Abstract
The OsFBT4 belongs to a small sub-class of rice F-box proteins called TLPs (Tubby-like proteins) containing the conserved N-terminal F-box domain and a C-terminal Tubby domain. These proteins have largely been implicated in both abiotic and biotic stress responses, besides developmental roles in plants. Here, we investigated the role of OsFBT4 in abiotic stress signalling. The OsFBT4 transcript was strongly upregulated in response to different abiotic stresses in rice, including exogenous ABA. When ectopically expressed, in Arabidopsis, under a constitutive CaMV 35S promoter, the overexpression (OE) caused hypersensitivity to most abiotic stresses, including ABA, during seed germination and early seedling growth. At the 5-day-old seedling growth stage, the OE conferred tolerance to all abiotic stresses. The OE lines displayed significant tolerance to salinity and water deficit at the mature growth stage. The stomatal size and density were seen to be altered in the OE lines, accompanied by hypersensitivity to ABA and hydrogen peroxide (H2O2) and a reduced water loss rate. Overexpression of OsFBT4 caused upregulation of several ABA-regulated/independent stress-responsive genes at more advanced stages of growth, showing wide and intricate roles played by OsFBT4 in stress signalling. The OsFBT4 showed interaction with several OSKs (Oryza SKP1 proteins) and localized to the plasma membrane (PM). The protein translocates to the nucleus, in response to oxidative and osmotic stresses, but failed to show transactivation activity in the yeast system. The OE lines also displayed morphological deviations from the wild-type (WT) plants, suggesting a role of the gene also in plant development.
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Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
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Funding
This research was funded by the Department of Science and Technology (DST), Government of India and J.C. Bose National Fellowship to JPK (SB/SR/JCB-13/2013) by the Science and Engineering Research Board (SERB), Government of India. Authors also acknowledge infrastructural support by the Department of Science and Technology (FIST and PURSE programs), Government of India, and the University Grants Commission (UGC-SAP), New Delhi. NJ thanks the Council of Scientific and Industrial Research (CSIR), New Delhi, for providing financial assistance in the form of RA Fellowship.
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NJ and JPK designed the research plan. NJ performed the experiments, analyzed the data and wrote the manuscript. JPK and PK critically supervised the progress of the research, gave vital inputs in the improvement of the research work, and edited the manuscript. JPK funded the cost of the entire research through his grants. All authors have read and approve the manuscript.
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Keynote
The rice Tubby-like protein (TLP) encoding gene, OsFBT4, plays role in both ABA-dependent and independent stress signalling and its overexpression confers stage-specific tolerance to salt, drought, and other abiotic stresses in Arabidopsis.
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Supplementary file1 (PDF 455 KB) Fig S1. Exon organization of OsFBT4 and its translated product and validation of the OsFBT4-OE lines in Arabidopsis. (A) and (B) represents the organization of the OsFBT4- cDNA and its translated product, respectively. (C) and (D) represents the genomic DNA PCR- based confirmation of the OE lines, using a combination of CaMV 35S promoter-F and OsFBT4-R primers (1.3 kb), and NPT-II gene specific primers, respectively. The transcript level of OsFBT4 in the OE lines quantified by RT-qPCR using Actin-II as internal control is presented in (E). The asterisk marks (*) indicate significant difference (P-value<0.05). Fig S2. Response of the OsFBT4-OE lines at 15-d old plant stage to ABA and paraquat. (A) depicts growth response of the OE lines to exogenous ABA (15 µM) and paraquat (2 µM) after 15 days on the stress media. Physiological measurements such as Fv/Fm values (B); total chlorophyll content (C); and final rosette diameter (D) after completion of 15 days on the stress media are graphically presented. The results are represented as mean ± SE, where n=3. The asterisk marks (*) indicate significant difference (P-value<0.05). Fig S3. Estimation of membrane injury parameters in 15-d old OsFBT4-OE plants in response to different abiotic stresses. (A) depicts overall ROS (H2O2) accumulation in 15-d old plants as seen by DAB staining; (B) graphically represents the Fv/Fm values; (C) shows the total chlorophyll content (represented in mg/g fresh wt.); the ion leakage percentages are presented graphically in (D). (E) and (F) shows the MDA and proline content of the plants, respectively, in response to different stresses. The results are represented as mean ± SE, where n=3. The asterisk marks (*) indicate significant difference (P-value<0.05).
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Jain, N., Khurana, P. & Khurana, J.P. Overexpression of a rice Tubby–like protein-encoding gene, OsFBT4, confers tolerance to abiotic stresses. Protoplasma 260, 1063–1079 (2023). https://doi.org/10.1007/s00709-022-01831-5
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DOI: https://doi.org/10.1007/s00709-022-01831-5