Gene 545 (2014) 61–71

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

Functional characterization of GhAKT1, a novel Shaker-like K+ channel gene involved in K+ uptake from cotton (Gossypium hirsutum) Juan Xu a, Xiaoli Tian a,⁎, A. Egrinya Eneji b, Zhaohu Li a a b

State Key Laboratory of Plant Physiology and Biochemistry, Department of Agronomy, China Agricultural University, Beijing 100193, China Department of Soil Science, Faculty of Agriculture, Forestry and Wildlife Resources Management, University of Calabar, Nigeria

a r t i c l e

i n f o

Article history: Received 12 February 2014 Received in revised form 16 April 2014 Accepted 2 May 2014 Available online 5 May 2014 Keywords: Expression profiles K+ uptake Overexpression Shaker-like K+ channel

a b s t r a c t Shaker-like potassium (K+) channels in plants play an important role in K+ absorption and transport. In this study, we characterized a Shaker-like K+ channel gene GhAKT1 from the roots of Gossypium hirsutum cv. Liaomian17. Phylogenetic analysis showed that the GhAKT1 belongs to the AKT1-subfamily in the Shaker-like K+ channel family. Confocal imaging of a GhAKT1-green fluorescent fusion protein (GFP) in transgenic Arabidopsis plants indicated that GhAKT1 is localized in the plasma membrane. Transcript analysis located GhAKT1 predominantly in cotton leaves with low abundance in roots, stem and shoot apex. Similarly, β-glucuronidase (GUS) activity was detected in both leaves and roots of PGhAKT1::GUS transgenic Arabidopsis plants. In roots, the GUS signals appeared in the epidermis, cortex and endodermis and root hairs, suggesting the contribution of GhAKT1 to K+ uptake. In leaves, GhAKT1 was expressed in differentiated leaf primordial as well as mesophyll cells and veins of expanded leaves, pointing to its involvement in cell elongation and K+ transport and distribution in leaves. Severe K+ deficiency did not affect the expression of GhAKT1 gene. GhAKT1-overexpression in either the Arabidopsis wild-type or akt1 mutant enhanced the growth of transgenic seedlings under low K+ deficiency and raised the net K+ influx in roots at 100 μM external K+ concentration, within the range of operation of the high-affinity K+ uptake system. The application of 2 mM BaCl2 resulted in net K+ efflux in roots, and eliminated the differences between GhAKT1-overexpression lines and their acceptors indicating that the K+ uptake mediated by GhAKT1 is also as Ba2+-sensitive as AtAKT1. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Potassium (K) is the most important and abundant cation in living plant cells and plays crucial roles in many physiological and biochemical processes, including enzyme activation, membrane transport, anion neutralization, co-transport of sugars, and osmoregulation (Clarkson and Hanson, 1980). Potassium ion (K+) concentrations in soil usually range from 0.04 to 3%, but the worldwide distribution of K+ is inconsistent. In the tropics and subtropics, one-quarter of the soil experienced a deficiency of K+ (Munson, 1985). Moreover, the release of exchangeable K+ is often slower than the rate of K+ acquisition by plants and, consequently, K+ content in some soils is very low (Johnston, 2005).

Abbreviations: bp, base pair; CBL, calcineurin B-like proteins; cDNA, complementary DNA; CIPK, CBL-interacting protein kinase; cNMP, cyclic nucleotide binding domain; EST, expressed sequence tag; GFP, green fluorescent protein; GUS, β-glucoronidase; MS, Murashige–Skoog media; NCBI, National Center for Biotechnology Information; NMT, non-invasive ion flux measuring technique; ORF, open reading frame; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RNA, ribonucleic acid; RT-PCR, reverse transcription PCR; WT, wild type; X-Gluc, 5-bromo-4-chloro-3-indolyl-β-Dglucuronic acid. ⁎ Corresponding author. E-mail address: [email protected] (X. Tian).

http://dx.doi.org/10.1016/j.gene.2014.05.006 0378-1119/© 2014 Elsevier B.V. All rights reserved.

Plant K status may further deteriorate in the presence of high levels of other monovalent cations such as Na+ and NH+ 4 that interfere with K+ uptake (Qi and Spalding, 2004; Rus et al., 2004; Spalding et al., 1999). Cotton (Gossypium hirsutum L.) is more sensitive to low K+ availability than most other major field crops, and often shows signs of K deficiency on soils not considered deficient in K+ (Cassman et al., 1989). Widespread K deficiency in cotton has occurred in many countries (Oosterhuis, 1994; Tian et al., 2008), because of the negative K+ balance in the soil, adoption of modern cultivars characterized by faster fruit set and greater boll load (Oosterhuis, 1994), and popularization of transgenic Bt (Bacillus thuringiensis Berliner) cotton (Tian et al., 2008), which is more susceptible to K deficiency (Yang et al., 2011; Zhang et al., 2007). To ensure an adequate supply, plants have a number of redundant mechanisms for K+ acquisition and translocation (Kochian and Lucas, 1988; Maser et al., 2001; Véry and Sentenac, 2003). In the past twenty years, a large number of genes encoding plant K+ transporters and channels, such as the KT/KUP/HAK family, the HKT family, and the Shaker-like K+ channel family, particularly for Arabidopsis (Arabidopsis thaliana), have been characterized (Fu and Luan, 1998; Kim et al., 1998; Quintero and Blatt, 1997; Santa-María et al., 1997). These K+

62

J. Xu et al. / Gene 545 (2014) 61–71

transporters vary in K+ affinity, kinetics, transcriptional modulation, and regulatory mechanism, etc. (Gambale and Uozumi, 2006; Gierth and Mäser, 2007; Lebaudy et al., 2007), and compose a complex system for plant K+ uptake and translocation. Among these K+ transporters and channels, the KT/KUP/HAK family of K+ transporters is involved in high-affinity K+ uptake into the roots, and the Shaker-like K+ channel family has been shown to code for voltage-gated highly K+-selective channels active at the plasma membrane, which provide major pathways for wholesale K+ uptake or secretion in most tissues and cell types (Gambale and Uozumi, 2006; Véry and Sentenac, 2003). In 1992, the Arabidopsis Shaker-like K+ channels AKT1 were isolated (Sentenac et al., 1992). AtAKT1 channel has been shown to mediate K+ uptake within the K+ concentrations that correspond to the highand the low-affinity K+ uptake systems described by Epstein in 1963 (Epstein et al., 1963; Hirsch et al., 1998; Ivashikina et al., 2001; Lagarde et al., 1996; Spalding et al., 1999). AtAKT1 expression was preferentially localized in the peripheral cell layers of the root mature regions, which was consistent with a role of AtAKT1 in root K+ uptake (Lagarde et al., 1996). After the cloning of AtAKT1, several cDNAs encoding K+ channels with homology to AKT1 were obtained from other species, such as SKT1 from potato (Solanum tuberosum, Zimmermann et al., 1998), LKT1 from tomato (Solanum lycopersicum, Hartje et al., 2000), TaAKT1 from wheat (Triticum aestivum, Buschmann et al., 2000), OsAKT1 from rice (Oryza sativa, Golldack et al., 2003), ZMK1 from maize (Zea mays, Philippar et al., 1999), DKT1 from carrot (Daucus carota, Formentin et al., 2004), CaAKT1 from pepper (Capsicum annuum, Martinez-Cordero et al., 2005), NKT1 from tobacco (Nicotiana tabacum, Sano et al., 2007), and VvK1.1 from grapevine (Vitis vinifera, Cuéllar et al., 2010). Up to now, however, little is known about the function of K+ channels in cotton. In this study, we identified and characterized GhAKT1, a novel member of Shaker-like family, from the root of cotton cv. Liaomian17. The results would be beneficial for elucidating how cotton acquires K+ and developing K+ efficient cotton genotypes by using biotechnological approaches.

gene was obtained through the 5′-and 3′-rapid amplification of cDNA ends (RACE) following the user manual of SMART RACE cDNA amplification kit (Clontech, Mountain View, CA, USA) by using the cDNA of Liaomian17 as the template. The gene-specific primers were as follows: QA-L (5′-ATGTTTCGAGGGTCAGTACTAT-3′) and ZA-R (5′-TTAAGGGTTT TGGGTGTCATTA-3′). The PCR product was cloned into the pGEM-T easy vector (Promega) and then sequenced. Phylogenetic analysis was performed with clustalX version 1.83 (Thompson et al., 1997) and MEGA4 (Tamura et al., 2007) by the neighbor-joining method. The amino acid sequence was analyzed with the SMART program (Schultz et al., 1998). Putative transmembrane spans were predicted by the TMPRED server (http://www.ch.embnet.org/software/TMPRED_form.htmL). 2.3. Subcellular localization of the GhAKT1 protein The open reading frame (ORF) excluding a stop codon of GhAKT1 was amplified by using primers with Sac1 and XbaI restriction sites; the sequences of these primers were as follows: AYL (5′-CGAG CTCATGTTTCGAGGGTCAGTACTAT-3′) and AYR (5′-GCTCTAGAAGGGTT TTGGGTGTCATTA-3′). The constructs, including 35S-GFP (control) and 35S-GhAKT1-GFP were transformed into Arabidopsis. For GFP localization in cells, the roots of seven-day-old transgenic Arabidopsis plants were transferred onto glass slides, covered with slips, and observed under a confocal laser microscope (FV1000, Olympus, Japan). The tissue samples were soaked in 500 mM mannitol on glass slides for 10 min at room temperature to plasmolyze cells, and then observed for GFP signal under the same confocal laser microscope as above. 2.4. Isolation of the GhAKT1 promoter and promoter::GUS assay

Cotton (G. hirsutum L.), cv. Liaomian17, developed and provided by Cash Crops Research Institute, Liaoning Academy of Agricultural Sciences, China, was used in this study to isolate GhAKT1. Seeds were surface-sterilized with 9% H2O2 for 30 min, then germinated in sand and cultured in nutrient solutions with 12 h light/12 h dark at 30 ± 2/22 ± 2 °C as described in Wang et al. (2012). The Arabidopsis (A. thaliana) WT of the ecotype, Columbia was also used. The T-DNA insertion line akt1 (SALK_071803) was ordered from the Arabidopsis Biological Resource Center (http://www.arabidopsis. org/abrc/). Low K+ (50–100 μM) MS medium was prepared by modification of the normal MS medium containing 20 mM K+ (KH2PO4 replaced by NH4H2PO4 and partial KNO3 replaced by NH4NO3) as described in Xu et al. (2006). For seed harvest, Arabidopsis plants were grown in potting soil mixture (rich soil: vermiculite = 2:1, v/v) and kept in growth chambers at 22 °C with illumination at 120 μmol m−2 s−1 for a 16 h light period. The relative humidity was ~70% (±5%).

Genomic DNA was extracted from cotton roots by the CTAB method. To isolate the GhAKT1 promoter, an adaptor-ligated genomic library was constructed by ligating digested genomic DNA with adaptors from the Universal Genome Walker Kit (Clontech, Mountain View, CA, USA) according to manufacturer protocol. Primers designed to amplify putative promoter sequence were corresponding to the 5′-untranslated region (UTR) and upstream sequences of GhAKT1 gene. Two genespecific primers, AGSP1 (5′-CTGCTGCTCTTTTGGAAATGCTCTCTT-3′) and AGSP2 (5′-AGAGAGAGAGGAACCAAAGGCTTTACC-3′) were derived from the mRNA sequence and used for PCR-based DNA walking. After obtaining the putative promoter fragment (2187 bp), it was amplified by using the common downstream primer PAL (5′-TAATTTCTTTCTCA CCCCACATTGT-3′) and PAR (5′-GCCTATGTTTACTGCTTCTCTTTTG-3′). The PGhAKT1::GUS construct was generated by fusing the promoter of GhAKT1 in the front of the β-glucuronidase (GUS) coding sequence in pBGWFS7,0 vector via the Gateway system. The PLACE database (Higo et al., 1999) and PlantCARE (Lescot et al., 2002) were used for promoter nucleotide sequence analysis. For GUS staining, the transgenic plants were incubated overnight at 37 °C in 1 mg mL−1 of 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc), 5 mM potassium ferrocyanide, 0.03% Triton X-100, and 100 mM sodium phosphate buffer (pH 7.0). The tissues were cleaned with 70% ethanol (Lagarde et al., 1996), and then observed and photographed with a stereoscope (SZ-16, Olympus, Japan). For examination of the detailed GUS staining, the tissues were observed and photographed with a bright-field microscope.

2.2. Cloning and sequence analysis of GhAKT1 gene

2.5. Construction of vectors and transformation of Arabidopsis

To identify the cotton homologue of the Arabidopsis Shaker-like K+ channel, AKT1, the total RNA was isolated from roots of Liaomian17 which were grown hydroponically in 2.5 mM K+ or 30 μM K+, as described in Wang et al. (2012). The amino acid sequences of AtAKT1 were used as probes to screen the cotton (G. hirsutum) EST database in the GenBank. The candidate ESTs' sequences were subjected to contig analysis with the SeqMan program. The full-length sequence of GhAKT1

The 35S::GhAKT1 construct was generated by cloning the coding sequence of GhAKT1 into the binary vector, pBI121 under the control of the CaMV 35S promoter. The SUPER::GhAKT1 construct was generated by cloning the coding sequence of GhAKT1 into pBIB vector under control of the SUPER promoter (Li et al., 2001). To generate GhAKT1overexpression lines, Arabidopsis WT plants were transformed with the 35S::GhAKT1 construct, and akt1 mutant plants with a

2. Materials and methods 2.1. Plant materials, growth conditions and treatments

J. Xu et al. / Gene 545 (2014) 61–71

SUPER::GhAKT1 construct. The constructs of Arabidopsis transformation with an Agrobacterium (strain GV3101) was carried out by the floral dip method (Clough and Bent, 1998).

63

Tech. Corp., Amherst, MA, USA). All measurements were repeated at least three times independently. 3. Results

2.6. Quantitative Real-Time PCR analyses Total RNA was extracted from at least three seedlings of Arabidopsis or cotton using the RN38 EASY spin plus Plant RNA kit (Aidlab Biotech, Beijing, China). The first strand cDNA was synthesized with 2 μg of purified total RNA using the RT-PCR system (Promega, USA) according to the manufacturer's protocol. Real-Time quantitative RT-PCR was performed on a 7500 Real-Time PCR system (Applied Biosystems, USA) using SYBR® Premix Ex Taq™ (Perfect Real Time) (TaKaRa Code: DRR041A). The expression levels of GhAKT1 gene were calibrated by the expression of actin (AtACT, Arabidopsis) or ubiquitin (GhUBQ7, cotton) genes. The relative gene expression was calculated by the 2−ΔΔCt method (Livak and Schmittgen, 2001), using the cotton ubiquitin gene, UBQ7 (GenBank No. DQ116441), as an internal control, each sample being analyzed three times. The quantitative primer pairs were as follows: 5′-ACAATGGGGCAAACATCAAT-3′(forward) and 5′-CGCCAT AACGAACGATTTCT-3′ (reverse) for GhAKT1; 5′-AAGAAGAAGACCTACA CCAAGCC-3′(forward) and 5′-GCCCACACTTACCGCAATA-3′(reverse) for GhUBQ7 and 5′-GGCAAGTCATCACGATTGG-3′(forward) and 5′CAGCTTCCATTCCCACAAAC-3′(reverse) for AtACT. 2.7. Identification of transgenic phenotype For seedling phenotype assays, four-day-old seedlings grown on vertical normal MS plates were transferred onto either fresh normal or low K+ (50 or 100 μM) MS plates placed vertically. After seven days, a batch of seedlings was harvested to determine K+ content. The remaining seedlings were photographed several days later. For determination of K+ content, the shoots and roots of seedlings were separated and washed with ddH2O, and then dried at 80 °C for 2 d before weighing. The dried samples were ashed in a muffle furnace at 575 °C for 5 h and then dissolved in 0.1 M HCl. K+ was measured with an atomic absorption spectrophotometer (model Z-2000, Hitachi, Japan). All assays were repeated three times independently. Germination assays were performed with Arabidopsis WT, akt1 mutant, and their GhAKT1-overexpression lines to understand the biological function of GhAKT1. At least 60 seeds of each genotype were sterilized and sown on either normal or low K+ (50 or 100 μM) MS plates (0.9% agar and 3% sucrose, w/v, pH 5.8). The media were kept at 4 °C for 3 d and then incubated in a growth room at 21–22 °C. After germination for 7 d, the seeds that developed green cotyledons were recorded. 2.8. Measurement of net K+ flux with Noninvasive Micro-test Technique (NMT) Net fluxes of K+ in intact roots of Arabidopsis plants were measured noninvasively with the NMT system (BIO-001B, Younger USA Sci. and Tech. Corp., Amherst, MA, USA) as described in Chen et al. (2010) in Xuyue Sci. & Tech. Co. (Beijing) (http://www.xuyue.net). Five-day-old seedlings grown on vertical MS medium were transferred onto fresh MS medium or low K+ (100 μM) MS medium containing 2 mM CsCl for three days. Excessive Cs+ (exceeding 200 μM) in the rhizosphere can inhibit K+ uptake entirely, and thus induce K+ starvation in plants (Hampton et al., 2004; White and Broadley, 2000). After 10 min of balance in measuring buffer (0.1 mM KCl, 0.1 mM CaCl2, 0.3 mM MES, pH 6.0), the net K+ fluxes in the meristematic zone (about 120 μm from the root tip) of intact roots were measured in fresh measuring so2+ lution. 2 mM NH+ (in the form 4 (in the form of NH4NO3) or 2 mM Ba of BaCl2) were added to the measuring buffer as indicated. The ion flux was calculated using SIET software Mageflux (Younger USA Sci. and

3.1. Characterization of the GhAKT1 cDNA clone The cotton homologue of the Arabidopsis Shaker-like K+ channel, AKT1, a full-length cDNA of 2,628 bp (GenBank accession no. KF294166) was obtained. The deduced polypeptide of 875 amino acids share sequence homology with AtAKT1 and other members of group I of the plant Shaker-like K+ channel family (Fig. 1). The levels of amino-acid sequence identity showed 75.9% similarity to AKT1 from castor bean (Ricinus communis, accession no. EQ974133), and 70.8% to the Arabidopsis AKT1 sequence (Sentenac et al., 1992). Furthermore, the deduced polypeptide exhibited all of the structural features that are shared by plants inwardly rectifying K+ channels, such as six transmembrane domains (S1–S6) (Uozumi et al., 1998), S5 and S6 connected by the P-loop, the K+ selectivity sequence of TxxTxGYGD in the P-loop, a putative cyclic nucleotide binding domain (cNMP), and five ankyrin repeat sequences that are present only in the AKT1 subfamily of plant inwardly rectifying K+ channels (Sentenac et al., 1992; Fig. 2). We therefore designated this gene GhAKT1 (G. hirsutum AKT1-like). 3.2. GhAKT1 is localized at the plasma membrane of plant cells As shown in Fig. 3A, the fluorescence derived from GFP in the control experiments was distributed throughout the cell, including the nucleus, while the green fluorescence was found on the surface of root tip cells of GhAKT1-GFP transgenic Arabidopsis plants (Fig. 3B). Because root tip cells do not contain large central vacuoles, the green fluorescence on cell surface reflects GFP expression on the plasma membrane or cell wall but not on the tonoplast or cytoplasm. After being plasmolyzed by mannitol treatment, it was observed that the green fluorescence was detached slightly from the cell wall of root tip cells (Fig. 3C), indicating a plasma membrane rather than a cell wall localization of the GhAKT1-GFP fusion protein. 3.3. Expression pattern profile of the GhAKT1 gene The highest levels of GhAKT1 transcripts occurred in cotton leaves, and they were also detected in shoot apex, roots, and stem (Fig. 4). In addition, a total of 18 independent transgenic Arabidopsis lines carrying the GhAKT1 promoter-GUS gene fusion all displayed the same pattern of GUS staining. The GhAKT1 promoter drove GUS expression in both leaves and roots (Fig. 5). In leaves, GhAKT1 was mainly expressed in mesophyll cells and veins (Fig. 5B) as well as leaf trichomes (Fig. 5C), which are large unicellular hair-like structures that extend from the epidermis of aerial tissues (Szymanski and Marks, 1998). Strong staining was also observed in differentiated leaf primordia at an early stage before cell elongation and leaf expansion (Fig. 5D). In roots, GhAKT1 was expressed in both primary and lateral root tips as well as mature regions (Fig. 5E, F and G). At the periphery of mature root, an intense staining was present in root hairs (Fig. 5E). Furthermore, we found that GUS staining was preferentially located in epidermis (EP) of mature root; and staining in cortex (CO) and endodermis (EN) was weaker than in EP (Fig. 5G). GhAKT1 expression did not change in response to low K+ stress. For example, no differences in GhAKT1 transcript levels between cotton control plants (grown in solutions containing 2.5 mM K+) and K+-stressed plants (grown in solutions containing 30 μM K+ for 0–48 h) could be detected (Supplementary Fig. S1). Also, the tissue specificity of GhAKT1 expression indicated by GUS activity in transgenic Arabidopsis grown on low K+ (100 μM) MS medium for 1 d and 3 d did not change when compared with control plants grown on normal MS medium (Supplementary Fig. S2).

64

J. Xu et al. / Gene 545 (2014) 61–71

Fig. 1. Phylogenetic analysis of polypeptide Shaker-like potassium channel sequences. The phylogenetic tree was constructed with a set of 23 protein sequences comprising the whole family (nine members) of Shaker channels in Arabidopsis. Bootstrap values of N50 of 100 replicates are reported near the nodes of the protein phylogram. The branch length is proportional to the evolutionary distance between the channels.

3.4. Overexpression of GhAKT1 in Arabidopsis WT The three homozygous GhAKT1-overexpression Arabidopsis lines (A5, A37 and A49) were selected to analyze the transcript level of GhAKT1 and to observe their phenotypes (Fig. 6A). In addition, the seedlings of these three lines grown either on normal MS medium for 14 d or on low K+ (100 μM) MS medium for 30 d were obviously bigger than those of WT (Fig. 6B). And the 11-day-old transgenic lines had significantly greater root and shoot biomass than WT on normal MS medium (Fig. 6C, D). Unexpectedly, there were no differences in root or shoot K+ contents between transgenic lines and WT (Fig. 6E, F). Therefore, the greater K+ accumulation in three transgenic lines grown on MS medium (Fig. 6G) was attributed to their higher biomass.

two GhAKT1-overexpression lines. Nevertheless, a8 and a17 showed absolute superiority to akt1 and WT when grown on low K+ (100 μM) MS medium for 14 d (Fig. 7B). The differences in biomass of 11-day-old seedlings among genotypes were similar to those in their phenotype (Fig. 7C, D). With regard to K+ content and K+ accumulation, a8 and a17 (11-day-old) had greater or significantly greater values than akt1 in most situations (Fig. 7E–H), but the shoot K+ contents of a8 and a17 were significantly lower than that of akt1 mutant on low K+ MS medium (Fig. 7F), being in agreement with their more severe K deficiency symptom (leaf chlorosis) than akt1 (Fig. 7B). The possible reason for this phenomenon was the faster growth of transgenic plants and therefore K+ dilution effects. In addition, the K+ contents of a8 and a17 did not exceed those of WT regardless of tissue types and K+ levels in the medium (Fig. 7E, F).

3.5. Overexpression of GhAKT1 in Arabidopsis akt1 mutant The two GhAKT1-overexpression lines (a8 and a17, seven-day-old) of akt1 mutant were selected to evaluate the transcript levels of GhAKT1 (Fig. 7A). When grown on normal MS medium for 14 d, there were no significant differences in phenotype among WT, akt1 and its

3.6. GhAKT1-overexpression increased the net K+ influx in Arabidopsis roots under K+ starvation In the present study, NMT was used to measure the steady flux profiles of K+ in root meristematic zone (120 μm from the root tip) of

J. Xu et al. / Gene 545 (2014) 61–71

65

Fig. 2. Predicted amino acid sequence of GhAKT1. S1–S6 indicate the predicted six transmembrane domains; P denotes the pore forming region; the putative cyclic nucleotide binding site cNMP is underlined; and the ankyrin-like repeats A1 to A5 are boxed.

Arabidopsis WT, akt1 mutant, and their GhAKT1-overexpression lines (A5 and a8). Grown on normal MS medium containing 20 mM K+, all the four genotypes showed K+ efflux because of lower K+ level

(100 μM) in the measuring buffer. Under K+ deprivation created by low K+ (100 μM) and 2 mM CsCl in growth medium, however, the net K+ influx was induced across genotypes except akt1 mutant (Fig. 8).

Fig. 3. Subcellular localization of the GhAKT1-GFP fusion protein in transgenic Arabidopsis plants. (A) Localization of control 35S-GFP fluorescence in young root cells. (B) Localization of 35S-GhAKT1-GFP fluorescence in young root cells. (C) Localization of 35S-GhAKT1-GFP fluorescence in root tip cells plasmolyzed with 0.5 M mannitol.

66

J. Xu et al. / Gene 545 (2014) 61–71

that the K+ uptake mediated by GhAKT1 is also Ba2 +-sensitive like AtAKT1. 3.7. GhAKT1-overexpression enhanced the seed germination of akt1 mutant under low K+ condition The GhAKT1-overexpression in Arabidopsis WT background did not affect the seed germination irrespective of K+ levels in the medium. However, GhAKT1-overexpression rescued the seed germination of akt1 mutant on low K+ (50–100 μM) MS medium. As shown in Fig. 9, there were no differences in seed germination among WT, akt1 and its two GhAKT1 transgenic lines (a8 and a17) on MS medium. When the K+ level in the medium reduced to 50–100 μM, the germination rate of akt1 mutant significantly decreased to less than 50%, whereas a8 and a17 had almost 100% germination rates similar to WT (Fig. 9). Fig. 4. Real-Time relative-quantitative RT-PCR analysis of GhAKT1 in various tissues of cotton seedlings, including root (R), shoot apex (A), unexpanded leaves (UL), stem (S), the first true leaf (FTL), the second true leaf (STL), and the third true leaf (TTL). The expression of GhAKT1 was calculated relative to GhUBQ7 expression activity. Data represent the average of three independent seedlings ± standard deviation. Standard errors are shown as bars above the columns.

Moreover, it was observed that GhAKT1-overexpression significantly enhanced the net K+ influx. A5 had 69% greater net K+ influx than WT, and a8 about 20 pmol cm−2 s−1 greater than akt1 mutant, thus suggesting that GhAKT1 can operate at the high-affinity range of K+ concentration (100 μM in measuring solution). When the high-affinity uptake system was inhibited in the presence of 2 mM NH+ 4 , the net K+ influx significantly decreased (except in akt1), but the differences among genotypes did not change (Fig. 8). Lastly, the addition of one of standard AtAKT1 inhibitor, 2 mM BaCl2 to the measuring buffer not only resulted in the net K+ efflux in root meristematic zone, but also eliminated the differences among genotypes (Fig. 8), demonstrating

4. Discussion In the present study, we described the isolation and characterization of GhAKT1 from G. hirsutum. The amino acid sequence and predicted protein structure of GhAKT1 (Fig. 2) strongly suggest that it is a member of the AKT1 subfamily (Group I) of Shaker-like K+ channels family, and is localized in the cell plasma membrane. Similar to AtAKT1 (Ivashikina et al., 2001; Lagarde et al., 1996), the GhAKT1 is expressed in root epidermis, cortex, endodermis cells and root hairs (Figs. 4, 5), where they may have a nutritional role in K+ uptake from the soil solution (Dennison et al., 2001). In aerial parts, the GhAKT1 promoter activity and GhAKT1 transcripts were detected in leaf primordia of Arabidopsis and shoot apex of cotton, respectively, suggesting the involvement of this channel in cell elongation and leaf expansion as AtAKT1 (Lagarde et al., 1996; Lew, 1991). In addition, the GhAKT1 is expressed at rather high levels in the first- (9-d-old) and second-leaf (6-d-old) of cotton plants (Fig. 4); GUS activity driven by

Fig. 5. Histochemical staining of PGhAKT1::GUS transgenic Arabidopsis plants. (A) Aerial parts of a 2-week-old plantlet. (B) Magnification of a leaf. (C) Leaf trichomes with three branches. (D) Leaf primordia around the shoot apex. (E–G) Roots. (H) Cross-section of a mature root zone. CO, cortex; EN, endoderm; EP, epiderm.

J. Xu et al. / Gene 545 (2014) 61–71

67

Fig. 6. Phenotype assays of GhAKT1-overexpression in Arabidopsis wild-type (Columbia). WT, wild-type; A5, A37 and A49, the three independent transgenic lines in WT background. (A) Real-Time PCR analysis of WT and its GhAKT1-overexpression lines grown on MS medium for seven days. (B) Phenotype comparison between WT and its transgenic plants grown on normal MS medium (left) for 14 days and on LK (low K+; 100 μM) medium (right) for 30 days. (C–H) Show the dry mass, K+ content and accumulation in 11-day-old seedlings. (C) and (D) Comparison of shoot and root dry mass on MS medium and LK medium, respectively. (E) and (F) Comparison of shoot and root K+ content on MS medium and LK medium, respectively. (G) and (H) Comparison of shoot and root K+ accumulation on MS medium and LK medium, respectively. Data are shown as means ± SE (n = 4).

68

J. Xu et al. / Gene 545 (2014) 61–71

J. Xu et al. / Gene 545 (2014) 61–71

Fig. 8. Comparison of net K+ flux measured by Noninvasive Micro-test Technique (NMT) among Arabidopsis WT (wild-type), akt1 mutant, and their GhAKT1-overexpression transgenic lines A5 and a8, respectively. All genotypes were starved of K+ by growing on low K+ (100 μM) medium containing 2 mM CsCl for 3 d, and then transferred to measuring 2+ buffer with 100 μM K+ in the absence or presence of 2 mM NH+ as indicat4 or 2 mM Ba ed. Standard errors are shown as bars above the columns (n = 5–7). Different lowercases indicate significant differences at P b 0.05.

promoter of GhAKT1 was also strong in expanded leaves of Arabidopsis, virtually covering all the mesophyll cells and vascular bundles (Fig. 5). Therefore, it is speculated that GhAKT1 may have a general function in regulating K+ transport and distribution in leaves. This type of wider expression profile of GhAKT1 is different with its Arabidopsis homologue, AtAKT1 (Lagarde et al., 1996; Pilot et al., 2003; Szyroki et al., 2001). In fact, it has been established that the expression profiles of AtAKT1 homologues varied to some extent with different species. For example, another group I Shaker-like gene, OsAKT1 from rice has been detected in mesophyll cells and vascular cells (Golldack et al., 2003). VvK1.1, the grapevine counterpart of the Arabidopsis AKT1 channel, its transcripts were also detected in phloem tissues, both in roots and in berries (Cuéllar et al., 2010). In Arabidopsis, neither RNA blot nor microarray experiments revealed an alteration in AKT1 transcription in K+-starved plants (Hampton et al., 2004; Maathuis et al., 2003; Pilot et al., 2003), suggesting that the activation of AKT1 by low K+ probably occurs posttranscriptionally. Later study demonstrated that a protein kinase, AtCIPK23, interacting with two calcineurin B-like proteins (AtCBL1 and AtCBL9), directly phosphorylates AKT1 (Xu et al., 2006). In the present study, the severe low K+ stress (30 μM) lasting for 0–48 h did not impact the accumulation of GhAKT1 transcripts in cotton plants (Fig. S1); and no changes in GUS activity driven by GhAKT1 promoter (Fig. S2) were observed in transgenic Arabidopsis subjected to 100 μM K+ for 1 d or 3 d. Therefore, we infer that the regulation of GhAKT1 also occurs post-transcriptionally as described for AtAKT1 (Hirsch et al., 1998; Lagarde et al., 1996; Spalding et al., 1999). Noninvasive Micro-test Technique (NMT) has become a useful tool in plant physiology research (Li et al., 2010; Shabala et al., 2005; Sun et al., 2010; Yang et al., 2010). In this study, NMT technique was applied to investigate the function of GhAKT1 by measuring K+ flux profiles in the root meristematic zone of Arabidopsis. No K+ influx in akt1 mutant was observed at K+ concentration of 100 μM, which is in line with the study of Hirsch et al. (1998) who found that the disruption of AtAKT1 gene resulted in no inward K+ currents in root cells by patch-clamp recordings. Considering the increased net K+ influx of A5 line relative to its GhAKT1-overexpression acceptor WT, and a8 line relative to its

69

acceptor akt1 mutant at low external K+ concentration (100 μM), we suggest that GhAKT1 can mediate K+ uptake from very low concentrations, within the range of operation of the high-affinity K+ uptake system, as its Arabidopsis homologue AtAKT1 (Hirsch et al., 1998). Moreover, when high-affinity transporters were inhibited by 2 mM + NH+ 4 and thereby K channel functioning was isolated, A5 and a8 still had greater K+ influx than WT and akt1, confirming the involvement of the Shaker-like K+ channel GhAKT1 at the high-affinity range of K+ concentration (100 μM). Also, we observed that the net K+ efflux occurred with no differences among genotypes following 2 mM BaCl2 application, indicating the domination of K+ efflux channel after the inhibition of influx channel by Ba2+ (Szczerba et al., 2009). Because of Shaker-like K+ channels are built of 4 identical or different subunits (Dreyer et al., 1997; Lebaudy et al., 2007), we cannot exclude the possibility that the heteromeric assembly of AtAKT1 and GhAKT1 result in the elevated net K+ influx in GhAKT1-overexpression Arabidopsis. Generally, the greater K+ influx in Arabidopsis roots led to the greater shoot or root K+ content (Figs. 6, 7). However, there were some exceptions. For example, grown on low K+ (100 μM) MS medium for 30 d, the shoot K+ content of A5 line was similar to that of WT (Fig. 6) despite its greater K+ influx in roots than the latter (Fig. 8). This odd perhaps comes from the different ages between seedlings used for growth analysis and for K+ NMT (30 vs. 8 d). In addition, a8 line (eight-day-old) had greater K+ influx than akt1 mutant under K starvation. Conversely, when grown on low K+ (100 μM) MS medium for 10 d, its shoot K+ content was even lower than that of akt1 mutant (Fig. 7). This contradiction is due to the K+ dilution effect in a8 plants by its greater biomass compared with akt1 (Fig. 7), and also suggests that a8 line had higher internal K+ utilization efficiency (dry mass produced per unit of K concentration) relative to akt1 mutant (albeit the underlying mechanism remained unclear). Seed germination depends on cell expansion, which is driven by passive water uptake during seed imbibition (Bewley and Black, 1994). Accumulation of mineral nutrients into the developing seeds is thought to be a prerequisite for efficient germination and seedling establishment. For example, K+ can contribute to the osmotic potential in germinating seeds and hence to water uptake (Bewley and Black, 1994). After exhaustion of K+ reserves in seeds, seedlings must absorb K+ from the external medium to sustain growth. In this study, the germination rate of akt1 mutants was not impaired on MS medium (Fig. 9) as reported previously (Pyo et al., 2010), but its radicle could not protrude well on low K+ (50–100 μM) MS medium due to defect in K+ uptake. GhAKT1overexpression in akt1 mutant complemented its germination ability completely, suggesting that GhAKT1 is involved in K+ uptake during seed germination. In conclusion, this study characterized a Shaker-like K+ channel gene, GhAKT1, from cotton. It encodes a plasma membrane-localized protein, which acts as a K+ influx channel. These results shed light on the mechanism of K+ uptake in cotton plants, and provide a candidate gene for improving the K nutrition of cotton. Conflict of Interest None. Acknowledgments The research was supported by the Genetically Modified Organisms Breeding Major Projects of China Grant (2011ZX005-004). We thank Dr. Chuanqing Sun (China Agricultural University) for the plasmid

Fig. 7. Phenotype assays of GhAKT1-overexpression in Arabidopsis akt1 mutant. WT, wild-type; a8 and 17, the two independent transgenic lines in akt1 background. (A) Real-Time PCR analysis of WT, akt1 mutant and its GhAKT1-overexpression lines grown on MS medium for seven days. (B) Phenotype comparison between the WT, akt1 mutant and its transgenic plants grown on normal MS (left) and LK (low K+; 100 μM) medium (right) for 14 days. (C–H) Show the dry mass, K+ content and accumulation in 11-day-old seedlings. (C) and (D) Comparison of shoot and root dry mass on MS medium and LK medium, respectively. (E) and (F) Comparison of shoot and root K+ content on MS medium and LK medium, respectively. (G) and (H) Comparison of shoot and root K+ accumulation on MS medium and LK medium, respectively. Data are shown as means ± SE (n = 4).

70

J. Xu et al. / Gene 545 (2014) 61–71

Fig. 9. GhAKT1-overexpression in Arabidopsis akt1 mutant rescues the germination ability. (A) Seed germination rates of wild-type (WT), akt1 mutant and its transgenic lines a8 and s17 at different K+ concentrations. (B) Phenotype of seed germination at various K+ concentrations. The germination rate was scored based on cotyledon emergence after 7 d of transferring to 22 °C. Data are shown as means ± SE (n = 4).

containing GFP gene. We also thank Dr. Jingping Hua (China Agricultural University) for providing us with pBI121 vector. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.006. References Bewley, J.D., Black, M., 1994. Seeds: Physiology of Development and Germination. Plenum Press, New York. Buschmann, P.H., Vaidyanathan, R., Gassmann, W., Schroeder, J.I., 2000. Enhancement of Na+ uptake currents, time-dependent inward-rectifying K+ channel currents, and K+ channel transcripts by K+ starvation in wheat root cells. Plant Physiology 122, 1387–1397. Cassman, K.G., Bryant, D.C., Higashi, S.L., Roberts, B.A., Kerby, T.A., 1989. Soil potassium balance and cumulative cotton response to annual potassium additions on a vermiculitic soil. Soil Science Society of America Journal 53, 805–812. Chen, J., Xiao, Q., Wu, F.H., Dong, X.J., He, J.X., Pei, Z.M., Zheng, H.L., 2010. Nitric oxide enhances salt secretion and Na+ sequestration in a mangrove plant, Avicennia marina, through increasing the expression of H+-ATPase and Na+/H+ antiporter under high salinity. Tree Physiology 30, 1570–1585. Clarkson, D.T., Hanson, J.B., 1980. The mineral nutrition of higher plants. Annual Review of Plant Physiology 31, 239–298. Clough, S.J., Bent, A.F., 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal 16, 735–743. Cuéllar, T., Pascaud, F., Verdeil, J.L., Torregrosa, L., Adam-Blondon, A.F., Thibaud, J.B., Sentenac, H., Gaillard, I., 2010. A grapevine Shaker inward K+ channel activated by the calcineurin B-like calcium sensor 1-protein kinase CIPK23 network is expressed in grape berries under drought stress conditions. Plant Journal 51, 58–69. Dennison, K.L., Robertson, W.R., Lewis, B.D., Hirsch, R.E., Sussman, M.R., Spalding, E.P., 2001. Functions of AKT1 and AKT2 potassium channels determined by studies of single and double mutants of Arabidopsis. Plant Physiology 127, 1012–1019. Dreyer, I., Antunes, S., Hoshi, T., Muller-Rober, B., Palme, K., Pongs, O., Reintanz, B., Hedrich, R., 1997. Plant K+ channel alpha-subunits assemble indiscriminately. Biophysical Journal 72, 2143–2150. Epstein, E., Rains, D., Elzam, O., 1963. Resolution of dual mechanisms of potassium absorption by barley roots. Proceedings of the National Academy of Sciences of the United States of America 49, 684–692. Formentin, E., Varotto, S., Costa, A., Downey, P., Bregante, M., Naso, A., Picco, C., Gambale, F., Schiavo, F.L., 2004. DKT1, a novel K+ channel from carrot, forms functional heteromeric channels with KDC1. FEBS Letters 573, 61–67. Fu, H.H., Luan, S., 1998. AtKUP1: a dual-affinity K+ transporter from Arabidopsis. Plant Cell 10, 63–73.

Gambale, F., Uozumi, N., 2006. Properties of Shaker-type potassium channels in higher plants. Journal of Membrane Biology 210, 1–19. Gierth, M., Mäser, P., 2007. Potassium transporters in plants — involvement in K+ acquisition, redistribution and homeostasis. FEBS Letters 581, 2348–2356. Golldack, D., Quigley, F., Michalowski, C.B., Kamasani, U.R., Bohnert, H.J., 2003. Salinity stress-tolerant and -sensitive rice (Oryza sativa L.) regulate AKT1-type potassium channel transcripts differently. Plant Molecular Biology 51, 71–81. Hampton, C.R., Bowen, H.C., Broadley, M.R., Hammond, J.P., Mead, A., Payne, K.A., Pritchard, J. , White, P.J., 2004. Cesium toxicity in Arabidopsis. Plant Physiology 136, 3824–3837. Hartje, S., Zimmermann, S., Klonus, D., Mueller-Roeber, B., 2000. Functional characterisation of LKT1, a K+ uptake channel from tomato root hairs, and comparison with the closely related potato inwardly rectifying K+ channel SKT1 after expression in Xenopus oocytes. Planta 210, 723–731. Higo, K., Ugawa, Y., Iwamoto, M., Korenaga, T., 1999. Plant cis-acting regulatory DNA elements (PLACE) database:1999. Nucleic Acids Research 27, 297–300. Hirsch, R.E., Lewis, B.D., Spalding, E.P., Sussman, M.R., 1998. A role for the AKT1 potassium channel in plant nutrition. Science 280, 918–921. Ivashikina, N., Becker, D., Ache, P., Meyerhoff, O., Felle, H.H., Hedrich, R., 2001. K+ channel profile and electrical properties of Arabidopsis root hairs. FEBS Letters 508, 463–469. Johnston, A.E., 2005. Understanding Potassium and Its Use in Agriculture. EFMA, Brussels. Kim, E.J., Kwak, J.M., Uozumi, N., Schroeder, J.I., 1998. AtKUP1: an Arabidopsis gene encoding high affinity potassium transport activity. Plant Cell 10, 51–62. Kochian, L.V., Lucas, W.J., 1988. Potassium transport in plants. Advances in Botanical Research 15, 93–178. Lagarde, D., Basset, M., Lepetit, M., Conejero, G., Gaymard, F., Astruc, S., Grignon, C., 1996. Tissue-specific expression of Arabidopsis AKT1 gene is consistent with a role in K+ nutrition. Plant Journal 9, 195–203. Lebaudy, A., Véry, A.A., Sentenac, H., 2007. K+ channel activity in plants: genes, regulations and functions. FEBS Letters 581, 2357–2366. Lescot, M., Dehais, P., Thijs, G., Marchal, K., Moreau, Y., Peer, Y.V., Rouze, P., Rombauts, S., 2002. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Research 30, 325–327. Lew, R.R., 1991. Substrate regulation of single potassium and chloride ion channels in Arabidopsis plasma membrane. Plant Physiology 95, 642–647. Li, X., Gong, Z., Koiwa, H., Niu, X., Espartero, J., Zhu, X., Veronese, P., Ruggiero, B., Bressan, R.A., Weller, S.C., Hasegawa, P.M., 2001. Bar-expressing peppermint (Mentha × Piperita L. var. Black Mitcham) plants are highly resistant to the glufosinate herbicide Liberty. Molecular Breeding 8, 109–118. Li, Q., Li, B.H., Kronzucker, H.J., Shi, W.M., 2010. Root growth inhibition by NH+ 4 in Arabidopsis is mediated by the root tip and is linked to NH+ 4 efflux and GMPase activity. Plant, Cell & Environment 33, 1529–1542. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using RealTime quantitative PCR and the 2−ΔΔCt method. Methods 25, 402–408. Maathuis, F.M., Filatov, V., Herzyk, P., Krijger, C., Axelsen, K.B., Chen, S.X., Green, B.J., Li, Y., Madagan, K.L., Sanchez-Fernandez, R., Forde, B.G., Palmgren, M.G., Rea, P.A., Williams, L.E., Sanders, D., Amtmann, A., 2003. Transcriptome analysis of root transporters reveals participation of multiple gene families in the response to cation stress. Plant Journal 35, 675–692.

J. Xu et al. / Gene 545 (2014) 61–71 Martinez-Cordero, M.A., Martinez, V., Rubio, F., 2005. High-affinity K+ uptake in pepper plants. Journal of Experimental Botany 56, 1553–1562. Maser, P., Thomine, S., Schroeder, J.I., Ward, J.M., Hirschi, K., Sze, H., Talke, I.N., Amtmann, A., Maathuis, F.J.M., Sanders, D., 2001. Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiology 126, 1646–1667. Munson, R.D., 1985. Potassium in Agriculture. American Society of Agronomy, Madison, WI. Oosterhuis, D.M., 1994. A post-mortem of the disappointing yields in the 1993 Arkansas cotton crop. In: Oosterhuis, D.M. (Ed.), Proc. of the 1994 Cotton Research Meeting and Summaries of Cotton Research in Progress, Ark. Agri. Exp. Sta., Special Report, pp. 22–26. Philippar, K., Fuchs, I., Luthen, H., Hoth, S., Bauer, C.S., Haga, K., Thiel, G., Ljung, K., Sandberg, G., Bottger, M., Becker, D., Hedrlch, R., 1999. Auxin-induced K+ channel expression represents an essential step in coleoptile growth and gravitropism. Proceedings of the National Academy of Sciences of the United States of America 96, 12186–12191. Pilot, G., Gaymard, F., Mouline, K., Cherel, I., Sentenac, H., 2003. Regulated expression of Arabidopsis shaker K+ channel genes involved in K+ uptake and distribution in the plant. Plant Molecular Biology 51, 773–787. Pyo, Y.J., Gierth, M., Schroeder, J.I., Cho, M.H., 2010. High-affinity K+ transport in Arabidopsis: AtHAK5 and AKT1 are vital for seedling establishment and postgermination growth under low-potassium conditions. Plant Physiology 153, 863–875. Qi, Z., Spalding, E.P., 2004. Protection of plasma membrane K+ transport by the salt overly sensitive1 Na+–H+ antiporter during salinity stress. Plant Physiology 136, 2548–2555. Quintero, F.J., Blatt, M.R., 1997. A new family of K+ transporters from Arabidopsis that are conserved across phyla. FEBS Letters 415, 206–211. Rus, A., Lee, B., Munoz-Mayor, A., Sharkhuu, A., Miura, K., Zhu, J.K., Bressan, R.A., Hasegawa, P.M., 2004. AtHKT1 facilitates Na+ homeostasis and K+ nutrition in planta. Plant Physiology 136, 2500–2511. Sano, T., Becker, D., Ivashikina, N., Wegner, L.H., Zimmermann, U., Roelfsema, M.R.G., Nagata, T., Hedrich, R., 2007. Plant cells must pass K+ threshold to re-enter the cell cycle. Plant Journal 50, 401–413. Santa-María, G.E., Rubio, F., Dubcovsky, J., Rodriguez-Navarro, A., 1997. The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter. Plant Cell 9, 2281–2289. Schultz, J., Milpetz, F., Bork, P., Ponting, C.P., 1998. SMART, a simple modular architecture research tool, identification of signaling domains. PNAS 95, 5857–5864. Sentenac, H., Bonneaud, N., Minet, M., Lacroute, F., Salmon, J.M., Gaymard, F., Grignon, C., 1992. Cloning and expression in yeast of a plant potassium ion transport system. Science 256, 663–665. Shabala, L., Cuin, T.A., Newman, I.A., Shabala, S., 2005. Salinity-induced ion flux patterns from the excised roots of Arabidopsis sos mutants. Planta 222, 1041–1050. Spalding, E.P., Hirsch, R.E., Lewis, D.R., Qi, Z., Sussman, M.R., Lewis, B.D., 1999. Potassium uptake supporting plant growth in the absence of AKT1 channel activity inhibition by ammonium and stimulation by sodium. Journal of General Physiology 113, 909–918. Sun, J., Wang, M.J., Ding, M.Q., Deng, S.R., Liu, M.Q., Lu, C.F., Zhou, X.Y., Shen, X., Zheng, X.J., Zhang, Z.K., Song, J., Hu, Z.M., Xu, Y., Chen, S.L., 2010. H2O2 and cytosolic Ca2+ signals

71

triggered by the PM H+-coupled transport system mediate K+⁄Na+ homeostasis in NaCl-stressed Populus euphratica cells. Plant, Cell & Environment 33, 943–958. Szczerba, M.W., Britto, D.T., Kronzucker, H.J., 2009. K+ transport in plants, physiology and molecular biology. Journal of Plant Physiology 166, 447–466. Szymanski, D.B., Marks, M.D., 1998. GLABROUS1 overexpression and TRIPTYCHON alter the cell cycle and trichome cell fate in Arabidopsis. Plant Cell 10, 2047–2062. Szyroki, A., Ivashikina, N., Dietrich, P., Roelfsema, M.R., Ache, P., Reintanz, B., Deeken, R., Godde, M., Felle, H., Steinmeyer, R., Palme, K., Hedrich, R., 2001. KAT1 is not essential for stomatal opening. Proceedings of the National Academy of Sciences of the United States of America 98, 2917–2921. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24, 1596–1599. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882. Tian, X.L., Wang, G.W., Yang, F.Q., Yang, P.Z., Duan, L.S., Li, Z.H., 2008. Differences in tolerance to low-potassium supply among different types of cultivars in cotton (Gossypium hirsutum L.). Acta Agronomica Sinica 34, 1770–1780. Uozumi, N., Nakamura, T., Schroeder, J.I., Muto, S., 1998. Determination of transmembrane topology of an inward rectifying potassium channel from Arabidopsis thaliana based on functional expression in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 95, 9773–9778. Véry, A.A., Sentenac, H., 2003. Molecular mechanisms and regulation of K+ transport in higher plants. Annual Review of Plant Biology 54, 575–603. Wang, Y., Li, B., Du, M.W., Eneji, A.E., Wang, B.M., Duan, L.S., Li, Z.H., Tian, X.L., 2012. Mechanism of phytohormone involvement in feedback regulation of cotton leaf senescence induced by potassium deficiency. Journal of Experimental Botany 63, 5887–5901. White, P.J., Broadley, M.R., 2000. Tansley Review No. 113. Mechanisms of caesium uptake by plants. New Phytologist 147, 241–256. Xu, J., Li, H.D., Chen, L.Q., Wang, Y., Liu, L.L., He, L., Wu, W.H., 2006. A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell 125, 1347–1360. Yang, Y.Q., Qin, Y.X., Xie, C.G., Zhao, F.Y., Zhao, J.F., Liu, D.F., Chen, S.Y., Fuglsang, A.T., Palmgren, M.G., Schumaker, K.S., Deng, X.W., Guo, Y., 2010. The Arabidopsis chaperone J3 regulates the plasma membrane H+-ATPase through interaction with the PKS5 kinase. Plant Cell 22, 1313–1332. Yang, F.Q., Wang, G.W., Zhang, Z.Y., Eneji, A.E., Duan, L.S., Li, Z.H., Tian, X.L., 2011. Genotypic variations in potassium uptake and utilization in cotton. Journal of Plant Nutrition 34, 83–97. Zhang, Z.Y., Tian, X.L., Duan, L.S., Wang, B.M., He, Z.P., Li, Z.H., 2007. Differential responses of conventional and Bt-transgenic cotton to potassium deficiency. Journal of Plant Nutrition 30, 659–670. Zimmermann, S., Talke, I., Ehrhardt, T., Nast, G., Müller-Röber, B., 1998. Characterization of SKT1, an inwardly rectifying potassium channel from potato, by heterologous expression in insect cells. Plant Physiology 116, 879–890.