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Photoperiod controls plant seed size in a CONSTANS-dependent manner

Nature Plants volume 9, pages 343–354 (2023)Cite this article

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Abstract

Photoperiodic plants perceive changes in day length as seasonal cues to orchestrate their vegetative and reproductive growth. Although it is known that the floral transition of photoperiod-sensitive plants is tightly controlled by day length, how photoperiod affects their post-flowering development remains to be clearly defined, as do the underlying mechanisms. Here we demonstrate that photoperiod plays a prominent role in seed development. We found that long-day (LD) and short-day (SD) plants produce larger seeds under LD and SD conditions, respectively; however, seed size remains unchanged when CONSTANS (CO), the central regulatory gene of the photoperiodic response pathway, is mutated in Arabidopsis and soybean. We further found that CO directly represses the transcription of AP2 (a known regulatory gene of seed development) under LD conditions in Arabidopsis and SD conditions in soybean, thereby controlling seed size in a photoperiod-dependent manner, and that these effects are exerted through regulation of the proliferation of seed coat epidermal cells. Collectively, our findings reveal that a crucial regulatory cascade involving CO-AP2 modulates photoperiod-mediated seed development in plants and provide new insights into how plants with different photoperiod response types perceive seasonal changes that enable them to optimize their reproductive growth.

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Fig. 1: Seed size determination of various plant species grown under LDs and SDs.
Fig. 2: CO mediates photoperiod-regulated seed size in Arabidopsis.
Fig. 3: The expression patterns of CO in developing Arabidopsis seeds.
Fig. 4: CO directly inhibits the transcription of AP2.
Fig. 5: CO controls epidermal cell proliferation of seed coat through AP2.
Fig. 6: GmCOL2b-GmAP2s mediates photoperiod-regulated seed size in soybean.
Fig. 7: A hypothetical model of photoperiod controlling seed size.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon request. The high-throughput sequencing data performed in this study were deposited in the NCBI Gene Expression Omnibus database with the accession numbers PRJNA827694 and PRJNA896767. Source data are provided with this paper.

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Acknowledgements

We thank Z. Dong of Guangzhou University for providing Arabidopsis seeds of different ecotypes. This work was supported by grants from Key Deployment Project of Chinese Academy of Sciences (ZDRW-ZS-2019-2 to X.H.), the National Natural Science Foundation of China (31871643 to X.H. and 31900212 to Y.H.), and the ‘Strategic Priority Research Program’ of the Chinese Academy of Sciences (XDA24010100 to X.H.).

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  1. These authors contributed equally: Bin Yu, Xuemei He, Yang Tang.

Authors and Affiliations

  1. Guangdong Provincial Key Laboratory of Applied Botany and Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China

    Bin Yu, Xuemei He, Zhonghui Chen, Limeng Zhou, Xiaoming Li, Chunyu Zhang, Xiang Huang, Yuhua Yang, Wenbin Zhang, Xingliang Hou & Yilong Hu

  2. University of the Chinese Academy of Sciences, Beijing, China

    Bin Yu, Xuemei He, Zhonghui Chen, Limeng Zhou, Xiaoming Li, Chunyu Zhang, Xiang Huang, Yuhua Yang, Wenbin Zhang, Xingliang Hou & Yilong Hu

  3. Guangdong Provincial Key Laboratory of Plant Adaptation and Molecular Design, Guangzhou Key Laboratory of Crop Gene Editing, Innovative Center of Molecular Genetics and Evolution, School of Life Sciences, Guangzhou University, Guangzhou, China

    Yang Tang & Fanjiang Kong

  4. School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

    Yansong Miao

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Contributions

Y.H. and X. Hou conceived and designed the project. B.Y., X. He, Y.T., Z.C., L.Z., X.L., C.Z., X. Huang, Y.Y. and W.Z. conducted the experiments. B.Y., X. He, Y.T., F.K., Y.M. and Z.C analysed the data. B.Y., X. He, X. Hou and Y.H. wrote the manuscript.

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Correspondence to Xingliang Hou or Yilong Hu.

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Nature Plants thanks Federico Valverde, Keqiang Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Flowering time and seed mass determination of various plant species grown under LDs and SDs.

a, Flowering time of various plants grown under LDs and SDs. DBF, days before flowering. Data are presented as means ± s.d. of 10 biological replicates. b, Hundred-seed mass of various plants grown under LDs and SDs. Seed mass is the average value of five sample batches with each containing 100 seeds. Data are presented as means ± s.d. of 5 biological replicates. Asterisks indicate significant difference compared with plants grown under LDs (two-tailed Student’s t-test, *P < 0.05, **P < 0.01). All exact P values are shown in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Flowering time and seed mass determination of various Arabidopsis ecotypes grown under LDs and SDs.

a, Flowering time of various Arabidopsis ecotypes grown under LDs and SDs. Data are presented as means ± s.d. of 10 biological replicates. b, Thousand-seed mass of various Arabidopsis ecotypes grown under LDs and SDs. Seed mass is the average value of five sample batches with each containing 1000 seeds. Data are presented as means ± s.d. of 5 biological replicates. Asterisks indicate significant difference compared with plants grown under LDs (two-tailed Student’s t-test, **P < 0.01). All exact P values are shown in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Phenotypes of various genetic materials of CO grown under LDs.

a, Comparison of representative 24-d-old plants with various genetic backgrounds grown under LDs. Scale bar, 5 cm. b, Flowering time of plants shown in a. Data are presented as means ± s.d. of 10 biological replicates. c, Plant stature of representative 60-d-old plants with various genetic backgrounds grown under LDs. Scale bar, 5 cm. d, Yield per plant of various genetic materials grown under LDs. Data are presented as means ± s.d. of 10 biological replicates. e, Comparison of representative 16 DAP siliques harvested from various genetic materials grown under LDs. Scale bar, 2 mm. f, Silique capacity per seed showing that big seeds occupy larger space than small seeds in siliques. The ratio was calculated by silique area against the seed number per silique in various genetic materials grown under LDs. Data are presented as means ± s.d. of 30 biological replicates. g, Comparison of dry mature seeds of various genetic materials grown under LDs. Scale bar, 500 µm. h, Seed size statistics of various genetic materials shown in g. Data are presented as means ± s.d. of 50 biological replicates. Different lowercase letters above the columns indicate the significant difference among different groups (one-way ANOVA, Tukey post-test, P < 0.05). All exact P values are shown in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 CO acts as the central regulator of photoperiod response pathway to promote flowering transition in Arabidopsis.

CO acts as the central regulator of photoperiod response pathway to integrate external and internal signals into the photoperiodic flowering pathway. The transcription and protein abundance of CO is tightly controlled by circadian clock and intrinsic clues for proper FLOWERING LOCUS T (FT) induction that triggers flowering transition. CO can also inhibit flowering by disordering the expression pattern of TERMINAL FLOWER 1 (TFL1), which functions antagonistic to FT. Photoperiod-dependent patterns of CO transcript abundance are tightly controlled by the FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1)-GIGANTEA (GI) complex, which mediates the degradation of CO transcriptional repressors known as CYCLING DOF FACTORs (CDFs). Degradation of CDFs facilitates access by transcriptional activators FLOWERING BHLHS (FBHs) to the CO promoter. Several kinds of photoreceptors, including FKF1, phytochrome A (phyA) and cryptochromes (CRYs), are involved in CO protein stabilization. Other factors, including phytochrome B (phyB), HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (HOS1), and CONSTITUTIVELY PHOTOMORPHOGENIC1 -SUPPRESSOR OF PHYTOCHROME A (COP1-SPA) complexes, are involved in the degradation of CO protein.

Extended Data Fig. 5 Photoperiod affects seed size independent of accumulative radiation.

Seed area of Col-0, 35S:CO, and co-9 grown under LDs-LDs, LDs-dSDs, and LDs-SDs. LDs-LDs, plants were grown under LDs until harvest; LDs-dSDs, plants were grown under LDs until bolting and then transferred to SD conditions under a double of normal light intensity; LDs-SDs, plants were grown under LDs until bolting and then transferred to SDs. Data are presented as means ± s.d. of 100 biological replicates. Different lowercase letters above the columns indicate the significant difference among different groups (one-way ANOVA, Tukey post-test, P < 0.05). All exact P values are shown in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 CO functions in early stage of seed development under LDs.

a, Representative unfertilized mature ovules of Col-0 and co-9 plants. Scale bar, 50 µm. b, Area of unfertilized mature ovules in Col-0 and co-9 plants. Data are presented as means ± s.d. of 10 biological replicates. c, Seed area of wild-type (Ler) and co-2 35S:CO-GR (CO-GR) treated with 10 μM Dex or mock. Different stages of silique at 2, 4, 6, 8 or 10 DAP were chosen for the first-time treatment, respectively, then once every 3 d until reaching maturity. Data are presented as means ± s.d. of 100 biological replicates. Different lowercase letters above the columns indicate the significant difference among different groups (one-way ANOVA, Tukey post-test, P < 0.05). All plants were grown under LDs. All exact P values are shown in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 The CO expression in developing Arabidopsis siliques and seeds.

a, GUS staining in developing siliques in pCO:GUS. Scale bar, 2 mm. b, Partial enlarged detail of GUS staining in 8 DAP siliques. Scale bar, 100 μm. CZSC, chalazal seed coat; H, hilum; SW, silique wall. All plants were grown under LDs.

Extended Data Fig. 8 gCO-3FLAG completely rescues the phenotype of co-9.

a–c, Flowering time, seed area and seed mass of Col-0, co-9 and 3 representative co-9 gCO-3FLAG lines (#1-3) grown under LDs. Seed mass is the average value of five sample batches with each containing 1000 seeds. For flowering time, seed area and seed mass, data are presented as means ± s.d. of 10, 100 and 5 biological replicates, respectively. Different lowercase letters above the columns indicate the significant difference among different groups (one-way ANOVA, Tukey post-test, P < 0.05). All exact P values are shown in Source Data Extended Data Fig. 8.

Source data

Extended Data Fig. 9 Localization of AP2 expression in developing seeds under LDs.

RNA in situ hybridization of AP2 in seeds at torpedo (left) and bent cotyledon (right) stages using the AP2 antisense or sense probe. Scale bar, 50 µm. EP, epidermis; EM, embryo. Each experiment was repeated three times with similar results.

Extended Data Fig. 10 AP2 mediates photoperiod-regulated seed size in Arabidopsis.

a, Seed area of Col-0 and ap2-12 grown under LDs and SDs. Data are presented as means ± s.d. of 100 biological replicates. b, RT–qPCR analysis of AP2 expression in developing seeds of Col-0 and co-9 grown under LDs and SDs. c, RT–qPCR analysis of AP2 expression in 4 DAP seeds of wild-type (Ler) and co-2 35S:CO-GR (CO-GR) treated with 10 μM Dex or mock at ZT16 under LDs and SDs, respectively. UBQ10 serves as an internal control. Data are presented as means ± s.d. of 3 biological replicates. Asterisks indicate significant difference compared with control groups (two-tailed Student’s t-test, **P < 0.01). All exact P values are shown in Source Data Extended Data Fig. 10.

Source data

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Reporting Summary

Supplementary Table

Supplementary Table 1. Differentially expressed genes in 4 DAP seeds of Col-0 and co-9 grown under LDs. Supplementary Table 2. Gene ontology (GO) analysis of genes regulated by CO. Supplementary Table 3. Differentially expressed genes in 4 DAP seeds of Col-0 and 35S:CO grown under LDs. Supplementary Table 4. Differentially expressed genes in 4 DAP seeds of Col-0 and ap2-12 grown under LDs. Supplementary Table 5. Differentially expressed genes in 4 DAP seeds of Col-0 and co-9 ap2-12 grown under LDs. Supplementary Table 6. Co-regulated genes in 4 DAP seeds of ap2-12 and co-9 ap2-12 grown under LDs. Supplementary Table 7. GO analysis of the genes co-regulated in 4 DAP seeds of ap2-12 and co-9 ap2-12 grown under LDs. Supplementary Table 8. KEGG analysis of the genes co-upregulated in 4 DAP seeds of ap2-12 and co-9 ap2-12 grown under LDs. Supplementary Table 9. KEGG analysis of the genes co-downregulated in 4 DAP seeds of ap2-12 and co-9 ap2-12 grown under LDs. Supplementary Table 10. List of primers and DNA sequences used in this study.

Supplementary Data

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Yu, B., He, X., Tang, Y. et al. Photoperiod controls plant seed size in a CONSTANS-dependent manner. Nat. Plants 9, 343–354 (2023). https://doi.org/10.1038/s41477-023-01350-y

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