Advanced transcriptome sequencing has revealed that the majority of eukaryotic genes undergo alternative splicing (AS). Nonetheless, little effort has been dedicated to investigating the functional relevance of particular splicing events, even those in the key developmental and hormonal regulators. Combining approaches of genetics, biochemistry and advanced confocal microscopy, we describe the impact of alternative splicing on the PIN7 gene in the model plant Arabidopsis thaliana. PIN7 encodes a polarly localized transporter for the phytohormone auxin and produces two evolutionarily conserved transcripts, PIN7a and PIN7b. PIN7a and PIN7b, differing in a four amino acid stretch, exhibit almost identical expression patterns and subcellular localization. We reveal that they are closely associated and mutually influence each other's mobility within the plasma membrane. Phenotypic complementation tests indicate that the functional contribution of PIN7b per se is minor, but it markedly reduces the prominent PIN7a activity, which is required for correct seedling apical hook formation and auxin-mediated tropic responses. Our results establish alternative splicing of the PIN family as a conserved, functionally relevant mechanism, revealing an additional regulatory level of auxin-mediated plant development.
We thank Claus Schwechheimer for the pin34 and pin347 seeds, Yuliia Mironova for technical assistance, Ksenia Timofeyenko and Dmitry Konovalov for help with the evolutional analysis, Konstantin Kutashev and Siarhei Dabravolski for assistance with FRET-FLIM, Huibin Han for advice with hypocotyl imaging, Karel Müller for the initial qRT-PCR on the tobacco cell lines, Stano Pekár for suggestions regarding the statistical analysis of the morphodynamic measurements, and Jozef Mravec, Dolf Weijers and Lindy Abas for their comments on the manuscript. This work was supported by the Czech Science Foundation (projects 16-26428S and 19-23773S to IK, MH and KRůžička, 19-18917S to JHumpolíčková and 18-26981S to JF), and the Ministry of Education, Youth and Sports of the Czech Republic (MEYS, CZ.02.1.01/0.0/0.0/16_019/0000738) to KRůžička and JHejátko. The imaging facilities of the Institute of Experimental Botany and CEITEC are supported by MEYS (LM2018129 – Czech BioImaging and CZ.02.1.01/0.0/0.0/16_013/0001775). The authors declare no competing interests.
Kashkan I, Hrtyan M, Retzer K, et al. Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana. New Phytologist. 2021. doi:10.1111/nph.17792
Kashkan, I., Hrtyan, M., Retzer, K., Humpolíčková, J., Jayasree, A., Filepová, R., … Růžička, K. (2021). Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana. New Phytologist. Wiley. https://doi.org/10.1111/nph.17792
Kashkan, Ivan, Mónika Hrtyan, Katarzyna Retzer, Jana Humpolíčková, Aswathy Jayasree, Roberta Filepová, Zuzana Vondráková, et al. “Mutually Opposing Activity of PIN7 Splicing Isoforms Is Required for Auxin-Mediated Tropic Responses in Arabidopsis Thaliana.” New Phytologist. Wiley, 2021. https://doi.org/10.1111/nph.17792.
I. Kashkan et al., “Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana,” New Phytologist. Wiley, 2021.
Kashkan I, Hrtyan M, Retzer K, Humpolíčková J, Jayasree A, Filepová R, Vondráková Z, Simon S, Rombaut D, Jacobs TB, Frilander MJ, Hejátko J, Friml J, Petrášek J, Růžička K. 2021. Mutually opposing activity of PIN7 splicing isoforms is required for auxin-mediated tropic responses in Arabidopsis thaliana. New Phytologist.
Kashkan, Ivan, et al. “Mutually Opposing Activity of PIN7 Splicing Isoforms Is Required for Auxin-Mediated Tropic Responses in Arabidopsis Thaliana.” New Phytologist, Wiley, 2021, doi:10.1111/nph.17792.
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