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Epigenetic silencing of callose synthase by VIL1 promotes bud-growth transition in lily bulbs

Nature Plants volume 9, pages 1451–1467 (2023)Cite this article

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Abstract

In plants, restoring intercellular communication is required for cell activity in buds during the growth transition from slow to fast growth after dormancy release. However, the epigenetic regulation of this phenomenon is far from understood. Here we demonstrate that lily VERNALIZATION INSENSITIVE 3-LIKE 1 (LoVIL1) confers growth transition by mediating plasmodesmata opening via epigenetic repression of CALLOSE SYNTHASE 3 (LoCALS3). Moreover, we found that a novel transcription factor, NUCLEAR FACTOR Y, SUBUNIT A7 (LoNFYA7), is capable of recruiting the LoVIL1–Polycomb Repressive Complex 2 (PRC2) and enhancing H3K27me3 at the LoCALS3 locus by recognizing the CCAAT cis-element (Cce) of its promoter. The LoNFYA7–LoVIL1 module serves as a key player in orchestrating the phase transition from slow to fast growth in lily bulbs. These studies also indicate that LoVIL1 is a suitable marker for the bud-growth-transition trait following dormancy release in lily cultivars.

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Fig. 1: The crucial role of intercellular communication during bud-growth transition in lily bulbs.
Fig. 2: LoVIL1 accelerates growth transition and promotes intercellular communication in lily.
Fig. 3: LoCALS3 delays growth transition by blocking intercellular communication in lily.
Fig. 4: LoVIL1 interactor, LoNFYA7, represses growth transition by blocking intercellular communication.
Fig. 5: LoNFYA7 directly binds the LoCALS3 promoter and controls its expression.
Fig. 6: LoNFYA7 recruits LoVIL1–PRC2 to enhance H3K27me3 modification at LoCALS3 locus.
Fig. 7: LoVIL1 is a reliable index for growth-transition trait in lily bulbs.

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Data availability

Data supporting the findings of this study are available from the corresponding author upon request. All primers used in this study are listed in Supplementary Table 1. RNA-sequencing data that support the findings of this study have been deposited in the NCBI Bioproject database under accession number PRJNA935924. Information on related genes in this study has been uploaded to GenBank in the NCBI Nucleotide database, including LoVIL1 (OQ469822), LoNFYA7 (OQ469823), LoCALS3 (OQ469824), LoFT1 (OQ469825), LoCALS5 (OQ469826), LoCALS7 (OQ469827), LoCALS10 (OQ469828), LoCALS12 (OQ469829) and LoHDA14 (OQ857021). Source data are provided with this paper.

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Acknowledgements

This work was funded by the Beijing Natural Science Foundation (6212012 to J.W.), National Natural Science Foundation projects (grants 32172617 and 31701952 to J.W.), Research Outstanding Talents Training Project of the Ministry of Agriculture and Rural Affairs, Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects (CEFF-PXM 2019_014207_000032), the 2115 Talent Development Program of China Agricultural University, the Project supported by the Strategic Development Department of China Association for Science and Technology, and 111 Project of the Ministry of Education (B17043).

Author information

Author notes

  1. These authors contributed equally: Wenqiang Pan, Jingru Li, Yunpeng Du.

Authors and Affiliations

  1. Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, Department of Ornamental Horticulture, China Agricultural University, Beijing, China

    Wenqiang Pan, Jingru Li, Yajie Zhao, Yin Xin, Shaokun Wang, Chang Liu, Mingfang Yi & Jian Wu

  2. Institute of Grassland, Flowers, and Ecology, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China

    Yunpeng Du & Xiuhai Zhang

  3. Institute of Biotechnology, Fujian Academy of Agricultural Sciences, Fuzhou, China

    Zhimin Lin & Shaozhong Fang

  4. Institute of Floriculture, Liaoning Academy of Agricultural Sciences, Shenyang, China

    Yingdong Yang

  5. Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel

    Michele Zaccai

  6. Department of Biological Sciences, University of Toronto Scarborough, Toronto, Ontario, Canada

    Sonia Gazzarrini

  7. Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada

    Sonia Gazzarrini

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  1. Wenqiang Pan
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Contributions

J.W. and M.Y. designed the research. W.P. and J.L. cloned constructs and performed most of the experiments and data analysis. Y.Z., Y.X., C.L. and S.W. helped perform RT–qPCR and lily transformation assays. W.P., J.L., M.Y. and J.W. conceived the research and designed the experiments. Y.D., Z.L., S.F., Y.Y. and X.Z. provided lily species and bulbs. W.P. and J.W. wrote the original paper. J.W., M.Z. and S.G. revised the paper. J.W. agreed to serve as the corresponding author and ensure communication.

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Correspondence to Jian Wu.

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Extended data

Extended Data Fig. 1 Effect of cold storage duration on lily sprouting and flower differentiation.

a, Bulb growth transition: cold storage (4 °C) accelerates bulb growth transition, while frozen storage (-1.5 °C) delays it for long-term storage. The distance between the bud and the bulb tip shows the bud elongation during storage. Data are presented as mean ± s.d. of three biological replicates (n = 5 bulbs per replicate). b, Bulb sprouting: effect of cold storage (4 °C) duration on bulb sprouting. Data are presented as mean ± s.d. of three biological replicates (n = 100 bulbs per sample; two-sided Student’s t-test). c, Plant height: effect of cold storage (4 °C) duration on plant height 90 days after planting. Data are presented as mean ± s.d. of three biological replicates (n = 100 bulbs per sample; two-sided Student’s t-test; ns: not significant and p > 0.05). d, Central bud development: central bud development at different stages using scanning electron microscopy. In DBs, the central bud was flat on the basal plate. In GTBs, the central bud grew up to2/3 bulb height and the SAM was evident. After sprouting, the SAM of the central bud was converted into FAM. The highlighted area in red indicates the tepal primordium. Consistent results were obtained in at least three independent bulbs. DB: dormant bulbs (cold storage at 4 °C for 0 weeks); GTB: growth transited bulbs (cold storage at 4 °C for 8 weeks); FAM: flower apical meristem; SAM: shoot apical meristem; SB: sprouting bulbs (cold storage at 4 °C for 8 weeks and then sprouting at 12 °C for 2 weeks); TP: Tepal primordium. Scale bar, 2 mm.

Source data

Extended Data Fig. 2 Simulation of symplastic transport in central buds of dormant bulbs under different treatments by using CFDA.

Deep dormant bulbs were cultivated on MS medium containing 1 mM N-Ethylmaleimide (NEM), 1 mM 2,3-Butanedione-monoxime (BDM), or 1 mM 2-Dioxy-D-glucose (DDG) for 4 weeks. MS medium was used as the control. Images showed the unloading of CFDA in central buds after importing a 3-hour tracer import. In the control and NEM treatment, CFDA fluorescence was primarily confined to the vascular bundles (Vb) with weak signals. However, in the DDG and BDM treatments, the fluorescence was spread to the surrounding parenchyma cells. Consistent results were obtained in at least three independent bulbs at the same stage. Scale bar, 20 μm.

Extended Data Fig. 3 Silencing of LoVIL1 affects bulb growth transition and flower development.

a, Phenotypes of TRV2-LoVIL1 silenced plants after the sprouting stage (cold storage at 4 °C for 8 weeks and then 12 °C for 2 weeks). Scale bar, 1 cm. b, Detecting the expression of virus COAT PROTEIN (CP) in SAMs of TRV2-LoVIL1 bulbs. FP (F-BOX FAMILY PROTEIN) served as the positive control. Noninfected plants were used as negative control (NC). c, Expression analysis of LoVIL1 in TRV2-LoVIL1 silenced plants using RT-qPCR. FP (F-BOX FAMILY PROTEIN) served as the internal reference gene. Data are presented as mean ± s.d. of three biological repeats (two-sided Student’s t-test). d,e, Short plants (d) with aborted or fewer flowers (e) were caused by silencing of LoVIL1. Images were captured 6 weeks after planting. Scale bars, 1 cm. f-i, Statistic analysis of flower number (f; n = 12 plants), flowering ratio (g; n = 12 plants), plant height (h; n = 14 plants), and leaf number (i; n = 10 plants) in LoVIL1-silenced plants 6 weeks after planting. TRV2 lines were used as controls. Data analysis in panels c, f-i employed a two-sided Student t-test. Data in panels f, h and i represent mean ± s.d. of the individual plants, while data in panel g are presented as mean ± s.d. of three biological replicates. M: DNA ladder marker.

Source data

Extended Data Fig. 4 Overexpression of LoVIL1 in lily.

a, Transgenic lilies with OE-LoVIL1 (35S:eGFP-LoVIL1) displayed accumulated anthocyanidin, early leaf senescence, and constant growth occurred. Scale bar, 1 cm. b, Expression of LoVIL1 in OE-LoVIL1 lilies using RT-qPCR. FP (F-BOX FAMILY PROTEIN) served as the internal reference gene. Data are presented as mean ± s.d. of three biological repeats (two-sided Student’s t-test). c, Detecting GFP-fused proteins in OE-LoVIL1 plants. GFP antibody was used for Western Blotting. Wildtype lily was used as the negative control. d, Quantification of scales number in OE-LoVIL1 and wildtype (WT) plants after 3 months of culture. Data are presented as mean ± s.d. of three independent plants (two-sided Student’s t-test).

Source data

Extended Data Fig. 5 LoVIL1 interacts with PRC2 complex but not LoNFYC6.

a, Validation of the interaction between LoVIL1 and PRC2 subunit LoMSI1 using yeast two-hybrid (Y2H) assay. AH109 yeast cells harbouring LoVIL1 and LoMSI1 were cultured on SD-TL (SD-Trp-Leu) and SD-TLH (SD-Trp-Leu-His) selecting media. Images were taken after 3 days of culture. At least five individual yeast colonies were tested for each construct, and one representative was shown. BD was used as the negative control; BD-53 was used as the positive control (same procedures in panels c and e). b, Split-luciferase complementation assay confirming the interaction between LoVIL1 and LoMSI1. The ORF of LoVIL1 was cloned into the nLUC vector, and LoMSI1 was cloned into the cLUC vector. cLUC is used as the negative control. Luciferase activities were determined by luminescence imaging after 3 days of N. benthamiana leaf agroinfiltration. Color bars indicate luminescence intensity from weak (blue) to strong (red or white). Three biological replicates were performed with consistent results (same procedures in d and f). c, Validation of the interaction between LoNFYA7 and LoNFYC6 using Y2H assay. AH109 yeast cells harbouring LoNFYA7 and LoNFYC6 were cultured on SD-TL and SD-TLH + 1 mM 3-AT selecting media for 3 days. d, Confirmation of the interaction between LoNFYA7 and LoNFYC6 using Split-luciferase complementation assay. e, LoVIL1 does not interact with LoNFYC6 in Y2H system. AH109 yeast cells harbouring LoNFYA7 and LoNFYC6 were cultured on SD-TL and SD-TLH + 1 mM 3-AT selecting media for 3 days. f, LoVIL1 does not interact with LoNFYC6 in the Split-luciferase complementation system.

Extended Data Fig. 6 Histone modification and subcellular localization of LoVIL1.

a, LoVIL1 enhances H3K27me3 modification. Nicotiana benthamiana leaves were transiently overexpressed with 35S:eGFP-LoVIL1. Samples were collected 3 days after agroinfiltration. Nuclei were collected and subjected to immunoprecipitation before confocal imaging. Blue, green, and red fluorescence represent the 4ʹ,6-diamidino-2-phenylindole (DAPI)-stained nuclei, eGFP-LoVIL1 overexpressed nuclei, and methylated histone (H3K27me3), respectively. Non-transfected nuclei are indicated by yellow arrowheads while eGFP-LoVIL1 overexpressed nuclei are indicated by purple arrowheads. Scale bars, 10 μm. b, Quantification of methylation marks shown in panel a. The staining intensity of histone modification was compared by analyzing 10 pairs of transfected nuclei versus non-transfected nuclei in the same field of view. Four fields were analyzed using the ImageJ tool. Data are presented as mean ± s.e. of four biological replicates (two-sided student’s t-test). c, Subcellular localization of LoVIL1 in N. benthamiana leaves. 35S:eGFP-LoVIL1 was transiently overexpressed in N. benthamiana leaves. Images were captured after 3 days of agroinfiltration. mCherry fused Arabidopsis PLASTID RIBOSOMAL PROTEIN L24B (AtRPL24B; AT5G54600) was used as a nuclear marker. Fluorescence signals were collected using a fluorescence microscope with excitation wavelengths of 405 nm, 488 nm, and 561 nm. Scale bars, 20 μm. Consistent results were obtained at least in three biological repeats.

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Extended Data Fig. 7 LoHDA14 antagonizes LoNFYA7 on LoCALS3 expression through histone modification.

a, Relative expression of LoCALS3 in calluses transiently overexpressing LoNFYA7, LoHDA14 or both (LoNFYA7 and LoHDA14). b, Levels of H3K27me3 at LoCALS3 locus in calluses transiently overexpressing LoNFYA7, LoHDA14 or both (LoNFYA7 and LoHDA14). c, H3K9ac levels at LoCALS3 locus in calluses transiently overexpressing LoNFYA7, LoHDA14 or both (LoNFYA7 and LoHDA14). Three independent calluses were used and the data in panels a-c represent the mean ± s.d. of three biological replicates. Different letters above bars in panels a-c indicate significant differences (p = 0.001) as determined by ANOVA Turkey’s HSD tests for pairwise comparisons.

Source data

Extended Data Fig. 8 Different flowering time in L. lancifolium and L. cultivar Must See.

a,b, Morphology of L. lancifolium (a) and Must See (b) 6 weeks after planting. Must See shows visible flower buds in while L. lancifolium remains in the vegetative stage. Scale bars, 5 cm. c, Distribution of cis-elements in the upstream region of the VIL1 start codon in L. lancifolium and Must See. The start codon refers to +1.

Extended Data Fig. 9 Effects of LoVIL1, LoNFYA7, and LoCALS3 on bulbil growth transition in L. lancifolium.

a, Phenotypes of LoVIL1-, LoNFYA7-, and LoCALS3- silenced bulbils 14 weeks after infiltration. Deep-dormant L. lancifolium bulbils without cold storage were used. Scale bar, 2 cm. b, Measurement of bud length in LoVIL1-, LoNFYA7-, and LoCALS3- silenced bulbils 14 weeks after infiltration. Data are presented as mean ± s.d. of five biological replicates (n = 10 bulbils per replicate; two-sided student t-test).

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Extended Data Fig. 10 Correlation between the dormancy trait and the expression levels of LoCALS3, LoNFYA7, and LoFT1.

a, The development of central bud and the expression of LoCALS3 in Oriental lilies 8 weeks of cold storage. b, Pearson correlation analysis of LoCALS3 expression and central bud development. c, The development of central bud and the expression of LoNFYA7 in Oriental lilies 8 weeks of cold storage. d, Pearson correlation analysis of LoNFYA7 expression and central bud development. e, The development of central bud and the expression of LoFT1 in Oriental lilies 8 weeks of cold storage. f, Pearson correlation analysis of LoFT1 expression and central bud development. Data in panels a, c, and e represent mean ± s.d. of three biological replicates (n = 30 bulbs for bud development; n = 3 bulbs for the expression analyses).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–14 and unprocessed gels or blots.

Reporting Summary

Supplementary Table 1

Primers used in this study.

Supplementary Data 1

Statistical source data for supplementary figures.

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Pan, W., Li, J., Du, Y. et al. Epigenetic silencing of callose synthase by VIL1 promotes bud-growth transition in lily bulbs. Nat. Plants 9, 1451–1467 (2023). https://doi.org/10.1038/s41477-023-01492-z

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