RETRACTED ARTICLE: SOG1 transcription factor promotes the onset of endoreduplication under salinity stress in Arabidopsis

As like in mammalian system, the DNA damage responsive cell cycle checkpoint functions play crucial role for maintenance of genome stability in plants through repairing of damages in DNA and induction of programmed cell death or endoreduplication by extensive regulation of progression of cell cycle. ATM and ATR (ATAXIA-TELANGIECTASIA-MUTATED and -RAD3-RELATED) function as sensor kinases and play key role in the transmission of DNA damage signals to the downstream components of cell cycle regulatory network. The plant-specific NAC domain family transcription factor SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1) plays crucial role in transducing signals from both ATM and ATR in presence of double strand breaks (DSBs) in the genome and found to play crucial role in the regulation of key genes involved in cell cycle progression, DNA damage repair, endoreduplication and programmed cell death. Here we report that Arabidopsis exposed to high salinity shows generation of oxidative stress induced DSBs along with the concomitant induction of endoreduplication, displaying increased cell size and DNA ploidy level without any change in chromosome number. These responses were significantly prominent in SOG1 overexpression line than wild-type Arabidopsis, while sog1 mutant lines showed much compromised induction of endoreduplication under salinity stress. We have found that both ATM-SOG1 and ATR-SOG1 pathways are involved in the salinity mediated induction of endoreduplication. SOG1was found to promote G2-M phase arrest in Arabidopsis under salinity stress by downregulating the expression of the key cell cycle regulators, including CDKB1;1, CDKB2;1, and CYCB1;1, while upregulating the expression of WEE1 kinase, CCS52A and E2Fa, which act as important regulators for induction of endoreduplication. Our results suggest that Arabidopsis undergoes endoreduplicative cycle in response to salinity induced DSBs, showcasing an adaptive response in plants under salinity stress.


Nuclei isolation and ploidy analysis by flowcytometry
Isolation of nuclei and subsequent analysis of the nuclear DNA content by flowcytometry were performed following previously described methods with minor modifications 1, 2 . Nuclei were isolated from root and first and second pair of rosette leaves of 7-days old wild type,OE-1, sog1-6 and sog1-1 mutant seedlings, either untreated or treated with increasing concentrations of NaCl for 12 hours by chopping method. Approximately 50-100 mg of excised plant tissue (roots or rosette leaves) was placed in a plastic 60-mm petri dish standing on a prechilled tile placed on ice in a rectangular plastic tray. Otto-I buffer [100mM citric acid and 0.5% (v/v) tween 20] was added in the proportions of 1 ml buffer per 100 mg tissue. Tissues were chopped using a new razor blade for 4-5 min. The homogenate was then filtered through a 40-μm nylon filter to remove tissue debris and then centrifuged subsequently at 500 g for 10 minutes. The nuclei pellet was resuspended in 500 µl of 1X PBS buffer (pH 7.4) with gentle tapping. The nuclear suspensions were stained with 4µg/ml DAPI and run through a FACS Calibur flowcytometer (BD) and about 10,000 flow cytometric events were recorded for each sample. The outputwas gated to eliminate signal from the debris Flowcytometry experiments were repeated at least three times for each genotype using independent biological replicates. FlowJo v. 10.0.6 (Tree Star, Inc.) was used for flow cytometric data analysis.

Study of root cell area expansion by propidium iodide staining
Roots of 7-days old wild type, OE-1, sog1-6 and sog1-1 mutant seedlings treated without or with different concentrations of NaCl for 12 hours were fixed in a solution of 50% (V/V) methanol and 10% (V/V) acetic acid and kept overnight at 4°C. On the next day, root samples were washed withsterile and incubated in 1% (W/V) periodic acid solution for 40 min. Roots were then stained with Schiff reagent containing 100 mM sodium metabisulfite, 0.15 M HCl and 10 mg/L propidium iodide. Stained samples were mounted in a chloral hydrate solution [chloral hydrate, glycerol, and water (8 g: 1 ml: 2 ml)]. Root tips were then visualized by confocal microscopy (Zeiss LSM-510 Meta). Root tip cell area and distance from the QC were measured with MBF ImageJ software by tracing cell contours.

Measurement of leaf cell area expansion
Expansion of Arabidopsis leaf epidermal and mesophyll cells were studied by Differential interference contrast (DIC) microscopy following the method described earlier 3, 4 . First and second pair of rosette leaves from untreated or NaCl treated 7-days old wild type, OE-1, sog1-6 and sog1-1 seedlings were fixed immediately in a solution of ethanol: glacial acetic acid (1:1) (v/v) for at least 12 hours following harvesting. Following incubation, leaves were dehydrated in a series of ethanol solutions (50%, 70%, 80%, 90%, and 100%) for at least 15 min in each step.
After dehydration, leaves were then submerged in a clearing solution containing chloral hydrate, glycerol and water in 4:2:1 proportion. Leaves were then visualized under a Zeiss Axio Scope A1 microscope using DIC optics and images were taken using the Axiocam 503 camera attached with the microscope. Cell areas were measured in ImageJ software. Trichomes of wild type and mutant plants were observed by SEM, as previously described 5 .

Total protein extraction and immunoblotting
Total protein was extracted following the procedure described previously 6 (Roy et al., 2013).
About 100 mg plant tissue from wild type, OE-1 and sog1-1 mutant seedlings, either untreated or treated with increasing concentrations of NaCl for 12 hours, were frozen in liquid nitrogen and homogenized in 1 ml of pre-chilled protein extraction buffer (50 mMTris-HCl pH 7.5, 5 mM MgCl2, 400 mM sucrose, 10% glycerol, 2.5 mM EDTA, pH 8.0, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM 2-mercaptoethanol, 5 mg/ml -1 leupeptin and 1 mg/ml -1 antipain). The homogenate was then transferred into a sterile centrifuge tube and centrifuged subsequently at 2,000 × g for 5 min at 4 ̊ C to pellet down the debris. The supernatant was transferred to a fresh tube and concentration of protein in each sample was determined by conventional Bradford assay 7 .
Western blot analysis was carried out following the method of Sambrook 8 . About 50 µg of the protein samples were separated on a 10% SDS-PAGE gel and then transferred on to a PVDF membrane (BioRad) using a Bio-Rad mini trans blot apparatus following the manufacturer's protocol. For assessment of expression of DNA damage response proteins and regulators of cell cycle and endoreduplication, we have used the respective antibodies developed in mouse system against the respective protein from mammalian system. All the proteins show considerable amount of sequence conservation between the plant and the corresponding mammalian counterpart (> 70% sequence identity). The antibodies against mammalian ATM (SIGMA-A6093), ATR (SIGMA-c515173), CDK1(SIGMA-SAB4500050), CDK2 (SIGMA-SAB4300388), Cyclin B1(SIGMA-C8831), WEE1(SCBT, sc-5285) and E2F1  showed specific cross-reactivity with plant ATM, ATR, CDKB1;1, CDKB2;1, CYCB1;1 and E2Fa respectively and this is also confirmed by immunoprecipitation assays. The antibody against mammalian CCS52A1 (SIGMA-SAB2105778) detects both plant CCS52A1 and A2 counterpart. The anti-AtSOG1 polyclonal antibody was generated against the Ni 2+ -NTA affinity resin purified full length recombinant AtSOG1 protein. His-tagged recombinant AtSOG1 protein was expressed in E. coli BL21 host strain and purified on nickel beads. The protein was separated by SDS-PAGE. Gel slices containing AtSOG1 protein were used to raise the antibody against the full-length AtSOG1 protein in rabbits by Abgene, Bhubaneswar, India. Titters of the antibody were assayed using ELISA. Affinity purification of immune serum was carried out using Protein A-Agarose Fast Flow resin (Sigma).
Primary antibodies were used in 1:1000 dilution and the specificity of different antibodies has been described earlier 9,10 . Alkaline phosphatase conjugated goat anti-rabbit IgG was used as secondary antibody (1:1000 dilutions). Immunospecific bands on the PVDF membrane were detected following incubation with NBT-BICP developer solution in dark for 5-10 min 8 . In case of blot analysis, for using the minimum volume of primary antibody, after the pilot experiment and determination of the position of respective specific bands, in most of the cases the blots in the relevant positions were cut and incubated with the antibody to minimize the use of excess antibodies.

Study of transcript accumulation under increasing salinity by semi-quantitative PCR
For expression analysis, total RNA was extracted from 7-days old wild type, OE-1, sog1-6 and sog1-1mutant seedlings, treated without or with increasing concentrations of NaCl for 12 hours.
Approximately 100 mg tissue was frozen immediately in liquid nitrogen after collection and then total RNA was extracted using RNeasyplant mini kit (Qiagen) following manufacturer's instructions. RNA concentration and purity were tested by spectrophotometric method. 1µg of total RNA was processed further to obtain cDNA using Superscript III Reverse Transcriptase (Invitrogen). Semi quantitative PCR was performed using the cDNA samples to examine the transcript accumulation of AtATM, AtSOG1, AtATR, AtBLT, AtSMR5 and AtSMR7 genes using specific expression primers (Supplementary table 2 For AtSMR7, a first cycle of 2 min at 94C, 45 s at 55C and 1 min at 72C was followed by 45 s at 94C, 45 s at 55C and 1 min at 72 C for 22 cycles. The conditions for semi-quantitative RT-PCR were chosen so that none of the mRNAs analyzed reached a plateau at the end of the amplification cycles, i.e. they were in the exponential phase of amplification, and that the two sets of primers (one set of gene specific primer and the other set for β-tubulin gene primers) used in each reaction did not compete with each other. Images of the RT-PCR products in ethidium bromide stained agarose gels were acquired in Bio-Rad Molecular Imager Gel Doc XR system with high resolution CCD camera and quantification of the bands was performed by Bio-Rad Image Densitometer GS-700 using Quantity One software. Band intensity was expressed as relative absorbance units. The ratio between the sample mRNA to be determined and β-tubulin was calculated to normalize for initial variations in sample concentration. In each set of samples, product quantity was normalized against β-tubulin after background subtraction. Mean values and standard deviation of all experiments performed were calculated after normalization to βtubulin. Mean values from three independent trials were used to represent the relative expression levels of transcripts (normalized to β-tubulin) in terms of percentage. Relative expression of each gene was determined by analyzing the band intensity using the ImageJ software.   Fig. S1: Determination of germination performance. (A-C) Analysis of germination frequencies in 3-days old SOG1 overexpressor line OE-1, wild-type, sog1-6 and sog1-1 mutant lines grown in absence or presence of increasing concentrations of NaCl. Three independent biological replicates for each genotype, each containing at least 30 seeds, were analysed. The mean value of germination frequency of individual experiment was determined and averaged subsequently for the three biological replicates. Error bar in the graphs represents ±SD. Asterisks represent statistically significant differences within a 5 and 1% confidence interval (*p < 0.05, **p < 0.01), respectively based on one-way ANOVA factorial analysis indicating the genotype that differs significantly in germination performance.   Densitometry analysis was carried out by ImageJ software (NIH). Asterisks indicate significant statistical differences (*p < 0.05 or **p < 0.01) from control (without NaCl treatment) using oneway ANOVA factorial analysis.  OE-1, sog1-6 and sog1-1 mutant seedlings grown in absence or presence of different concentrations of NaCl. Densitometry analysis was carried out by ImageJ software (NIH). Each bar represents the mean value of three independent replicates. Error bar indicates standard deviation. Asterisks indicate significant statistical differences (*p < 0.05 or **p < 0.01) from control (without NaCl treatment) using one-way ANOVA factorial analysis. Asterisks indicate significant statistical differences (*p < 0.05 or **p < 0.01) from control (without NaCl treatment) using one-way ANOVA factorial analysis.  protein gel blots using total protein extracted from 7-days old wild-type, sog1-6 and sog1-1 mutant seedlings. Affinity purified custom made anti-SOG1 polyclonal antibody (1:250 dilutions) was used to detect the SOG1 protein which is detected in WT and has been mentioned on the right-hand side of the gel image. Portion of the blots containing the specific bands of interest were cut prior to incubation with the primary antibody. Respective loading control of total protein extracts from wild-type, sog1-6, and sog1-1 seedlings, respectively has been presented below the blot image. Representative gel blot images from at least three independent experiments are shown. Transcript level of β-tubulin has been shown as the internal control. (E) Representative triplicates of SOG1 protein gel blots using total protein extracted from 7-days old wild-type, OE-1, OE-2 and OE-3 seedlings. Affinity purified custom made anti-SOG1 polyclonal antibody (1:250 dilutions) was used to detect the SOG1 protein which is detected in wild-type and has been mentioned on the right-hand side of the gel image. The blots were cut prior to hybridization with the primary antibody. Respective loading control of total protein extracts from wild-type, OE-1, OE-2 and OE-3 seedlings respectively has been presented below the blot image. Representative gel blot images from at least three independent experiments are shown.    triplicates of ATM and ATR protein gel blots, respectively using the total protein isolated from 7-days old OE-1, wild-type and sog1-1mutant plants respectively in response to increasing NaCl.

Supplementary Figures
(D-F) Full length gel images of respective loading controls.