TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation

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Abstract

The Arabidopsis genome contains three genes encoding proteins of the TRANSPARENT TESTA GLABRA 1 (TTG1) WD-repeat (WDR) subfamily. TTG1 is a known regulator of epidermal cell differentiation and pigment production, while LIGHT-REGULATED WD1 and LIGHT-REGULATED WD2 are known regulators of the circadian clock. Here, we discovered a new central role for TTG1 WDR proteins as regulators of the circadian system, as evidenced by the lack of detectable circadian rhythms in a triple lwd1lwd2ttg1 mutant. This shows that there has been subfunctionalization via protein changes within the angiosperms, with some TTG1 WDR proteins developing a stronger role in circadian clock regulation while losing the protein characteristics essential for pigment production and epidermal cell specification, and others weakening their ability to drive circadian clock regulation. Our work shows that even where proteins are very conserved, small changes can drive big functional differences.

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Fig. 1: Evolution of the TTG1 and LWD1/LWD2 clades.
Fig. 2: lwd1lwd2ttg1 has arrhythmic CCA1:LUC and leaf movement.
Fig. 3: CCA1:LUC rhythms in WDR family single, double and triple mutant lines.
Fig. 4: Two of the three M. polymorpha WDR genes rescue the ttg1-1 mutant.
Fig. 5: TTG1 and LWD1/LWD2 proteins show differential ability to rescue the circadian defect of the lwd1lwd2ttg1 triple mutant.
Fig. 6: Different WDR proteins show differential abilities to rescue the circadian defect of the lwd1lwd2ttg1 triple mutant.

Data availability

All data that support the findings of this study are available in the University of Cambridge data repository, with the identifier https://doi.org/10.17863/CAM.44078. Source data for Figs. 2, 3, 5 and 6 and Extended Data Figs. 2–7 and 9 are provided with the paper.

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Acknowledgements

We thank M. Dorling for laboratory and greenhouse support, and E. Moyroud for helpful discussions. E. Matthus and J. Davies provided help with the root hair analysis, M. Stancombe and X. Wang provided help with the circadian clock experiments, and N. Albert and K. Davies provided guidance on anthocyanin extraction. We thank S.-H. Wu (Institute of Plant and Microbial Biology, Academia Sinica, Taipei) for providing the lwd1lwd2 CCA1:LUC line. We thank the Cambridge University Botanic Garden for supplying A. trichopoda tissue for RT-PCR. C. A. Lugo provided support with the statistical analysis, and Q. Wang provided support with the figures and dot plot presentations. C.A.A. acknowledges support from the Cambridge University Botanic Garden Research Fund. T.J.H. was supported by BBSRC UK grant BB/M006212/1, awarded to A.A.R.W.

Author information

B.J.G., C.A.A. and A.A.R.W. conceived the project and designed the experiments. C.A.A. and T.J.H. conducted all of the experiments. S.F.B. conducted all of the phylogenetic analyses. B.J.G., A.A.R.W. and C.A.A. wrote the manuscript. All authors commented on the manuscript before submission.

Correspondence to Alex A. R. Webb or Beverley J. Glover.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Magnus Ekland and the other, anonymous, reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Alignment of TTG1- WDR proteins.

The alignment was performed with the MAFFT algorithm using the cloud-based informatics platform benchling. The colours indicate the degree of amino acid conservation between the six proteins from dark red for the most conserved to blue for the least conserved.

Extended Data Fig. 2 Transgenic rescue of the ttg1-1 phenotype.

a,b, Amount of anthocyanin in mg/g of dry weight, bars represent standard deviation of up to three different extractions. Comparison between WT, ttg1-1 mutant and ttg-1-1 mutant ectopically expressing a, TTG1, LWD1, LWD2, MpWDR1, MpWDR2. b, MpWDR3, AmLWD. Graphs show mean values for two biological replicates, each replicate pooled several seedlings. Source data

Extended Data Fig. 3 Transgenic rescue of the ttg1-1 phenotype.

a,b,c, Boxplots of root hair count in 2.5 mm of the first 5 mm of the root in the same genotypes used for the anthocyanin assay in Extended Data Fig. 2. Number of plants analyzed in a is WT = 13, ttg1 = 7, ttg1 35S:TTG1 = 9, ttg1 35S:LWD1 = 8, ttg1 35S:MpWDR1 = 10; in b is WT = 13, ttg1 = 10 ttg1 35S:TTG1 = 11, ttg1 35S:LWD2 = 14, ttg1 35S:MpWDR2 =12; in c is WT = 9, ttg1 = 6, ttg1 35S:AmLWD = 6, ttg1 35S:MpWDR3 = 6. Additional details about the statistics can be found in Supplementary Table 3. d, Table illustrating p values for pairwise comparisons. p values were calculated using a non parametric anova using the Kruskal-Wallis test, followed by a post hoc analysis of the means using the Conover test. ttg1 35S:TTG1/WDR1/WDR2 are all significantly different from the ttg1 mutant. ttg1 35S:LWD1/AmLWD are significantly different from the WT and ttg1 35S:WDR3 is significantly different from both, with higher support to be different from the WT. Additional details about the statistics can be found in Supplementary Table 3. Source data

Extended Data Fig. 4 Semi quantitative RT-PCR in plants ectopically expressing TTG1 WDR genes in the ttg1-1 mutant and in the triple mutant lwd1lwd2ttg1.

The figure shows for each sample 5 ul of the same amplification reaction after 20-25-35 PCR cycles. DNA ladder is 1kb hyperladder (Bioline). a, PCR of WT and ttg1 overexpressing lines (35S:TTG1, LWD1, LWD2, MpWDR1, MpWDR2) and reference gene (EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (EIF4A1)). b, PCR of WT, triple mutant lwd1lwd2ttg1 and ttg1 overexpressing lines (35S:AmLWD, TTG1, LWD1, LWD2, MpWDR1, MpWDR2). Negative control samples indicated with “-“. Given the big differences observed, this experiment was performed only once, using multiple lines for most of the transgenic plants. Source data

Extended Data Fig. 5 Flowering time phenotype of single and double and triple mutant combinations.

a, WT and triple mutant plants grown in the same tray in long day conditions show a dramatically different flowering time. b, c, The graphs represent the mean number of rosette and cauline leaves at bolting in different mutant combinations, error bars represent standard deviation. Number of plants in b is WT = 57, lwd1lwd2ttg1 = 47 in c is WT = 22, lwd1ttg1 = 23, lwd1lwd2 = 16, lwd2ttg1 = 17, ttg1 = 14. Source data

Extended Data Fig. 6 Comparison of lwd1 mutant with lwd1ttg1 double mutant.

a, CCA1:LUC luminescence measured from Col-0, lwd1, ttg1 and lwd1ttg1 seedlings. Seedlings were entrained in 12:12 light dark cycle and transferred to camera chamber on day 9. Luminescence was measured for one 12:12 light dark cycle and 96 hours in constant light. Mean luminescence shown with SEM for n = 7, except ttg1 where n = 3. FFT-NLLS was used to estimate period values implemented using Biodare 2. Student’s t test was used to identify whether genotypes were significantly different for period values with RAE<0.5. * denotes p<0.5. b, Expression analysis of LWD2 in ttg1lwd1 mutant. The graph shows mean relative expression of LWD2 in the ttg1lwd1 double mutant compared to WT in three biological replicates, data obtained by qRT-PCR with LWD2 specific primers and reference gene UBQ10. Error bars represent standard deviation on three biological replicates. Source data

Extended Data Fig. 7 lwd1lwd2ttg1 triple mutant phenotype.

a, Rosettes of Arabidopsis plants with mutant combinations of different TTG1 WDR genes. Plants were germinated at the same time and grown in the same LD conditions. In the triple mutant lwd1lwd2ttg1 leaf morphology is perturbed, whereas single and double mutant combinations have wild type leaf morphologies. These lines were grown repeatedly with no variations on these observations. b, Leaf margins of ttg1 mutant, lwd1ttg1 double mutant and the triple mutant lwd11lwd2ttg1. These differences were observed in a minimum of 6 plants in each of at least three independent batches. c, Boxplot of trichome numbers on the leaf edge of the ttg1 mutant, double mutants and the triple mutant. Data represent total trichome number on a plant with 9 leaves (number of plants counted lwd1lwd2ttg1 17, ttg1 18, ttg1lwd1 13, ttg1lwd2 11). d, Table illustrating p values for pairwise leaf trichome number comparisons. p values were calculated using a non parametric anova using the Kruskal-Wallis test, followed by a post hoc analysis of the means using the Conover test (the null hypothesis that the population medians of all of the groups are equal). Additional details about the statistics can be found in Supplementary Table 3. Source data

Extended Data Fig. 8 Semi quantitative RT- PCR in plants ectopically expressing TTG1 WDR genes in the triple mutant lwd1lwd2ttg1.

DNA ladder is 1kb hyperladder (Bioline). The figure shows for each sample 5ul of the same amplification reaction after 20-25- 35 or 22-27-35 PCR cycles. a, PCR with gene specific primers for TTG1 and reference gene (EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (EIF4A1)) on cDNA of triple mutant lwd1lwd2ttg1 plants overexpressing TTG1 in the triple mutant lwd1lwd2ttg1. b, PCR with gene specific primers for MpWDR3 and housekeeping gene on ttg1 and triple mutant lwd1lwd2ttg1 plants expressing Marchantia polymorpha gene MpWDR3. Data for MpWDR2 are included for comparison to a gene expression level that was capable of transgenic rescue. Negative control samples indicated with “-“. The figure show all line analyzed in semi quantitative RT-PCR. Source data

Extended Data Fig. 9 Flowering time of WT, triple mutant and triple mutant plants overexpressing TTG1-like WDR proteins.

a, Plants in each panel were sown at the same time and grown alongside each other in long day conditions. WT, triple mutant plants lwd1lwd2ttg1, triple mutant overexpressing LWD1, TTG1, MpWDR1, MpWDR2, MpWDR3 and AmLWD. All plants that were analyzed (WT 66, lwd1lwd2 41, triple 48, triple 35S:AmLWD1 26, triple 35S:LWD1 39, triple 35S:MpWDR1 23, triple 35S:MpWDR2 30, triple 35S:MpWDR3 54, triple 35S:TTG1 66) show the same pattern, with small variations that are reported quantitatively in b. b, Mean number of rosette and cauline leaves at bolting (error bars indicate standard deviation) and relative p values obtained using Post hoc pairwise test for multiple comparisons of mean rank sums (Dunn’s test) used after Kruskal-Wallis one-way analysis of variance by ranks to do pairwise comparisons. Triple 35S:LWD1 and triple 35S:MpWDR1 have the same flowering time as wild type; triple 35S:TTG1, triple 35S:MpWDR2 and triple 35S:AmLWD flower slightly earlier than the wild type and triple 35S:MpWDR3 flowers later than the WT. 35S:MpWDR3 flowers at the same time as the triple mutant (p value 1). lwd1lwd2 flowering time is significantly different from the wild type and most of the transgenics but the p value is higher when we compare lwd1lwd2 to triple 35S:TTG1 and triple 35S:AmLWD (p value respectively 0.09, 0.1). Number of plants in the analysis: WT 66, lwd1lwd2 41, triple 48, triple 35S:AmLWD1 26, triple 35S:LWD1 39, triple 35S:MpWDR1 23, triple 35S:MpWDR2 30, triple 35S:MpWDR3 54, triple 35S:TTG1 66. Additional details about the statistics can be found in Supplementary Table 3. Source data

Extended Data Fig. 10 Transgenic rescue of ttg1-1 mutant with 35S:AmLWD.

AmLWD is not able to rescue the seedcoat and trichome phenotypes of the ttg1-1 mutant. 6 of 6 independent transgenic lines all showed the same phenotype.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2, and Supplementary Tables 1–3.

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Airoldi, C.A., Hearn, T.J., Brockington, S.F. et al. TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation. Nat. Plants 5, 1145–1153 (2019) doi:10.1038/s41477-019-0544-3

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