LIST OF TABLES
5. CHARACTERIZATION OF VUNAC1/2 TFS IN RESPONSE TO ABIOTIC STRESSES UNDER STARVATION
5.2 METHODOLOGY .1 Vector preparation
The VuNAC1/2 genes were cloned from a drought-hardy cowpea genotype (Kannando White) [252], as described in our previous work [372]. The 35S:VuNAC1/2 constructs used to generate the constitutive overexpressor Arabidopsis lines were prepared by cloning the complete ORF (888 bp each) at the SfiI site of pBEAB, a T-DNA vector (provided from Gifu University, Japan). The constructs were mobilized in the EHA105 strain of Agrobacterium tumefaciens, independently using tri-parental mating through pRK2013 helper plasmid.
5.2.2 Floral-dip transformation
The wild type Arabidopsis seeds (Col-0 variety) were imbibed in water at 4˚C for two days.
Around 50 seeds were sprinkled per pot containing soilrite mix supplemented with MS media.
The plants were grown at 24˚C, 16/8 light/dark photoperiod conditions at 85% relative humidity, for around five weeks until the bolting stage arrived with the maximum number of unopened buds. The agrobacterium strain harboring the 35S:VuNAC1 and 35S:VuNAC2 plasmid were grown till O.D.600 reached 0.8 in LB media. The cells were harvested and suspended in 10 mM MgCl2, 5% sucrose, and 0.02 % Silwet-L-77 [411]. The plants were dipped in the agrobacterium suspension for 10 seconds with gentle swirling and incubated in the dark for one day, followed by transfer in the growth chamber. The T0 generation seeds were harvested upon maturation. The seeds were sterilized in 1% (v/v) sodium hypochlorite for 10 minutes with vigorous vortexing followed by washing with de-ionized water (5X, 2 min each).
For screening, the seeds were germinated in ½X MS supplemented with 0.8% agar, 0.5%
sucrose, and 50 µg/ml kanamycin. After seven days of selection, the green seedlings were transferred to soil to further analyze the transgene insertion.
5.2.3 Growth conditions for stress analysis
For the study in the seedling stage, healthy seeds of wild type (Col-0) and T2 generation transgenic seeds were sterilized in 1% (v/v) sodium hypochlorite for 10 minutes followed by washing with de-ionized water and kept for stratification at 4°C for 48 hrs. The seeds were
transferred to plates containing ½ X MS media supplemented with 1% (w/v) sucrose (added as indicated), 0.8% agar, (pH 5.8) and grown in long-day photoperiod condition (16 hr of light, 8 hr of dark) at 25 °C, with white light illumination (110 μmol photons m-2s-1). For various stress treatments, the growth media was supplemented with the chemicals as per the indicated concentration. For stress analysis of soil-grown plants, 1-week old germinated seedlings were transferred to soilrite mix and grown for four weeks while supplementing MS media every week. For imposing nutrient stress, only water was supplied throughout the vegetative growth of 4 weeks. For drought assay, water was withdrawn, and plants were maintained at 40%
humidity for a week until recovery. To inflict high salinity, 400 mM of NaCl solution was supplied twice for a week.
5.2.4 Physiological parameters and biochemical assays
5.2.4.1 Determination of photosynthetic parameters
To determine chlorophyll fluorescence and gas exchange, we chose the fourth fully expanded trifoliate from the top to record the associated parameters using a portable infrared gas analyzer photosynthetic system Li-COR 6800 (Li-COR, U. S. A). To measure attributes such as net photosynthetic rate (A), stomatal conductance (Gsw), internal CO2 concentration (Ci), quantum-yield of photosystem II (ϕ PSII) and electron transport rate (ETR), the air- temperature, relative-humidity, CO2 concentration, fan-speed, light-intensity, and color- spectrum were maintained at 25° C, 50%, 400 µmol mol-1, 10000 rpm, 100 µmol m-2 s-1, red/blue 0.9/0.1 respectively.
5.2.4.2 Determination of relative water content
Fresh leaves taken from mid-canopy were cut into 3x3 cm segments. Ten segments were weighed together to record the fresh weight (FW). The turgid weight (TW) was determined by measuring fully hydrated leaf segment weight, kept immersed in the sealed flask at 10 0 C for 4 hours. The dry weight (DW) was determined by drying the leaf segments at 700 C in an oven for 36 hrs to obtain consistent weight. Finally, the relative water content (RWC) was calculated for each sample using the formula [412]:
RWC (%) = {(FW-DW) / (TW-DW)} x 100
5.2.4.3 Determination of electrolyte leakage rate
The leaf samples were cut into 1 cm discs and dipped in de-ionized water. The electrical conductivity (ECa) was determined in terms of µS at 25°C using a conductivity meter (CON 700, Eutech). Next, the samples were incubated at 50° C water bath for 30 min in sealed test tubes, and the electrical conductivity (ECb) was measured. The samples were then boiled at 100° C for 10 min to measure the electrical conductivity (ECc). The electrolytic leakage was calculated as [413]:
Electrolyte leakage (%) = {(ECb − ECa) ×100} / ECc 5.2.4.4 Determination of lipid peroxidation
The level of membrane lipid peroxidation was estimated by the method described by Heath and Packer (1968) in terms of malondialdehyde (MDA) content [414]. 200 mg leaf tissue was homogenized in 5 ml of TBA reagent: 0.25% (v/v) thiobarbituric acid and 10% (v/v) trichloroacetic acid. The homogenate mixture was boiled for 30 min in a water bath at 95° C and cooled to RT. After centrifugation (10,000 g, 10 min), the absorbance of the clear supernatant was measured at 532 nm and 600 nm. The lipid peroxidation rate equivalents were as:
MDA equivalent nmol/gFW = [(A532-A600)/155000] x 1000000
5.2.4.5 Determination of proline content and non-enzymatic ROS scavengers (glutathione and ascorbate)
Proline concentration was determined following the method of Bates et al. (1973) [415].
100 mg of each leaf sample was homogenized in 5 ml of 3% aqueous sulfosalicylic acid (v/v) and left for 3 hrs for extraction. The mixture was centrifuged (3000 g, 10 min) to separate the supernatant. To the 0.5 ml supernatant, 0.5 ml glacial acetic acid and 0.5 ml acidic ninhydrin was added and boiled in a 90° C water bath for 30 min. After cooling, 1.5 ml of toluene was added and mixed vigorously. The absorbance was read at 520 nm. The proline concentration was determined in terms of µg/ml from a standard curve. Total glutathione and ascorbate contents were determined as per the method Griffith (1980) and Oser (1979), respectively [416, 417]. 200 mg of each leaf sample was homogenized in 5% (w/v) sulphosalicylic acid and centrifuged (10000 g, 15 min at 4º C) to collect the supernatant. To determine ascorbate content, 0.2 ml of plant extract was mixed with 0.4 ml of 2% sodium molybdate, 0.4 ml 0.15
N H2SO4 and 0.2 ml 1.5 mM Na2HPO4, and incubated at 60 ºC for 40 minutes. The mixture was centrifuged at 3000 g, and the clear supernatant was collected to record the absorbance at 660 nm. For the determination of total glutathione content, plant extract was neutralised with 0.1 M phosphate buffer. The 0.4 ml neutralised extract was mixed with 0.25 µl of 0.1 M phosphate buffer, 80 µl of 6 mM 5,5´-dithiobis (2-nitrobenzoic acid). The absorbance was recorded at 412 nm. The concentration was determined from the standard curve.
5.2.5 Visualization of stomata by FESEM
The plant tissue was pre-fixed with 2.5 % glutaraldehyde solution overnight. The sample was then dehydrated with an ethanol gradient (10%, 20%, 30%, 50%, and 70%, once for 10 min at each step), and then immersed in 100% ethanol (twice, 30 minutes each step). One-cm- sections were mounted on carbon tape. The specimen was coated with gold and analyzed at 2000X magnification using FESEM (Gemini 300, Zeiss, Germany), operating under an accelerating voltage of 3 kV.
5.2.6 Gene regulatory models
The orthologous Arabidopsis NAC TFs were used to predict the VuNAC1/2 coexpression network generated by ATTED II v. 10.1 (https://atted.jp/) [338]. The genes in the constructed network were subjected to ontology analysis to annotate the ATAF-like VuNAC TFs using the panther tool (http://go.pantherdb.org/genelistanalysis.do) [339]. The upstream regulators were predicted using the RnR (Regulatory Network Research) database of Arabidopsis T87 culture cells (http://webs2.kazusa.or.jp/kagiana/rnr0912/indexff.html).