-mediated injuries in plants . IL is also an important indicator of membrane injury. Thus, MDA content and IL were measured to assess the role of MaPIP1;1 overexpression in reducing membrane injury under drought or salt conditions. MaPIP1;1 overexpression resulted in decreased IL and MDA content relative to WT, indicating that MaPIP1;1-overexpressing plants may experience less lipid peroxidation and membrane injury under salt or drought conditions. Consistent with our findings, TaAQP7-overexpressing tobacco plants show lower levels of MDA and IL when compared to WT under drought stress and BjPIP1-overexpressing plants exhibit reduced MDA and IL under Cd stress [26, 40]. Overexpression of OsPIP2;7 in rice results in decreased IL under chilling stress and TaAQP8-overexpressing tobacco plants exhibit reduced MDA and IL relative to WT plants under salt stress [25, 41]. Collectively these studies indicate that AQPs play a vital role in decreasing IL and MDA, thereby reducing membrane injury under various abiotic stresses. AQPs participate in the rapid transmembrane water flow during growth and development in plants. When plants are subjected to drought or salt conditions, increased transport of water across membranes is crucial to maintain a healthy physiological status. We also observed that MaPIP1;1-overexpressing plants subjected to drought or salinity treatments exhibited better growth than the WT plants. We surmise that physiological improvements conferred by MaPIP1;1 overexpression contribute to plants maintaining the protein machinery and hence reducing membrane injury.
A large number of different ion transporters and channel proteins, such as SOS1, NHX and HKT, are situated in the plasma membrane. These proteins play crucial roles in maintaining ion homeostasis during a variety of abiotic stresses. For example, AtSOS1 and SlSOS1 are membrane-bound Na+/H+ antiporters that improve salt stress tolerance by exporting Na+. The reduced membrane injury observed in MaPIP1;1-overexpression lines led us to examine K+ and Na+ accumulation in WT plants and transgenic lines. MaPIP1;1 overexpression decreases the accumulation of cellular K+ and Na+ and improves the K+/Na+ ratio under salt stress. Previous studies have also reported that aquaporins regulate the distribution of Na+ and K+ under salt stress. TaNIP-overexpressing plants exhibit higher K+ and lower Na+ levels compared to WT plants under salt stress . TaAQP8-overexpressing tobacco plants have elevated Na+ and K+ levels in roots, reduced Na+ and increased K+ in stems compared to WT plants under salt treatment . Although overexpression of aquaporins appears to cause different patterns of altered Na+ and K+ distribution, the evidence suggests that these lead to improved K+/Na+ ratios under salt conditions. In recent years, a high cytosolic K+/Na+ ratio has become an accepted marker of salinity tolerance . Therefore, the increased salt stress tolerance conferred by MaPIP1;1 overexpression may be due to not only decreased membrane injury but also the increased K+/Na+ ratio in transgenic lines.
Maintaining the ability to retain water is vital for plants to combat drought stress. AQPs function in rapid transmembrane water flow during growth and development and play important roles in maintaining plant water relations under drought conditions. We observed that MaPIP1;1-overexpressing plants exhibited better growth and a lower rate of water loss compared to WT plants under drought conditions, indicating a positive influence of MaPIP1;1 on water retention. Consequently, we investigated the physiological mechanisms involved in improved water retention conferred by MaPIP1;1. When plants experience drought conditions, the accumulation of compatible osmolytes is employed as a strategy to maintain osmotic adjustment. One such compatible solute is the amino acid proline, whose accumulation functions to decrease the cellular osmotic potential and to enhance cellular protection . MaPIP1;1-overexpressing transgenic plants maintained higher levels of proline and osmotic potential compared to WT plants subjected to similar drought treatment, implying that MaPIP1;1 may function in maintaining osmotic adjustment under drought stress. The reduced membrane injury conferred by overexpression of MaPIP1;1 may also contribute to improved osmotic adjustment under drought stress.
Dehydration can lead to inhibition of physiological processes; therefore plants initiate adaptive mechanisms to survive osmotic stresses [44, 45]. ABA-dependent signal transduction pathways play crucial roles in the adaptation of plants to stress . When Arabidopsis plants were subjected to water stress, some ABA-responsive genes, such as RD29A, RD29B, KIN2 and RAB18 showed increased transcript levels, indicating that the injury resulted from water stress induces the expression of ABA-responsive genes . We examined the expression of these ABA-responsive genes in MaPIP1;1-overexpressing transgenic seedlings in relation to WT seedlings. The ABA-responsive genes were downregulated in the transgenic seedlings subjected to dehydration or salt treatments in comparison to similarly treated WT seedlings. This result suggests that the MaPIP1;1-overexpressing transgenic plants were less responsive to ABA signaling compared to WT plants, implying that MaPIP1;1-overexpressing plants have improved physiological status under drought and salt stress conditions.
The findings of this study demonstrated a role for MaPIP1;1 function in improving tolerance to drought and salt stresses. MaPIP1;1 overexpression resulted in enhanced tolerance to salt stress not only by reducing membrane injury but also by maintaining a higher cellular K+/Na+ ratio. Enhanced drought stress tolerance conferred by MaPIP1;1 is related to decreased membrane injury and improved osmotic balance. These findings further our understanding of the mechanisms of environmental stress on plants and highlight the role of AQPs in reducing membrane injury, improving ion distribution and maintaining osmotic balance. It is necessary to point out that heterologous expression of banana MaPIP1;1 in Arabidopsis results in these conclusions that are valid for such a heterologous system, but may not be the same in other plants. Further studies are required to characterize the function of MaPIP1;1 in banana.
Young banana (Musa acuminata L. AAA group, cv. Brazilian) seedlings were obtained from the banana tissue culture centre (Danzhou, Institute of Banana and Plantain, Chinese Academy of Tropical Agricultural Sciences). Banana seedlings were grown in soil supplied with half-strength Hoagland’s solution under greenhouse conditions (28°C; 200 μmol m−2 s−1 light intensity; 16 h light/8 h dark cycle; 70% relative humidity). Seedlings with uniform growth at the five-leaf stage were selected for stress treatment. For NaCl treatment, banana seedlings grown in soil were irrigated with half-strength Hoagland’s solution supplemented with 350 mM NaCl for up to 6 h . Hsiao (1973) proposed that extent of drought stress that plant suffered can be divided to three levels according the water potential in soil . For drought stress assays, water was withheld from banana seedlings grown in soil and samples were collected when the soil moisture content reached different stress degrees as outlined by Hsiao (1973). The soil moisture content was measured using an instrument according to manual instructions (TZS-1, TOP, Zhejiang, China). For low temperature treatments, banana seedlings were transferred into a growth chamber, in which the temperature was maintained at 28, 15, 10, 7 or 5°C for 12 h.
The full-length cDNA encoding MaPIP1;1 was amplified by RACE using sequence information from a cDNA fragment previously identified by suppression subtractive hybridization (SSH) . Single-stranded cDNA was used as a source template and was generated from banana fruit 2 d after harvest. For 5′ RACE, the forward primer sequence was 5′-catctcgccgaggtgctccttgtgc-3′ and the reverse primer sequence was 5′-ccttgcctcaacaacacgatc-3′. For 3′ RACE, the forward primer sequence was 5′- cagcggtggcggttggcagcggaggc-3′, and the reverse primer sequence was 5′-ctccgagatctggacgagc-3′. Amplified products were inserted into the pGEM-T easy vector (Promega, Madison, WI, USA). A pair of specific primers was used (5′- tcggccattacggccgggga-3′ and 5′-cttatttttaagggtttttgatac-3′) to amplify the entire open reading frame (ORF) based on the sequences of the 5′ and 3′ ends. The resulting full-length cDNA encoding MaPIP1;1 was assessed by DNAMAN software and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
The MaPIP1;1 ORF, including engineered NcoI/SpeI restriction sites, was obtained using gene-specific primers. The PCR products were inserted into pCAMBIA1304-GFP expression vector to generate a MaPIP1;1-GFP fusion protein under the control of the CMV35S promoter. The pCAMBIA1304-MaPIP1;1-GFP construct and the pm-rk used as a plasma membrane-localized maker were transiently co-expressed in onion epidermal cells using a gene gun to deliver the expression plasmids (PDS-1000, BIO-RAD) . After a 48 h incubation at 25°C on Murashige and Skoog medium (MS), fluorescence was examined by fluorescence microscopy (LSM700, Carl Zeiss, Germany). The exitation/emission wavelengths are 485/515 nm for GFP, 585/615 nm for RFP and 460/490 nm for GFP and RFP in the same well.
The pCAMBIA1304-MaPIP1;1-GFP construct was transferred into Agrobacterium strain GV3101. Transgenic Arabidopsis plants were generated using the floral dip-mediated infiltration method . Seeds from T0 transgenic plants were selected on half-strength MS medium containing 50 mg/L of hygromycin B. Homozygous T3 lines were used for further functional investigation of MaPIP1;1. Five hygromycin-resistant transgenic lines from the T3 generation were used to determine the integration of MaPIP1;1 to Arabidopsis genome by Southern analysis. The transcriptional levels of MaPIP1;1 in the 5 independent T3 lines was examined by qRT-PCR analysis, in which the AtActin gene was used as an internal control.
Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type control for these experiments. Seeds were sterilized in 75% (v/v) ethanol for 10 minutes, vernalized for 2 d at 4°C in the dark and then germinated on half-strength MS medium or directly on soil. Plants were grown under a chamber (22°C; 120 μmol m−2 s−1 light intensity; 16 h light/8 h dark cycle; 70% relative humidity). For phenotype analysis in early seedlings under normal conditions, four day-old seedlings were used to determine the root hairs, then the seedlings were transferred to half-strength MS medium for 15 days, and then the photos were taken and roots length were measured. For salt stress tolerance analysis in early seedlings, four day-old seedlings were transferred to half-strength MS or the same medium supplemented with 50–150 mM NaCl for 15 days, then the photos were taken, and then root length and root hairs were measured. For salt stress tolerance analysis in adult plants, Arabidopsis plants at four weeks of age were irrigated with 350 mM NaCl for 15 days, then the photos were taken, and then survival rates were assessed. For osmotic stress tolerance analysis, four day-old seedlings were transferred to half-strength MS or the same medium supplemented with 100–300 mM mannitol for 15 days, then the photos were taken, and then root length and root hairs were measured. For drought stress tolerance analysis, plants were grown in pots filled with a mixture of soil and sand (3:1) at 22°C for four weeks. Water was withheld from the treatment group for 20 days, then the photos were taken and survival rates were calculated. For expression analysis of ABA-responsive genes in WT and transgenic lines, fifteen day-old seedlings were transferred to half-strength MS agar plates supplemented with 350 mM NaCl for up to 10 h or 300 mM mannitol for up to 6 h. Whole seedlings were used to quantify relative gene expression.
Thirty fully expanded leaves from each line were detached from four week-old Arabidopsis plants and weighed immediately (Fresh Weight, FW). The leaves were placed on open Petri dishes, which were then placed in an incubation chamber (humidity 45%, 22°C). Samples were weighed at different time intervals (Desiccated Weights, DW). The water loss rates was calculated according to the formula: water loss rate (%) = (FW – DW) / FW × 100 .
Four week-old plants were well watered or irrigated with 350 mM NaCl treatment for 15 days and leaf samples were collected to examine MDA and IL. Fifteen day-old seedlings were transferred to fresh half-strength MS agar plates supplemented with or lacking 350 mM NaCl and whole seedlings were used to measure MDA and IL. Four week-old plants were either well-watered or subjected to simulated drought treatment by withholding water for 20 days. Leaves of WT and transgenic lines were collected to examine MDA, IL, proline and osmotic potential. Ion leakage (IL) was measured according to the method described by Jiang and Zhang (2001) . Leaf samples were cut into strips and incubated in 10 ml of distilled water at 25°C for 8 h. The initial conductivity (C1) was determined with a conductivity meter (DDBJ-350). The samples were then boiled for 10 min to yield complete IL. After cooling down, the electrolyte conductivity (C2) was measured. IL was calculated according to the equation: IL (%) = C1/C2 × 100. Proline content was measured according to Bates (1973) . Malondialdehyde (MDA) content was measured according to the thiobarbituric acid colorimetric method as described by Heath and Packer (1968) . The osmotic potential was measured using a dewpoint PotentiaMeter according to the manufacturer’s instruction (WP4C, DECAGON, USA).
Four week-old Arabidopsis plants were well watered or irrigated with 350 mM NaCl for 15 days. The leaves and roots from WT plants and the transgenic lines were collected to determine ion content. Plant materials were washed with ultrapure water, then treated at 105°C for 10 min and baked at 80°C for 48 h. Samples consisting of 50 mg of dry material were dissolved in 6 ml of nitric acid and 2 ml H2O2 (30%) and then heated at 180°C for 15 min. The digested samples were diluted in a total volume of 50 ml with ultrapure water, transferred into new tubes and analyzed by atomic absorption spectroscopy (Analyst 400, Perkin Elmer, USA).
Genomic DNA isolated from Arabidopsis leaves was digested with EcoRI restriction enzyme, separated on 0.8% agarose gels, and transferred to nylon membranes. cDNA probes used in Southern blotting were amplified using a MaPIP1;1 primer set: F- 5′ATGTGTAATCCCAGCAGC and R- 5′CAAGGAGGACGGAAACAT. The probe was labeled using random primer labeling system. Hybridizations were performed according to the manufacturer’s instructions (Roche11745832910, DIG High Prime DNA Labeling and Detection Starter Kit, USA).
Expression of MaPIP1;1 in banana organs and leaves after various treatments as well as ABA-responsive genes in Arabidopsis were measured by qRT-PCR using SYBR® Premix Ex Taq™ (TaKaRa) chemistry on a Stratagene Mx3000P (Stratagene, CA, USA) instrument. Total RNA was extracted from Arabidopsis and banana tissues using a plant RNA extraction kit (QIAGEN) according to the manufacturer’s instructions. 3 μg of total RNA from each sample was converted into cDNA using SuperScript II reverse transcriptase (Invitrogen). In all qRT-PCR experiments, 2–ΔΔCt method was employed to assess relative expression of the tested genes with three replicates of each condition . Prior to quantification experiments, a series of template and primer dilutions were conducted to obtain the optimal template and primer concentrations for amplifying the target genes. Primers used in qRT-PCR analysis had high efficiency and specificity based on melting curve analysis and agarose gel electrophoresis. The sequences of these primers were included in Additional file 1: Table S1. To confirm the specificity of primer pairs, PCR products were subsequently subjected to sequence analysis. Amplification efficiencies of primer pairs were between 90% and 110%. MaRPS2 (HQ853246) and MaUBQ2 (HQ853254) were used as the internal controls to normalize expression of target genes in banana and β-ACTIN2 (At3g18780) and β-ACTIN8 (At1g49240) were selected as reference genes to normalize transcript levels of target genes in Arabidopsis. All the selected reference genes were verified to be constitutive expression and suitable to be used as internal controls [56–58].
This work was supported by the earmarked fund for Modern Agro-industry Technology Research System (nycytx-33), the Ministry of Science and Technology of the People’s Republic of China (2011AA10020605) and the Major Technology Project of Hainan (ZDZX2013023-1-07; ZDZX2013023-1-16).
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