Amino acids
4.2. METHODS 4. EXPERIMENTAL
4.2.1.3 In vitro culture of A. thaliana seedlings
Sterilized Arabidopsis seeds were germinated and grown on solid MSAR medium (Koncz et al., 1994, section4.1.8.5), under short day conditions at 20 °C using 200 µE·m−2·s−1 to 400 µE·m−2·s−1 of irradiation energy.
4.2.1.4 Measurement of root length
For measuring the root length, sterilized Arabidopsis seeds were germinated in vertically-positioned squared petri dishes containing MSAR solid medium in the culture room. The germination rate was recorded and the root growth was measured by the help of a piece of grid paper every day.
4.2.1.5 Selection of Arabidopsis transformants
T1 seeds were sterilized in RNeasy Mini Spin columns (QIAGEN) as described in section4.2.1.2 and germinated either on solid MSAR medium containing the appropriate antibiotics and a mixture of cefotaxime and ticarcillin to avoid Agrobacterium growth, or directly on soil for selection by Basta™ solution (250 mg/ml of phosphinothricin; 0,1 % Tween-20). Antibiotic-resistant transformants were transferred first onto non-selective sterile medium, and subsequently into soil.
T1 plants were cultivated in the greenhouse in order to set seeds. T2 seeds from several primary transformants were germinated in sterile conditions on MSAR medium containing ap-propriate antibiotics for selection. Seedlings from those T2 lines showing a 3:1 segregation of resistant:sensitive progeny were grown and screened for homozygous T3 offspring.
4.2.1.6 Crosses of Arabidopsis plants
To perform crosses of Arabidopsis plants, open flowers and siliques were removed from the selected stem after bolting. Three to four closed flowers were emasculated with a pair of tweezers by removing petals, sepals and immature anthers. Pollen from a selected mature plant was used to dust the stigma. Seed maturation was monitored regularly and fully grown siliques were collected and dried.
4.2.1.7 Sub-culture of Arabidopsis cell suspensions
Every 7 days, 10 ml to 15 ml of cell suspension were taken from 50 ml 7 day-old Arabidopsis thaliana (ecotype Col-0) cell suspension cultures and added to 35 ml to 40 ml of fresh liquid medium (Mathur and Koncz,1998, section4.1.8.3). Cells were grown under constant agitation with 120 rpm to 150 rpm at 18 °C to 22 °C. Photosynthetic light-grown cells suspensions were maintained under 200 µE·m−2·s−1 to 400 µE·m−2·s−1 of irradiation energy.
4.2.1.8 Histochemical assay of β-glucuronidase (uidA) reporter enzyme activity in planta Freshly harvested seedlings or plant organs were immersed in X-Gluc solution and vacuum treated for 3 min in an desiccator, followed by incubation at 37 °C of 2 to 16 hours. The stained tissue was washed 4× in 70 % ethanol to remove chlorophylls (Jefferson et al.,1987).
X-Gluc solution:
X-Gluc (50 mg/ml in DMSO) 200 µl/ml
Triton X-100 0,1 %
Phosphate (Na) buffer (pH 7,0) 0,1 M
K3Fe(CN)6 0,5 mM
K4Fe(CN)6 0,5 mM
When staining Arabidopsis embryos, siliques of different ages were removed form the plant, opened with a needle under a stereo-microscope. Immature seeds were isolated, fixed in acetone for 30 min on ice and subsequently washed with 0,2 M Na-phosphate buffer. Seeds were stained overnight at 37 °C submerged in X-Gluc solution. The next day, seeds were washed in 0,2 M Na-phosphate buffer, mounted on a microscope slide and cleared with freshly-prepared chloral hydrate solution on a glass slide for at least 1 h. GUS-stained embryos were inspected under the light microscope.
Chloral hydrate solution:
chloral hydrate 2,5 g
H2O 0,7 ml
glycerol 0,3 ml
4.2.2 DNA handling methods
4.2.2.1 DNA extraction from plant material
Plant DNA isolation was performed using a modified method fromKoncz and Schell(2002). Two to four leaves (50–200 mg) were ground for 3 min in presence of 750 µl of freshly-made DNA extraction buffer in a 2 ml eppendorf tube containing 3 small steel spheres using a TissueLyser (QIAGEN) at maximal speed. The tubes were briefly centrifuged and 250 µl of 5 M potassium acetate (pH 6,0) was added. The samples were pelleted by centrifugation for 10 min, the cleared supernatant (1 ml) was transferred to a clean eppendorf tube and 700 µl of iso-propanol was added to precipitate the DNA for 20 min at room temperature. The DNA pellet was washed with 70 % ethanol (section4.2.2.7) and the ethanol was completely removed. The DNA was dissolved in 400 µl of HTE solution and incubated following by addition of 100 µl of RNase A solution for 30 min at 37 °C. Subsequently, 100 µl of pre-digested proteinase K solution was added and the DNA sample was incubated for 1 h at 37 °C. DNA was further purified extracting twice with 600 µl of phenol/chloroform/iso-amyl-alcohol (25/24/1) on ice (section4.2.2.6). Upon addition of
1⁄10volumes of 3 M sodium acetate (pH 6,0), DNA was precipitated with 400 µl of iso-propanol for 30 min at 0 °C. DNA was pelleted by centrifugation for 10 min at 13 000 rpm, washed with 1 ml of 70 % ethanol and dried. Purified DNA was finally dissolved in 50 µl of LTE solution.
4.2. METHODS 4. EXPERIMENTAL
DNA extraction buffer:
Tris-HCl (pH 8,5) 200 mM
EDTA 50 mM
NaCl 500 mM
β-mercaptoethanol 14 mM
SDS 1 %
HTE solution:
Tris-HCl (pH 8) 100 mM
EDTA 50 mM
LTE solution:
Tris-HCl (pH 7,5) 10 mM
EDTA 1 mM
RNase A solution (in LTE, boiled for 10 min and kept on ice until needed):
RNase A 10 mg/ml
Proteinase K solution (in LTE, pre-digested at 37 °C for 10 min and used immediately):
Proteinase K 2 mg/ml
4.2.2.2 Plant genotyping by PCR
TaKaRa LA Taq™ polymerase (TaKaRa Bio Europe) was used for diagnostic amplification of plant DNA samples, in order to verify the presence of studied trans-gene or T-DNA insertion. A master mix was prepared for multiple PCR reactions, using the following components:
10× LA PCR™ Buffer Ⅱ (Mg2+ free) 1 µl
25 mM MgCl2 1 µl
10 mM dNTP mixture 1,6 µl
Primer mix (10 µM each) 2 µl
Template DNA 1–2 µl
TaKaRa LA Taq™ (5 U/µl) 0,1 µl
Autoclaved distilled water up to 10 µl
A Bio-Rad DNA Engine Tetrad®2 cycler was used for PCR amplification. The reactions were performed in 10 µl final volume using the following program:
1. Denaturation: 94 °C for 5 min 2. Denaturation: 94 °C for 30 s 3. Extension: 68 °C for 1 min/kb 4. End: 4 °C for ever
30× or 35×
4.2.2.3 Preparation of plasmid DNA by alkaline lysis
Isolation of plasmid DNA from E. coli was performed using the classical method of alkaline lysis (Birnboim and Doly,1979). Briefly, a single bacterial colony, grown overnight on selective LB medium, was inoculated into 2–5 ml of liquid LB medium supplemented with the appropriate
antibiotics. The bacterial culture was grown overnight at 37 °C with vigorous shaking (250 rpm) and subsequently transferred into a 2 ml eppendorf tube and pelleted at maximum speed for 1 min in an eppendorf centrifuge. The supernatant was removed and the bacterial pellet was re-suspended in 280 µl of ice-cold solution Ⅰ. To lyse the cells, 360 µl of freshly-prepared solution Ⅱ was added to the tube and gently mixed by inversion. After 5 min of incubation, 540 µl of ice-cold solution Ⅲ was added to neutralize the lysate and the mixture was incubated at 4 °C for 10 min.
The cell debris was removed by centrifugation at 13 000 rpm for 10 min. One ml of supernatant was transferred into a new eppendorf tube and nucleic acids were precipitated with 500 µl of iso-propanol at −20 °C for at least 10 min.
Nucleic acids were pelleted by centrifugation at 13 000 rpm for 10 min and the supernatant was removed. The DNA pellet was re-suspended in 500 µl of 25 µg/µl RNase A solution and incubated at 37 °C for 1 h. Subsequently, DNA was precipitated by addition of 600 µl of solution Ⅳ and pelleted by centrifugation. The pellet was washed with 1 ml of 70 % ethanol and, after removing the ethanol, the purified DNA was dissolved in 50 µl of water or TE buffer.
Solution Ⅰ:
Tris-HCl (pH 8,0) 50 mM
Glucose 50 mM
EDTA 10 mM
Solution Ⅱ (freshly prepared):
NaOH 200 mM
SDS 1 %
Solution Ⅲ:
NaOAc (pH 4,8) or KOAc (pH 6,0) 3 M Solution Ⅳ:
Iso-propanol 88 %
KOAc 0,2 M
When DNA was subjected to Sanger sequencing, it was purified using the NucleoSpin® Plasmid kit (Macherey-Nagel) or the E.Z.N.A.®Plasmid miniprep kit Ⅰ (PeqLab Biotechnologie) to obtain high quality samples.
4.2.2.4 DNA sequencing
Plasmids and DNA fragments were sequenced in collaboration with the Automatic DNA Isolation and Sequencing (ADIS) service at the MPIPZ. An Applied Biosystems 3730XL Genetic Analyser was used for this purpose.
4.2.2.5 Clone identification by PCR
Recombinant Taq DNA polymerase (Invitrogen) was used for diagnostic identification of recom-binant plasmid carrying specific DNA fragments. A master mix was prepared for multiple PCR reactions from the following components on ice:
4.2. METHODS 4. EXPERIMENTAL
10× PCR buffer (−MgCl2) 2 µl
10 mM dNTP mixture 0,4 µl
50 mM MgCl2 0,6 µl
Primer mix (10 µM each) 1 µl
Template DNA:
plasmid DNA 0,5 µl
colony-PCR bacteria
Taq DNA polymerase (5 U/µl) 0,2 µl Autoclaved distilled water up to 20 µl
For colony-PCR screening, the DNA template was added by transferring a bacterial colony from selective medium into the PCR tube using a toothpick. The PCR amplification was performed using a Bio-Rad DNA Engine Tetrad®2 cycler. The reactions were performed in a final volume of 20 µl using the following program:
1. Denaturation: 95 °C for 10 min 2. Denaturation: 95 °C for 30 s 3. Annealing: 58–62 °C for 30 s
(primer-pair dependent) 4. Extension: 72 °C for 1 min/kb 5. End: 4 °C for ever
25× to 30×
4.2.2.6 Phenol/chloroform extraction
Phenol/chloroform extraction was performed to remove contaminating proteins. DNA samples were mixed by gently vortexing with equal volume of phenol/chloroform solution (1/1) and subsequently centrifuged at 13 000 rpm for 5 min. The aqueous upper phase was collected in a new eppendorf tube and the extraction step was repeated until there was no more visible protein ring between the water and the organic solvent phases. To remove phenol traces, the sample was extracted with equal volume of chloroform/iso-amyl-alcohol (24/1) mixture on ice. Phases were separated by centrifugation and DNA from the supernatant was precipitated by adding 2 volumes of ethanol after the addition of1⁄10volumes of 3 M sodium acetate solution (pH 5,2).
4.2.2.7 DNA precipitation
DNA was precipitated using either ethanol or iso-propanol. For ethanol precipitation, 1/10 volumes of 3 M sodium acetate (pH 5,2) and 2 volumes of absolute ethanol were added and samples were incubated at −20 °C for at least 30 min. Precipitated DNA was pelleted by centrifugation at 13 000 rpm in an eppendorf centrifuge for 10 min. Pelleted DNA was washed with 70 % ethanol, centrifuged again and following the removal of ethanol, samples were dried in a desiccator.
For iso-propanol precipitation,1⁄10volumes of 3 M sodium acetate (pH 5,2) and 0,7 volumes of iso-propanol were added to the samples and the DNA was precipitated by incubation on ice for at least 20 min. DNA was pelleted by centrifugation at 13 000 rpm for 10 min. The DNA pellet was washed with 1 ml of 70 % ethanol, dried and dissolved in water or LTE solution (page109).
4.2.2.8 Agarose gel electrophoresis
Plasmids, DNA fragments and PCR products were separated by agarose gel electrophoresis. The gel agarose concentration depended on the size of the DNA fragments being analysed. Agarose was dissolved in 0,5× TBE buffer supplemented with 0,1 µg/ml of ethidium bromide and heated in a microwave oven until boiling. The agarose solution was poured into an electrophoresis tray and once gelled, submerged into a gel tank filled with 0,5× TBE buffer. DNA samples were mixed (5:1) with 6× loading dye and loaded into the gel wells. For size estimation, 5 µl of 1 kb DNA ladder (NEB) was used. Electrophoresis was performed at 50 V to 150 V. DNA was visualized using an UV light trans-illuminator. Gel images were captured using a Kodak DC-920 digital camera and processed with the Kodak Digital Science 2D v3.0.2 program.
5× TBE buffer:
Tris base 54 g/l
Boric acid 27,5 g/l
EDTA 4,65 g/l
6× loading dye:
Bromophenol blue 2,5 mg/ml
Xylene cyanol 2,5 mg/ml
Sucrose 400 mg/ml
4.2.2.9 Measurement of nucleic acid concentration
DNA concentration was determined using a Nanodrop ND-1000 spectrophotometer (PeqLab Biotechnologie). OD260 value of 1 corresponds to 50 µg/ml of double stranded DNA or 40 µg/ml of RNA. Pure preparations should have a OD260/OD280 ratio between 1,8 and 2,0 (Sambrook and Russell,2001).
4.2.3 General cloning techniques
4.2.3.1 Reliable DNA amplification
AccuPrime™ Taq DNA Polymerase High Fidelity (Invitrogen) was used for cloning purposes in order to have exact copies from genomic or cDNA templates. This enzyme mix has a component that provides a proof-reading 3’→5’ exonuclease activity, producing high fidelity copies. The following components were mixed in 200 µl PCR tubes:
10× AccuPrime™ PCR buffer Ⅰ 5 µl
Primer mix (10 µM each) 2 µl
Template DNA 1 µl
AccuPrime™ Taq High Fidelity 0,2 µl Autoclaved distilled water up to 50 µl
After a denaturation step (94 °C for 2 min), a three-step cycling program was used:
4.2. METHODS 4. EXPERIMENTAL
1. Denaturation: 94 °C for 15 s 2. Annealing: 55 °C for 30 s 3. Extension: 68 °C for 1 min/kb 4. End: 4 °C for ever
25× to 30×
4.2.3.2 Purification of DNA fragments from agarose gels
After gel electrophoresis, DNA fragments were excised from the agarose gel placed on an UV light trans-illuminator. DNA was extracted and purified using QIAquick®Gel Extraction Kit (QIAGEN) following the manufacturer’s instructions.
4.2.3.3 Digestion with restriction endonucleases
Digestions of DNA samples were performed with restriction endonucleases according to the manufacturer’s instructions. Typically, reactions were carried out in 1,5 ml eppendorf tubes in a final volume of 20 or 50 µl using 1–5 U of enzyme per µg of DNA. The duration of digestions varied from 2 h to overnight.
4.2.3.4 Blunt-end generation of digested DNA fragments
Overhanging DNA ends were blunted with T4 DNA polymerase (NEB) which can be used for removing 3’-overhanging nucleotides and fill-in 3’-recessed ends. The digested vector or isolated DNA fragment was dissolved in 1× restriction enzyme reaction NEBuffer supplemented with 100 µM dNTPs. One U of enzyme per µg of DNA was added to the reaction and incubated for 15 min at 12 °C. T4 DNA polymerase was inactivated by adding EDTA (10 mM final concentration) to the mixture followed by heating to 75 °C for 20 min.
4.2.3.5 Dephosphorylation of DNA ends
In order to prevent self-ligation of linearized vectors, digested plasmids were dephosphorylated at their 5’ end with Antarctic phosphatase (NEB). As this enzyme is heat inactivated, no further purification step is necessary. Linearized vectors were purified by gel electrophoresis.1⁄10volume of Antarctic phosphatase reaction buffer and 5 U of Antarctic phosphatase were added. The reaction mixture was incubated for 1 h at 37 °C followed by inactivation of the enzyme at 65 °C for 5 min to 10 min.
4.2.3.6 DNA ligation
T4 DNA ligase (NEB) was used for the ligation of linearized vectors with DNA fragments.
Normally, vector and fragment were mixed at a molar ration of 1:3. The ligase reactions were prepared by addition of 2 µl T4 DNA Ligase Reaction buffer and 1 µl of T4 DNA ligase and performed in a final volume of 20 µl with an overnight incubation at 12 °C. As a control for re-circularization of the cloning vector, a similar ligation reaction was carried out with the linearized vector.
4.2.4 Gateway®cloning
The Gateway®technology (Invitrogen) is based on the use of site-specific recombination reactions that mediate the integration and excision of the lysogenic λ phage. Integration of DNA fragments flanked by attP attachment sites in a carrier plasmid into a target (i.e. donor) vector harboring the suicide ccdB gene between the attB sequences is mediated by the phage encoded Integrase (Int) and bacterial IHF a/b proteins (BP Clonase). Homologous exchange recombination at the att sites transfers the DNA fragment into the target donor vector and converts the attB to attL sites, while the original carrier plasmid acquires the lethal ccdB gene flanked by attP-derived attR sequences.
Using the reverse excision reaction catalysed by Int, IHF a/b and Xis (“excisionase” or LP Clonase) enzymes, the attL-flanked fragment can be moved from the constructed “entry” vector to any destination vector (including expression or gene fusion vectors) by exchange recombination with the attP sites that flank in these vectors the suicide ccdB marker gene (Hartley et al.,2000).
4.2.4.1 BP cloning reaction
The BP cloning reaction promotes recombination between the attB sites flanking a PCR product and the attP sites of the “donor” to create an attL-containing entry clone. This reaction is catalysed by the BP Clonase™ Ⅱ enzyme mix (Invitrogen). The following components were mixed in a 1,5 ml eppendorf tube:
attB-PCR product (20 fmol to 50 fmol) 1 µl to 7 µl
pDONR™ vector (150 ng/µl) 1 µl
TE buffer, pH 8,0 up to 8 µl
BP Clonase™ Ⅱ enzyme mix 2 µl
Reactions were performed for 1 h at 25 °C, and stopped by addition of 1 µl of Proteinase K solution and incubation at 37 °C for 10 min.
4.2.4.2 LR cloning reaction
The LR Clonase™ Ⅱ enzyme mix (Invitrogen) facilitates the recombination reaction between the attL-containing entry clone and the attR substrate (destination vector) to create an attB-containing expression clone. This expression vector can be a binary vector to be transformed by Agrobacteriuminto plants. The following components were mixed in a 1,5 ml eppendorf tube:
Entry clone (50 ng to 150 ng) 1 µl to 7 µl Destination vector (150 ng/µl) 1 µl
TE buffer, pH 8,0 up to 8 µl
LR Clonase™ Ⅱ enzyme mix 2 µl
Reactions were incubated for 1 h at 25 °C, stopped by addition of 2 µl of Proteinase K solution and incubation at 37 °C for 10 min.
4.2. METHODS 4. EXPERIMENTAL
4.2.5 Transformation of bacteria and Arabidopsis
4.2.5.1 Preparation of electro-competent bacterial cells
Preparation of electro-competent E. coli cells was carried out according to the protocol ofDower et al.(1988). A single colony was inoculated in 10 ml of liquid LB and grown overnight at 37 °C with constant shaking (200 rpm). One ml of this culture was inoculated into 300 ml of liquid LB and incubated overnight at 37 °C with shaking. Subsequently, the bacterial culture was diluted to an OD600 value of 0,1–0,2 and cells were grown at 16 °C until the OD600 reached a value of 0,5 to 0,6. Cells were pelleted by centrifugation in Sorwall GSA tubes at 5 000 rpm for 20 min and re-suspended in 200 ml of cold water. This washing step was repeated 3 times to remove salts, then cells were re-suspended in 50 ml of water, transferred into a 50 ml falcon tube and pelleted again for 10 min at 5 000 rpm. Finally, cells were re-suspended in 800 µl of 7 % DMSO solution, aliquoted in eppendorf tubes containing 50 µl of cell suspension and immediately snap-frozen in liquid nitrogen. Aliquots were stored at −70 °C until further use.
Electro-competent A. tumefaciens cells were prepared using a similar protocol. Agrobacteria were grown in liquid YEB medium at 21 °C. After the final washing step, cells were re-suspended in 1 ml sterile 10 % glycerol solution, aliquoted, snap-frozen in liquid nitrogen and stored at −70 °C.
4.2.5.2 Electroporation of bacterial cells
An aliquot of competent E. coli or A. tumefaciens cells was thawed on ice. Cells were mixed with 0,5 µl to 3 µl of either plasmid DNA or pre-dialysed ligation mixture and transferred into pre-chilled 0,2 cm-wide electroporation cuvettes (Bio-Rad). Transformation was performed in a Bio-Rad Gene Pulser set to 2,5 kV voltage, 25 µF capacitance and 200 Ω resistance. After electroporation, bacteria were suspended in 1 ml of liquid LB or YEB medium, transferred into an eppendorf tube and incubated for 1 h at 37 °C –for E. coli– or 2 h at 28 °C –for A. tumefaciens–.
After the recovery phase, E. coli was spread on solid LB media supplemented with antibiotics, whereas A. tumefaciens was plated on selective YEB medium.
4.2.5.3 Agrobacterium-mediated transformation of Arabidopsis
Agrobacterium-mediated transformation of Arabidopsis was performed by the flower dipping method or by co-cultivation of cell suspensions with Agrobacterium.
The flower dipping method (Clough and Bent, 1998) is based on genetic transformation of ovules. Five hundred ml of Agrobacterium cells were grown overnight in the presence of antibiotics to select for the marker of the binary vector. The Agrobacterium culture was pelleted at 5 000 rpm for 10 min followed by re-suspension in 300 ml of transformation medium. Open flowers and siliques were removed from bolting plants cultivated in 10 cm-diameter pots –10–12 plants per pot– and then their inflorescences were submerged in the bacterial suspension for at least 5 min. For gradual adaption to lower humidity, plants were covered with a plastic bag, which was removed 2 days later. Seeds were collected in paper bags and T1 transformants were selected on solid MSAR medium containing antibiotics corresponding to the selectable marker of the T-DNA, as well as ticarcillin and cefotaxime to avoid Agrobacterium growth.
Transformation medium:
MS salts 1⁄2
B5 vitamins 1×
Sucrose 5 %
BAP 0,044 µM
Silwet L-77 0,005 %
Arabidopsis cell suspensions were transformed with Agrobacterium using the protocol of Ferrando et al.(2000) with minor modifications. Briefly, Agrobacteria were grown in petri dishes with selective media until covering the whole plate, then, the Agrobacterii layer was transferred with the help of a sterile spatula into an Erlenmeyer flask containing 50 ml of 3 day-old sub-cultured cell suspension of Arabidopsis. Following 2 days of co-cultivation, Agrobacterium growth was stopped by addition of ticarcillin and cefotaxime (50 µl each). Transformed cell suspensions were sub-cultured using the 7 day-cycle, and at the 2nd sub-culture, the medium was supplemented with antibiotics to select for the T-DNA-encoded resistance marker.
4.2.6 Construction of binary vectors for transformation
4.2.6.1 pPCV002PRL1::PRL1-PIPL
The pPCV002-PRL1::PRL1-PIPL construct was designed with the help of Dr. D. Szakonyi for expression of a modified PRL1 protein in fusion with the triple PIP-L tag (see below), Strep and HiA tags under the control of the native PRL1 promoter, and to test the functionality of this protein by genetic complementation in the Arabidopsis prl1 mutant. D. Szakonyi has previously demonstrated that essential regulatory elements of the PRL1 promoter are localized between
The pPCV002-PRL1::PRL1-PIPL construct was designed with the help of Dr. D. Szakonyi for expression of a modified PRL1 protein in fusion with the triple PIP-L tag (see below), Strep and HiA tags under the control of the native PRL1 promoter, and to test the functionality of this protein by genetic complementation in the Arabidopsis prl1 mutant. D. Szakonyi has previously demonstrated that essential regulatory elements of the PRL1 promoter are localized between