Abstract
Key message
The Arabidopsis Tubby-like protein (TLP) encoding gene, AtTLP2, plays intricate roles during ABA-dependent abiotic stress signalling, particularly salt and dehydration stress responses.
Abstract
TLPs (Tubby-like proteins) are a small group of eukaryotic proteins characterized by the presence of a Tubby domain. The plant TLPs have been widely shown to play important roles during abiotic stress signaling. In this study, we investigated the role of an Arabidopsis TLP, AtTLP2, in mediating abiotic stress responses. Both attlp2 null mutant and overexpression (OE) lines, in Arabidopsis, were studied which indicated the role of the gene also in development. The attlp2 mutant showed an overall dwarfism, while its overexpression caused enhanced growth. AtTLP2 localized to the plasma membrane (PM) and showed nuclear translocation in response to dehydration stress. The protein interacted with ASK1 and ASK2, but failed to show transactivation activity in yeast. AtTLP2 was transcriptionally induced by stress, caused by salt, dehydration and ABA. The attlp2 mutant was insensitive to ABA, but hypersensitive to oxidative stress at all stages of growth. ABA insensitivity conferred tolerance to salt and osmotic stresses at the germination and early seedling growth stages, but caused hypersensitivity to salt and drought stresses at advanced stages of growth. The OE lines were more sensitive to ABA, causing increased sensitivity to most stresses at the seed germination stage, but conferring tolerance to salt and osmotic stresses at more advanced stages of development. The stomata of the attlp2 mutant were less responsive to ABA and H2O2, while that of the OE lines exhibited greater sensitivity. Several ABA-regulated stress responsive marker genes were found to be downregulated in the mutant, but upregulated in the OE lines. The study establishes that AtTLP2 plays intricate roles in abiotic stress signaling, and the response may be largely ABA dependent.
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The datasets generated in the current study are available from the corresponding author on reasonable request.
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Acknowledgements
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 programmes), 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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JC BOSE NATIONAL FELLOWSHIP, SB/SR/JCB-13/2013, Jitendra Paul Khurana.
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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 research, gave vital inputs in 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 approved the manuscript.
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299_2022_2953_MOESM1_ESM.pdf
Fig S1. PCR-based confirmation of the attlp2 mutant and the AtTLP2-OE lines. (A) depicts diagrammatically the AtTLP2 genomic DNA, exon organization and position of T-DNA insertion. The primers used for the PCR validation, using genomic DNA from mutant plants, have also been indicated. The combination of AtTLP2-specific FP and RP primers and T-DNA specific LBA primer produced PCR-bands of 1100 bp in the mutant and 2.1 kb in WT, proving homozygous mutation (B). (C) shows RT-PCR using AtTLP2 CDS-specific cFP and cRP primers, depicted in the figure, amplifying the 1100 bp AtTLP2-CDS in WT but no product in the mutant, confirming null mutation. (D) depicts the PCR-based confirmation of the AtTLP2-OE lines, using genomic DNA and NPT-II (750 bp) specific primers. Fig S2. Response of the attlp2 mutant and the AtTLP2-OE lines at 15-d old plant stage to ABA and oxidative stress. (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). Supplementary file1 (PDF 143 KB)
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Jain, N., Khurana, P. & Khurana, J.P. AtTLP2, a Tubby-like protein, plays intricate roles in abiotic stress signalling. Plant Cell Rep 42, 235–252 (2023). https://doi.org/10.1007/s00299-022-02953-z
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DOI: https://doi.org/10.1007/s00299-022-02953-z