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Tissue-specific clocks in Arabidopsis show asymmetric coupling

Abstract

Many organisms rely on a circadian clock system to adapt to daily and seasonal environmental changes. The mammalian circadian clock consists of a central clock in the suprachiasmatic nucleus that has tightly coupled neurons and synchronizes other clocks in peripheral tissues1,2. Plants also have a circadian clock, but plant circadian clock function has long been assumed to be uncoupled3. Only a few studies have been able to show weak, local coupling among cells4,5,6,7. Here, by implementing two novel techniques, we have performed a comprehensive tissue-specific analysis of leaf tissues, and show that the vasculature and mesophyll clocks asymmetrically regulate each other in Arabidopsis. The circadian clock in the vasculature has characteristics distinct from other tissues, cycles robustly without environmental cues, and affects circadian clock regulation in other tissues. Furthermore, we found that vasculature-enriched genes that are rhythmically expressed are preferentially expressed in the evening, whereas rhythmic mesophyll-enriched genes tend to be expressed in the morning. Our results set the stage for a deeper understanding of how the vasculature circadian clock in plants regulates key physiological responses such as flowering time.

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Figure 1: Direct tissue isolation from cotyledons.
Figure 2: Vasculature and mesophyll have different gene expression profiles.
Figure 3: Tissue-specific luciferase assay (TSLA).
Figure 4: The vasculature clock is robust and dominant to other clocks.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

All microarray data are available from the Gene Expression Omnibus database under accession code GSE50438.

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Acknowledgements

We thank H. Fukuda and Y. Sugisawa for processing raw microarray data; S. Yonehara for providing c-Jun and A-Fos plasmids; G. Breton, K. Hitomi, T. Oyama, T. Muranaka and Y. Kondo for advice; T. Koto, K. Katayama and B. Y. Chow for technical assistance; J. A. Hejna and T. R. Endo for English proofreading. This work was supported by an HFSP long-term Fellowship LT00017/2008-L (to M.E.), a JST PRESTO 11103346 (to M.E.), JSPS KAKENHI grants 22770036 and 25650097 (to M.E.), a Sumitomo Foundation and Nakatani Foundation (to M.E.), Grants-in-Aid for Scientific Research on Priority Areas 19060012 and 19060016 (to T.A.), and National Institutes of Health (NIH) Grants R01 GM056006 and GM067837 (to S.A.K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Authors and Affiliations

Authors

Contributions

M.E. and S.A.K. planned the experiments. M.E. and H.S. performed experiments. M.E., M.A.N., T.A. and S.A.K. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Motomu Endo.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Optimization and validation of gene expression analysis in isolated tissues.

a, b, Relationship between enzyme concentration and opacity of the enzyme solutions (a) or processing time for vasculature and epidermis isolation (b). Higher concentrations of enzyme (>2%) lead to lower handling ability during tissue isolation because of its opacity (grey box). Mean ± s.d.; n = 6. c, The expression levels of the 10 reference genes in whole leaves, mesophyll and vasculature were detected under long-day (LD) and short-day (SD) conditions, and then the average expression stabilities M were calculated according to Vandesompele’s method9. We chose ACTIN2 (ACT2), TUBULIN5 (TUB5), POLYUBIQUITIN10 (UBQ10), ELONGATION FACTOR1α (EF1α), ASCORBATE PEROXIDASE 3 (APX3)33 and ISOPENTENYL PYROPHOSPHATE:DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2 (IPP2)33 as commonly used reference genes, and we also chose ARABIDOPSIS TRITHORAX 3 (ATX3), REGULATORY PARTICLE TRIPLE-A 1A (RPT1a), THIOREDOXIN 3 (TRX3) and ASPARTIC PROTEINASE A1 (APA1) based on microarray analysis19. d, The expression of tissue-specific marker genes was detected in vasculature from plants grown under long-day conditions for 10 days. SULTR2;1 was used as a marker of phloem companion cells. WUSCHEL RELATED HOMEOBOX 4 (WOX4) and HOMEOBOX GENE 8 (AtHB8) were used as markers of procambium/cambium. IRREGULAR XYLEM 3 (IRX3) was used as a marker of xylem. Gene expression levels were calculated relative to LHCB2.1 expression. e, Total RNA was extracted from 10 cotyledons and 10 vasculatures grown under long-day conditions for 10 days. Extracted RNA was quantified and RNA content per single cotyledon and vasculature was estimated. n = 23. f, Expression of TOC1 and CCA1 in whole leaves, mesophyll, vasculature and epidermis. Plants were grown under long-day conditions for 10 days, and whole leaves, mesophyll, vasculature and epidermis were collected and/or isolated every 4 h. g, The expression level of the stress-induced genes COLD-REGULATED 15A (COR15A), ALCOHOL DEHYDROGENASE 1 (ADH1) and RESPONSIVE TO DESSICATION 29A (RD29A) in isolated mesophyll and vasculature with or without 50 µg ml−1 of α-Amanitin (an inhibitor of RNA polymerase II). d, f, g, The geometric mean of APA1 and IPP2 was used as a control. Mean ± s.e.m.; n = 3.

Extended Data Figure 2 Models used to identify cycling transcripts.

Models used in the HAYSTACK analysis were named Spike, Rigid, Cos, Mt, AsyMt1, AsyMt2, hBox, Box1, Box1.5 and Box2. All models are shifted in 1-h increments, and diel peaks at ZT0 (black) and ZT2 (grey) are shown as examples. Underlined models were used in a previous study19.

Extended Data Figure 3 Number of cycling genes, percentage of adopted models, and relationship between amplitude and genes called cycling in each condition.

a, b, Number of genes that cycle under long-day or short-day conditions in each tissue. c, Percentage of genes called cycling in a number of conditions. Four per cent of genes were not rhythmic in any condition. The remaining 96% of genes were broken down by the number of conditions for which they were called cycling. d, Frequency of model name adopted by the HAYSTACK analysis. Mt, AsyMt1 and AsyMt2 are integrated as Mt; and hBox, Box1, Box1.5 and Box2 are integrated as Box. e, Comparison of the percentage of genes called cycling versus genes not called cycling, by amplitude. Amplitude was estimated by dividing the maximum by the mean expression value across the time course.

Extended Data Figure 4 Validation of the sensitivity and specificity of the microarray analysis.

a, b, Expression profiles of mesophyll- (a) and vasculature-specific marker genes (b) under long-day (left) and short-day (right) conditions. CAB3, CARBONIC ANHYDRASE 1 (CA1)34 and KANADI 1 (KAN1) were applied as mesophyll markers. SUC2, FT and EARLY NODULIN-LIKE PROTEIN 9 (ENODL9)35 were applied as vasculature markers. c, Diel and inter-tissue variations in the expression of the reference genes APA1 and IPP2.

Extended Data Figure 5 Relative gene expression levels, percentage of phase shift genes and per cent of overlapping genes.

a, b, Relative gene expression levels in whole leaf, mesophyll and vasculature under short-day (a) and long-day (b) conditions. The average expression level in vasculature at ZT16 (a) and ZT0 (b) was set to 0. Blue- and green-coloured genes indicate higher and lower expression than average, respectively. c, Gene expression patterns of the PRR7, TOC1 and ELF4 in whole leaf, mesophyll and vasculature under long-day and short-day conditions. d, Percentage of genes showing a given phase shift when comparing two given tissues under long-day and short-day conditions. Phase shifts plotted as positive are phase delay. e, Phase shift topology graph with phase shift of the target tissue on the y axis and the reference tissue phase bin on the x axis. Heat-map indicates per cent of genes that are rhythmic between both conditions. f, Percentage of overlapping genes (POG) between any two tissues under long-day and short-day conditions. The P value resulting from the HAYSTACK analysis was used for gene ranking.

Extended Data Figure 6 Z-score profiles of cis-regulatory elements in each tissue.

Z-score profiles of the long-day vasculature element (LVE), short-day vasculature element (SVE), evening element (EE), Gbox, telo-box (TBX), starch box (SBX), and protein box (PBX) under long-day and short-day conditions are shown. The horizontal dotted line indicates the threshold (FDR < 1%).

Extended Data Figure 7 Luciferase complementation assay of TOC1/CaMV35S, TOC1/SUC2, CCA1/CaMV35S, CCA1/SUC2, and CaMV35S/SUC2 TSLA.

ac, Real-time monitoring of the luminescence of 10-day-old TOC1/SUC2 TSLA #2 (n = 9) and TOC1/CaMV35S TSLA #4 (n = 12) (a), CCA1/SUC2 TSLA #11 (n = 18) and CCA1/CaMV35S TSLA #1 (n = 12) (b), and CaMV35S/SUC2 TSLA #9 (n = 12) (c) seedlings under L/D or free running conditions. CT, circadian time. Signals after subtraction of background noise are shown. Mean ± s.d.; c.p.s., counts per second; c.p.30S., counts per 30 s. d, Period length of the TSLA lines shown in Fig. 3e, f and Extended Data Fig. 7a, b are calculated using fast Fourier transform non-linear least square analysis (FFT-NLLS)36. Mean ± 95% confidence interval.

Extended Data Figure 8 Clock genes expression in whole leaf and vasculature under L/D and continuous light free running conditions.

a, Ratio between the amplitude in the vasculature with respect to amplitude in whole leaf extracted from Fig. 4a (V(peak−trough)/W(peak−trough)). Mean ± s.e.m. b, c, TOC1, ELF4 and CCA1 expression under L/D and free running conditions in whole leaves (b) and vasculature (c). Plants were entrained for 5 days and shifted into the continuous light condition for 1 week. ZT, zeitgeber time; CT, circadian time. Mean ± s.d.; n = 3. To validate the robustness of each gene, the highest expression level in each gene in each tissue is set to 1. d, Ratio between the amplitude of TOC1::LUC with respect to the amplitude of TOC1::LUC; SUC2::CCA1 #18 extracted from Fig. 4b. Mean ± s.e.m.

Extended Data Figure 9 Organ- and tissue-specific expression of CCA1-GFP driven by tissue-specific promoters.

Expression levels of CCA1-GFP in a specific organ (a) and tissue (b). Plants were grown for 10 days under L/D conditions and seedlings were separated into each organ and tissue at ZT0. Based on contamination rate obtained from Fig. 1d, cross-contamination adjusted signals were shown (b). For the CCA1-GFP detection, GFP expression was measured by qPCR and the geometric mean of APA1 and IPP2 was used as a control. The highest values are set as 1. Mean ± s.e.m.; n = 3.

Extended Data Table 1 Gene ontology slim term enrichment analysis

Supplementary information

Supplementary Table 1

This file contains predefined models for the HAYSTACK. (XLS 129 kb)

Supplementary Table 2

This file contains a list of genes that are candidates of internal control. (XLS 103 kb)

Supplementary Table 3

This file contains a list of genes that are expressed dominantly in mesophyll. (XLS 488 kb)

Supplementary Table 4

This file contains a list of genes that are expressed dominantly in vasculature. (XLS 546 kb)

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Endo, M., Shimizu, H., Nohales, M. et al. Tissue-specific clocks in Arabidopsis show asymmetric coupling. Nature 515, 419–422 (2014). https://doi.org/10.1038/nature13919

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