LIST OF TABLES
4. CLONING OF NOVEL COWPEA TRANSCRIPTION FACTORS AND THEIR FUNCTIONAL CHARACTERIZATION IN YEAST
4.2 MATERIALS AND METHODS
4.2.1 Gene isolation and construction of expression vectors
The trifoliate leaves from 15-day-old healthy cowpea seedlings of Kannanado White (IITA, Nigeria), a drought hardy genotype [252], were subjected to PEG 6000 (20% w/v) and NaCl (200 mM) stress for 24 h and sampled for RNA extraction to prepare the cDNA library. Nested PCR was performed to isolate two partial NAC encoding gene sequences from the library, using a set of degenerate primers targeting conserved regions of previously reported stress- responsive NAC genes [27, 31, 33]. The full-length ORFs were retrieved by the 5' and 3’- RACE PCR (Invitrogen), mentioned in Table A2.10, Appendix 2. For their phylogenetic clustering, the ESTs available for cowpea NAC family and Arabidopsis NAC genes were downloaded from Plant TFDB 4.0 database (http://planttfdb.gao-lab.org/index.php) [52]. The promoter sequences were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov), and the regulatory elements were identified using the PLACE tool (https://www.dna.affrc.go.jp/PLACE/?action=newplace) [332].
For the localization study, the 35S:VuNAC1/2:GFP constructs were generated by fusing an eGFP sequence (isolated from pBI221) at the 3’-end of the VuNAC ORFs lacking the stop codon, by overlapping PCR, followed by cloning in pBEAB plant binary expression vector (provided from Gifu University, Japan) at the SfiI sites. The constructs were transformed in the EHA105 Agrobacterium strain. For protein expression, the full-length ORFs were cloned in the pET28a (+) vector (GE Healthcare) to fuse the 6×His tag at the N’-end, using BamH1/SalI site (VuNAC1) and BamH1/XhoI sites, followed by the transformation in BL21(DE3) bacterial strain (GE Healthcare). For the yeast-based reporter assay, GAL4BD was fused with the full- length VuNAC ORFs (GAL4BD:VuNAC1/2) and with the VuNAC ORFs having truncated C’-part (GAL4BD:VuNAC1/2ΔAD) and cloned in pGBKT7 vector (Clontech) at NdeI/BamHI sites. All the constructs were individually transformed in the Y2H Gold yeast strain, as described in the Yeast transformation manual (Clontech). The transformants were screened on SD-/Trp plates incubated at 30ºC for 3-4 days. The GAL4BD:VuNAC1/2ΔAD constructs were also used as bait plasmids for the yeast two-hybrid (Y2H) assay. To generate the prey plasmids, GAL4AD was fused with the full-length VuNAC ORFs (GAL4AD:VuNAC1/2) and cloned in pGADT7 vector (Clontech) at NdeI/BamHI sites. The constructs were transformed in the Y187 yeast strain (Clontech). The transformants were screened on SD-/Leu plates incubated at 30 ºC for 3-4 days. The primer sequences used in this study are listed in Table A2.11, Appendix 2.
4.2.2 Expression analysis and stress treatment
Healthy cowpea seeds (Kannando White) were germinated by soaking in water for two days in the dark followed by a long-day photoperiod condition (16 hr of light, 8 hr of dark) at 28°C, with white light illumination (110 µmol photons m-2s-1). Four-day-old germinated seedlings were transferred to gauzed hydroponics container supplied with modified Hoagland hydroponics media for approximately ten days until the first trifoliate leaves expanded [333].
For various stress treatments, the hydroponics media was supplemented with 20% PEG 6000, 200 mM NaCl, 50 µM of AlCl3 (pH 5.0), 50 µM (±) ABA, and 50 µM methyl jasmonate (MeJA), individually. For heat and cold treatments, the plants were transferred to the respective growth chambers maintained at 45°C and 4°C. For dehydration stress, the leaves were dried at 26°C until 10% fresh weight loss. The trifoliate leaves from the stressed and the control plants were sampled at the indicated time intervals for RNA extraction using the RNeasy mini kit (Qiagen), followed by cDNA synthesis (Applied Biosystems, USA). The semi-quantitative and quantitative real-time PCR was performed in triplicates for each sample on Thermocycler Dice, Real-time system II (Takara, Japan). VuUbiquitin2 was used as a reference to determine the relative expression. The experiments were repeated three times with similar results.
4.2.3 Localization, transactivation, and dimerization
To determine the subcellular localization of GFP-tagged VuNAC1 and VuNAC2 proteins, healthy onion epidermis was transiently transformed by infiltration method, with the Agrobacterium strain carrying the 35S:VuNAC1/2:GFP constructs (O.D.600 ~0.8), suspended in the infiltration buffer (41.65 mM D-glucose, 10 mM MgCl2, 10 mM MES-KOH (pH 5.6), and 100 µM Acetosyringone). The strain expressing the eGFP alone driven by a 35S promoter was used as the positive control. After three days of co-cultivation, the infiltrated section was stained with 1 μg/mL DAPI (4',6-diamidino-2-phenylindole) in 10% glycerol and kept in darkness for 10 min. The fluorescence was captured by Zeiss Axio (Germany) under the excitation filters 470 nm (for eGFP) and 400 nm (for DAPI). For the yeast-based reporter assay, the 10-fold serial dilutions of the overnight cultures (O.D. ~0.6) of Y2H transformants carrying the pGKT7 plasmid (negative control), GAL4BD:VuNAC1/2, and GAL4BD:VuNAC1/2ΔAD constructs were spotted on agar plates with appropriate selection media, SD/-Trp, SD-Trp + 40 µg/ml of X α-gal (SD/-Trp +X) and SD/-Trp + 40 µg/ml of X α-gal + 2 µg/ml of Aureobasidin A (SD/-Trp + XA), and incubated at 30 ºC for 2-3 days. The formation of blue colonies and growth in the presence of Aureobasidin A antibiotic indicated the expression of MEL1 and AUR1C reporter genes, respectively, through transactivation. For the Y2H assay, Y2H gold
strain harboring the bait plasmid, i.e., pGBKT7-53 (positive control), pGBKT7-Lam (negative control), and GAL4BD:VuNAC1/2ΔAD constructs and Y187 strain carrying the prey plasmid, i.e., pGADT7-T (positive control) and GAL4AD:VuNAC1/2 constructs, were mated as described in the Y2H manual (Clontech), and the colonies were screened in the double drop- out media (SD/-Trp-Leu) for the presence of both bait and prey plasmids. The 10-fold serial dilutions of the overnight cultures (O.D. ~0.6) of selected colonies were spotted on agar plates with appropriate selection media SD/-Trp-Leu, SD/-Trp-Leu-Ade-His (quadruple drop out), SD/-Trp-Leu + 40 µg/ml of X α-gal (SD/-Trp-Leu + XA), and incubated at 30 ºC for 2-3 days.
The formation of blue colonies on SD/-Trp-Leu-Ade-His media in the presence of Aureobasidin A indicated the expression of MEL1, HIS3, ADE2, and AUR1C reporter genes, due to prey-bait interaction. To avoid the false reporter expression due to the transactivation ability of VuNAC1/2, the bait plasmids with truncated C’-part (GAL4BD:VuNAC1/2ΔAD) were used for the Y2H assay.
4.2.4 Protein purification, EMSA assay, and structure prediction
The BL21 (DE3) strains harboring the His-tagged VuNAC ORFs were grown in LB media + 50 µg/ml Kanamycin (O.D.600 ~0.4). The culture was induced with 1mM IPTG followed by incubation for 16 hours at 30 ˚C, 180 rpm. The cells were harvested, the pellet was re- suspended in 50 ml of suspension buffer (50 mM Na2HPO4, 50 mM NaH2PO4, 300 mM NaCl, and 1 mM PMSF), added with 0.5 mg/ml lysozyme, pH 7.4), and incubated on a rocker at 4 ˚C for 1 hour. The lysate was homogenized by sonication and centrifuged at 11000 rpm at 4 ˚C for 30 min to separate the soluble and insoluble fractions. The His-tagged protein was purified from both the fractions by Ni-NTA affinity chromatography and eluted in suspension buffer supplemented with 250 mM imidazole. The eluted protein was diluted in 50 ml of dialysis buffer (5 mM Tris-HCl and 15 mM NaCl, pH 7.4) and dialyzed overnight to purify and regenerate the native structure. The protein was further concentrated with a centrifugal filter (10 kDa MWCO). The concentration of the purified protein was estimated using Bradford’s assay to be around 20 mg/ml. 20 µl of the protein was electrophoresed on 12% SDS -PAGE gel at 120 V to evaluate the purity and molecular weight. The purified VuNAC proteins were tested for their ability to bind 40 bp NACBS probe made of 4X repeats of 5'-CATGTCCACG-3' motifs NACBS using electrophoretic mobility shift assay (EMSA) [369]. The HPLC-purified oligonucleotide and its reverse complementary strands were annealed by heating at 70 ºC for 5 min and gradual cooling at room temperature in 50 mM NaCl solution to form a double- stranded DNA probe. The NACBS motif was substituted with polyAs to generate the mutated
probe to serve as a negative control. The different concentrations of the protein sample were incubated with 0.01 nmol of DNA probes at 25 ˚C for 30 min in 1 X binding buffer (20 mM Tris–HCl, pH 8.0, 100 mM KCl, 5 mM MgCl2, 5% glycerol, and 1 mM DTT) in a 25 µl reaction volume. After incubation at 25˚C for 30 min, the reaction mixture was electrophoresed in 1%
agarose gel in 0.5X TBE buffer at 110 V, and the DNA shift was visualized in the UV light after ethidium bromide staining. Furthermore, the secondary structures and 3D protein folds were predicted using Phyre v 2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) 4.2.5 HPLC analysis
The Y2H Gold strains expressing GAL4BD:VuNAC1/2 (transgenic) and pGBKT (wild type) were cultured in a shaking flask (200 rpm) at 30ºC in YPD media. The supernatant was collected followed by 10000 rpm spin for 30 s at the indicated time-intervals and filtered through a 0.2 µm membrane filter. 20 µl of the sample was injected in a Shimadzu Prominence HPLC System with SPD-M20A DAD (Shimadzu, Japan), equipped with an automatic injector.
The Aminex HPX-87H column (300 χ 7.8 mm) (Bio-Rad Labs, CA) was used at the oven temperature of 50°C, and the sample and reference cells were maintained at 25°C. The isocratic elution was done in a de-gassed mobile-phase solvent of 3.0 mM of sulfuric acid in water at a flow rate of 0.6 ml/min. The analytes were detected by an RI detector (Model 410). The serial dilutions of ethanol, glycerol, and glucose were run as standard for quantitative estimation.
4.2.6 FTIR spectroscopy
The yeast strains were cultured in a shaking flask (200 rpm) at 30ºC in YPD media. The samples were collected at two different growth phases, the late-log phase (OD600 ∼1.2) and late- stationary phase (O.D.600 ∼2.0), in biological replicates. The metabolism was cold- arrested in ice. The cells were harvested, washed in PBS buffer, and then dried to powder using the Scanvac Coolsafe Freeze dryer (Electronex, Mumbai). The ATR-FTIR spectra for the powdered sample were recorded with an ATR unit (Specac, U.K.) combined with an IRAffinity 1-S spectrophotometer (Shimadzu, Japan) with a resolution of 4 cm-1 and 64 scans in the range of 4500-400 cm-1. The acquired spectra were analyzed using OMNIC software. The absorption spectra of the wild type strain were subtracted from that of the transgenic strains and plotted to project variations between the two samples.
4.2.7 FESEM and Light microscopy
The yeast cells were fixed with 1.0% (v/v) glutaraldehyde solution for 3 h [370]. After fixation, the cells were spotted on an aluminum surface mounted on carbon tape and gradually air-dried. The specimen was coated with gold and analyzed at 2000X magnification using GeminiSEM 300 (Zeiss, Germany), operating under an accelerating voltage of 2 kV. For light microscopy, the yeast culture was spotted on a glass slide and visualized under Nikon E100 LED Binocular Microscope (Nikon, Japan) under the bright field.
4.2.8 LC-MS profiling of metabolome
50 mg powder of each sample was taken, and metabolite quenching was carried out using 2 ml of methanol: water (1:1) mixture, followed by ultrasonication for 30 min and centrifugation at 14000 rpm for 3 min. The polar-phase supernatant was dried by lyophilization.
The residue was re-suspended in 0.5 ml of LC mobile phase A (0.1% formic acid in water) and filtered through a 0.2 µm filter before injection [371]. The samples were analyzed by LC-MS in Agilent 1260 binary LC System (Agilent Technologies, D.E.) using Agilent Zorbax Eclipse Plus C18 column (50 x 2.1 mm, 1.8 μm). The injection volume was 2.0 µl and the flow rate was 0.3 ml/min. The LC solvents were 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). The gradient elution was as follows: t = 0 min, 5% B; t = 18 min, 95% B; t = 27 min, 95% B; t = 27.1 min, 5% B; t = 30 min, 5% B. All MS acquisitions were performed in both positive and negative electrospray ionization (ESI) mode at a scan rate of 3.0 Hz in the mass range 60-1600 m/z. The capillary voltage, cone voltage, fragmentor voltage were 4 kV, 45 V, and 150 V, respectively. The gas temperature was set at 350 °C. The data were analyzed with Agilent Masshunter Qualitative Analysis v. B.06.00 and Agilent Mass Profile professional v. 12.6.1 to detect peaks and de-convolute the mass spectra. The Yeast Metabolite Database (YMD) and METLIN database were used to annotate the metabolites.