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The auxin-responsive transcription factor SlDOF9 regulates inflorescence and flower development in tomato

Nature Plants volume 8, pages 419–433 (2022)Cite this article

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Abstract

Understanding the mechanisms underlying differentiation of inflorescence and flower meristems is essential towards enlarging our knowledge of reproductive organ formation and to open new prospects for improving yield traits. Here, we show that SlDOF9 is a new modulator of floral differentiation in tomato. CRISPR/Cas9 knockout strategy uncovered the role of SlDOF9 in controlling inflorescence meristem and floral meristem differentiation via the regulation of cell division genes and inflorescence architecture regulator LIN. Tomato dof9-KO lines have more flowers in both determinate and indeterminate cultivars and produce more fruit upon vibration-assisted fertilization. SlDOF9 regulates inflorescence development through an auxin-dependent ARF5-DOF9 module that seems to operate, at least in part, differently in Arabidopsis and tomato. Our findings add a new actor to the complex mechanisms underlying reproductive organ differentiation in flowering plants and provide leads towards addressing the diversity of factors controlling the transition to reproductive organs.

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Fig. 1: Heatmap representation of the expression patterns of 205 transcription factor genes preferentially expressed in meristem tissues.
Fig. 2: SlDOF9 is the most highly expressed among all DOF genes in tomato inflorescence and flower meristems.
Fig. 3: Tomato dof9-KO lines display altered inflorescence and flower development.
Fig. 4: The dof9-KO mutation results in enhanced fruit number and fruit yield regardless of the genetic background.
Fig. 5: SIMs differentiation is associated with transcriptomic reprogramming of cell division and differentiation genes in dof9-KO lines.
Fig. 6: SlDOF9 is regulated by auxin and displays an expression pattern in IMs that correlates with that of SlARF5.
Fig. 7: SlDOF9 is under direct regulation of SlARF5.

Data availability

Sequence data from this article can be found in the GenBank/Sol Genomics data libraries under the following accession numbers: SlDof1 (Solyc01g096120), SlDof2 (Solyc02g065290), SlDof3 (Solyc02g067230), SlDof4 (Solyc02g076850), SlDof5 (Solyc02g077950), SlDof6 (Solyc02g077960), SlDof7(Solyc02g078620), SlDof8 (Solyc02g088070), SlDof9 (Solyc02g090220), SlDof10 (Solyc02g090310), SlDof11 (Solyc03g082840), SlDof12 (Solyc03g112930), SlDof13 (Solyc03g115940), SlDof14 (Solyc03g121400), SlDof15 (Solyc04g070960), SlDof16 (Solyc04g079570), SlDof17 (Solyc05g007880), SlDof18 (Solyc05g054510), SlDof19 (Solyc06g005130), SlDof20 (Solyc06g062520), SlDof23 (Solyc06g071480), SlDof24 (Solyc06g075370), SlDof25 (Solyc06g076030), SlDof26 (Solyc08g008500), SlDof27 (Solyc08g082910), SlDof28 (Solyc09g010680), SlDof29 (Solyc10g009360), SlDof30 (Solyc10g086440), SlDof31 (Solyc11g010940), SlDof32 (Solyc11g066050), SlDof33 (Solyc11g072500), SlDof34 (Solyc00g024680). The raw datasets supporting the conclusions of this article are available (study PRJEB41426) at the European Nucleotide Archive with the following accession numbers: ERR4862988–ERR4862999. The homologue protein sequences from Solanaceae species and Arabidopsis are available from SOL Genomics database (https://solgenomics.net/) and EnsemblPlants protein database (http://plants.ensembl.org). Source data are provided with this paper.

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Acknowledgements

We are grateful to L. Lemonnier and D. Saint-Martin for the cultivation of tomato plants. We are also grateful to GetPlage for deep sequencing and GenoToulBioinfo for giving access to the computing facilities. G.H. was supported by the Chinese Scholarship Council. The research was supported by the European Union grants H2020 TomGEM 679796 and HARNESSTOM 101000716 and by the Labex TULIP ANR-10-LABX-41.

Author information

Author notes

  1. These authors contributed equally: Guojian Hu, Keke Wang.

Authors and Affiliations

  1. Université de Toulouse, INRAe/INP Toulouse, Génomique et Biotechnologie des Fruits—UMR990, Castanet-Tolosan, France

    Guojian Hu, Keke Wang, Baowen Huang, Isabelle Mila, Pierre Frasse, Elie Maza, Anis Djari, Mohamed Zouine & Mondher Bouzayen

  2. Laboratoire de Recherche en Sciences Végétales—UMR5546, Université de Toulouse, CNRS, UPS, Toulouse-INP, Toulouse, France

    Guojian Hu, Keke Wang, Baowen Huang, Pierre Frasse, Elie Maza, Anis Djari, Mohamed Zouine & Mondher Bouzayen

  3. Biologie du Fruit et Pathologie—UMR 1332, Université Bordeaux, INRAE, Villenave d’Ornon, France

    Michel Hernould

  4. Key Laboratory of Plant Hormones and Development Regulation of Chongqing, School of Life Sciences, Chongqing University, Chongqing, China

    Zhengguo Li & Mondher Bouzayen

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Contributions

M.B. directed the project. G.H., M.B. and K.W. conceived the project and designed the experiments. G.H. performed the experiments and contributed to the drafting of the article. G.H. and K.W. contributed to the implementation of the experiments and performed the analysis of the RNA-seq data. I.M., K.W. and G.H. conducted the subcellular localization and transactivation assay work. G.H. and B.H generated the transgenic lines. P.F. contributed to the ChIP experiment. E.M. and A.D. helped to perform the bioinformatic analyses. K.W. and B.H. contributed to the design of the CRISPR/Cas9 strategies. M.H., Z.L. and M.Z. contributed to the critical analysis of the results and discussion. M.B. supervised the work and modified the manuscript input from all co-authors.

Corresponding author

Correspondence to Mondher Bouzayen.

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Nature Plants thanks Esther van der Knaap and 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 Tomato dof9-KO lines display altered inflorescence and flower development.

(a) Generation of dof9-KO mutant lines via CRISPR/Cas9 strategy in WVA.106 (WVA) and Ailsa Craig (AC) undetermined tomato cultivars. The same guide RNAs (sgRNA1 and sgRNA2; green bars) located in the vicinity of ZFM motif were used for editing the SlDOF9 gene sequence via CRISPR/Cas9 strategy. Mutations within SlDOF9 coding sequences corresponding to nucleotide insertions or deletions are pasted red and the predicted resulting proteins are schematically illustrated (lower panel). (b) Impaired flower organ development in AC and WVA dof9-KO mutants (upper panel). White arrows indicate petalloid sepals in dof9-KO mutants and red arrow heads point to protruded stigmas or split stamens. Scale bar=1 cm. The frequency of altered flower organs phenotypes in sepals, petals and stamens were shown in lower panel. (c) Frequency of altered flower organs phenotypes in sepals, petals and stamens in Micro-Tom cultivar. (d) Leafy inflorescence phenotype displayed by AC and Micro-Tom dof9-KO mutants. Branched and leafy inflorescence are occasionally observed in AC and Micro-Tom dof9-KO mutants. Branching events are shown by red arrows and leaves differentiated from inflorescence meristems by white arrows.

Source data

Extended Data Fig. 2 Downregulation of SlDOF9 results in early flowering in Micro-Tom cultivar.

(a) dof9 mutation promotes earlier flower development compared to WT, while DOF9 overexpression delays flowering. Percentage of flowering plants at 31, 38 and 45 days after germination in WT, dof9 mutants and OE-DOF9 lines. (b) Number of vegetative leaves before flowering in WT, dof9 mutants and OE-DOF9 line. Dots indicate individual plants (n = 12). Statistical significance compared to WT was determined by two-sided t-test (***P < 0.001). Pdof9A = 1.6e-04; Pdof9B = 4.08e-08; Pdof9C = 5.58e-05; POE-DOF9 = 1.37e-09. Box edges represent the 0.25 and 0.75 quantiles, the bold lines indicate median values and Whiskers indicate 1.5 times the interquartile range. (c) Faster meristem maturation process in dof9-KO compared to WT and OE-DOF9 lines. The apical meristem in WT plants starts doming at 14 days after germination (DAG), whereas this differentiation process occurs as early as 9-12 DAG in dof9-KO lines resulting in early flowering. By contrast, in OE-DOF9 lines meristem transition from VM to TM is strongly delayed (30 DAGs) compared to WT (14 DAGs) or to dof9-KO lines (9-12 DAGs). The figure shows that in WT until the 7th leaf is initiated, the status of the meristem remains at early vegetative meristem (EVM) or late vegetative meristem (LVM) stages and the transition meristem (TM) appears when the 8th leaf is initiated. The differentiation of the TM occurs earlier in dof9-KO lines (6th leaf) while it is delayed in OE-DOF9 lines (10th leaf). L5, L6, L7, L8 and L10 refer to leaf numbering started from the 1st leaf. The pictures are representative of ten independent meristem samples all showing similar results. Scale bar = 100 µm.

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Extended Data Fig. 3 Phenotypes of SlDOF9 overexpressing lines.

(a) SlDOF9 transcript levels in WT and in two independent overexpression lines (OE-DOF9) representative of the phenotypes displayed by these overexpressing tomato plants. Statistical significance compared to WT was determined by two-sided t-test. Error bars mean ± SEM of three biological replicates. (b) Representative abaxially curling leaves with retarded growth observed in OE-DOF9 lines. Higher expression level of the SlDOF9 transgene in Line 1 (L1) results in more severe phenotypes than in L2 lines that exhibit lower expression level of the transgene. Scale bars=2 cm.

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Extended Data Fig. 4 Fruit number and fruit yield in WT and dof9-KO tomato plants assessed in the absence of manual pollination.

Fruit yield is determined by assessing the total fruit weight produced by WT and dof9-KO mutants in Micro-Tom (a) and WVA106 (b) cultivars. Dots indicate individual plants (nmicro-Tom = 8; nwva-WT = 5; nwva-L4 = 4). Statistical significance compared to WT was determined by two-sided t-test. Box edges represent the 0.25 and 0.75 quantiles, the bold lines indicate median values and Whiskers indicate 1.5 times the interquartile range.

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Extended Data Fig. 5 Fruit diameter in dof9-KO Micro-Tom mutant lines.

Dots indicate individual plants (n = 6). Statistical significance compared to WT was determined by two-sided t-test. Box edges represent the 0.25 and 0.75 quantiles, the bold lines indicate median values and Whiskers indicate 1.5 times the interquartile range.

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Extended Data Fig. 6 qRT–PCR validation of differentially expressed genes (DEGs) in WT and dof9 initially revealed by RNA-seq profiling.

(a) Transcript accumulation levels corresponding to genes related to cell division/differentiation, hormone metabolism and inflorescence development were assessed by qRT–PCR in three independent lines to validate their expression profile revealed by RNA-seq. Statistical significance compared to WT was determined by two-sided t-test. Error bars mean ± SEM of three biological replicates performed using WT and dof9A mutant lines. Two biological replicates were performed in dof9A and dof9B mutant lines. (b) Assessing the correlation levels between RNA-seq and qRT–PCR expression data. Statistical significance between RNA-seq expression data and qRT–PCR data was determined by two-sided Pearson’s Correlation test.

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Extended Data Fig. 7 Phylogenetic analysis and protein structure of SlDOF2 and SlDOF9.

(a) The phylogenetic tree of tomato and Arabidopsis DOF proteins was constructed by neighbour-joining algorithm. Tomato SlDOF9 and SlDOF2 group in the same clade than Dof5.8 and OBP1 Arabidopsis orthologues (emphasized in red). (b) Protein sequence alignment of SlDOF9 and SlDOF2 with the conserved C2C2-zinc-finger motif (ZFM) framed with dash lines and Bipartite NLS peptide underlined in green.

Extended Data Fig. 8 SlDOF9 is a transcriptional activator involved in meristem differentiation.

(a) SlDOF9 is exclusively localized in the nuclear compartment as assessed by transient expression in tobacco protoplasts of the YFP fused to the N-terminal of tomato SlDOF9 protein. Three independent experiments are performed giving similar results. Scale bar = 20 μm. (b) SlDOF9 is a transcription activator as revealed by transient expression assays in tobacco protoplasts using the GFP reporter gene under the control of the Cyclin SlCycU3;2 promoter or a synthetic promoter containing 7 repeats of the conserved DOF-binding sites (‘AAAAG’ motif). SlCycU3;2 was selected as putative SlDOF9 target gene based on its strong downregulation in dof9-KO lines (see Supplementary Table 1). The two reporter constructs were co-transformed in tobacco protoplasts with an effector construct corresponding to SlDOF9 driven by 35S promoter. The control assay was performed with an empty vector (vector) lacking the SlDOF9 CDS. Statistical significance compared to control was determined by two-sided t-test. (c) Reduced ability to differentiate shoots from dof9-KO mutant callus. Cotyledons (white arrows) from WT and dof9-KO tomato lines were grown 30 days in shoot regeneration medium to form Calli and subsequently shoots and leaves (red arrows). In contrast to WT, the dof9-KO calli are unable to regenerate shoots. The number of regenerated shoots per total number of cotyledon explants is indicated at the top. The data are representative of three independent experiments with independent mutant lines. Scale bar = 1 cm.

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Supplementary information

Supplementary Information

Supplementary Discussion.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–4: 1, List of transcription factor genes preferentially expressed in meristem tissues; 2, Complete list of DEGs in SlDOF9-KO lines; 3, Genes differentially expressed (DEGs) in dof9-KO lines that are related to cell division and differentiation, auxin and cytokinin homoeostasis, flowering time, inflorescence and flower development; 4, List of primers used in this study.

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Hu, G., Wang, K., Huang, B. et al. The auxin-responsive transcription factor SlDOF9 regulates inflorescence and flower development in tomato. Nat. Plants 8, 419–433 (2022). https://doi.org/10.1038/s41477-022-01121-1

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