Functional and biochemical characterization of GmCLC1, a vacuolar chloride channel from soybean (Glycine max L. Merr.)

Table of contents

1 Introduction
1.1 Chloride channels
1.2 AtCLCa, the workhorse in plant sciences
1.3 Soybean production on saline soils
1.4 GmCLC1 confers salinity tolerance
1.5 Objectives of this project

2 Methods and Materials
2.1 BY-2 cell studies
2.1.1 BY-2 cell lines
2.1.2 Cultivation of BY-2 callus culture
2.1.3 BY-2 cell suspension culture
2.1.4 Treatment of samples
2.1.4.1 Trypan blue for viability studies
2.1.4.2 6-CFDA for vacuolar pH studies
2.1.5 Microscopy
2.1.5.1 Trypan blue by light microscopy
2.1.5.2 6-CFDA by confocal microscopy
2.1.6 Result analysis
2.1.6.1 Trypan blue
2.1.6.2 6-CFDA
2.2 Transgenic Arabidopsis thaliana
2.2.1 Cultivation of Arabidopsis thaliana
2.2.2 Preparation of Agrobacterium tumefaciens
2.2.2.1 GmCLC1-V7 binary vector
2.2.2.2 Transformation and selection of transformed E. coli
2.2.2.3 Plasmid verification by Polymerase Chain Reaction (PCR)
2.2.2.4 Plasmid purification
2.2.2.5 Transformation of A. tumefaciens with GmCLC1-V7
2.2.3 Preparation of the Agrobacterium infiltration medium
2.2.4 Vacuum infiltration of A. thaliana
2.2.5 Harvest and selection of A. thaliana seeds
2.2.6 Propagation of A. thaliana
2.2.7 Collection of plant samples
2.2.8 Preparation of the samples
2.2.9 Measuring the NO3- and Cl- concentrations by colorimetric methods
2.2.9.1 Calibration curves
2.2.9.2 Samples’ NO3- and Cl- concentrations
2.2.9.3 Statistical analysis

3 Results
3.1 Influence of pH on GmCLC1
3.1.1 Viability assay of BY-2 cells
3.1.2 Vacuolar pH of BY-2 cells
3.2 Complementation studies of GmCLC1 and AtCLCa
3.2.1 Transformation of A. thaliana
3.2.1.1 Selection of A. tumefaciens carrying GmCLC1-V7
3.2.1.2 Infiltration of A. thaliana with Agrobacterium
3.2.1.3 Selection of transformed Arabidopsis seeds
3.2.2 Analysis of nitrate and chloride concentrations
3.2.2.1 Analyzed plants
3.2.2.2 NO3- and Cl- calibration curves
3.2.2.3 NO3- and Cl- in Arabidopsis samples

4 Discussion
4.1 GmCLC1 is pH dependent
4.2 Cl- substitutes NO3- in AtCLCa knock-down mutant

5 References

6 Appendix
I. List of Abbreviations
II. Chemicals
III. Laboratory Equipment
IV. Growth Media
V. AtCLCa expression in A. thaliana mutant lines
VI. AtCLCa and GmCLC1 sequences’ similarity
VII. Software

1 Introduction

1.1 Chloride channels

Chloride channels (CLCs) build up a protein family with orthologs that can be found ubiqui­tously in all phyla. The first protein of the CLC family being discovered was CLC-0 by Miller and White in 1980 [1] who studied the electric organ of the marine ray Torpedo marmorata. With the successful cloning and structural analysis of CLC-0, done by Thomas J. Jentsch [2] in 1990, the scientific community’s interest in chloride channels grew. 20 years later, the knowl­edge of CLCs has dramatically increased and several important discoveries unravelled that chloride channels, all identified by “a complex transmembrane transport domain and a soluble regulatory domain” [3], are strongly varying in their cellular location, substrate specificity and function - even within the same species. For example, the well studied model plant Arabidop­sis thaliana carries genes for 7 different CLCs (AtCLCa – AtCLCg) in its genome [4]. While AtCLCa [5] and AtCLCb [6] are located on the tonoplast [5], AtCLCe was localized in the thylakoid mem­brane [4], AtCLCd in the trans -Golgi network [7], and AtCLCf in the cis -Golgy vesicles [4]. Some CLCs’ substrate is chloride, but also specificity for nitrate is reported: AtCLCa has even a higher affinity to nitrate than to chloride [5]. These differences in CLCs’ occurrence and substrate specificity come along with a diversity of functions they execute. In plants, turgor maintenance [5, 8], stomatal movement [9, 10] and nutrient storage [5, 11] are just a few examples. Besides their diverse physiological functions, CLCs also differ in their biochemical function and mode of action.

1.2 AtCLCa, the workhorse in plant sciences

Among plant chloride channels the probably most intensively studied one is AtCLCa from Arabidopsis thaliana. Being one of seven chloride channels found in Arabidopsis, AtCLCa is proofed to be located in the tonoplast [5]. Achieved was this discovery by harnessing AtCLCa–GFP (Green Fluorescent Protein) fusion proteins which were transiently expressed in protoplasts from Arabidopsis cell suspensions. Confocal microscopy studies and protein immunodetection by specific antibodies confirmed AtCLCa’s tonoplast localization [5]. There it functions as a nitrate/proton antiporter which sequesters NO3- from the cytosol into the vacuole [5].

AtCLCa is a NO₃^-/H⁺ antiporter

Patch clamp studies of Arabidopsis vacuoles demonstrated AtCLCa’s antiporter mechanism. Two NO3- ions were transported from the cytosol into the vacuolar lumen by pumping one H+ in the opposite direction. In contrast to other CLCs with anion/proton antiporter function [12 – 14], AtCLCa shows a higher affinity for nitrate than for chloride. Studies with site directed mutated AtCLCa proteins (E203A) revealed that a glutamate residue at position 203 of the primary structure is important for substrate specificity [15]. Another conserved residue in CLCs is suggested to be of importance for the mode of action. Like AtCLCa, human hCLC4 [13] and bacterial EcCLC-1 [12] were found to transport anions as an anion/proton antiporter. Comparison of the anion/proton antiporter’s sequences with the channels’ sequences showed that a glutamate or a valine residue, respectively, are present after helix H of the tertiary structure [16] (see Figure 1).

Abbildung in dieser Leseprobe nicht enthalten

Figure 1: Chloride channels of different species and their conserved critical amino acid residues. The anion/proton antiporter of the CLC family have a glutamate residue at the position equivalent to E203 of EcCLC-1. In contrast, valine at the same position is responsible for the mode of action as a channel.

The influence of pH on AtCLCa and other CLCs

AtCLCa’s antiporter mechanism was unravelled by patch clamp studies of Arabidopsis vacuoles [5]. In other experiments, AtCLCa was expressed in Xenopus oocytes and NO3- and Cl- were solved in solutions of different pH and applied extracellular [15]. Higher pH resulted in higher nitrate and chloride flux, which can be explained by the antiporter function. Protons are pumped across the oocytes’ membrane into the extracellular lumen, while anions are pumped into the cytosol. When the concentration of H+ in the extracellular lumen is high already, CLC mediated transport is slowed down [15].

Studies on CLCs functioning as a channel came to inconsistent results. Mammalian CLC-2 opened at acidic extracellular pH [17]. This is in line with findings about CLC-0 which was activated by lowering extracellular pH [18]. In contrast, patch clamp studies with CLC-K1 from rat kidney displayed a reduced chloride current when extracellular pH was lowered [19].

As recent studies showed, low pH leads to an uncoupling of the Cl-/H+ antiporter Ad‑CLC‑3. This uncoupling resulted in a completely chloride dependent current. The authors say “at pH 4.0 ClC-3 behaves as an anion-selective channel” and suggest Ad‑CLC‑3 to have a regulating function on the pH in acidic intracellular compartments [20].

1.3 Soybean production on saline soils

The world production of soybeans was over 220 million tonnes in 2007 [21] and soybean is of exceptional importance in the production of oil, protein rich food and cattle feed. However, in many areas of the world the production of soybeans is negatively effected by insufficient natural precipitation which often is tried to overcome by irrigation. When water is applied in dry areas where the evaporation exceeds the run-off, ions solved in the water remain on the soil where they accumulate and finally lead to salinity induced stress in the growing plants [22]. Soybean is a moderately NaCl tolerant plant [23] that’s protein content is negatively influenced by the salt [24]. Alleviating this negative influence and enabling the plant to grow and in a final stage develop seeds - the beans human desire - under conditions of high salinity is of great economic and humanitarian interest.

1.4 GmCLC1 confers salinity tolerance

Salinity stress of plants has been extensively studied in the model plant Arabidopsis thaliana and led to the identification of several genes related to increased tolerance. Most of these genes play a major role in Na+ transport, such as AtSOS1 [25], AtNHX [26] and AtHKT1;1 [27]. Also in soybean, a NHX (GmNHX1) was cloned and its biochemical proper­ties analyzed and found to have positive effects on salinity stress [8]. Potassium’s counter ion Cl- is far less well studied and the research focus lies on proteins of the chloride channel family. In Arabidopsis are 7 members of the CLC family known, whereas in soybean only one such protein is identified yet: GmCLC1 [8]. This was achieved by Li et al. in 2006, who created transgenic GmCLC1-YFP (Yellow Fluorescent Protein) tobacco Bright Yellow (BY)-2 cell lines and found GmCLC1 located in the tonoplast. Moreover, the protein conferred salinity tolerance to the transgenic BY-2 cells by sequestering Cl- from the cytosol into the vacuole. Little is yet known about GmCLC1’s function in the in planta level and its biochemical properties. Comparison of the protein sequence with other CLCs showed that is has a glutamate residue at the similar position as CLC orthologs known for their anion/proton antiporter function (see Figure 1). With AtCLCa, the best studied plant CLC – that is also located in the tonoplast – which was identified to have a higher affinity to nitrate than to chloride and to function as a NO3-/H+ antiporter [5], the soybean chloride channel shares 79 % of its sequence (see Appendix VI.). Therefore, in this project studies were conducted of GmCLC1 in an approach to find similar functions as are reported for AtCLCa.

1.5 Objectives of this project

In this research project I was working on two different approaches to identify the functional and biochemical properties of GmCLC1:

(i) Complementation studies of GmCLC1 transgenic Arabidopsis AtCLCa knock-down mutants to see whether GmCLC1 transports nitrate and/or chloride
(ii) The influence of pH on the function of GmCLC1 in transgenic BY-2 cells
under salt (NaCl) stress

For the complementation studies, three Arabidopsis thaliana mutants showing suppressed AtCLCa expression were ordered (Appendix V.). Together with a wild type line (Col-0), the plants’ chloride and nitrate concentration was measured by colorimetric methods. As AtCLCa is knocked down, diminished concentrations of nitrate, for which’s storage in the vacuole AtCLCa is known [5], were expected. As AtCLCa’s selectivity for nitrate is higher than for chloride, the Cl- concentration of the mutant lines compared to the WT line was expected to be equal or only slightly reduced. All four Arabidopsis lines were transformed with GmCLC1 by the aid of Agrobacterium tumefaciens in a vacuum infiltration process [28], which became a standard method in the work with Arabidopsis. Successfully transformed organisms were screened on MS medium containing 0.5 % kanamycin, as coupling of GmCLC1 to a gene for kanamycin resistance introduced this marker. Measurements of the NO3- and Cl- concentrations in the GmCLC1 transgenic Arabidopsis lines (three AtCLCa knock-down mutants and the WT line) were expected to show significantly altered concentrations of nitrate and/or chloride. Until now, only the chloride transport of GmCLC1 is reported [8], but nothing was published about its transport of nitrate. Therefore, the transgenic plants’ chloride concentration was expected to be increased. Nitrate concentrations could either be similar or increased.

Bright Yellow (BY)-2 cells from Nicotiana tabacum, a wild type and several transgenic lines constitutively expressing GmCLC1, were treated with 100 or 125 mM NaCl and analyzed with two different methods: light microscope studies of trypan blue, a dye utilized for viability assays; and confocal microscope studies of the pH sensitive fluorescent dye 6-CFDA.

In the viability assay, BY-2 cells were treated for 24 h with 100 or 125 mM NaCl at different pH (3.7, 5, and 6.3). The cells were stained with trypan blue [28], a dye that only can penetrate dead cells and thus made it possible to distinguish living (uncoloured) from dead (bluish coloured) cells under a light microscope. In case that GmCLC1 functions as an anion/proton antiporter, the cells treated at high pH were expected to show greater survival rates as a result from the cells’ capability of sequestering chloride from the cytosol into the vacuole [8]. At low pH the antiporter might be uncoupled [20] and chloride transport into the vacuole stops after equilibrium between cytosol and vacuole is reached. This would result in toxic cytosolic chloride concentrations.

Stained with the fluorescent dye 6-CFDA, BY-2 cells were treated with 100 mM or 125 mM NaCl solution for 1 h. Then, under a confocal microscope, the cells were excited with 458 and 488 nm and the emission of both was measured. After converting the emission into a greyscale, the ratio of 488 and 458 nm was calculated, indicating the vacuolar pH. In another experiment a real time measurement was conducted in which the emission at t = 0‑5 min after application of 100 mM NaCl was recorded. Compared to WT cells, GmCLC1- transgenic cells would show a lower vacuolar pH if GmCLC1 pumps H+ from the vacuole into the cytosol while Cl- is pumped in the other direction.

2 Methods and Materials

2.1 BY-2 cell studies

2.1.1 BY-2 cell lines

Tested in this experiment were a wild type line and the transgenic lines C1.169 and C218 of BY-2 cells from Nicotiana tabacum. PCR and northern blotting were performed prior to this experiment, and verified that the transgenic line carried and expressed the gene GmCLC1 from soybean. The cell lines were available in the laboratory and used for a viability assay and the measurement of the vacuolar pH concentration.

2.1.2 Cultivation of BY-2 callus culture

For easy storage and handling, the BY-2 cell lines were cultivated as persistent callus culture, growing on MS + 50 mg * ml-1 kanamycin and 1.5 g * 500 ml-1 gelrite plates at 25 °C in darkness. With the callus’ growth their size exceeded the plates’ space and the calluses were subcultured under sterile conditions every 7‑14 days.

2.1.3 BY-2 cell suspension culture

Calluses from the cell lines were used as inoculum of suspension cultures which were raised as the starting material for further treatment and tests.

A piece of callus, about the size of 1 cm³, was added to a flask containing 10 ml MS medium. By pipetting up and down with the help of an electric pipette, the piece was chopped down, ideally to single cells. These suspension cultures were incubated in darkness for 3 d at room temperature and 110 rpm. Then the solution was poured in flasks containing 40 ml fresh MS medium and incubated under the same conditions as before for another 3‑4 days.

All steps of raising the suspension culture and handling it until the collection of cells for microscopic analysis were performed under sterile conditions in an air flow clean bench. Flasks (Erlenmeyer flasks) with a capacity of 250 ml and 10 ml were used, depending on the volume which was handled. To avoid contaminations, flasks were covered with double layered aluminium foil and plates were sealed with Parafilm®.

2.1.4 Treatment of samples

Depending on the test which was performed (viability assay or vacuolar pH studies), the suspension cultures needed a different treatment.

2.1.4.1 Trypan blue for viability studies

The 50 ml suspension culture was poured into a 50 ml falcon tube and time given for the cells to settle down. From the solution, the upper 40 ml were discarded and 10 ml cell culture suspension remained, from which 1 ml was given into two flasks, one with 9 ml MS solution (pH 6.3 by TRIS buffer) as negative control and the other with 9 ml MS solution (pH 6.3 by TRIS buffer) + 100 (or 125) mM NaCl. Afterwards, the cells were incubated at room temperature for 24 h in darkness and 150 rpm. From these solutions were 100 µl transferred into a tube, 100 µl trypan blue (0.4 %) dye added, and stained for 15 min [28].

2.1.4.2 6-CFDA for vacuolar pH studies

Staining was performed according to the supplier’s instructions; incubation time and amount of dye were adjusted. Cells stained with the fluorescent dye were allowed to settle down twice prior to the microscopic measurement. That procedure resulted in samples with higher amounts of living cells, by this accelerating the work with the light sensitive dye and ensuring consistent results. See Figure 2 for the detailed steps.

Abbildung in dieser Leseprobe nicht enthalten

Figure 2. Procedures for the treatment of BY-2 cells for vacuolar pH measurements. Explanations of the single steps are presented from 1 – 7. [Own figure with pictures from http://www.openclipart.org/user-detail/olagosta]

2.1.5 Microscopy

20 µl of the samples were given on microscopic glass slides for the microscopic measurements.

2.1.5.1 Trypan blue by light microscopy

Fields on the slides were selected at random in the light microscope. In average about 10 cells were in one field, 11-13 fields from every sample were photographed.

2.1.5.2 6-CFDA by confocal microscopy

Two different wavelengths, 488 nm (green yellow) and 458 nm (red), were sequentially used to excite the samples, and the emission of 500 – 530 nm was measured. Cells were zoomed 20 x with an oil lens, typically a picture showed 5-7 Cells. 10-13 pictures from every sample were taken. Each picture consisted of three individual ones, one with normal light, one with 458 nm excitation and one with 488 nm excitation. As light bleached the fluorescent dye, all measurements were consequently performed rapidly.

2.1.6 Result analysis

The pictures taken with the microscope’s camera were analyzed in different ways depending on the test approaches’ requirements. For the viability assay the survival rates, and for the pH studies the vacuolar pH represented by fluorescent emission ratios, were measured. The resulting data was reviewed with a one-way ANOVA and Tukey’s range test as post-hoc test.

2.1.6.1 Trypan blue

Straight away from the shot pictures, the living and dead cells were counted, indicated by colourless (unstained) or bluish (stained) appearance, respectively, and summed up for every sample. The living cells were divided by the total amount of cells to receive the survival rate. Results of the transgenic lines were paired to strengthen the role of GmCLC1 in the assay, and compared with the wild type.

2.1.6.2 6-CFDA

Not every cell on the pictures was analysed. Instead, they were only selected if they did not overlap with other cells. Before the pictures of the emission strength at 458 nm and 488 nm irradiation could be compared, they first were converted to a greyscale by using Adobe Photoshop® where the red and green intensity values correspond directly to greyscale values. WCIF Image J was then utilized to measure the cells’ brightness at 256 different intensities (8 bit), ranging from black to white. For this, initially the non-specific background was measured and afterwards deducted from every cell’s data. The brightness was determined for every cell and the ratio of their data from pictures of 458 nm and 488 nm excitation calculated. To alleviate the identification of the cells, their surface area was measured.

2.2 Transgenic Arabidopsis thaliana

Seeds of the Arabidopsis thaliana ecotype Columbia (Col-0) and three AtCLCa knock down mutants (561H12 and 624E03.7 and 624E03.11) were available in the laboratory and used in this study.

2.2.1 Cultivation of Arabidopsis thaliana

Arabidopsis thaliana seeds germinated on MS medium and were transferred to potting soil after about 10 days. Growing seeds on a medium such as MS bears the risk of contaminations with bacteria and fungi. Preventing this made working under sterile conditions in an air flow clean bench necessary. During the plants’ development were water and nutrients supplied continuously.

Seeds were sterilized in an air flow clean bench according to standard laboratory protocol. 1 ml Clorox® was added to about 25‑50 seeds in an Eppendorf tube. 3 min of vigorous shaking were followed by 1 min thoroughly washing with 1 ml autoclaved milliQ water. This step was repeated three times. Stored in 250‑500 ml milliQ water (depending on the amount of seeds) the seeds’ dormancy was broken after 3 d of stratification at 4 °C and darkness. After that, again under sterile conditions, disposable glass dropper were used to plate the seeds on squared plates with MS-medium. Aligned in two columns, 10‑12 seeds per line were plated on the medium. Avoidance of contaminations and maintenance of high humidity within the plates were achieved by wrapping them with Parafilm®. The plates were stored in a vertical position in a growth chamber with a constant temperature of 22 °C and 18 h light per day.

Arabidopsis’ seeds germinated soon and after 8-10 days the seedlings had reached a root length of about 5 cm and were transferred onto autoclaved potting soil in ordinary plastic cups, where they continued growing without limiting space. A forceps was used to carefully transfer the fragile plants from the plate to soil. Three seedlings were planted in every cup. On trays with 24 cups and covered with shrinkage foil to maintain humidity in the plants’ microclimate for the first days, the young plants were raised at 20 °C and exposed to 9 h light per day. The short illumination period let Arabidopsis develop a long shoot with many flower buds. Sub-irrigation was applied when necessary and the commercial fertilizer Hyponex® was added for several times.