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PHOTOTROPIN1 lysine 526 functions to enhance phototropism in Arabidopsis

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Abstract

Main conclusion

After blue-light exposure, ubiquitination of PHOTOTROPIN1 lysine 526 enhances phototropic responses.

Abstract

Arabidopsis blue-light photoreceptor, PHOTOTROPIN1 (PHOT1) mediates a series of blue-light responses that function to optimize photosynthesis efficiency. Blue-light sensing through the N-terminal sensory domain activates the C-terminal kinase activity of PHOT1, resulting in autophosphorylation. In addition to phosphorylation, PHOT1 lysine residue 526 (Lys526), after blue-light exposure, was found to carry a double glycine attachment, indicative of a possible ubiquitination modification. The functionality of PHOT1 Lys526 was investigated by reverse genetic approaches. Arginine replacements of PHOT1 Lys526, together with Lys527, complemented phot1-5 phot2-1 double mutant with attenuated phototropic bending, while blue-light responses: leaf expansion and stomatal opening, were restored to wild type levels. Transgenic seedlings were not different in protein levels of phot1 Lys526 527Arg than the wild type control, suggesting the reduced phototropic responses was not caused by reduction in protein levels. Treating the transformants with proteosome inhibitor, MG132, did not restore phototropic sensitivity. Both transgenic protein and wild type PHOT1 also had similar dark recovery of kinase activity, suggesting that phot1 Lys526 527Arg replacement did not affect the protein stability to cause the phenotype. Together, our results indicate that blocking Lys526 ubiquitination by arginine substitution may have caused the reduced phototropic phenotype. Therefore, the putative ubiquitination on Lys526 functions to enhance PHOT1-mediated phototropism, rather than targeting PHOT1 for proteolysis.

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Data availability

The data underlying this article will be available upon reasonable request to the corresponding author. Accession numbers: Arabidopsis PHOT1, At3g45780.

Abbreviations

NPH3:

NON-PHOTOTROPIC HYPOCOTYL 3

PHOT1:

Wild type PHOTOTROPIN1 protein

PHOT1 GFP :

Wild type PHOTOTROPIN1 cDNA with green fluorescent protein tag

phot1 Lys526 527Arg:

Mutated phototropin1 protein with lysine residues 526, 527 replaced with arginine

References

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Acknowledgements

We thank Dr. Lynn Hartweck, University of Minnesota, for critical reading of the manuscript; and Professor Akira Nagatani, Kyoto University, for the gift of the PHOT1 cDNA transgenic plasmid, Pp1:P1G-nosT/pPZP211. We thank late Professor Winslow Briggs, Carnegie Institution, Stanford University, for generous supports.

Funding

This work was financially supported by a grant from the Ministry of Science and Technology, Taiwan, (Grant number MOST110WFA1610040) to TST.

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Authors and Affiliations

  1. Department of Agricultural Biotechnology, National Chiayi University, 300 Syuefu Road, Chiayi, 600, Taiwan

    Tong-Seung Tseng, Chih-An Chen & Ming-Hung Lo

Authors
  1. Tong-Seung Tseng
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  2. Chih-An Chen
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  3. Ming-Hung Lo
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Contributions

TST conceived and drafted this project for research. CAC, and MHL helped with material preparations, experiment set up, and data collecting; TST together with help from CAC and MHL conducted and carried out the experiments. TST drafted the manuscript. All authors reviewed and approved the final manuscript.

Corresponding author

Correspondence to Tong-Seung Tseng.

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The authors declare that they have no competing financial interests.

Additional information

Communicated by Dorothea Bartels.

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Supplementary Information

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425_2024_4332_MOESM1_ESM.pptx

Supplementary file1 Supplementary Fig. S1 Representation of the PHOTOTROPIN1 protein. PHOT1: PHOTOTROPIN1 protein; LOV: LOV domain; J-alpha: J-alpha helix; number1: the starting methionine of PHOT1; K526: lysine residue at position 526 of PHOT1. Lower part shows detail amino acid residues (one letter) from position 501 to 560. The LOV2 core sequence was underlined, following by the double lysine pair at position 526 (underlined), and the other double lysine pair at position 550, used for mutagenesis to replace with arginine. Supplementary Fig. S2 a Phototropic responses after 8.5 h of blue light (1.5 μmol m-2 s-1) irradiance. Three-day-old etiolated seedlings of both gl1 and phot2-1 (functional PHOT1 only) were wild type controls. phot1-5/phot2-1double mutant exhibited residual bending angles. PHOT1 GFP complementation resorted the phototropic response of the phot1-5/phot2-1double mutant to the wild type level, while phot1 Lys526Arg GFP (phot1 K526R GFP), phot1 Lys527Arg GFP (phot1 K527R GFP), or phot1 Lys526 527Arg GFP (phot1 K526 527R GFP) transgenic lines had smaller bending angles than phot2-1. The asterisks above the bars indicate individual transgenic lines with significant smaller bending angles than phot2-1 (data represent: mean +SE; n>20; Student’s t test; P < 0.05). b Attenuated phototropic responses of phot1 Lys526Arg GFP and phot1 Lys527Arg GFP transgenic lines. Three-day-old etiolated seedlings were treated with 1 μmol m-2 s-1 of blue light and bending angles of hypocotyls were imaged for analyses at the time indicated (mean +SE; n>25). The phot2-1 mutant and PHOT1 GFP line were wild type controls. phot1 K526R GFP: phot1-5/phot2-1double mutant complemented with phot1 Lys526Arg GFP; phot1K527R GFP: phot1-5/phot2-1double mutant complemented with phot1 Lys527Arg GFP. The asterisks above the bars indicate the individual transgenic line with significant smaller bending angles than the phot2-1 control (functional PHOT1 only) (Student’s t test; P < 0.05). Similar results were obtained in two other experiments. Supplementary Fig. S3 phot1 Lys526 527Arg GFP replacements did not change the ubiquitination pattern of PHOT1. Etiolated three-day-old seedlings expressing PHOT1 GFP or phot1 Lys526 527Arg GFP (phot1 526 527R GFP) were irradiated with 100 μmol m-2 s-1 of blue light for two hours. Microsomal fractions made from these seedlings were immuno-precipitated with antibodies against GFP (IP: α -GFP). The immuno-precipitated proteins were resolved with SDS-PAGE and blotted for protein detection with PHOT1-specific antibodies (IB: α -PHOT1), or with P4D1 antibodies (IB: α -P4D1) that recognize ubiquitination modifications on pulled-down phot1 protein. Supplementary Fig. S4 Etiolated hypocotyl elongation and gravitropic responses of phot1Lys526 527Arg GFP individual, or double replacements transgenic lines. a Etiolated hypocotyl elongation. Hypocotyl elongation of etiolated seedlings grown on half-strength MS medium was imaged for measurement at the time indicated (mean +SE; n>20). b Gravitropic responses of etiolated seedlings. Seeds were germinated vertically on half-strength MS medium for three days in darkness. Medium plates were turned horizontally and hypocotyl bending angles were imaged for analyses at the time indicated (mean +SE; n>25). PHOT1 GFP: PHOT1 GFP line. phot1 K526R GFP: phot1-5/phot2-1double mutant complemented with phot1 Lys526Arg GFP. phot1 K527R GFP: phot1-5/phot2-1double mutant complemented with phot1 Lys527Arg GFP. phot1 K526 527R GFP: phot1-5/phot2-1double mutant complemented with phot1 Lys526 527Arg GFP. Similar trends were seen in two different experiments. Supplementary Fig. S5 phot1 Lys550 551Arg GFP transformant lines exhibited complementation in blue light responses of phototropic bending and stomatal opening. a Etiolated three-day-old seedlings of phot1 Lys550 551Arg GFP transformant line (phot1 K550 551R GFP) were treated with blue light (1 μmol m-2 s-1) for eight hours; the bending angles were determined (mean +SE; n>30). phot2-1 mutant was used as a control of PHOT1 functional only. b Stomatal opening in response to blue light treatments. Stomata from abaxial leaf surface of mature (5-week-old) phot1 Lys550 551Arg GFP transformants (phot1 K550 551R GFP) were treated with blue light (1 and 10 μmol m-2 s-1), superimposed with red light (50 μmol m-2 s-1) for two hours before harvesting for measurements (mean +SE; n>25). phot2-1 mutant was used as a control. Similar results were seen in two different experiments. No significant difference between the phot2-1 control and individual transgenic lines (Student’s t test; P < 0.05)

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Tseng, TS., Chen, CA. & Lo, MH. PHOTOTROPIN1 lysine 526 functions to enhance phototropism in Arabidopsis. Planta 259, 56 (2024). https://doi.org/10.1007/s00425-024-04332-2

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