Calmodulin-like gene MtCML40 is involved in salt tolerance by regulating MtHKTs transporters in Medicago truncatula

Environmental and Experimental Botany 157 (2019) 79–90

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Calmodulin-like gene MtCML40 is involved in salt tolerance by regulating MtHKTs transporters in Medicago truncatula Xiuxiu Zhanga,b, Tianzuo Wanga,b, Min Liua, Wei Sunc, Wen-Hao Zhanga,b,d,e,

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State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, Beijing, China College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China c Key Laboratory of Vegetation Ecology, Ministry of Education, Institute of Grassland Science, Northeast Normal University, Changchun, China d Research Network of Global Change Biology, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China e Inner Mongolia Research Center for Prataculture, Chinese Academy of Sciences, Beijing 100093, China b

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Keywords: Medicago truncatula Calmodulin-like proteins MtCML40 Salt stress MtHKTs Na+ accumulation

Calcium (Ca2+) is a universal messenger mediating numerous physiological processes in responses to developmental and environmental cues in plant cells. Calmodulin (CaM) and calmodulin-like proteins (CMLs) are important plant Ca2+ sensors involved in decoding Ca2+ signatures to execute downstream physiological responses. Despite the involvements of CML proteins in the regulation of developmental processes, little is known about the function of CMLs in response to abiotic stresses in plants. To characterize CML proteins, we isolated and functionally characterized a gene encoding a CML protein from legume model plant Medicago truncatula, referred to as MtCML40. The MtCML40 belonged to subgroup VI of CML family. Expression of MtCML40 was upregulated by salt, cold and osmotic stress as well as ABA treatment, suggesting a role of MtCML40 in abiotic stress. To test this hypothesis, we generated MtCML40 overexpressing transgenic lines in M. truncatula. Overexpression of MtCML40 rendered seed germination more sensitive to salt stress as evidenced by greater inhibition of seed germination of transgenic lines than wild-type seeds when exposed to NaCl, while seed germination of WT and transgenic lines was comparable under control conditions. In addition to seed germination, exposure to salt stress led to greater inhibition of shoot and root growth, reduction in chlorophyll and carotenoid concentrations and photosynthetic rates in the transgenic lines than WT plants, suggesting a negative regulation of salt tolerance by MtCML40. The greater accumulation of Na+ in shoots of transgenic lines may account for the greater sensitivity to salt stress. We further found that overexpression of MtCML40 resulted in down-regulation of MtHKT1;1 and MtHKT1;2 that encoded proteins associated with removal of Na+ from shoots. Taken together, our results demonstrate that MtCML40 is involved in the regulation of salt tolerance by targeting MtHKT-dependent Na+ accumulation in M. truncatula.

1. Introduction Calcium (Ca) is one of the mineral nutrients essential for plant growth and development. In addition, Ca ion (Ca2+) is also an ubiquitous signaling molecule involved in the moderation of numerous developmental and environmental cues by evoking a transient elevation of cytosolic Ca2+ activity ([Ca2+]cyt) in plant cells (Dodd et al., 2010; Kudla et al., 2010). Membrane transporters, including channels, carriers and pumps are invovled in the generation of specific Ca2+ signals in response to specific stimuli (Dodd et al., 2010; Kudla et al., 2010). Most Ca2+ sensors bind to Ca2+ via the EF-hand motif, a helix-loophelix structure, leading to a conformational change (Gifford et al., 2007). EF-hand proteins act as transducers of Ca2+ signals in plants,

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and include calmodulin (CaM) and calmodulin-like proteins (CMLs), calcineurin B-like proteins (CBLs) and Ca2+ dependent protein kinases (CDPKs) (Hrabak et al., 2003; Ranty et al., 2006; Weini and Kudla, 2009). The involvements of SOS pathway in the salt stress has been clearly documented. Salt stress elicits an increase in [Ca2+]cyt sensed by a calcineurin B-like protein CBL4 (SOS3), which subsequently interacts with a CBL-interacting protein kinase CIPK24 (SOS2). The SOS3/SOS2 complex then targets to the plasma membrane activating the membrane-bound Na+/H+ antiporter (SOS1) via phosphorylation (Shi et al., 2002; Qiu et al., 2003). The calmodulin-like proteins (CMLs) do not contain known functional motifs, but they do possess EF-hand motif (s), in which more than 16% of the amino acid sequences can match CaM (McCormack and

Correspondence author at: State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, Beijing, China. E-mail address: [email protected] (W.-H. Zhang).

https://doi.org/10.1016/j.envexpbot.2018.09.022 Received 24 July 2018; Accepted 26 September 2018 Available online 06 October 2018 0098-8472/ © 2018 Elsevier B.V. All rights reserved.

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been made to understand the molecular mechanisms by which M. truncatula responds and adapts to saline conditions. Merchan et al. (2007) identified several regulatory genes associated with root growth in response to salt stress using suppressive subtractive hybridizations (SSH) and the microarray technique. de Lorenzo et al. (2007) identified differentially expressed genes in salt-tolerant and salt-sensitive M. truncatula genotypes in response to salt stress. Li et al. (2011) conducted a detailed transcriptomic analysis of M. truncatula in response to salt stress. However, the mechanisms by which M. truncatula responds to salt are rarely explained. As for the SOS pathway, upregulation of SOS1 has been shown to play a role in salt tolerance in M. truncatula (Liu et al., 2015; Sandhu et al., 2017). On the other hand, Sandhu et al. (2018) identified six NHX genes in M. truncatula and confirmed their important roles in sequestering Na+ into vacuoles. Based on the results reported in the literature, few studies have linked the Ca2+ sensors to Na+ transporters under salt stress in M. truncatula. Although the CML family has been extensively investigated in Arabidopsis, little is known about the CML members in M. truncatula. In the present study, we first isolated a CML gene, MtCML40, which encoded a calmodulin-like protein consisting of four predicted Ca2+ binding sites and overexpression of MtCML40 leads to Na+ accumulation, rendering the transgenic plants more sensitive to salt stress. We further dissected the signaling pathways associated with the function of MtCML40 in response to salt stress.

Braam, 2003). In Arabidopsis, several CMLs have been proposed to be related with different physiological processes. The first characterized CML, AtCML24, has been shown to be involved in ionic homeostasis, photoperiod response, abscisic acid-mediated inhibition of germination and seedling growth (Delk et al., 2005). In addition, CML23 and CML24 are involved in flowering (Tsai et al., 2007), while CML42 plays a role in trichome branching (Dobney et al., 2009). Wang et al. (2015) reported the involvement of CML25 in pollen germination and pollen tube elongation. CML7 is associated with elongation of root hairs (Won et al., 2009; Lin et al., 2011). CML20 and CML39 are involved in microtubule organization (Azimzadeh et al., 2008), and seedling establishment (Bender et al., 2013), respectively. But few studies have functionally characterized CMLs in the context of responses to abiotic stresses in plants (Bender and Snedden, 2013). Genes encoding AtCML8 (Park et al., 2010) and AtCML24 (Delk et al., 2005) were found to be induced by salt and cold treatment respectively. Further studies suggested that atcml9 mutants exhibited enhanced tolerance to salt and drought stress through ABA-mediated pathway (Magnan et al., 2008). Moreover, the rice and tuber mustard CML genes, OsMSR2 and BjAAR1 were also suggested to be involved in ABA-mediated salt and drought tolerance (Xu et al., 2011; Xiang et al., 2013). Yamaguchi et al. (2005) found that AtCML18 binding to AtNHX1 significantly lowered its Na+/ H+ exchange activity, leading to a decrease in the Na+/K+ ratio. In addition to the above mentioned CMLs, AtCML37, AtCML38, and AtCML39 are also responsive to various stimuli, including salt, drought, and ABA (Vanderbeld and Snedden, 2007). More recently, Yin et al. (2017) suggested a novel rice CML gene, OsDSR-1, played important roles in conferring tolerance to drought stress by decreasing the occurrence of oxidative damage. However, the roles of CML proteins in plants are still largely unknown. Salinity is one of the major abiotic factors limiting crop yield worldwide (Julkowska and Testerink, 2015). Plants growing in saline soils have to cope with osmotic stress, sodium toxicity and the associated oxidative stress (Zhu, 2002; Munns and Tester, 2008). Upon exposure to salt stress, plants firstly encounter osmotic stress due to the presence of NaCl in soil solution (Julkowska and Testerink, 2015). As salt stress persists, accumulation of Na+ in the cytosol of plant cells disrupts cellular ion homeostasis, exerting a toxic effect on plants. Plants have evolved mechanisms to counteract Na+ toxicity (Munns and tester, 2008). Tonoplast-localized Na+/H+ exchanger1 (NHX1) (Blumwald and Poole, 1985) and plasma membrane-localized SALT OVERLY SENSITIVE 1 (SOS1) (Qiu et al., 2002; Yamaguchi et al., 2013) are two major players at the cellular level responsible for low cytoplasmic Na+ concentrations in plants. Among them, NHXs mange Na+ detoxification via sequestration of Na+ within the vacuole, while the SOS pathways are involved in exporting Na+ out of the cells (Deinlein et al., 2014). Regardless of the mechanisms mentioned above, HKT1 is an another major membrane transporter responsible for the Na+ transportation in Arabidopsis and wheat (Rus et al., 2001; Laurie et al., 2002; Mäser et al., 2002; Rus et al., 2004). The common roles that AtHKT1;1 and its rice ortholog OsHKT1;5 play are transferring Na+ from the xylem into the surrounding xylem parenchyma cells, thereby protecting plants from Na+ toxicity (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006). Further studies showed that AtHKT1 may directly retrieve Na+ from the xylem and unload Na+ into the root vacuoles (Davenport et al., 2007). However, Berthomieu et al. (2003) performed genetic and molecular analyses of the sas2 mutants and concluded that AtHKT1 is involved in recirculation Na+ from shoots to roots, probably by mediating Na+ loading into the phloem in shoots and then unloading in roots. Medicago truncatula has emerged as a model plant in legume genetics and genomics due to its small diploid genome, short life cycle, prolific seed production and easy transformation (Tang et al., 2014). Legumes are particularly important because of their symbiotic relationship with nitrogen-fixing bacteria. Like other crops, many leguminous crops are sensitive to salt stress (Kang et al., 2010). Efforts have

2. Materials and methods 2.1. Plant materials, growth conditions, and stress treatments M. truncatula ecotype R108-1 and three transgenic lines were used in this study. Plants were grown in petri dishes in the green house with 25 °C (day)/20 °C (night), 14 h – day/10 h – night periods with light intensity of 140 μmol m−2 s−1. Sulfuric acid treated seeds were surface sterilized by incubation for 10 min in 10% (v/v) sodium hypochlorite, and then washed with sterile water. After stratification for 2 day at 4 °C in darkness, the seeds were placed on appropriate medium containing 0.8% (w/v) sugar, 0.8% (w/v) agar with or without NaCl. The composition of medium was as followed: 0.5 mM KH2PO4, 1 mM MgSO4, 0.25 mM CaCl2, 0.1 mM Fe-Na2-EDTA, 1 mM NH4NO3, 2.5 mM KNO3, 30 μM H3BO3, 5 μM MnSO4, 1 μM ZnSO4, 1 μM CuSO4 and 0.7 μM Na2MoO4. Seed germination is defined as the emergence of the radicles through the seed coat. Thirty-five seeds per sample in each treatment were tested for the determination of seed germination rate. For the analysis of stress responses, roots growth was monitored in the same medium as mentioned before. Sulfuric acid treated seeds were surface-sterilized for 10 min in 10% (v/v) sodium hypochlorite, and then washed with sterile water. After washing with sterilized water, seeds were sown on 0.8% water-agar plates and stored for 2 days at 4 °C before incubating overnight at 25 °C in the dark to ensure uniform germination. Germinated seedlings were transferred to square plates containing appropriate medium and grown vertically in a growth chamber. To analyze salt stress responses in mature plants, germinated seedlings with about 2-cm radicles were transferred to plastic buckets filled with tap water. Three days later, changed the water with fully aerated nutrient solution and then changed the nutrient solution every three days. Each bucket contained wild type (R108-1) and transgenic lines for 12 plants and repeated for three buckets. After grown in the culture solution for 3 weeks, half of the plants were transferred to culture solution with 100 mM NaCl. The pH of the hydroponic solution was adjusted to 6.0. 2.2. Constructs and transformation of M. truncatula To obtain transgenic materials, the coding region of MtCML40 was amplified using primers 5′-TGG CGC GCC ATG AAG AAT GCG GGA-3′ 80

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Microsoft Excel (Livak and Schmittgen, 2002).

(AscI site underlined) and 5′-CTT AAT TAA TCA AGC GTA ATC TGG AAC ATC GTA TGG GTA CTG CAT CAT TGT TAT G-3′ (PacI site underlined). The AscI/PacI-digested product was inserted in the downstream of cauliflower mosaic virus 35S (CaMV 35S) promoter of PMDC32. After PMDC32:MtCML40 was transformed to Agrobacterium tumefaciens EHA105 by electroporation, transformation of Medicago truncatula (R108-1) was performed using Agrobacterium tumefaciensmediated method (Cosson et al., 2006). Transgenic plants were selected using real time qPCR (RT-qPCR) with primers MtCML40 (5′-GCT AAG ACA AAG GCT AA-3′ and 5′-CCT CCA AAC TCA AAT AA-3′) and MtActin gene (5′-ACG AGC GTT TCA GAT G-3′ and 5′-ACC TCC GAT CCA GAC A-3′). Three independent lines of the T2 generation were randomly chosen for further physiological studies.

2.6. Measurements of pigments concentration To determine chlorophyll concentration in wild-type and transgenic plants, newly formed leaves were harvested, weighed, and extracted with aqueous ethanol (95% v/v). The absorbance (A) of the supernatant was recorded at wavelengths of 665 and 649 nm. Total chlorophyll concentration was calculated as 6.64 A665+18.08 A649. At the same time, the absorbance of the supernatant was also recorded at wavelength of 470 and used for the calculation of the carotenoids concentration, which was calculated as (1000*A470-2.05*Ca-114.8*Cb)/ 245. All pigments were expressed as mg g−1 fresh weight.

2.3. Expression and purification of MtCML40 2.7. Measurements of photosynthetic characteristics To express and purify the MtCML40 in Escherichia coli, the coding region was amplified by PCR using the primers 5′-AAT CGG ATC TGG TTC CGC GTG GAT CCA TGA AGA ATG CGG GAT TCG A-3′ containing a BamHI site (underlined), and 5′-TCA GTC AGT CAC GAT GCG GCC GCT CAC TGC ATC ATT GT-3′ containing a NotI site (underlined). The PCR product was ligated to the C-terminal of glutathione S-transferase (GST) in pGEX-4T-1 (GE) through Seamless Assembly Cloning Kit. The recombinant plasmid was transformed into E.coil BL21. Protein expression was induced with 0.5 mM isopropyl thiogalactopyranoside (IPTG) at 30 °C. IPTG was added when the optical density at 600 nm of the culture was reached 0.8. Bacterial cells were harvested after induction for 6 h by centrifuging the culture at 4000 g for 10 min. The purification of recombinant protein was carried out by GST Resin (TransGen).

Photosynthetic rates of wild-type and transgenic plants were measured between 8.30 and 12.30 with a LI-6400 XT portable photosynthesis system equipped with a LED leaf cuvette (Li-Cor, Lincoln, NE, USA). Artificial illumination was applied to leaves in the chamber from a red-blue 6400-02B LED light source attached to the sensor head giving continuous light (1000 μmol m−2 s−1 photosynthetic photon flux density); ambient CO2 concentration was ∼500 μmol CO2 mol−1. 2.8. Determination of Na+ and K+ contents in shoots and roots Three-week-old plants of transgenic and WT plants were changed into hydroponic nutrient solution with 0 and 100 mM NaCl for 5 days then shoots and roots of plants were separated post-harvested. Following a wash with distilled water to remove surface containing ions, the shoots and roots samples were left to fix at 105 °C for 10 min and to dry at 80 °C for 48 h, and then triturated. About 20 mg of dry materials were weighed and placed in a digestion tube, and suspended it in 6 ml of concentrated nitric acid overnight. 1 ml of hydrogen peroxide were added to the digested tissue powder and allowed to completed digest for 2 h. The digested solution was made up to 100 ml with double distilled water. The Na+ and K+ concentrations in the aciddigested samples that represent total Na+ and K+ in the tissue samples were determined using an ICP-AES (Thermo).

2.4. Ca2+-dependent electrophoretic mobility shift assay Ca2+-dependent electrophoretic mobility shift assay (EMSA) was performed according to the methods described previously (Takezawa, 2000). CaCl2 at 5 mM or ethylene glycol-bis-(b-amino-ethylether) N, N, N’, N’-tetra-acetic acid (EGTA) was added into the sample buffer of GST protein and GST-MtCML40 recombinant protein. Proteins were analyzed on 10% polyacrylamide gel electrophoresis under non-denaturing conditions, and stained with Coomassie Brilliant Blue R-250. 2.5. RNA isolation and real-time quantitative PCR

2.9. Determination of Na+ in xylem and phloem Total RNA was isolated using RNAiso Plus reagent (Takara). The total RNA was reverse-transcribed into first-strand cDNA with PrimeScript® RT reagent Kit (TaKaRa). RT-qPCR was performed using ABI Stepone Plus instrument to study the expression patterns of genes responsible for ion uptake and transport. Gene-specific primers used for RT-qPCR were designed using software Primer Premier 5, and were as follows: for MtSOS1 (5′-GCT GAC TTT CCC GTA TG-3′ and 5′-TGG CAC CCA GTT CTT TC-3′), for MtSOS3 (5′-TCT GAG GCA AAC AGG GTA3′and 5′-CTG GGA AAT GCT AAG GTA AT-3′), for MtHKT1;1 (5′-TCT TCT GCC ATA TTG GTG CTC TT-3′ and5′-CTC CCA AAG AAC ATT ACC AAG AT-3′), for MtHKT1;2 (5′-CCT TCC TCC TTA CAC CTC ATT CC-3′ and5′-ATC TTA CCC TCA TCA CTC CAT TTC-3′). MtActin gene (accession no. BT141409) was used as internal control with primers ( 5′-ACGAGCGTT TCA GAT G-3′ and 5′-ACC TCC GAT CCA GAC A-3′). The primer sequence of MtActin has been used previously and MtActin has proved to be consistent due to its lower variation compared with other four constitutive genes under salt stress (Merchan et al., 2007; Song et al., 2012). Each reaction contained 5.0 μL of the SYBR Green Master Mix reagent, 0.4 μL cDNA samples and 0.6 μL of 10 μM gene-specific primers in a final volume of 10 μL. The thermal cycle used was 95 °C for 2 min, 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The relative expression level was analyzed by the comparative CT method using the

Xylem sap was collected by a pressure chamber (PMS, Instruments, Corvallis, OR) following the protocols described by Li et al. (2009). Briefly, the excised roots from WT and transgenic plants grown in medium supplemented with and without 100 mM NaCl for varying periods were put into the chamber. A pressure of about 1.1 MPa was applied for 15 min to allow for collection of the xylem sap. The collected saps without the first few drops were diluted directly in 5% HNO3 solution. Sodium contents in the saps were measured by ICP-AES (Thermo). Phloem saps from M. truncatula seedlings were collected following the protocols described by Berthomieu et al. (2003) and Corbesier et al. (2003). Briefly, four mature leaves were detached from M. truncatula seedlings at their petiole bases. The petioles were recut under 20 mM EDTA-K2 (pH 7.5). Four leaves collected from one plant were placed in a 2 ml microcentrifuge tube with their petioles immersed in 1 ml of 15 mM EDTA-K2 (pH 7.5). Then the tubes were placed in an illuminated growth room in airtight transparent plastic containers for 4 h, in which the atmosphere was saturated with steam to prevent uptake of EDTA solution by the leaves, to dissolve the phloem sap in EDTA solution. Then the EDTA solution was diluted with equal volumes of 10% HNO3 solution and used to determine Na+ concentrations by ICP measurements. 81

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Fig. 1. MtCML40: a member of the calmodulin-like (CML) family. (A) Alignment of amino acid sequences of CML40 with that of CaMs in Arabidopsis thaliana. (B) Phylogenetic tree of Medtr3g089090 by comparing with 20 AtCML proteins using MEGA 6 sofware. The sequences were obtained from NCBI. (http://www.ncbi.nlm. nih.gov/). The subgroups are highlighted with different colors, where blue signifies group Ⅴ, red signifies group III, green signifies VII and purple signifies group VI (C) Purified CML protein was run on a 10% non-denaturing polyacrylamide gel in the presence of 5 mM CaCl2 (right) or EGTA (left). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

3. Results

relay function, we examined its structural similarity by comparing amino acid sequences to the typical CaMs. Medtr3g089090 differed from a typical CaM because its EF-hand motifs were not completely conserved with the typical EF-hands of CaMs and shared 30% identity with Arabidopsis CaM2 (Fig. 1A). To identify its homologous proteins in Arabidopsis, phylogenetic trees based on the full-length amino acid sequences of CML proteins were constructed using MEGA6 software

3.1. MtCML40 was a Ca2+-binding protein As a representative Ca2+-binding sensor relay protein, CaM had four EF-hand motifs and its conformation was changed in the presence of Ca2+. To verify whether Medtr3g089090 possesses a Ca2+ sensor

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in leaves, stems and roots (Fig. 2E).

(Fig. 1B). The amino acid sequence of MtCML was highly homologous to the AtCML40, and thus designated as MtCML40 accordingly. Based on the segment sequence of MtCML40, RACE was performed to obtain the full-length cDNA. The results showed that MtCML40 had a 420 bp open reading frame that encodes a protein of 139 amino acid residues, with a calculated molecular mass of about 16 kDa. To determine whether MtCML40 can bind to Ca2+ and undergo the conformational changes observed in CaM proteins, we analyzed the MtCML40 protein with native-PAGE in the absence and presence of Ca2+. Recombination of MtCML40 with GST (glutathione S-transferase) was expressed in E.coli BL21 and purified using GST Resin (TransGen). A native-PAGE mobility-shift assay was performed to confirm the ability of the MtCML40 to bind to Ca2+. As shown in Fig. 1C, MtCML40 exhibited the characteristics of Ca2+ shift, whereas no shift was observed for GST, a non-Ca2+-binding protein.

3.3. Overexpression of MtCML40 rendered M. truncatula hypersensitive to salt stress To functionally characterize MtCML40, we overexpressed the MtCML40 in M. truncatula (R108-1) under the control of a CaMV 35 s promoter. Compared with the untransformed WT plants, the abundance of MtCML40 transcript was higher in the T2 generation of transgenic lines (Fig. 2F). Three independent transgenic lines, L1, L2 and L3, were chosen for further physiological studies. To evaluate the roles of MtCML40 in salt stress, phenotypes of transgenic lines and WT at varying developmental stages were compared in the absence and presence of NaCl. In the absence of NaCl in the medium, seeds from WT and the transgenic line fully germinated within 2 d after sowing (Fig. 3A, B). However, overexpression of MtCML40 led to greater suppression of seed germination in the presence of 100 mM NaCl. For example, a seed germination rate of 90% was observed after 4 days of exposure to 100 mM NaCl, whereas the transgenic lines exhibited a seed germination rate of 44%, 50%, 53%, respectively under the same conditions (Fig. 3C, D). In addition to seed germination, we also compared the effects of salt stress on growth of seedlings from WT and transgenic lines in the absence and presence of NaCl. Root length of both WT and transgenic lines were significantly reduced after exposure to salt stress (Fig. 4A, B), however, the NaCl-induced inhibition of root growth in transgenic lines was more profound than that in WT plants

3.2. Expression of MtCML40 was responsive to various stimuli and ABA Transcripts of MtCML40 were rapidly increased upon exposure to salt, cold, dehydration and ABA treatments. For example, treatment with NaCl led to a 10-fold increase in MtCML40 transcript in leaves and 20-fold in roots (Fig. 2A). The cold- and dehydration-induced increase in MtCML40 transcripts was rapid (Fig. 2B, C). An exogenous application of ABA to M. truncatula seedlings also resulted in a significant upregulation of MtCML40 (Fig. 2D). Moreover, we found that MtCML40 was expressed mainly in flowers, with nearly identical expression levels

Fig. 2. Real-time qPCR analysis of MtCML40 gene expression in M. truncatula seedlings treated with varying stresses or exogenous application of ABA. Total RNA from 3-week-old seedlings and leaves was analyzed by real-time qPCR with gene-specific primers for MtCML40. (A) The transcript levels of MtCML40 were plotted as the relative expression (fold) of plants exposed to NaCl (200 mM) for the indicated period (A), dehydration (B), cold (4 °C) (C) and 100 μM ABA (D) compared with control plants. (E) Real-time qPCR analysis of MtCML40 expression in different tissues. Data are means ± SE for three independent experiments. (F) Expression of MtCML40 in wildtype (WT) and transgenic plants overexpressing the MtCML40.

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Fig. 3. MtCML40 is involved in seed germination under salt conditions. Three independent lines were used for the germination rate study. Seeds were sown on mediums with or without NaCl after 2-day stratification. Data are means ± SE for three replicates with each replicate containing 35 seeds. (A) Seed germination and seed germination rates (B) of WT and transgenic lines in the absence of NaCl. (C) Seed germination and seed germination rates (D) of WT and transgenic lines in the presence of 100 mM NaCl.

higher shoot Na+ concentrations in transgenic plants than in WT plants (Fig. 6A). Unlike Na+, K+ concentrations in WT and transgenic plants were comparable under both control and salt-stressed conditions despite a marked reduction in shoot K+ concentrations in both WT and transgenic plants under conditions of salt stress (Fig. 6B). The differential changes in shoot Na+ and K+ concentrations led to a significantly higher shoot Na+/K+ ratio in transgenic plants than in WT plants under salt stressed conditions (Fig. 6C). In contrast to shoots, no differences in root Na+ and K+ concentrations and Na+/K+ ratios between WT and transgenic plants were observed under control and salt stressed conditions (Fig. 6D–F). Given the important role of SOS proteins in the regulation of Na+ acquisition (Zhu et al., 2003), the effects of salt stress on expression of MtSOS1 and MtSOS3 were studied by RT-qPCR. The expressional level of MtSOS1 was lower in transgenic plants than in WT under both control and salt stressed conditions (Fig. 6G). However, the salt

(Fig. 4B, C). Furthermore, we have examined biomass of the mature plants in response to salt stress. Similarly, salt treatment reduced both the shoot and root biomass more noticeably for the transgenic plants (Fig. 5A–D). No significant differences were, however, found for the ratio of root to shoot under salt stress (Fig. 5E).

3.4. Effects of salt stress on Na+ and K+ accumulation in shoots and roots Given that plants suffering from salt stress have to cope with excess toxic Na+ in the growth medium, we compared the effects of salt stress on Na+ and K+ concentrations as well as Na+/K+ ratios in shoots and roots of WT and transgenic plants. Shoot Na+ concentrations between WT and transgenic plants under control conditions were comparable (Fig. 6A). There were marked increases in shoot Na+ concentrations of WT and transgenic plants upon exposure to NaCl supplement, and the increase was greater in transgenic plants than in WT plants, leading to a

Fig. 4. MtCML40-overexpressing (OE) seedlings are more sensitive to salt stress than WT. (A) WT and transgenic seedlings grown vertically in agarose media without (A) and with 100 mM NaCl (B) for 10 days. (C) Effects of 100 mM NaCl on primary root length of WT and three transgenic lines. Data are means ± SE (n = 3–5). “*” indicates a statistically significant difference at P < 0.05 between WT and the three transgenic lines under the same treatment. 84

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Fig. 5. Effects of salt stress on shoot biomass (A), root biomass (B) and root/shoot ratio of WT and transgenic lines. Three-week-old seedlings of WT and transgenic lines grown hydroponically were exposed to solution supplemented with and without 100 mM NaCl for 5 days. (A, B) Phenotypes of WT and transgenic plants treated with and without 100 mM NaCl for 5 days. (C) Dry weight of WT and transgenic plants propagated as in (A) and (B). Shoot biomass (C), root biomass (D) and root/ shoot ratio (E). Data are means ± SE (n = 4). “*” indicates a statistically.

treatment did not affect MtSOS1 expressional pattern between transgenic lines and WT (Fig. 6G). On the other hand, no significant differences were found in MtSOS3 expressional levels between transgenic lines and WT under either control or salt conditions (Fig. 6H). The results suggested that the SOS pathways may not participate in the regulation of different Na+ allocation between WT and transgenic plants.

responsive to salt stress, as evidenced by less increases in the abundance of MtHKT1;1 and MtHKT1;2 transcripts after salt treatments (Fig. 7C, D). We also analyzed the promoters of MtHKT1;1 and MtHKT1;2 and figured out that they contained the ABI4-binding motifs (Fig. 8A). The expression levels of MtABI4 were higher in transgenic lines than in WT (Fig. 8B).

3.5. Effects of salt stress on Na+ concentrations in xylem and phloem saps

3.6. Effects of salt stress on pigment contents and photosynthetic rates (Pn)

To dissect the mechanisms underlying the greater accumulation of Na in shoots by overexpression of MtCML40, we analyzed Na+ concentration in the xylem saps of WT and transgenic plants under control and salt stressed conditions. Na+ concentrations in the xylem were comparable between WT and transgenic lines in the control medium without NaCl (Fig. 7A). A marked increase in Na+ concentrations in the xylem of both WT and transgenic lines was observed when exposed to medium supplemented with NaCl, and the increase was significantly greater in the xylem of transgenic plants than that of WT plants (Fig. 7A). In addition, we also compared the effects of salt stress on Na+ concentrations in the phloem of WT and transgenic plants. In contrast to Na+ in xylem, no significant differences in Na+ concentrations in the phloem of both WT and transgenic plants were observed in the absence and presence of NaCl (Fig. 7B). These results indicate that overexpression of MtMCL40 disrupts Na+ translocation in the xylem, while it has no effect on Na+ transport in the phloem. AtHKT1 is a candidate protein involved in the removal of Na+ from shoots, thereby playing a role in salt tolerance in Arabidopsis (Alberts et al., 2002). To test whether a similar mechanism underpins the observed results in M. truncatula, we cloned AtHKT1 homologs from M. truncatula, including MtHKT1;1 and MtHKT1;2, and monitored their expression patterns in response to salt stress. The expression patterns of MtHKT1;1 and MtHKT1;2 in WT and transgenic plants were comparable under control conditions (Fig. 7C, D). However, overexpression of MtCML40 rendered the expression of MtHKT1;1 and MtHKT1;2 less

We further compared the contents of pigments between WT and transgenic plants under control and salt stressed conditions. No differences in chlorophyll and carotenoids contents were observed between WT and transgenic plants under control conditions (Fig. 9A, B). However, salt stress led to a significant reduction in foliar chlorophyll and carotenoids contents in WT and transgenic leaves, with WT plants maintaining a relatively higher foliar chlorophyll and carotenoids concentrations than those of transgenic plants (Fig. 9A, B). In addition, transpiration rates of both WT and transgenic plant were significantly reduced after salt treatment, with the transgenic plants showing lower transpiration rates than the WT plants (Fig. 9C). Photosynthetic rates in transgenic plants under control conditions were similar to that in WT, while the photosynthetic rates were significantly lower in transgenic plants than those in WT plants under salt stress (Fig. 9D).

+

4. Discussion Plants are equipped with a repertoire of distinct but related proteins known as calmodulin-like proteins (CMLs). The Arabidopsis and M. truncatula genomes harbor 50 CMLs and 50 EF-hand Ca2+-binding proteins, respectively (McCormack and Braam, 2003; Zhu et al., 2015). The involvement of CMLs in the regulation of developmental processes in Arabidopsis has been reported (See review by Zhu et al., 2015). However, no studies have evaluated the functions of CMLs in response to abiotic stress. In the present study, we identified a CML (MtCML40) 85

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Fig. 6. Na+ and K+ content in shoots and roots of WT and transgenic plants under control and salt conditions. 3-week-old plants grown hydroponically were exposed to a new nutrient solution either with or without 100 mM NaCl for another 5 days. Na+ and K+ concentrations in leaves and roots from WT and transgenic plants were determined by inductively coupled plasma mass spectrometry (ICP-MS). (A) Shoot Na+ and K+ (B) contents under control and salt treatment of WT and transgenic plants. (C) Shoot Na+ / K+ ratio under control and salt treatment of WT and transgenic plants. (D) Root Na+ and K+ (E) contents under control and salt treatment of WT and transgenic plants. (F) Root Na+ / K+ ratio under control and salt treatment of WT and transgenic plants. (G) Expression patterns of MtSOS1 and MtSOS3 (H) under control and salt treatment of WT and transgenic plants. Data are means ± SE (n=3). Asterisks indicate a statistically significant difference (*P < 0.05, **P < 0.01, ***P < 0.001) between salt treatments.

charged amino acids, glutamic acids and aspartic acids (Gifford et al., 2007). Binding to Ca2+ can reduce negative charges and change the migration rate. However, the native-PAGE assay used in our study cannot allow us to test structural changes. Further studies on Ca2+induced conformational change are needed by binding to hydrophobic phenyl-sepharose in a Ca2+- dependent manner (Vanderbeld and Snedden, 2007). In addition, MtCML40 was a highly regulated gene as evidenced by that its expression was sensitive to diverse stimuli, including salt, drought, cold stress as well as ABA (Fig. 2A–D). These stimuli have been reported to evoke increases in cytosolic Ca2+ activity (Reddy et al., 2011). Taken together, these results may suggest that MtCML40 expression is induced by the stimuli that use Ca2+ as a second messenger, and that MtCML40 may serve as a potential Ca2+ sensor in plant cells. The MtCML40-overexpressing (MtCML40-OE) plants displayed more hyper-sensitivity to salt stress than WT in terms of seed germination, root and shoot growth (Figs. 3D, 4C and 5C, D). Unlike previous studies (de Lorenzo et al., 2007), no significant differences were found for the ratio of root to shoot dry weight between WT and transgenic plants (Fig. 5E). This result may suggest the same sensitivity of roots and shoots to salt stress in the overexpression of MtCML40. Plants suffering from salt stress have to cope with excessive

gene in the legume model plant M. truncatula, and characterized its function in response to salt stress by generating transgenic plants overexpressing the MtCML40 in M. truncatula. Our results showed that overexpression of MtCML40 rendered the transgenic plants more sensitive to salt stress. We further discovered that the hypersensitivity to salt stress of the overexpression lines was likely to result from greater accumulation of Na+ in shoots of the transgenic plants due to downregulation of MtHKT1;1 and MtHKT1;2. These findings highlight the regulatory roles of CML proteins in salt stress response in M. truncatula. MtCMLs were classified into seven separated subgroups according to their divergent forms with the conserved CaM (Zhu et al., 2015). The MtCML40 identified in the present study had a low degree of identity (30%) with CaM (Fig. 1A), and belonged to subgroup VI (Fig. 1B). The diversity of EF-hand proteins, which are able to bind to Ca2+ using their helix-loop-helix structure (Gifford et al., 2007), has been suggested to allow for differential responses to Ca2+ signaling (Bender et al., 2013). Several Arabidopsis CMLs, including CML37, 38, 39, and 42 displayed an electrophoretic mobility shift in the presence of Ca2+ (Vanderbeld and Snedden, 2007; Dobney et al., 2009). We found that MtCML40 was capable of binding to Ca2+ as indicated by a decreased rate of native-PAGE migration in the presence of Ca2+ (Fig. 1C). Most Ca2+-binding motifs, especially the EF-loops, are rich in negatively

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Fig. 7. Na+ content in xylem and phloem sap under control and salt conditions. Three-weekold plants grown hydroponically were exposed to a new nutrient solution either with or without 100 mM NaCl for various days. Na+ content in the xylem sap and in the phloem sap extracted from WT (n = 3) and transgenic plants (n = 5) was determined by inductively coupled plasma mass spectrometry (ICP-MS). (A) Na+ content in xylem sap and in phloem sap(B)of WT and transgenic plants under control and salt conditions. (C) Differing expression levels of MtHKT1;1 and MtHKT1;2 (D) under control and salt conditions. Data are means ± SE (n=3). “*” indicates a statistically significant difference (P < 0.05) between different lines.

enzymes’ activity (Bhandal and Malik, 1988). Therefore, the ability to maintain a low level of Na+/ K+ ratio in shoots under salt stress is an important trait for plants to tolerate to salt stress (Zhu et al., 2003). We found that transgenic plants overexpressing MtCML40 exhibited higher Na+ concentrations in their shoots than their counterpart WT plants when exposed to salt stress, while no significant differences in shoot K+ concentrations between the transgenic plants and WT plants, thus leading to a higher Na+/K+ ratio in transgenic plants than in WT plants under salt-stressed conditions (Fig. 6A–C). The greater accumulation of Na+ in the transgenic plants would make the plants to suffer from Na+ toxicity, thus rendering them more sensitive to salt stress. In contrast to shoots, overexpression of MtCML40 did not alter Na+ accumulation in roots (Fig. 6D–F), implying that MtCML40 may control Na+ translocation between roots and shoots, rather than Na+ uptake by roots. We further explored the mechanisms by which overexpression of MtCML40 enhanced Na+ accumulation in shoots. The Na+ transporters SOS1 has often been reported to play a role in the regulation of root Na+ accumulation (Shi et al., 2003). As a putative Na+/H+ antiporter in the plasma membrane, SOS1 also mediates Na+ extrusion from plant cells and regulates long distance Na+ transport between shoots and roots (Yamaguchi et al., 2013). Similarly, SOS3 in Arabidopsis is a calcineurin-like protein involved in control of Na+ loading into the xylem (Zhu, 2002), which includes four EF-hand Ca2+-binding motifs and forms dimers when Ca2+ concentrations increased (SánchezBarrena et al., 2005). However, we found that overexpression of MtCML40 did not alter the expression patterns of MtSOS1 and MtSOS3 in M. truncatula (Fig. 6G, H), suggesting that MtCML40-mediated Na+ accumulation is independent of MtSOS1 and MtSOS3. In addition to SOSs, HKT transporters have been demonstrated to be involved in Na+ accumulation in plants under salt stress (Mäser et al., 2002; Berthomieu et al., 2003; Horie et al., 2006; Davenport et al., 2007). In Arabidopsis, AtHKT1 was initially proposed to control Na+ entry into roots (Rus et al., 2001). Loss-of-function mutations in AtHKT1 rendered plants Na+ hypersensitive and disturbed distribution of Na+ between roots and shoots, causing Na+ overaccumulation in leaves and chlorosis under salt stress (Mäser et al., 2002). Furthermore, reduced AtHKT1 activity has been shown to cause growth inhibition and Na+ overaccumulation in shoots under salt stress through loading Na+ into the phloem and unloading in roots (Berthomieu et al., 2003). These findings reveal that AtHKT1 is an important player to minimize Na+ accumulation in Arabidopsis and that AtHKT1-mediated Na+ transport is

Fig. 8. Putative cis-elements identified in MtHKTs promoters that can be combined by MtABI4. The promoter sequences are defined as 3000 bp upstream of the starting site. (A) Cis-elements identified in MtHKT1;1 (above) and MtHKT1;2 (below) promoters. (B) Differing expression levels of MtABI4 under control and salt conditions. Data are means ± SE (n=3). Asterisks indicate a statistically significant difference (*P < 0.05, ***P < 0.001) between different lines.

accumulation of Na+ in their shoots. The toxic effect of Na+ is largely due to its ability to compete with K+ at binding sites essential for cellular function. Potassium is essential for more than 50 enzymes to function in plants, and excessive accumulation of Na+ and concurrent suppression of K+ accumulation under salt stress would inhibit the 87

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Fig. 9. Effects of salt stress on chlorophylls (A), carotenoids (B), transpiration (c) and photosynthetic rates (D) of WT and transgenic plants. Data are means ± SE (n = 4). Asterisks indicate a statistically significant difference (*P < 0.05, **P < 0.01) between different lines.

and transgenic plants, with the greater reduction in transgenic plants than WT plants (Fig. 9A, B). This result can be explained by greater decrease in chlorophyll concentrations in transgenic plants than WT plants due to their greater accumulation of toxic Na (Fig. 6A). In addition to pigments, we also found significantly lower transpiration rates in transgenic lines than in WT plants under salt stress (Fig. 9C), suggesting a more pronounced stomatal closure and lower gas exchange in transgenic plants. The greater inhibition of photosynthetic rates by salt stress in MtCML40-overexpresing plants would reduce their growth, thus leading to their poorer phenotypes compared to WT plants under salt stress. In conclusion, we isolated and functionally characterized a gene encoding a calmodulin-like protein in the legume model plant M. truncatula, MtCML40, by generating transgenic M. truncatula plants overexpressing MtCML40. One important finding is that overexpression of MtCML40 rendered the transgenic lines more sensitive to salt stress. We further discovered that overexpression of MtCML40 down-regulated expression of MtHKTs that encode proteins involved in Na+ translocation, thus leading to Na+ toxicity due to less efficient removal of Na+ from shoots.

essential for protection of plant leaves against salt stress in Arabidopsis (Mäser et al., 2002; Berthomieu et al., 2003; Sunarpi et al., 2005). The involvement of AtHKT1 in salt stress by regulating Na+ translocation prompts us to determine Na+ concentrations in both the xylem and phloem as well as the expression levels of MtHKTs in M. truncatula in the absence and presence of NaCl. We found that overexpression of MtCML40 did not alter Na+ concentration in the phloem (Fig. 7B), but an increase in Na+ concentration in the xylem was detected in the transgenic plants relative to WT plants (Fig. 7A). AtHKTs gene expression analysis (Fig. 7C, D) further supports the complementation results in Fig. 7A, suggesting the reduced expression of AtHKTs in transgenic lines as the cause of enhanced Na+ accumulation in xylem sap, thus elevating Na+ accumulation in shoots (Fig. 6A). These results indicate that the hypersensitivity of MtCML40-OE plants to salt stress is likely to be caused by less Na+ unloading in the xylem, thus leading to greater Na+ accumulation in shoots. It has been shown that ABI4 can bind the HKT1;1 promoter termed as ABEs, resulting in reduced target gene expression (Shkolnik-Inbar et al., 2013). ABI4 as a transciption factor, CE1 was the first identified ABI4 DNA-binding sites (Niu et al., 2002). Recent reports also demonstrat that S-box, G-bos and CE1-like motifs might function as the core ABI4-binding elements (Acevedo-Hernández et al., 2005; Bossi et al., 2009; Reeves et al., 2011). We analyzed the promoters of MtHKT1;1 and MtHKT1;2 and found that they contained the core recognition motifs that MtABI4 can bind (Fig. 8A). Expression levels of MtABI4 were analyzed between WT and transgenic lines, and significantly enhanced expression levels in MtCML40-OE plants were detected (Fig. 8B), supporting our suggestion that MtABI4 may bind to the recognition sequences in the MtHKTs promoters to inhibit the expression of MtHKTs. This conclusion is consistent with previous study, which showed that HKT1;1 expression level increased in the abi4 mutants. Therefore abi4 mutants accumulated a lower level of Na+ in the shoots than WT plants following exposure to NaCl, whereas similar Na+ concentrations were accumulated in their roots (Shkolnik-Inbar et al., 2013). Previous studies have shown that salinity reduced the photosynthetic rates in plants by reducing the photosynthetic pigments, blocking the photosynthetic electron transport and inhibiting PSII activity (Chaves et al., 2009, 2011). Carotenoids as non-enzymatic antioxidants take part in photoprotection during photosynthesis (Ashraf and Harris, 2013). In the present study, we found that concentrations of the pigments were reduced markedly by exposure to NaCl for both WT

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