Physiologia Plantarum 153: 355–364. 2015
© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
A cold-induced myo-inositol transporter-like gene confers tolerance to multiple abiotic stresses in transgenic tobacco plants Mame Abdou Nahr Sambe† , Xueying He† , Qinghua Tu† and Zhenfei Guo* State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, South China Agricultural University, Guangzhou 510642, China
Correspondence *Corresponding author, e-mail: [email protected] Received 25 January 2014; revised 30 March 2014 doi:10.1111/ppl.12249
A full length cDNA encoding a myo-inositol transporter-like protein, named as MfINT-like, was cloned from Medicago sativa subsp. falcata (herein falcata), a species with greater cold tolerance than alfalfa (M. sativa subsp. sativa). MfINT-like is located on plasma membranes. MfINT-like transcript was induced 2–4 h after exogenous myo-inositol treatment, 24–96 h with cold, and 96 h by salinity. Given that myo-inositol accumulates higher in falcata after 24 h of cold treatment, myo-inositol is proposed to be involved in cold-induced expression of MfINT-like. Higher levels of myo-inositol was observed in leaves of transgenic tobacco plants overexpressing MfINT-like than the wild-type but not in the roots of plants grown on myo-inositol containing medium, suggesting that transgenic plants had higher myo-inositol transport activity than the wild-type. Transgenic plants survived better to freezing temperature, and had lower ion leakage and higher maximal photochemical efficiency of photosystem II (Fv /Fm ) after chilling treatment. In addition, greater plant fresh weight was observed in transgenic plants as compared with the wild-type when plants were grown under drought or salinity stress. The results suggest that MfINT-like mediated transport of myo-inositol is associated with plant tolerance to abiotic stresses.
Introduction It is a common phenomenon that plants accumulate sugars and other compatible solutes when exposed to drought, salinity and low-temperature stresses. Sugars, such as sucrose, raffinose family oligosaccharides (RFOs) and sugar alcohols, accumulate to maintain cell turgor and to stabilize cell proteins and structures in response to abiotic stresses (Bartels and Sunkar 2005). Accumulation of sugars (such as RFO, galactinol, trehalose †
These authors contributed equally to the work.
and fructan) and sugar alcohols (such as myo-inositol, D-pinitol and D-ononitol) shows a strong correlation to abiotic stress tolerance (Loewus and Murthy 2000, Taji et al. 2002, Bartels and Sunkar 2005). Stress tolerance can be improved by manipulation of genes associated with the metabolism of sugars and sugar alcohols (Miyazaki et al. 2004, Schneider et al. 2008, Zhuo et al. 2013, Tan et al. 2013a). myo-Inositol plays an essential role in cell metabolism and plant growth, and serves as a precursor to a large variety of compounds. myo-Inositol can be converted to galactinol, which in turn is converted to raffinose (Taji et al. 2002). myo-Inositol induces the
Abbreviations – GFP, green fluorescent protein; INT, myo-inositol transporter; MfGolS1, galactinol synthase gene from Medicago falcata; MfINT-like, myo-inositol transporter-like gene; MS, Murashige and Skoog medium; MST, monosaccharide transporter; PCR, polymerase chain reaction; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RFOs, raffinose family of oligosaccharides.
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galactinol synthase gene (GolS) expression and participates in cold-induced MfGolS1 (galactinol synthase gene from Medicago falcata) expression (Zhuo et al. 2013). It is also involved in regulation of programmed cell death (PCD) and basal resistance to pathogens in Arabidopsis (Meng et al. 2009, Chaouch and Noctor 2010, Donahue et al. 2010). Sugars are synthesized in mature source leaves and transported to different sink tissues or organs. The relative amounts of sugars and sugar alcohols that are transported depend on monosaccharide transporters (MSTs) of the major facilitator superfamily (MFS). MST consists of 12 transmembrane domains that form a central pore and shuttles soluble monosaccharides across hydrophobic membranes. Many members of the MST superfamily are capable of transporting more than one species of monosaccharide, although they have higher affinities for certain substrates than for others (Slewinski 2011). Transcription of many MST genes is induced by abiotic stresses such as cold, drought and salinity, correlating with the significant sugar accumulation in plant tissues (Wormit et al. 2006, Yamada et al. 2010). The coordinated increases in MST and sugars allow the altered partitioning of soluble carbohydrates and protect plant cells by stabilizing membranes and conducting osmotic adjustment. myo-Inositol transporter belongs to MST superfamily and plays an important role in inositol uptake and partitioning. An increased transport of myo-inositol from leaves to roots through phloem and simultaneously increased myo-inositol transport from roots to leaves in xylem was observed in salt-stressed ice plant (Mesembryanthemum crystallinum) (Nelson et al. 1999). MITR1 and MITR2 from ice plants (M. crystallinum) are the first cDNAs of putative plant myo-inositol transporters. Yeast (Saccharomyces cerevisiae) cells overexpressing MITR1 could grow on lower concentrations of myo-inositol than control cells (Chauhan et al. 2000). There are four putative myo-inositol transporter genes (INT) in Arabidopsis (Schneider et al. 2006). Three of them, AtINT1, AtINT2 and AtINT4 have been functionally characterized (Schneider et al. 2006, 2007, 2008), whereas AtINT3 is a pseudogene (Schneider et al. 2007). AtINT2 and AtINT4 are plasma membrane-localized proteins transporting myo-inositol and several inositol epimers (Schneider et al. 2006, 2007). AtINT4 is strongly expressed in pollen and phloem companion cells in Arabidopsis with a high affinity for myo-inositol (Schneider et al. 2006), while AtINT2 is weakly expressed in the anther tapetum, vasculature and leaf mesophyll with a lower affinity for myo-inositol than AtINT4 (Schneider et al. 2007). AtINT1 is localized in tonoplast and mediates the efflux of inositol from vacuole (Schneider et al. 2008). 356
Although these transporters have been identified and characterized in Arabidopsis, knowledge on their physiological roles in plants is limited. Studies overexpressing INT genes for improved tolerance to abiotic stresses have not been reported. Alfalfa (Medicago sativa subsp. sativa) is the most important forage legume. Accumulation of sugars associated with cold acclimation has been well documented in alfalfa. Sugar concentrations are consistently higher in cold-tolerant alfalfa cultivars than in cold-sensitive cultivars (Cunningham and Volenec 1998). The tolerant cultivars tend to accumulate RFOs in crown and roots earlier than the sensitive cultivars during cold acclimation under the field conditions (Castonguay et al. 1995, Castonguay and Nadeau 1998, Cunningham et al. 2003). Medicago sativa subsp. falcata has better cold tolerance than alfalfa (Riday and Brummer 2002, Pennycooke et al. 2008), which is associated with higher accumulation of sucrose, myo-inositol, galactinol and RFOs during cold acclimation (Tan et al. 2013a, Zhuo et al. 2013). However, there has been no report on sugar transporters associated with cold tolerance in alfalfa plants. A partial-length cDNA clone encoding a sugar transporter has been obtained in a suppression subtractive hybridization (SSH) cDNA library of falcata responsive to cold (Pang et al. 2009), implying its potential role in cold tolerance of falcata. The objectives of this study were to isolate a full-length cDNA of the sugar transporter gene [myo-inositol transporter-like gene (MfINT-like)] from falcata and to reveal its role in abiotic stress tolerance. The expression of MfINT-like in response to cold and myo-inositol was analyzed. Transgenic tobacco plants overexpressing MfINT-like gene were generated and characterized for tolerance to diverse abiotic stresses.
Materials and methods Plant materials, growth conditions and treatments Plants of falcate, Medicago truncatula, and transgenic and wild type (WT) tobacco (Nicotiana tabacum) were grown in 15-cm diameter plastic pots containing a mixture of peat and perlite (3:1, v/v) in a greenhouse under natural light with temperatures ranging from 20 to 28∘ C for 10–12 weeks, as described previously (Tan et al. 2013a, 2013b, Zhuo et al. 2013). For analysis of gene expression, 10-week-old falcata and M. truncatula plants were exposed to low temperature treatment at 5∘ C for 4 days in a growth chamber with 12-h photoperiod and 200 μmol m−2 s−1 light intensity. For dehydration treatment, the detached leaves from falcata were placed in a hood for gradual dehydration for 12 h; for salt treatment, the potted plants were irrigated daily with 100 ml of 0.25 M NaCl solution for
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4 days (Tan et al. 2013a). For treatment with sugars, the detached falcata leaves were placed in beakers containing 50–300 mM myo-inositol, respectively, under light of 80 μmol photons m−2 s−1 (Zhuo et al. 2013). For myo-inositol transport experiment, sterilized seeds were germinated on Murashige and Skoog (MS) basal salt medium with no vitamin and phytohormones. One-week-old seedlings were transplanted to a new MS medium containing 10 mM myo-inositol (no myo-inositol as a control), and grown for four additional weeks in a growth chamber at 25∘ C with 12-h photoperiod and light intensity of 200 μmol photon m−2 s−1 . For measurement of myo-inositol as affected by cold treatment, 10-week-old falcata plants and tobacco plants were treated by low temperature at 5∘ C for 2 days in a growth chamber as described above, with those in another growth chamber at 25∘ C as a control. The experiments were biologically repeated three times. Isolation of a full-length cDNA of MfINT-likebreak from falcata Leaves (0.1 g) of falcata were harvested at the time points as indicated in Fig. 2A with the cold treatment, and ground in liquid nitrogen. Total RNA was isolated from cold-treated falcata leaves (0.1 g), using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Two micrograms of total RNA was used for reverse transcription with 160 U of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega, Madison, WI) in the presence of oligo (dT)18 in a 20 μl reaction mixture (Zhuo et al. 2013). For amplification of MfINT-like, primers RT79 (5′ -CATGGAAGGAGGTGTACCAGAA-3′ ) and RT80 (5′ TGACACGGTAACTCGGACAAACT-3′ ) were designed after an assembly of expressed sequence tag (EST) sequences using SeqMan (DNASTAR Inc, Madison, WI) based on sequence data in the GenBank. Polymerase chain reaction (PCR) was conducted in a reaction mixture containing the first-strand cDNA as the template, primers RT79 and RT80, and KOD-Plus DNA polymerase (TOYOBO, Osaka, Japan). MfINT-like cDNA and the deduced amino acid sequences were analyzed using DNAMAN software (Lynnon Biosoft, Vaudreuil, Canada).
(Allium cepa) epidermal cells using the PDS-1000 System (Bio-Rad, Hercules, CA) at 1100 psi helium pressure. The expression of the MfINT-like-GFP fusion protein in the onion epidermal cells was observed by confocal laser scanning microscopy (Leica SP, Solms, Germany) after the transformed epidermal cells were incubated on 1/2 MS medium at 22∘ C for 30 h in the dark. The vector p35S-GFP was used as a control for the bombardment. DNA and RNA blot hybridization Genomic DNA was extracted from leaves (1 g) by using the hexadecyltrimethylammonium bromide (CTAB) method (Murray and Thompson 1980). DNA and RNA blot hybridization were conducted according to standard procedures, using [𝛼-32 P] dCTP labeled MfINT-like coding sequence (from nt 637 to 1040 of the cDNA clone) as probes. The hybridization signals were detected using Typhoon Trio (General Electric Company, Fairfield, CT). Real-time quantitative reverse transcription PCR Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed as previously described (Tan et al. 2013b), using the Mini Option Real-Time PCR System (Bio-Rad) according to the manufacturer’s instructions. Primers ZG1755 (5′ -TGGC TTCAATGGCATCTC-3′ ) and ZG1756 (5′ -CAACTCCTAA CATCCACCTC-3′ ) were used for amplification of MfINTlike, while the primers ZG1613 (5′ -ATTCACGAGACCAC CTAC-3′ ) and ZG1614 (5′ -GAGCCACAACCTTAATCTTC -3′ ) were used for amplification of actin as an internal control. The primer specificity was validated by melting profiles before qRT-PCR was conducted. Two biological and three technical replicates were performed in each experiment. Generation of transgenic tobacco plants The MfINT-like coding sequence was cloned into the pBI121 binary vector, under control of the CaMV 35S promoter, to construct an expression plasmid pBIMfINT-like. Transgenic tobacco plants were generated as described previously (Tan et al. 2013b), using Agrobacterium tumefaciens strain EHA105 harboring pBI-MfINT-like.
Subcellular localization of MfINT-like:GFP in onion epidermal cells
Measurement of myo-inositol
The coding sequence of MfINT-like cDNA was fused in frame to the N-terminus of green fluorescent protein (GFP) in the vector p35S-GFP. After the fusion gene plasmid (6 μg) was co-precipitated with 3 mg of gold particles, the particles were used for bombarding onion
For myo-inositol transport experiment, sterilized seeds were germinated for 1 week on MS basal salt medium without phytohormones. The seedlings were then transplanted to a new MS medium containing 10 mM myoinositol, with no myo-inositol as a control, and grown
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for 4 weeks in a growth chamber at 25∘ C with a 12-h photoperiod under light of 200 μmol photon m−2 s−1 . myo-Inositol was extracted from leaves or roots using heated 80% ethanol and measured using Waters high-performance liquid chromatography system (HPLC) as described previously (Tan et al. 2013a). Measurements of freezing and chilling tolerance For freezing treatment, 5-week-old tobacco plants were moved into a growth chamber with temperature decreasing from 25 to −3∘ C linearly within 2 h and maintained for 10 h under light of 700 μmol photon m−2 s−1 . The experiments were performed five times using 20–25 plants each line per replicate. Plant survival rate was calculated at the end of the experiments. For chilling treatment, tobacco plants were treated in a growth chamber at 3∘ C for 3 days with a 12-h photoperiod under light of 700 μmol photon m−2 s−1 . Ion leakage and maximal photochemical efficiency of photosystem II (Fv /Fm ) were measured for five plants of each line (Wan et al. 2009). Measurements of drought and salinity tolerance Drought and salinity stress tolerance was assessed based on biomass in response to drought or salt stress as described previously (Tan et al. 2013a, Zhuo et al. 2013). Sterilized seeds were placed on MS medium for 1 week for germination. The seedlings were transplanted to a fresh MS medium containing 0.1 M NaCl for salinity treatment or 0.1 M mannitol for drought treatment with no supplement as a control. The plants were growing under light of 200 μmol photon m−2 s−1 with 12-h photoperiod. Twenty plants were transplanted in a Petri dish (15-cm in diameter). Each treatment consisted of three dishes as replicates. Fresh weight of shoots and roots was measured at the sixth week after the treatments. Statistical analysis All data from three biological replicates were subjected to analysis of variance (ANOVA) according to the model for completely randomized design using an SPSS program (SPSS Inc., Chicago, IL). Significance of difference was evaluated by Duncan’s test at 0.05 probability level.
Results MfINT-like sequence analysis A 2007-bp length cDNA sequence (GenBank accession number KC986375) was cloned from falcata leaves by RT-PCR, which contains an open reading frame (ORF) of 1719 bp, encoding a protein of 572 amino acids 358
(Fig. 1A). The deduced polypeptide of 62 kDa has an isoelectric point (pI) of 8.34. Sequence BLAST analysis showed that it was highly homologous to Arabidopsis inositol transporters, but not sucrose transporters. Thus, we named it MfINT-like. A phylogenetic tree of MfINT-like and other INTs from Arabidopsis and M. truncatula is given in Fig. S1, Supporting information. MfINT-like was most homologous (97%) in AA sequence to a hypothetical protein (MTR_2g048720) in M. truncatula, and 74 and 65% identical to a carbohydrate transporter (MTR_2g049020) and inositol transporter (MTR_5g077580) in M. truncatula, respectively. In addition, MfINT-like was 68% identical to AtINT2 (At1g30220), compared with 47, 54 and 58% to AtINT1 (At2g43330), AtINT3 (At2g35740) and AtINT4 (At4g16480), respectively (Fig. S1). Twelve transmembrane helices were predicted according to the deduced protein sequence of MfINT-like protein by using two membrane protein topology servers, TMHMM 2.0 (http://www.cbs.dtu.dk/ services/TMHMM) and HMMTOP (http://www.enzim.hu/ hmmtop), indicating that MfINT-like belonged to MST superfamily. The 12 transmembrane regions are indicated in Fig. 1A. There is one consensus sequence for N-glycosylation (Asn376-Asn377-Thr378) in MfINT-like. MfINT-like localization was analyzed using CELLO v.2.5 (subCELlular LOcalization predictor, http://cello.life.nctu.edu.tw/) and cross checked with PSORT prediction software (http://wolfpsort.org/). The results showed that MfINT-like was predicted to be localized in plasma membrane (data not shown). For verification of this prediction, transient expression of MfINT-like-GFP fusion protein was performed in onion (A. cepa) epidermal cells. Compared with the whole cellular distribution of GFP, MfINT-like-GFP fusion protein fluorescence was observed at the perimeter of the transformed cells (Fig. 1B), indicating that MfINT-like is localized on the plasma membrane. Closely related homologs of MfINT-like exist in the genome of falcata. Three hybridization signals were observed by DNA blot hybridization after the genomic DNA of falcata was digested by EcoRV or XbaI (Fig. 1C). Expression of MfINT-like in response to abiotic stress and myo-inositol Transcript of MfINT-like was initially induced at 24 h, and reached the maximum level at 48 h after cold treatment (Fig. 2A). In contrast, the transcript levels of three homologs in M. truncatula that are most similar to MfINT-like were not affected by cold treatment (Fig. S2). MfINT-like was not responsive to dehydration until leaf water content decreased to 45% (data not
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Fig. 1. Amino acid sequence (A), subcellular localization of MfINT-like (B) and DNA blot hybridization analysis (C). The predicted transmembrane regions in sequence are underlined, and the putative N-glycosylation sequence (Asn376 -Asn377 -Thr378 ) is boxed (A). MfINT-like-GFP fusion protein and the control (GFP only) were transient expressed in onion epidermal cells to show the subcellular localization (B). For DNA hybridization, the falcata genomic DNA (15 μg) was digested with EcoRV or XbaI, respectively.
shown), but it was induced at 4 days after salinity treatment (Fig. 2B). MfINT-like was also induced by treatment with myo-inositol. MfINT-like transcript increased significantly at 2 and 4 h after treatment with myo-inositol (Fig. 2C); myo-inositol had a dose-effect on the induced MfINT-like transcript level (Fig. 2D). In addition, the data showed that expression of MfINT-like was more rapidly induced by myo-inositol than by cold or salinity. Analysis of transgenic tobacco plants DNA hybridization revealed that MfINT-like was integrated into the genomes of transgenic tobacco plants, likely with two transgene copies in lines S1 and S3 and one copy in line S6 (Fig. 3A). In addition, transgenic plants expressed MfINT-like gene, with more abundant transcript in line S1 than in lines S3 and S6 (Fig. 3B). In order to measure transporter activity of MfINT-like, transgenic tobacco and the wild-type plants were grown on medium without or with myo-inositol. myo-Inositol levels in roots were higher (13–15%) in the transgenic tobacco lines S1 and S3 than in the wild-type when plants were growing on the medium without myo-inositol (Fig. 3C), but showed no significant difference in shoot (Fig. 3D). After growing on the medium containing 10 mM myo-inositol, myo-inositol levels in roots were significantly increased in both transgenic plants and the wild-type (Fig. 3C); however, the increase rate was similar in transgenic plants (20–38%) and the wild-type (25%) (Fig. 3E). Compared with the
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wild-type (Fig. 3D), myo-inositol levels in shoot were significantly increased in transgenic plants by 30–67% (Fig. 3D, E). Analysis of abiotic stress tolerance in transgenic plants Most of the wild-type plants died after freezing treatment, while transgenic plants were little affected by freezing treatment (Fig. 4A). Compared with the 10% survival rate of the wild-type, transgenic plants had 77–83% survival rate, with no significant difference among transgenic plants (Fig. 4B). Similar ion leakage or Fv /Fm was observed in all plants under control conditions. Chilling treatment resulted in increased ion leakage and decreased Fv /Fm in all plants, while lower levels of ion leakage and higher levels of Fv /Fm were observed in transgenic plants than in the wild-type (Fig. 5A, B). For example, Fv /Fm decreased by 18–24% in the transgenic lines, but reduced by 38% in the wild-type (Fig. 5B). Drought and salinity tolerance were also assessed. The plants cultured on medium containing 0.1 M mannitol (drought treatment) or 0.1 M NaCl (salinity treatment) showed significant difference in fresh weight of whole plant. Drought decreased fresh weight by 2–11% and salinity stress reduced fresh weight by 33–40% for the transgenic plants (Fig. 5C), while fresh weight was reduced by 46 or 59% in the wild-type exposed to drought or salinity stress, respectively (Fig. 5C). 359
Fig. 2. MfINT-like transcripts as affected by cold (A), salinity (B) and myo-inositol (C, D). Plants were exposed to 5∘ C in a growth chamber for cold treatment (A), or irrigated with 0.15 M NaCl solution for salinity treatment (B). Leaflets were excised and placed in 200 mM myo-inositol for 24 h (C), or in different concentrations of myo-inositol solutions for 4 h (D). The expression levels were determined by qRT-PCR and normalized to actin expression. The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
Fig. 3. Analysis of transgenic plants (lines S1, S3 and S6) in comparison to the wild-type control (W); 15 μg of genomic DNA digested with EcoRI and 20 μg total RNA was used for DNA (A) or RNA blot hybridization (B), respectively. myo-Inositol concentrations in roots (C) and leaves (D) were determined after plants were grown on MS medium containing 10 mM myo-inositol or without myo-inositol as control for 4 weeks. The increased rate of myo-inositol (E) indicates the relatively increased amounts in MS medium containing myo-inositol compared with those in myo-inositol-free medium. The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
Analysis of myo-inositol levels in responsebreak to cold treatment myo-Inositol contents as affected by cold treatment were measured in transgenic and wild-type tobacco plants as well as in falcata plants. Ten-fold higher levels of myo-inositol were detected in leaves than in roots of both 360
tobacco and falcata plants; however, no significant differences in myo-inositol content were observed between transgenic plants and the wild-type under control conditions. While significant accumulation of myo-inositol was observed in falcata leaves and roots after cold treatment, no significant change of myo-inositol was
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Fig. 4. Analysis of freezing tolerance of transgenic lines (S1, S3 and S6) in comparison to the wild-type (W). Photos were taken after plants were treated by freezing at −3∘ C for 10 h, followed by 2-day recovery at room temperature (A) and the survival rates were calculated (B). The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
Fig. 5. Analysis of tolerance to chilling (A, B), drought and salinity (C) in transgenic tobacco plants (S1, S3 and S6) in comparison to the wild-type (W). Ion leakage (A) and Fv /Fm (B) were determined after plants were treated at 3∘ C for 3 days. With those growing on MS medium at 25∘ C as control, plant seedlings were grown for 6 weeks on MS medium containing 0.1 M mannitol (drought stress) or 0.1 M NaCl (salt stress) at 25∘ C, and the whole plant fresh weight was measured (C). The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
observed in the wild-type tobacco plants. Nevertheless, myo-inositol was significantly increased in leaves and roots of the transgenic tobacco plants after cold treatment (Fig. 6). There was a 1.9-fold and 95% increase in leaves and roots of falcata, and 1.2- to 1.6-fold and 47–66% increase in leaves and roots of transgenic tobacco plants, respectively.
Discussion A putative INT encoding gene (MfINT-like) belonging to the MST superfamily was isolated from leaves of falcata in this study. Among annotated Arabidopsis proteins, MfINT-like has the highest AA sequence identity with
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AtINT2 (68%), a plasma membrane-localized protein mediating myo-inositol efflux (Schneider et al. 2007), as compared with AtINT1 (a tonoplast-localized protein) and AtINT4 (another plasma membrane-localized protein) (Schneider et al. 2006, 2008). MfINT-like was predicted by sequence analysis and confirmed to be located in plasma membrane by transient expression of MfINT-like-GFP fusion protein in onion epidermal cells. Three MfINT-like hybridization signals were observed in the genomes of falcata, indicating that isoforms of MfINT-like gene exist in falcata. Transport of sugars is an important phenomenon in plants. Sugars and derivatives including myo-inositol are usually synthesized in source organ like mature 361
Fig. 6. myo-Inositol concentrations in transgenic plants (S1, S3 and S6) in comparison to the wild-type tobacco (W) and falcata (MF) as affected by chilling treatment. Fresh weight of leaves (A) or roots (B) was used to calculate the concentration. The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
leaves, and transported to sink organ such as young leaves and roots. Accumulation of total non-structure carbohydrates in roots and crowns is associated with overwintering and subsequent spring growth of alfalfa (Cunningham and Volenec 1998). More RFOs are accumulated in winter-hardy cultivars than in winter-sensitive cultivars (Cunningham et al. 2003). Medicago sativa subsp. falcata accumulates more sucrose, myo-inositol, galactinol and RFOs than M. sativa and M. truncatula (Zhang et al. 2011, Tan et al. 2013a, Zhuo et al. 2013). The increased myo-inositol may function as a precursor of RFO synthesis and compatible solute for protection against abiotic stress (Loewus and Murthy 2000, Loewus 2006). Transport of myo-inositol is important in salt tolerance in ice plants; its level goes up by 10-fold in xylem under salinity (Nelson et al. 1999). MfINT-like transcript was induced 2–4 h after treatment with myo-inositol, consistent with a report that all seven myo-inositol transporter genes in Cryptococcus neoformans are induced by myo-inositol (Xue et al. 2010). Moreover, MfINT-like transcript was induced at 24 h after cold treatment and maintained high up to 96 h, which was slower than the induction by myo-inositol treatment, but well-coordinated with the accumulation of myo-inositol during cold treatment (Tan et al. 2013a). myo-Inositol concentration is initially increased at 24 h and maintained high in falcata leaves during the cold treatment, which results from the induced expression of 362
MfMIPS1 by cold (Tan et al. 2013a). The coordination between cold induced myo-inositol accumulation and MfINT-like transcription suggested that myo-inositol is involved in cold induced MfINT-like expression. Combined with the previous reports (Tan et al. 2013a, Zhuo et al. 2013), our results revealed an interesting chain reaction: cold causes myo-inositol accumulation, which, in turn, induces expression of MfINT-like to promote its own transportation, and expression of MfGolS1 (Zhuo et al. 2013) for its conversion to the downstream metabolite RFOs. This reflects a sophisticated control of metabolite concentrations in plant cells for adaption to the changing environments. Association of MfINT-like with abiotic stress tolerance was revealed in this study using transgenic tobacco plants overexpressing MfINT-like. Physiological assessments indicated that transgenic plants had enhanced tolerance to multiple abiotic stresses including freezing, chilling, drought and salinity. This is the first observation that overexpressing INT resulted in elevated abiotic stress tolerance. Compared with the plants growing on myo-inositol-free medium, more myo-inositol was observed in roots of transgenic plants and the wild-type after growing on the medium containing myo-inositol, with no significant difference in the increase rate between the two types of plants. This result indicated the similar capacity for uptake of myo-inositol from medium among plant lines. Moreover, an enhanced myo-inositol concentration was only observed in shoot of transgenic plants growing on the medium containing myo-inositol but not in the wild-type, indicating an enhanced myo-inositol transport in transgenic plants as a result of overexpression of MfINT-like. In addition, more myo-inositol was observed in roots of the transgenic lines S1 and S3 than in the wild-type roots when plants were grown on myo-inositol-free medium. This observation indicates that an enhanced myo-inositol transport might have a positive feed-back effect on myo-inositol biosynthesis. The speculation was supported by the observation that enhanced myo-inositol was accumulated in the cold-treated transgenic tobacco plants as compared with the wild-type. myo-Inositol accumulates in many plants in response to cold, drought and salinity stresses through an induced expression of MIPS expression (Nelson et al. 1998, Majee et al. 2004, Tan et al. 2013a). Our results revealed that MfINT-like also confers abiotic stress tolerance in plants by enhancing myo-inositol transport and accumulation. In conclusion, a MfINT-like in falcata was identified. Overexpression of MfINT-like leads to improved tolerance to multiple abiotic stresses through enhanced accumulation of myo-inositol.
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Acknowledgements – This work was supported by the National Basic Research Program of China (2014CB1 38701) and the Natural Science Foundation of China (30830081).
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Miyazaki S, Rice M, Quigley F, Bohnert HJ (2004) Expression of plant inositol transporters in yeast. Plant Sci 166: 245–252 Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight DNA. Nucleic Acids Res 8: 4321–4325 Nelson DE, Rammesmayer G, Bohnert HJ (1998) Regulation of cell-specific inositol metabolism and transport in plant salinity tolerance. Plant Cell 10: 753–764 Nelson DE, Koukoumanos M, Bohnert HJ (1999) myo-Inositol-dependent sodium uptake in ice plant. Plant Physiol 119: 165–172 Pang C, Wang C, Chen H, Guo Z, Li C (2009) Transcript profiling of cold responsive genes in Medicago falcata. In: Yamada T, Spangenberg G (eds) Molecular Breeding of Forage and Turf. Springer, New York, pp 141–149 Pennycooke JC, Cheng H, Stockinger EJ (2008) Comparative genomic sequence and expression analyses of Medicago truncatula and alfalfa subspecies falcata COLD-ACCLIMATION-SPECIFIC genes. Plant Physiol 146: 1242–1254 Riday H, Brummer EC (2002) Forage yield heterosis in alfalfa. Crop Sci 42: 716–723 Schneider S, Schneidereit A, Konrad KR, Hajirezaei MR, Gramann M, Hedrich R, Sauer N (2006) Arabidopsis inositol transporter 4 mediates high-affinity H+ symport of myo-inositol across the plasma membrane. Plant Physiol 141: 565–577 Schneider S, Schneidereit A, Udvardi P, Hammes U, Gramann M, Dietrich P, Sauer N (2007) Arabidopsis inositol transporter2 mediates H+ symport of different inositol epimers and derivatives across the plasma membrane. Plant Physiol 145: 1395–1407 Schneider S, Beyhl D, Hedrich R, Sauer N (2008) Functional and physiological characterization of Arabidopsis inositol transporter1, a novel tonoplast-localized transporter for myo-inositol. Plant Cell 20: 1073–1087 Slewinski TL (2011) Diverse functional roles of monosaccharide transporters and their homologs in vascular plants: a physiological perspective. Mol Plant 4: 641–662 Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K (2002) Important roles of drought- and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana. Plant J 29: 417–426 Tan J, Wang C, Xiang B, Han R, Guo Z (2013a) Hydrogen peroxide and nitric oxide mediated cold- and dehydration-induced myo-inositol phosphate synthase that confers multiple resistances to abiotic stresses. Plant Cell Environ 36: 288–299 Tan J, Zhuo C, Guo Z (2013b) Nitric oxide mediates coldand dehydration-induced expression of a novel
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MfHyPRP that confers tolerance to abiotic stress. Physiol Plant 149: 310–320 Wan X, Tan J, Lu S, Lin C, Hu Y, Guo Z (2009) The increased tolerance to oxidative stress in transgenic tobaccos expressing a wheat oxalate oxidase gene via induction of antioxidant enzymes is mediated by H2 O2 . Physiol Plant 136: 30–44 Wormit A, Trentmann O, Feifer I, Lohr C, Tjaden J, Meyer S, Schmidt U, Martinoia E, Neuhaus HE (2006) Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport. Plant Cell 18: 3476–3490 Xue C, Liu T, Chen L, Li W, Liu I, Kronstad JW, Seyfang A, Heitman J (2010) Role of an expanded inositol transporter repertoire in Cryptococcus neoformans sexual reproduction and virulence. mBio 1: e00084–10 Yamada K, Osakabe Y, Mizoi J, Nakashima K, Fujita Y, Shinozaki K, Yamaguichi-Shinozaki K (2010) Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides. J Biol Chem 285: 1138–1146 Zhang L-L, Zhao M-G, Tian Q-Y, Zhang W-H (2011) Comparative studies on tolerance of Medicago
truncatula and Medicago falcata to freezing. Planta 234: 445–457 Zhuo C, Wang T, Lu S, Zhao Y, Li X, Guo Z (2013) A cold responsive galactinol synthase gene from Medicago falcata (MfGolS1) is induced by myo-inositol and confers multiple tolerances to abiotic stresses. Physiol Plant 149: 67–78
Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Phylogenetic analysis of MfINT-like in falcata with homologous INTs or proteins in Arabidopsis and Medicago truncatula. Fig. S2. Effect of cold on transcript levels of MTR_ 2g048720, MTR_5g077580 and MTR_2g049020 in Medicago truncatula. Plants were exposed to 5∘ C in growth chamber as cold treatment. The expression levels were normalized to that of actin using qRT-PCR. The same letter above a column indicates no significant difference by Duncan’s test at P < 0.05.
Edited by M. Uemura
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© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317
A cold-induced myo-inositol transporter-like gene confers tolerance to multiple abiotic stresses in transgenic tobacco plants Mame Abdou Nahr Sambe† , Xueying He† , Qinghua Tu† and Zhenfei Guo* State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, South China Agricultural University, Guangzhou 510642, China
Correspondence *Corresponding author, e-mail: [email protected] Received 25 January 2014; revised 30 March 2014 doi:10.1111/ppl.12249
A full length cDNA encoding a myo-inositol transporter-like protein, named as MfINT-like, was cloned from Medicago sativa subsp. falcata (herein falcata), a species with greater cold tolerance than alfalfa (M. sativa subsp. sativa). MfINT-like is located on plasma membranes. MfINT-like transcript was induced 2–4 h after exogenous myo-inositol treatment, 24–96 h with cold, and 96 h by salinity. Given that myo-inositol accumulates higher in falcata after 24 h of cold treatment, myo-inositol is proposed to be involved in cold-induced expression of MfINT-like. Higher levels of myo-inositol was observed in leaves of transgenic tobacco plants overexpressing MfINT-like than the wild-type but not in the roots of plants grown on myo-inositol containing medium, suggesting that transgenic plants had higher myo-inositol transport activity than the wild-type. Transgenic plants survived better to freezing temperature, and had lower ion leakage and higher maximal photochemical efficiency of photosystem II (Fv /Fm ) after chilling treatment. In addition, greater plant fresh weight was observed in transgenic plants as compared with the wild-type when plants were grown under drought or salinity stress. The results suggest that MfINT-like mediated transport of myo-inositol is associated with plant tolerance to abiotic stresses.
Introduction It is a common phenomenon that plants accumulate sugars and other compatible solutes when exposed to drought, salinity and low-temperature stresses. Sugars, such as sucrose, raffinose family oligosaccharides (RFOs) and sugar alcohols, accumulate to maintain cell turgor and to stabilize cell proteins and structures in response to abiotic stresses (Bartels and Sunkar 2005). Accumulation of sugars (such as RFO, galactinol, trehalose †
These authors contributed equally to the work.
and fructan) and sugar alcohols (such as myo-inositol, D-pinitol and D-ononitol) shows a strong correlation to abiotic stress tolerance (Loewus and Murthy 2000, Taji et al. 2002, Bartels and Sunkar 2005). Stress tolerance can be improved by manipulation of genes associated with the metabolism of sugars and sugar alcohols (Miyazaki et al. 2004, Schneider et al. 2008, Zhuo et al. 2013, Tan et al. 2013a). myo-Inositol plays an essential role in cell metabolism and plant growth, and serves as a precursor to a large variety of compounds. myo-Inositol can be converted to galactinol, which in turn is converted to raffinose (Taji et al. 2002). myo-Inositol induces the
Abbreviations – GFP, green fluorescent protein; INT, myo-inositol transporter; MfGolS1, galactinol synthase gene from Medicago falcata; MfINT-like, myo-inositol transporter-like gene; MS, Murashige and Skoog medium; MST, monosaccharide transporter; PCR, polymerase chain reaction; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RFOs, raffinose family of oligosaccharides.
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galactinol synthase gene (GolS) expression and participates in cold-induced MfGolS1 (galactinol synthase gene from Medicago falcata) expression (Zhuo et al. 2013). It is also involved in regulation of programmed cell death (PCD) and basal resistance to pathogens in Arabidopsis (Meng et al. 2009, Chaouch and Noctor 2010, Donahue et al. 2010). Sugars are synthesized in mature source leaves and transported to different sink tissues or organs. The relative amounts of sugars and sugar alcohols that are transported depend on monosaccharide transporters (MSTs) of the major facilitator superfamily (MFS). MST consists of 12 transmembrane domains that form a central pore and shuttles soluble monosaccharides across hydrophobic membranes. Many members of the MST superfamily are capable of transporting more than one species of monosaccharide, although they have higher affinities for certain substrates than for others (Slewinski 2011). Transcription of many MST genes is induced by abiotic stresses such as cold, drought and salinity, correlating with the significant sugar accumulation in plant tissues (Wormit et al. 2006, Yamada et al. 2010). The coordinated increases in MST and sugars allow the altered partitioning of soluble carbohydrates and protect plant cells by stabilizing membranes and conducting osmotic adjustment. myo-Inositol transporter belongs to MST superfamily and plays an important role in inositol uptake and partitioning. An increased transport of myo-inositol from leaves to roots through phloem and simultaneously increased myo-inositol transport from roots to leaves in xylem was observed in salt-stressed ice plant (Mesembryanthemum crystallinum) (Nelson et al. 1999). MITR1 and MITR2 from ice plants (M. crystallinum) are the first cDNAs of putative plant myo-inositol transporters. Yeast (Saccharomyces cerevisiae) cells overexpressing MITR1 could grow on lower concentrations of myo-inositol than control cells (Chauhan et al. 2000). There are four putative myo-inositol transporter genes (INT) in Arabidopsis (Schneider et al. 2006). Three of them, AtINT1, AtINT2 and AtINT4 have been functionally characterized (Schneider et al. 2006, 2007, 2008), whereas AtINT3 is a pseudogene (Schneider et al. 2007). AtINT2 and AtINT4 are plasma membrane-localized proteins transporting myo-inositol and several inositol epimers (Schneider et al. 2006, 2007). AtINT4 is strongly expressed in pollen and phloem companion cells in Arabidopsis with a high affinity for myo-inositol (Schneider et al. 2006), while AtINT2 is weakly expressed in the anther tapetum, vasculature and leaf mesophyll with a lower affinity for myo-inositol than AtINT4 (Schneider et al. 2007). AtINT1 is localized in tonoplast and mediates the efflux of inositol from vacuole (Schneider et al. 2008). 356
Although these transporters have been identified and characterized in Arabidopsis, knowledge on their physiological roles in plants is limited. Studies overexpressing INT genes for improved tolerance to abiotic stresses have not been reported. Alfalfa (Medicago sativa subsp. sativa) is the most important forage legume. Accumulation of sugars associated with cold acclimation has been well documented in alfalfa. Sugar concentrations are consistently higher in cold-tolerant alfalfa cultivars than in cold-sensitive cultivars (Cunningham and Volenec 1998). The tolerant cultivars tend to accumulate RFOs in crown and roots earlier than the sensitive cultivars during cold acclimation under the field conditions (Castonguay et al. 1995, Castonguay and Nadeau 1998, Cunningham et al. 2003). Medicago sativa subsp. falcata has better cold tolerance than alfalfa (Riday and Brummer 2002, Pennycooke et al. 2008), which is associated with higher accumulation of sucrose, myo-inositol, galactinol and RFOs during cold acclimation (Tan et al. 2013a, Zhuo et al. 2013). However, there has been no report on sugar transporters associated with cold tolerance in alfalfa plants. A partial-length cDNA clone encoding a sugar transporter has been obtained in a suppression subtractive hybridization (SSH) cDNA library of falcata responsive to cold (Pang et al. 2009), implying its potential role in cold tolerance of falcata. The objectives of this study were to isolate a full-length cDNA of the sugar transporter gene [myo-inositol transporter-like gene (MfINT-like)] from falcata and to reveal its role in abiotic stress tolerance. The expression of MfINT-like in response to cold and myo-inositol was analyzed. Transgenic tobacco plants overexpressing MfINT-like gene were generated and characterized for tolerance to diverse abiotic stresses.
Materials and methods Plant materials, growth conditions and treatments Plants of falcate, Medicago truncatula, and transgenic and wild type (WT) tobacco (Nicotiana tabacum) were grown in 15-cm diameter plastic pots containing a mixture of peat and perlite (3:1, v/v) in a greenhouse under natural light with temperatures ranging from 20 to 28∘ C for 10–12 weeks, as described previously (Tan et al. 2013a, 2013b, Zhuo et al. 2013). For analysis of gene expression, 10-week-old falcata and M. truncatula plants were exposed to low temperature treatment at 5∘ C for 4 days in a growth chamber with 12-h photoperiod and 200 μmol m−2 s−1 light intensity. For dehydration treatment, the detached leaves from falcata were placed in a hood for gradual dehydration for 12 h; for salt treatment, the potted plants were irrigated daily with 100 ml of 0.25 M NaCl solution for
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4 days (Tan et al. 2013a). For treatment with sugars, the detached falcata leaves were placed in beakers containing 50–300 mM myo-inositol, respectively, under light of 80 μmol photons m−2 s−1 (Zhuo et al. 2013). For myo-inositol transport experiment, sterilized seeds were germinated on Murashige and Skoog (MS) basal salt medium with no vitamin and phytohormones. One-week-old seedlings were transplanted to a new MS medium containing 10 mM myo-inositol (no myo-inositol as a control), and grown for four additional weeks in a growth chamber at 25∘ C with 12-h photoperiod and light intensity of 200 μmol photon m−2 s−1 . For measurement of myo-inositol as affected by cold treatment, 10-week-old falcata plants and tobacco plants were treated by low temperature at 5∘ C for 2 days in a growth chamber as described above, with those in another growth chamber at 25∘ C as a control. The experiments were biologically repeated three times. Isolation of a full-length cDNA of MfINT-likebreak from falcata Leaves (0.1 g) of falcata were harvested at the time points as indicated in Fig. 2A with the cold treatment, and ground in liquid nitrogen. Total RNA was isolated from cold-treated falcata leaves (0.1 g), using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Two micrograms of total RNA was used for reverse transcription with 160 U of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega, Madison, WI) in the presence of oligo (dT)18 in a 20 μl reaction mixture (Zhuo et al. 2013). For amplification of MfINT-like, primers RT79 (5′ -CATGGAAGGAGGTGTACCAGAA-3′ ) and RT80 (5′ TGACACGGTAACTCGGACAAACT-3′ ) were designed after an assembly of expressed sequence tag (EST) sequences using SeqMan (DNASTAR Inc, Madison, WI) based on sequence data in the GenBank. Polymerase chain reaction (PCR) was conducted in a reaction mixture containing the first-strand cDNA as the template, primers RT79 and RT80, and KOD-Plus DNA polymerase (TOYOBO, Osaka, Japan). MfINT-like cDNA and the deduced amino acid sequences were analyzed using DNAMAN software (Lynnon Biosoft, Vaudreuil, Canada).
(Allium cepa) epidermal cells using the PDS-1000 System (Bio-Rad, Hercules, CA) at 1100 psi helium pressure. The expression of the MfINT-like-GFP fusion protein in the onion epidermal cells was observed by confocal laser scanning microscopy (Leica SP, Solms, Germany) after the transformed epidermal cells were incubated on 1/2 MS medium at 22∘ C for 30 h in the dark. The vector p35S-GFP was used as a control for the bombardment. DNA and RNA blot hybridization Genomic DNA was extracted from leaves (1 g) by using the hexadecyltrimethylammonium bromide (CTAB) method (Murray and Thompson 1980). DNA and RNA blot hybridization were conducted according to standard procedures, using [𝛼-32 P] dCTP labeled MfINT-like coding sequence (from nt 637 to 1040 of the cDNA clone) as probes. The hybridization signals were detected using Typhoon Trio (General Electric Company, Fairfield, CT). Real-time quantitative reverse transcription PCR Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed as previously described (Tan et al. 2013b), using the Mini Option Real-Time PCR System (Bio-Rad) according to the manufacturer’s instructions. Primers ZG1755 (5′ -TGGC TTCAATGGCATCTC-3′ ) and ZG1756 (5′ -CAACTCCTAA CATCCACCTC-3′ ) were used for amplification of MfINTlike, while the primers ZG1613 (5′ -ATTCACGAGACCAC CTAC-3′ ) and ZG1614 (5′ -GAGCCACAACCTTAATCTTC -3′ ) were used for amplification of actin as an internal control. The primer specificity was validated by melting profiles before qRT-PCR was conducted. Two biological and three technical replicates were performed in each experiment. Generation of transgenic tobacco plants The MfINT-like coding sequence was cloned into the pBI121 binary vector, under control of the CaMV 35S promoter, to construct an expression plasmid pBIMfINT-like. Transgenic tobacco plants were generated as described previously (Tan et al. 2013b), using Agrobacterium tumefaciens strain EHA105 harboring pBI-MfINT-like.
Subcellular localization of MfINT-like:GFP in onion epidermal cells
Measurement of myo-inositol
The coding sequence of MfINT-like cDNA was fused in frame to the N-terminus of green fluorescent protein (GFP) in the vector p35S-GFP. After the fusion gene plasmid (6 μg) was co-precipitated with 3 mg of gold particles, the particles were used for bombarding onion
For myo-inositol transport experiment, sterilized seeds were germinated for 1 week on MS basal salt medium without phytohormones. The seedlings were then transplanted to a new MS medium containing 10 mM myoinositol, with no myo-inositol as a control, and grown
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for 4 weeks in a growth chamber at 25∘ C with a 12-h photoperiod under light of 200 μmol photon m−2 s−1 . myo-Inositol was extracted from leaves or roots using heated 80% ethanol and measured using Waters high-performance liquid chromatography system (HPLC) as described previously (Tan et al. 2013a). Measurements of freezing and chilling tolerance For freezing treatment, 5-week-old tobacco plants were moved into a growth chamber with temperature decreasing from 25 to −3∘ C linearly within 2 h and maintained for 10 h under light of 700 μmol photon m−2 s−1 . The experiments were performed five times using 20–25 plants each line per replicate. Plant survival rate was calculated at the end of the experiments. For chilling treatment, tobacco plants were treated in a growth chamber at 3∘ C for 3 days with a 12-h photoperiod under light of 700 μmol photon m−2 s−1 . Ion leakage and maximal photochemical efficiency of photosystem II (Fv /Fm ) were measured for five plants of each line (Wan et al. 2009). Measurements of drought and salinity tolerance Drought and salinity stress tolerance was assessed based on biomass in response to drought or salt stress as described previously (Tan et al. 2013a, Zhuo et al. 2013). Sterilized seeds were placed on MS medium for 1 week for germination. The seedlings were transplanted to a fresh MS medium containing 0.1 M NaCl for salinity treatment or 0.1 M mannitol for drought treatment with no supplement as a control. The plants were growing under light of 200 μmol photon m−2 s−1 with 12-h photoperiod. Twenty plants were transplanted in a Petri dish (15-cm in diameter). Each treatment consisted of three dishes as replicates. Fresh weight of shoots and roots was measured at the sixth week after the treatments. Statistical analysis All data from three biological replicates were subjected to analysis of variance (ANOVA) according to the model for completely randomized design using an SPSS program (SPSS Inc., Chicago, IL). Significance of difference was evaluated by Duncan’s test at 0.05 probability level.
Results MfINT-like sequence analysis A 2007-bp length cDNA sequence (GenBank accession number KC986375) was cloned from falcata leaves by RT-PCR, which contains an open reading frame (ORF) of 1719 bp, encoding a protein of 572 amino acids 358
(Fig. 1A). The deduced polypeptide of 62 kDa has an isoelectric point (pI) of 8.34. Sequence BLAST analysis showed that it was highly homologous to Arabidopsis inositol transporters, but not sucrose transporters. Thus, we named it MfINT-like. A phylogenetic tree of MfINT-like and other INTs from Arabidopsis and M. truncatula is given in Fig. S1, Supporting information. MfINT-like was most homologous (97%) in AA sequence to a hypothetical protein (MTR_2g048720) in M. truncatula, and 74 and 65% identical to a carbohydrate transporter (MTR_2g049020) and inositol transporter (MTR_5g077580) in M. truncatula, respectively. In addition, MfINT-like was 68% identical to AtINT2 (At1g30220), compared with 47, 54 and 58% to AtINT1 (At2g43330), AtINT3 (At2g35740) and AtINT4 (At4g16480), respectively (Fig. S1). Twelve transmembrane helices were predicted according to the deduced protein sequence of MfINT-like protein by using two membrane protein topology servers, TMHMM 2.0 (http://www.cbs.dtu.dk/ services/TMHMM) and HMMTOP (http://www.enzim.hu/ hmmtop), indicating that MfINT-like belonged to MST superfamily. The 12 transmembrane regions are indicated in Fig. 1A. There is one consensus sequence for N-glycosylation (Asn376-Asn377-Thr378) in MfINT-like. MfINT-like localization was analyzed using CELLO v.2.5 (subCELlular LOcalization predictor, http://cello.life.nctu.edu.tw/) and cross checked with PSORT prediction software (http://wolfpsort.org/). The results showed that MfINT-like was predicted to be localized in plasma membrane (data not shown). For verification of this prediction, transient expression of MfINT-like-GFP fusion protein was performed in onion (A. cepa) epidermal cells. Compared with the whole cellular distribution of GFP, MfINT-like-GFP fusion protein fluorescence was observed at the perimeter of the transformed cells (Fig. 1B), indicating that MfINT-like is localized on the plasma membrane. Closely related homologs of MfINT-like exist in the genome of falcata. Three hybridization signals were observed by DNA blot hybridization after the genomic DNA of falcata was digested by EcoRV or XbaI (Fig. 1C). Expression of MfINT-like in response to abiotic stress and myo-inositol Transcript of MfINT-like was initially induced at 24 h, and reached the maximum level at 48 h after cold treatment (Fig. 2A). In contrast, the transcript levels of three homologs in M. truncatula that are most similar to MfINT-like were not affected by cold treatment (Fig. S2). MfINT-like was not responsive to dehydration until leaf water content decreased to 45% (data not
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Fig. 1. Amino acid sequence (A), subcellular localization of MfINT-like (B) and DNA blot hybridization analysis (C). The predicted transmembrane regions in sequence are underlined, and the putative N-glycosylation sequence (Asn376 -Asn377 -Thr378 ) is boxed (A). MfINT-like-GFP fusion protein and the control (GFP only) were transient expressed in onion epidermal cells to show the subcellular localization (B). For DNA hybridization, the falcata genomic DNA (15 μg) was digested with EcoRV or XbaI, respectively.
shown), but it was induced at 4 days after salinity treatment (Fig. 2B). MfINT-like was also induced by treatment with myo-inositol. MfINT-like transcript increased significantly at 2 and 4 h after treatment with myo-inositol (Fig. 2C); myo-inositol had a dose-effect on the induced MfINT-like transcript level (Fig. 2D). In addition, the data showed that expression of MfINT-like was more rapidly induced by myo-inositol than by cold or salinity. Analysis of transgenic tobacco plants DNA hybridization revealed that MfINT-like was integrated into the genomes of transgenic tobacco plants, likely with two transgene copies in lines S1 and S3 and one copy in line S6 (Fig. 3A). In addition, transgenic plants expressed MfINT-like gene, with more abundant transcript in line S1 than in lines S3 and S6 (Fig. 3B). In order to measure transporter activity of MfINT-like, transgenic tobacco and the wild-type plants were grown on medium without or with myo-inositol. myo-Inositol levels in roots were higher (13–15%) in the transgenic tobacco lines S1 and S3 than in the wild-type when plants were growing on the medium without myo-inositol (Fig. 3C), but showed no significant difference in shoot (Fig. 3D). After growing on the medium containing 10 mM myo-inositol, myo-inositol levels in roots were significantly increased in both transgenic plants and the wild-type (Fig. 3C); however, the increase rate was similar in transgenic plants (20–38%) and the wild-type (25%) (Fig. 3E). Compared with the
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wild-type (Fig. 3D), myo-inositol levels in shoot were significantly increased in transgenic plants by 30–67% (Fig. 3D, E). Analysis of abiotic stress tolerance in transgenic plants Most of the wild-type plants died after freezing treatment, while transgenic plants were little affected by freezing treatment (Fig. 4A). Compared with the 10% survival rate of the wild-type, transgenic plants had 77–83% survival rate, with no significant difference among transgenic plants (Fig. 4B). Similar ion leakage or Fv /Fm was observed in all plants under control conditions. Chilling treatment resulted in increased ion leakage and decreased Fv /Fm in all plants, while lower levels of ion leakage and higher levels of Fv /Fm were observed in transgenic plants than in the wild-type (Fig. 5A, B). For example, Fv /Fm decreased by 18–24% in the transgenic lines, but reduced by 38% in the wild-type (Fig. 5B). Drought and salinity tolerance were also assessed. The plants cultured on medium containing 0.1 M mannitol (drought treatment) or 0.1 M NaCl (salinity treatment) showed significant difference in fresh weight of whole plant. Drought decreased fresh weight by 2–11% and salinity stress reduced fresh weight by 33–40% for the transgenic plants (Fig. 5C), while fresh weight was reduced by 46 or 59% in the wild-type exposed to drought or salinity stress, respectively (Fig. 5C). 359
Fig. 2. MfINT-like transcripts as affected by cold (A), salinity (B) and myo-inositol (C, D). Plants were exposed to 5∘ C in a growth chamber for cold treatment (A), or irrigated with 0.15 M NaCl solution for salinity treatment (B). Leaflets were excised and placed in 200 mM myo-inositol for 24 h (C), or in different concentrations of myo-inositol solutions for 4 h (D). The expression levels were determined by qRT-PCR and normalized to actin expression. The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
Fig. 3. Analysis of transgenic plants (lines S1, S3 and S6) in comparison to the wild-type control (W); 15 μg of genomic DNA digested with EcoRI and 20 μg total RNA was used for DNA (A) or RNA blot hybridization (B), respectively. myo-Inositol concentrations in roots (C) and leaves (D) were determined after plants were grown on MS medium containing 10 mM myo-inositol or without myo-inositol as control for 4 weeks. The increased rate of myo-inositol (E) indicates the relatively increased amounts in MS medium containing myo-inositol compared with those in myo-inositol-free medium. The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
Analysis of myo-inositol levels in responsebreak to cold treatment myo-Inositol contents as affected by cold treatment were measured in transgenic and wild-type tobacco plants as well as in falcata plants. Ten-fold higher levels of myo-inositol were detected in leaves than in roots of both 360
tobacco and falcata plants; however, no significant differences in myo-inositol content were observed between transgenic plants and the wild-type under control conditions. While significant accumulation of myo-inositol was observed in falcata leaves and roots after cold treatment, no significant change of myo-inositol was
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Fig. 4. Analysis of freezing tolerance of transgenic lines (S1, S3 and S6) in comparison to the wild-type (W). Photos were taken after plants were treated by freezing at −3∘ C for 10 h, followed by 2-day recovery at room temperature (A) and the survival rates were calculated (B). The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
Fig. 5. Analysis of tolerance to chilling (A, B), drought and salinity (C) in transgenic tobacco plants (S1, S3 and S6) in comparison to the wild-type (W). Ion leakage (A) and Fv /Fm (B) were determined after plants were treated at 3∘ C for 3 days. With those growing on MS medium at 25∘ C as control, plant seedlings were grown for 6 weeks on MS medium containing 0.1 M mannitol (drought stress) or 0.1 M NaCl (salt stress) at 25∘ C, and the whole plant fresh weight was measured (C). The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
observed in the wild-type tobacco plants. Nevertheless, myo-inositol was significantly increased in leaves and roots of the transgenic tobacco plants after cold treatment (Fig. 6). There was a 1.9-fold and 95% increase in leaves and roots of falcata, and 1.2- to 1.6-fold and 47–66% increase in leaves and roots of transgenic tobacco plants, respectively.
Discussion A putative INT encoding gene (MfINT-like) belonging to the MST superfamily was isolated from leaves of falcata in this study. Among annotated Arabidopsis proteins, MfINT-like has the highest AA sequence identity with
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AtINT2 (68%), a plasma membrane-localized protein mediating myo-inositol efflux (Schneider et al. 2007), as compared with AtINT1 (a tonoplast-localized protein) and AtINT4 (another plasma membrane-localized protein) (Schneider et al. 2006, 2008). MfINT-like was predicted by sequence analysis and confirmed to be located in plasma membrane by transient expression of MfINT-like-GFP fusion protein in onion epidermal cells. Three MfINT-like hybridization signals were observed in the genomes of falcata, indicating that isoforms of MfINT-like gene exist in falcata. Transport of sugars is an important phenomenon in plants. Sugars and derivatives including myo-inositol are usually synthesized in source organ like mature 361
Fig. 6. myo-Inositol concentrations in transgenic plants (S1, S3 and S6) in comparison to the wild-type tobacco (W) and falcata (MF) as affected by chilling treatment. Fresh weight of leaves (A) or roots (B) was used to calculate the concentration. The same letter above columns indicates no significant difference by Duncan’s test at P < 0.05.
leaves, and transported to sink organ such as young leaves and roots. Accumulation of total non-structure carbohydrates in roots and crowns is associated with overwintering and subsequent spring growth of alfalfa (Cunningham and Volenec 1998). More RFOs are accumulated in winter-hardy cultivars than in winter-sensitive cultivars (Cunningham et al. 2003). Medicago sativa subsp. falcata accumulates more sucrose, myo-inositol, galactinol and RFOs than M. sativa and M. truncatula (Zhang et al. 2011, Tan et al. 2013a, Zhuo et al. 2013). The increased myo-inositol may function as a precursor of RFO synthesis and compatible solute for protection against abiotic stress (Loewus and Murthy 2000, Loewus 2006). Transport of myo-inositol is important in salt tolerance in ice plants; its level goes up by 10-fold in xylem under salinity (Nelson et al. 1999). MfINT-like transcript was induced 2–4 h after treatment with myo-inositol, consistent with a report that all seven myo-inositol transporter genes in Cryptococcus neoformans are induced by myo-inositol (Xue et al. 2010). Moreover, MfINT-like transcript was induced at 24 h after cold treatment and maintained high up to 96 h, which was slower than the induction by myo-inositol treatment, but well-coordinated with the accumulation of myo-inositol during cold treatment (Tan et al. 2013a). myo-Inositol concentration is initially increased at 24 h and maintained high in falcata leaves during the cold treatment, which results from the induced expression of 362
MfMIPS1 by cold (Tan et al. 2013a). The coordination between cold induced myo-inositol accumulation and MfINT-like transcription suggested that myo-inositol is involved in cold induced MfINT-like expression. Combined with the previous reports (Tan et al. 2013a, Zhuo et al. 2013), our results revealed an interesting chain reaction: cold causes myo-inositol accumulation, which, in turn, induces expression of MfINT-like to promote its own transportation, and expression of MfGolS1 (Zhuo et al. 2013) for its conversion to the downstream metabolite RFOs. This reflects a sophisticated control of metabolite concentrations in plant cells for adaption to the changing environments. Association of MfINT-like with abiotic stress tolerance was revealed in this study using transgenic tobacco plants overexpressing MfINT-like. Physiological assessments indicated that transgenic plants had enhanced tolerance to multiple abiotic stresses including freezing, chilling, drought and salinity. This is the first observation that overexpressing INT resulted in elevated abiotic stress tolerance. Compared with the plants growing on myo-inositol-free medium, more myo-inositol was observed in roots of transgenic plants and the wild-type after growing on the medium containing myo-inositol, with no significant difference in the increase rate between the two types of plants. This result indicated the similar capacity for uptake of myo-inositol from medium among plant lines. Moreover, an enhanced myo-inositol concentration was only observed in shoot of transgenic plants growing on the medium containing myo-inositol but not in the wild-type, indicating an enhanced myo-inositol transport in transgenic plants as a result of overexpression of MfINT-like. In addition, more myo-inositol was observed in roots of the transgenic lines S1 and S3 than in the wild-type roots when plants were grown on myo-inositol-free medium. This observation indicates that an enhanced myo-inositol transport might have a positive feed-back effect on myo-inositol biosynthesis. The speculation was supported by the observation that enhanced myo-inositol was accumulated in the cold-treated transgenic tobacco plants as compared with the wild-type. myo-Inositol accumulates in many plants in response to cold, drought and salinity stresses through an induced expression of MIPS expression (Nelson et al. 1998, Majee et al. 2004, Tan et al. 2013a). Our results revealed that MfINT-like also confers abiotic stress tolerance in plants by enhancing myo-inositol transport and accumulation. In conclusion, a MfINT-like in falcata was identified. Overexpression of MfINT-like leads to improved tolerance to multiple abiotic stresses through enhanced accumulation of myo-inositol.
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Acknowledgements – This work was supported by the National Basic Research Program of China (2014CB1 38701) and the Natural Science Foundation of China (30830081).
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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Phylogenetic analysis of MfINT-like in falcata with homologous INTs or proteins in Arabidopsis and Medicago truncatula. Fig. S2. Effect of cold on transcript levels of MTR_ 2g048720, MTR_5g077580 and MTR_2g049020 in Medicago truncatula. Plants were exposed to 5∘ C in growth chamber as cold treatment. The expression levels were normalized to that of actin using qRT-PCR. The same letter above a column indicates no significant difference by Duncan’s test at P < 0.05.
Edited by M. Uemura
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