Plant Mol Biol DOI 10.1007/s11103-015-0305-2

Constitutive and stress‑inducible overexpression of a native aquaporin gene (MusaPIP2;6) in transgenic banana plants signals its pivotal role in salt tolerance Shareena Sreedharan1 · Upendra K. Singh Shekhawat1 · Thumballi R. Ganapathi1 

Received: 17 September 2014 / Accepted: 28 February 2015 © Springer Science+Business Media Dordrecht 2015

Abstract  High soil salinity constitutes a major abiotic stress and an important limiting factor in cultivation of crop plants worldwide. Here, we report the identification and characterization of a aquaporin gene, MusaPIP2;6 which is involved in salt stress signaling in banana. MusaPIP2;6 was firstly identified based on comparative analysis of stressed and non-stressed banana tissue derived EST data sets and later overexpression in transgenic banana plants was performed to study its tangible functions in banana plants. The overexpression of MusaPIP2;6 in transgenic banana plants using constitutive or inducible promoter led to higher salt tolerance as compared to equivalent untransformed control plants. Cellular localization assay performed using transiently transformed onion peel cells indicated that MusaPIP2;6 protein tagged with green fluorescent protein was translocated to the plasma membrane. MusaPIP2;6-overexpressing banana plants displayed better photosynthetic efficiency and lower membrane damage under salt stress conditions. Our results suggest that MusaPIP2;6 is involved in salt stress signaling and tolerance in banana.

Shareena Sreedharan and Upendra K Singh Shekhawat have contributed equally to this work. Electronic supplementary material  The online version of this article (doi:10.1007/s11103-015-0305-2) contains supplementary material, which is available to authorized users. * Thumballi R. Ganapathi [email protected] 1



Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India

Keywords  Banana · Abiotic stress · Aquaporin · MusaPIP2;6 · Agrobacterium-mediated genetic transformation

Introduction High soil salinity is a major constraint in enhancement of agricultural productivity worldwide (Boyer 1982). Almost 20 % of the total cultivable land worldwide is affected by salinity, which is a major concern for agricultural scientists and planners. Salt stress negatively affects every physiological process of a growing plant and ultimately leads to a compromise in the overall productivity. Understanding the salt stress response signaling and studying its vital components in detail has therefore become an important requisite in agricultural research today (Huang et al. 2012). Since high salt concentration in soil directly affects the availability of water in addition to the accompanying toxic effects, studies on ubiquitous water-selective channel proteins, the aquaporins (AQPs) which regulate and mediate rapid trans-membrane water flow during different physiological processes, have become crucial in context of salt stress response pathways (Cabot et al. 2014). Plant AQPs have been broadly categorized into four families based on the amino acid sequence homology and their subcellular localization: plasma membrane intrinsic proteins (PIPs), tonoplast membrane intrinsic proteins (TIPs), nodulin 26-like intrinsic proteins (NIPs) and small basic intrinsic protein (SIPs) (Baiges et al. 2002). The recently released banana genome database provides 50 unique loci when probed with the query “aquaporin”, indicating there are close to 50 AQP gene loci in banana genome (D’Hont et al. 2012).

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Several studies have reported transcriptional activation of AQP genes by different environmental stresses indicating a positive correlation between AQP regulation and abiotic stress tolerance in plants (Bohnert et al. 1995). Indeed, recent studies conducted on important monocot species have reported that the heterologous overexpression of AQP genes in transgenic plants imparts improved tolerance to cold, salt and drought stress. TaAQP8 overexpression in transgenic tobacco improved salt stress tolerance (Hu et al. 2012) whereas ectopic expression of TaAQP7 improved the drought tolerance in tobacco plants indicating specific roles and functions of each AQP isoform in the overall management of water balance in plant cells (Zhou et al. 2012). Similarly, constitutive overexpression of OsPIP1;1 improved salt tolerance in transgenic rice (Liu et al. 2012) and constitutive and stress-inducible overexpression of a native plasma membrane AQP in banana improved drought, salt and cold tolerance in transgenic banana plants (Sreedharan et al. 2013). Banana is a highly salt sensitive crop. High soluble salt contents in soil causes accelerated collapse of banana root system (Turner 2005). Since, AQPs have been known to be involved in abiotic stress response pathways of banana and related monocot species, we studied contrasting EST datasets of banana available with NCBI and identified a AQP gene from banana (EST accession number FL667907) which displayed differential expression between tissues subjected to osmotic stress and untreated control samples. To understand the significance of this differential expression and to investigate whether this gene can ameliorate the incidence and consequences of salt stress in banana plants, we generated transgenic banana plants which overexpressed the full length coding sequence of this gene either under the control of a constitutive promoter or under the control of an abiotic stress inducible promoter. The transgenic banana plants so generated showed marked improvement in salt stress tolerance.

Plant Mol Biol

before (Shekhawat et al. 2011a; Sreedharan et al. 2012). Using this total RNA, full length coding sequence of EST accession no. FL6670907 was obtained using 3′ RACE. ExPASy translate tool (http://au.expasy.org/tools/dna.html) was employed to predict the putative protein sequence for MusaPIP2;6 and the sequence so obtained was aligned to its best homologs using ClustalW2 program (http://www. ebi.ac.uk/Tools/clustalw2/index.html) and box shade server (http://www.ch.embnet.org/software/BOX_form.html). Evolutionary associations for MusaPIP2;6 protein were investigated using MEGA 5 software. Genomic compliment of MusaPIP2;6 coding region was amplified from banana cv. Karibale Monthan genomic DNA isolated using GenElute Plant Genomic DNA Miniprep kit (Sigma, USA). Expression profiling of MusaPIP2;6 in banana leaves and roots in different stress conditions

Primers

Banana cv. Karibale Monthan in vitro maintained plantlets were hardened in greenhouse for 2–3 months. Uniform sized banana plants with 4–5 leaves were irrigated with 250 mM NaCl and 100 µM ABA. Cold stress was applied by exposing the plants to 10 ± 2 °C inside a growth chamber maintained at 16 h light/8 h dark cycle. For drought stress, plants were washed properly to remove soil particles and then left to dry on blotting sheets. Plants normally growing in the greenhouse under optimal conditions were taken as experimental controls. Leaves and root samples obtained from treated plants at different time points were frozen in liquid nitrogen and stored at −80 °C. Total RNA was isolated from these samples as described above. The first-strand cDNA synthesis was performed using 5 µg total RNA, Oligo (dT)12–18 primer and ThermoScript Reverse Transcriptase (Invitrogen, USA). Three independently treated samples from separate plants for each treatment were pooled before total RNA isolation. The cDNAs obtained were used in semi-quantitative PCR reactions (comprising 28 cycles) to determine the expression level of MusaPIP2;6 in the respective samples. Musa Actin gene was also amplified along side from the cDNA samples to validate equivalence of the cDNA content among different samples.

Primers used in the present study are listed in Supplemental Table S1.

Determination of cellular localization of MusaPIP2;6 protein

Amplification and sequence analysis of MusaPIP2;6 gene

MusaPIP2;6 cDNA (amplified from banana cv. Karibale Monthan) without the stop codon was inserted in-frame at the N-terminal end of GFP in the plant binary vector pCAMBIA-1302 (GenBank accession no. AF234298) by using NcoI and SpeI restriction sites. The newly constructed binary vector (pMusaPIP2;6-1302) was then employed to transform onion peels using Agrobacterium tumefaciens strain EHA105 as essentially described before

Materials and methods

Total RNA was isolated from young banana leaves of cv. Karibale Monthan by using Concert Plant RNA Reagent (Invitrogen, USA) followed by RNA clean up by employing RNeasy Plant Mini Kit (Qiagen, Germany) together with RNase Free DNase Set (Qiagen, Germany) as described

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Plant Mol Biol

(Sreedharan et al. 2012). Onion peels were also transformed with unmodified pCAMBIA-1302 vector using A. tumefaciens strain EHA105. Three days after inoculation with Agrobacterium, cellular localization of the MusaPIP2;6::GFP fusion protein in onion cells was examined by a fluorescence microscope (Eclipse 80i, Nikon, Japan). MusaPIP2;6::GFP fusion protein was visualized in the transiently transformed cells by using GFP filter set (excitation 460–500 nm, emission 515–550 nm) and the representative cells were photographed. Plant binary vector construction for constitutive and stress‑inducible overexpression of MusaPIP2;6 Expression cassettes meant for achieving either constitutive or stress-inducible overexpression of MusaPIP2;6 in transgenic banana plants were inserted into the MCS of pCAMBIA-1301 binary vector. pCAMBIA-1301-nos (Shekhawat et al. 2011a) vector was restricted with HindIII and BamHI and Zea mays polyubiquitin promoter amplified from Zea mays genomic DNA (flanked by HindIII and PstI restriction site) and the MusaPIP2;6 CDS amplified using banana cv. Karibale Monthan leaf cDNA (flanked by SbfI and BamHI restriction sites) were inserted in the nos containing linearised binary vector backbone in a three-way ligation reaction. The newly constructed plant expression vector (denoted as Ubi-MusaPIP2;6) with the expression cassette (pZmUbi-MusaPIP2;6-nos), was then sequenced with appropriate primers to confirm the sequence of MusaPIP2;6 coding region. Similarly, another vector was constructed wherein MusaDHN-1 promoter was used in place of Zea mays polyubiquitin promoter and this vector [having expression cassette (pMusaDHN-1-MusaPIP2;6-nos)] was denoted as DhnMusaPIP2;6. Both these vectors were then electroporated into A. tumefaciens strain EHA 105 and later used for transformation of banana suspension culture embryogenic cells. Agrobacterium‑mediated genetic transformation of banana embryogenic cells and generation of transgenic banana plants Agrobacterium-mediated genetic transformation of banana (cv. Rasthali) suspension culture embryogenic cells and generation of putatively transformed banana plants was performed as essentially described before (Shekhawat et al. 2011a; Ganapathi et al. 2001).

construct were chosen for further molecular and biochemical analysis based on luxuriant growth and subsequent healthy multiple shoot development on hygromycin supplemented medium. Genomic DNA (isolated as described above) sourced from leaves of the selected transgenic lines was used for performing Southern blot analysis (using DIG-labeled probes targeting hygromycin phosphotransferase gene) to confirm the transgenic nature of these lines and to determine the copy number of the T-DNA introduced in banana genome in these lines. The exact quantum of overexpression of MusaPIP2;6 in Ubi-MusaPIP2;6 lines and its inducible expression in Dhn-MusaPIP2;6 lines (after 24 h of treatment with 250 mM NaCl) was ascertained using real time quantitative RT-PCR. Rest-MCS software utility was used to derive relative expression values (Pfaffl et al. 2002). For these RT-PCR reactions total RNA extraction and the subsequent first strand cDNA synthesis was performed as described before. cDNAs derived from leaves derived from untransformed banana plants were used as control in these RT-PCR reactions. Assessment of abiotic stress responses of Ubi‑MusaPIP2;6 and Dhn‑MusaPIP2;6 banana lines and evaluation of biochemical and physiological parameters Stress responses of Ubi-MusaPIP2;6 and Dhn-MusaPIP2;6 derived transgenic banana plants were studied by exposing the 3–4 months old greenhouse maintained whole transgenic plants together with equivalent controls to simulated salt stress. To determine response towards salt stress, both set of plants with the controls were irrigated with 250 mM NaCl every alternate day for 10 days followed by recovery from salt stress (for 1.5 months) by irrigation with water at alternate days. For detached leaf assays, uniform sized transgenic and control leaves were treated with 1/10 MS basal medium added with 250 mM NaCl for 7 days such that only their petioles were dipped in the salt supplemented media. All the stress assays performed in triplicates were repeated twice and representative samples were photographed. Malondialdehyde (MDA) equivalents were estimated in the salt stressed tissues to analyse the membrane damage in the transgenic tissues upon salt stress. Continuous excitation plant efficiency analyzer (Hansatech Instruments make Model Handy-Pea) was used to analyze photosynthetic efficiency (Fv/Fm) in the salt stressed leaf samples.

Molecular analysis of transgenic banana plants

Results

Out of a total of twelve and nine putatively transformed lines generated respectively from Ubi-MusaPIP2;6 and Dhn-MusaPIP2;6 constructs, three lines derived from Ubi-MusaPIP2;6 construct and two lines derived from Dhn-MusaPIP2;6

Isolation and sequence analysis of MusaPIP2;6 A novel PIP (Plasma membrane Intrinsic Protein) gene was identified in the banana EST libraries maintained at NCBI

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(Genbank accession no. FL667907). Since this EST was incomplete at the 3′ end, the complete cDNA sequence was deciphered using 3′ RACE (Rapid Amplification of cDNA Ends). Homology searching revealed this PIP gene to be most homologous with OsPIP2;6 among the rice PIPs and therefore it was named as MusaPIP2;6. The coding region of MusaPIP2;6 consisted of 849 nucleotides encoding a protein of 282 amino acids (Fig. 1). MusaPIP2;6 coding sequence was interrupted by three introns in its genomic complement. Sequence alignment of MusaPIP2;6 predicted protein sequence with its best homologs indicated high conservation of these protein sequences across different species which indicated conserved functionality of this AQP in these species (Figs. 2, 3). Expression profiling of MusaPIP2;6 gene in different abiotic stress conditions Semi quantitative RT-PCR performed using cDNA derived from stressed and non-stressed tissues of banana cv. Karibale Monthan plants clearly showed inducibility of MusaPIP2;6 in all the four abiotic stress conditions tested (Fig. 4A). This result revalidated our earlier

Fig. 1  Sequence of the coding region together with the predicted amino acid sequence for MusaPIP2;6 gene

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Plant Mol Biol

identification and selection of EST FL667907 from the banana EST libaray database. Further, the induction in response to these stress stimuli was almost on similar pattern in both leaves and roots indicating the importance of this aquaporin in abiotic stress response pathways in banana plant. Cellular localization of MusaPIP2;6 protein A 227 amino acid domain predicted to form a transmembrane water channel and possessing Asn-Pro-Ala signature motifs which are highly unique in the members of major intrinsic protein superfamily were conserved in MusaPIP2;6 predicted protein sequence. When a plant expression cassette comprising of MusaPIP2;6::GFP fusion cDNA driven by CaMV 35S promoter was transiently transformed into onion peel cells, most of the GFP fluorescence was found to be localized in the plasma membrane of the onion cells thereby confirming the localization of MusaPIP2;6 protein. In pCAMBIA-1302 transformed onion peels, GFP fluorescence was more evenly distributed throughout the whole cell including the nuclei owing to its smaller size (Fig. 4 B, C, D).

Plant Mol Biol

Fig. 2  Alignment of MusaPIP2;6 protein sequence with sequences from Hedychium (AEF32110), Oryza (Q7XLR1), Petunia (AAL49750), Gossypium (ACB42441), Manihot (ACB87734), Jat-

ropha (ABM54183). Note the high homology towards the C-terminal regions of the aligned proteins

Generation and analysis of transgenic banana plants

cocultivation. These embryos later gave rise to secondary embryos after subculture on the fresh medium of the same composition (Fig. 5B, C). The newly formed embryos differentiated radicle and plumule when they were subcultured on embryo germination medium (Fig.  5D, E). These germinating embryos were then induced to form multiple shoots on banana multiplication medium to facilitate generation of clonal copies of single embryonic event. The shoots so obtained were rooted on medium supplemented with NAA. These rooted transgenic plantlets derived from the two binary

Banana cv. Rasthali embryogenic suspension culture cells were transformed using Agrobacteria harboring the two binary vectors respectively with constitutive and inducible MusaPIP2;6 expression cassettes (Fig. 5A). The cocultivated embryogenic cell cultures were aspirated onto glass fibre filters. White colored embryos started originating from the transformed banana cells cultured on banana embryo induction medium supplemented with hygromycin (5 mg l−1) within 4 weeks of

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Plant Mol Biol

Fig. 3  Phylogenetic relationship of MusaPIP2;6 with closely related PIP sequences belonging to different species. The accession numbers of the respective protein sequences used for constructing the phylogenic tree are given alongside. This bootstrapped tree with 1000 replicates was made using ClustalW2 and MEGA 5 tools

vector constructs were hardened and acclimatized in a contained greenhouse (Fig. 5F, G). Three lines out of the twelve putatively transformed lines derived from Ubi-MusaPIP2;6 and two lines out of nine derived from Dhn-MusaPIP2;6 construct were selected based on proficient growth on hygromycin supplemented medium. T-DNA copy numbers were determined in these transgenic lines by Southern blotting (Fig. 6A). DIG-labeled probes directed against the hygromycin phosphotransferase gene coding sequence present in the T-DNA region were hybridised with BamHI restricted genomic DNAs derived from the different transgenic lines. As the

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restriction enzyme BamHI cuts the T-DNA of the overexpression binary vector only once, the number of hybridization bands that appear on the auto radiographs directly correlate with the T-DNA copy number integrated in the genome of these transgenic lines. T-DNA copy numbers ranging from 1 to 5 were determined in the five lines analyzed. The lines analyzed here were derived from independent transformation events as indicated by the different positions and numbers of the hybridization bands detected in southern blot. The five selected transgenic lines derived from the two binary constructs were also analyzed by quantitative

Plant Mol Biol

Fig. 4  Expression profiling of MusaPIP2;6 gene transcript and cellular localization of MusaPIP2;6 in transformed onion peels. A MusaPIP2;6 gene transcript expression in response to different abiotic stress conditions at different time points. Banana Actin gene transcript was amplified along side to demonstrate equivalence of cDNA

content B onion peels transformed with pCAMBIA-1302 showing GFP fluorescence well distributed in the cells. C and D pMusaPIP2;6-1302 transformed onion peels wherein the GFP is seen predominantly in the plasma membranes

real-time RT-PCR to estimate the exact level of overexpression/ inducible expression of MusaPIP2;6 in these transgenic lines (Fig. 6B, C). Musa EF1α gene was used for expression normalization in these RT-PCR reactions (Chen et al. 2011; Podevin et al. 2012). Quantitative real-time RTPCR analysis showed the overexpression of MusaPIP2;6 was 3.67 times (relative to the expression of MusaPIP2;6 in untransformed control) in U1, 20.99 times in U2, 14.75 times in U3. The expression of MusaPIP2;6 in unstressed Dhn-MusaPIP2;6 plants was higher that the unstressed controls showing strong background transcription activity from the MusaDHN-1 promoter. This may be due to the fact that only ~1 kb proximal promoter region upstream of MusaDHN-1 gene has been used as promoter here (Shekhawat et al. 2011a). Under salt stressed conditions, the difference in MusaPIP2;6 expression in untransformed controls

and the Dhn-MusaPIP2;6 plants was 28.84 times (relative to the expression of MusaPIP2;6 in stressed untransformed control) in D1 line and 53.44 times in D2 line. Abiotic stress responses of Ubi‑MusaPIP2;6 and Dhn‑MusaPIP2;6 transgenic lines Transgenic banana plants overexpressing MusaPIP2;6 with either of the two promoters were observed to be phenotypically normal in growth and development stance in in vitro as well as in greenhouse conditions. The transgenic lines were in fact indistinguishable from the untransformed lines in normal growing conditions (Figs. 7A, 8A). When 3–4 months old uniform greenhouse growing plants derived from the two constructs were exposed to salt stress, all the transgenic plants as well as the untransformed

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Plant Mol Biol

Fig. 5  Generation of transgenic banana plants overexpressing MusaPIP2;6 with constitutive or inducible promoter. A Schematic diagram of T-DNA region of binary vector UbiMusaPIP2;6 and Dhn-MusaPIP2;6 designed to overexpress MusaPIP2;6 constitutively or in an inducible fashion in transgenic banana plants. B and C embryo cultures of banana transformed respectively with the two vectors. D and E transformed germinating embryos developing on hygromycin supplemented medium. F and G transgenic hardened plants in greenhouse

controls suffered extensive damage as indicated by severe chlorosis in the leaves and partial wilting (Figs. 7B, 8B). Upon return to normal unstressed optimal physiological conditions, the two groups of transformed plants demonstrated swift recovery in the form of regeneration of new leaves from the growing apex thereby regaining a healthy growing posture within 1.5 months from the start of the recovery period (Figs. 7C, 8C). The untransformed control plants which were treated in a similar manner were not able to fully recover from the salt stress damage. This observation indicated that although the incidence of salt stress conditions was uniformly damaging and deleterious for the transgenic and the untransformed control plants as seen at the end of the stress application period, the difference was in the ability of the transgenic plants expressing MusaPIP2;6 either constitutively or in an inducible manner to recover faster after the stress treatment was lifted. This ability also resonates positively with the present environmental paradigm wherein the continually varying weather patterns make the ability to recover faster from abiotic stress conditions a desirable trait. The recovery potential of both the Ubi-MusaPIP2;6 and Dhn- MusaPIP2;6 plants was underscored by the better health and density of their roots as compared to those of untransformed control plants after the recovery period was over (Figs. 7D, 8D). Better

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root health conferred upon the MusaPIP2;6 overexpressing plants probably enabled these transgenic plants to resume optimal physiological activities much earlier than the control plants. Study of root health under stress conditions is especially relevant in banana which has a shallow adventitious root system and hence is affected by physiological water insufficiency under salt stress almost immediately. The higher expression of MusaPIP2;6 in the roots of the two groups of transgenic plants led to early resumption of water balance in the transformed plants after the salt stress was withdrawn. In detached leaf stress assays, wherein the leaves were directly exposed to simulated salt stress, we observed differential damage to the untransformed and transgenic leaves during the course of stress application. In the control untransformed leaves the chlorosis and browning along the midribs were more prominent (Figs. 7E, 8E). This indicated positive contribution of MusaPIP2;6 in overcoming these salt stress conditions in the transformed leaves. Photosynthetic efficiency estimated as the maximum quantum efficiency of Photosystem II and expressed in the form of a widely accepted physiological parameter Fv/ Fm [ratio of variable fluorescence (Fv) over the maximum fluorescence value (Fm)] is considered to be a sensitive parameter of overall plant health and exposure to any sort

Plant Mol Biol

Fig. 6  Molecular analysis of transformed banana plants. A Southern blot analysis of Ubi-MusaPIP2;6 (U1, U2 and U3) and DhnMusaPIP2;6 (D1 and D2) transformed banana lines. B real-time quantitative RT-PCR analysis of the three selected Ubi-MusaPIP2;6 derived transgenic lines (U1, U2 and U3) for determination of the exact quantum of MusaPIP2;6 overexpression in transgenic banana lines. C real-time quantitative RT-PCR analysis of the two selected

Dhn-MusaPIP2;6 derived transgenic lines (D1 and D2) for determination of the exact quantum of MusaPIP2;6 untreated (white bars) and salt stress inducible (grey bars) expression. All gene expression values have been normalized against Musa EF1α cDNA expression levels. Expression of MusaPIP2;6 in untransformed plants has been assumed to be one for estimating the level of overexpression of MusaPIP2;6 in different transgenic lines. Values are mean ±SE

of biotic and abiotic stress stimuli causes reduction in the capacity for photochemical quenching of energy within Photosystem II thus lowering the Fv/Fm. In our detached leaf salt stress assays, the stressed leaves of untransformed controls showed Fv/Fm ratios significantly lower than what was recorded in the transgenic stressed leaves signifying deficient photosynthetic functions in the untransformed banana leaves as compared to the transgenic leaves overexpressing MusaPIP2;6 (Figs. 7F, 8F). The relatively better performance of the transformed leaves was further established by analyzing the MDA equivalents in leaves. MDA levels were the highest in the untransformed salt stressed leaves in effect showing that significant membrane damage has occurred in untransformed leaves as compared to the transgenic ones (Figs. 7G, 8G).

stimuli (Hachez et al. 2006). Since most of these characterization studies on AQPs have been performed using model plants like Arabidopsis or rice owing to the easy availability of proven experimental procedures, AQPs native to important fruit plants like banana (which is the most important fruit worldwide) have scarcely been studied. This imbalance in the importance and the relative global research priorities can lead to undesirable consequences since such a significant crop needs robust research support to tackle any contingencies that may arise in future. Several studies conducted recently have focused on banana abiotic stress responses and the associated genes such as MpRCI (Feng et al. 2009), MusaWRKY71 (Shekhawat et al. 2011b; Shekhawat and Ganapathi 2013), MpAsr (Liu et al. 2010; Dai et al. 2011), MusaDHN-1 (Shekhawat et al. 2011a), MusaSAP1 (Sreedharan et al. 2012), MusabZIP53 (Shekhawat and Ganapathi 2014). We recently characterized an AQP from banana and showed that when overexpressed using a constitutive and inducible promoter, this AQP provides multiple abiotic stress tolerance in transgenic banana plants (Sreedharan et al. 2012). Another study has recently showed that overexpression of MaPIP1;1 in transgenic Arabidopsis plants improves the drought and salt stress tolerance (Xu et al. 2014). The present report

Discussion AQPs being the most vital water channel proteins in plants, they have been extensively investigated to elucidate the roles they undertake in different abiotic stress responses. Several such studies have concluded that overexpression of specific AQPs results in tolerance to select abiotic stress

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Plant Mol Biol

Fig. 7  Abiotic stress tolerance assays and physiological and biochemical analysis of Ubi-MusaPIP2;6 transgenic lines A untransformed control plants (C) and Ubi-MusaPIP2;6 transgenic plants (U1, U2 and U3) before being subjected to salt stress. B test plants after being exposed to salt stress. C test plants 45 days after being recovered from salt stress in a controlled greenhouse. D roots of control and transgenic Ubi-MusaPIP2;6 lines 45 days after recovery from salt stress. E detached leaves derived from greenhouse maintained control and transgenic plants after exposure to simulated salt stress. F photosynthetic efficiency (measured as Fv/ Fm ratio) of untransformed and Ubi-MusaPIP2;6 transgenic leaves salt stress. G MDA levels in untransformed and UbiMusaPIP2;6 transgenic leaves exposed to salt stress

describes cloning and characterization of another AQP gene from banana. This gene when overexpressed using a constitutive or an inducible promoter imparted improved salt stress tolerance in transgenic banana plants. Transcriptome sequencing of several plant species has indicated that a good number of AQP isoforms are expressed in a plant at any given time. This redundancy makes the functional studies on a single AQP in plants a difficult exercise to undertake especially if the overexpression studies are being conducted in heterologous expression systems. Thus characterisation of AQP genes in their native plants by overexpression using constitutive and stress inducible promoters is the most acceptable option. Expression in native milieu

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allows the protein in question to interact with its original cellular partners thus giving an accurate estimate of its actual roles in different cellular processes. MusaPIP2;6 was identified from banana sequence databases maintained at NCBI using OsPIP2;6 sequence as query. OsPIP2;6 has been reported to be closely allied with low temperature tolerance in rice (Matsumoto et al. 2009). In this study, rice seedlings of the two varieties differing in low temperature tolerance were exposed to 4 °C for 24 h followed by recovery at 25 °C for 24 h. All the PIP isoforms were found to be down regulated at the end of cold stress period but when these seedlings were recovered at 25 °C, OsPIP2;6 was among a few genes which regained

Plant Mol Biol Fig. 8  Abiotic stress tolerance assays and physiological and biochemical analysis of DhnMusaPIP2;6 transgenic lines A untransformed control plants (C) and Dhn-MusaPIP2;6 transgenic plants (D1 and D2) before being subjected to salt stress. B test plants after being exposed to salt stress. C test plants 45 days after being recovered from salt stress in a controlled greenhouse. D roots of control and transgenic Dhn-MusaPIP2;6 lines 45 days after recovery from salt stress. E detached leaves derived from greenhouse maintained control and transgenic plants after exposure to simulated salt stress. F photosynthetic efficiency (measured as Fv/ Fm ratio) of untransformed and Dhn-MusaPIP2;6 transgenic leaves salt stress. G MDA levels in untransformed and DhnMusaPIP2;6 transgenic leaves exposed to salt stress

the pre-cold exposure transcript levels in the cold tolerant variety whereas in the cold sensitive variety its expression decreased even further post recovery. Considering broad functional conservation between rice and banana, we postulated that overexpression of close homologs of OsPIP2;6 in transgenic banana plants using either constitutive or inducible promoter may improve abiotic stress tolerance. MusaPIP2;6 has good similarity with its closest homologs from related species indicating high sequence conservation in these AQP genes. Further, we observed that this aquaporin gene transcript is induced in response to cold,

drought, salt stress and also ABA application. For characterizing its exact roles in abiotic stress responses in banana plant, binary vector expression constructs were designed for the determination of its subcellular location and for its overexpression in transgenic banana plants with constitutive or stress inducible promoters. The GFP fluorescence in MusaPIP2;6::GFP fusion construct transformed onion cells was predominantly localized in the plasma membrane reconfirming its active role in water transport. Both the constitutive and the inducible promoters enabled substantial overexpression of MusaPIP2;6 in transgenic banana plants

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as shown by results of quantitative real time RT-PCR reactions performed using primers which amplified transcripts derived from both the native and the T-DNA copy of this gene. Both groups of transgenic banana plants, that is, UbiMusaPIP2;6 and Dhn-MusaPIP2;6 derived plants showed better endurance to salt stress as indicated by their faster recovery from salt injury and positive biochemical parameters (MDA levels and photosynthetic efficiency—Fv/Fm ratio) of these transgenic plants as compared to untransformed controls. In these transgenic plants, improved cellular water levels resulting presumably from higher AQP numbers reduced damage to cellular membranes (including chloroplastic membranes) under salt stressed conditions. The study of important ecophysiological parameters like root hydraulic resistance and stomatal conductance in these transgenic plants is currently being planned and will be suitably reported in near future. The present study is important as it shows the importance of AQPs in abiotic stress especially the salt stress response in a major plant species like banana. This study along with our earlier investigations (Sreedharan et al. 2013) signifies the pivotal role of AQPs in the stress physiology of banana. Acknowledgments  Authors thank Dr. S P Kale, Head, Nuclear Agriculture and Biotechnology Division, BARC for his continuous support and encouragement.

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