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A molecular framework underlying the compound leaf pattern of Medicago truncatula

Nature Plants volume 6, pages 511–521 (2020)Cite this article

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Abstract

Compound leaves show more complex patterns than simple leaves, and this is mainly because of a specific morphogenetic process (leaflet initiation and arrangement) that occurs during their development. How the relevant morphogenetic activity is established and modulated to form a proper pattern of leaflets is a central question. Here we show that the trifoliate leaf pattern of the model leguminous plant Medicago truncatula is controlled by the BEL1-like homeodomain protein PINNATE-LIKE PENTAFOLIATA1 (PINNA1). We identify PINNA1 as a determinacy factor during leaf morphogenesis that directly represses transcription of the LEAFY (LFY) orthologue SINGLE LEAFLET1 (SGL1), which encodes an indeterminacy factor key to the morphogenetic activity maintenance. PINNA1 functions alone in the terminal leaflet region and synergizes with another determinacy factor, the C2H2 zinc finger protein PALMATE-LIKE PENTAFOLIATA1 (PALM1), in the lateral leaflet regions to define the spatiotemporal expression of SGL1, leading to an elaborate control of morphogenetic activity. This study reveals a framework for trifoliate leaf-pattern formation and sheds light on mechanisms generating diverse leaf forms.

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Fig. 1: Phenotypic characterization of pinna1 mutants.
Fig. 2: Positional cloning and characterization of PINNA1.
Fig. 3: Expression pattern of PINNA1 and subcellular localization of the encoded protein.
Fig. 4: PINNA1 negatively regulates SGL1 expression.
Fig. 5: PINNA1 synergizes with PALM1 to regulate lateral leaflet development.
Fig. 6: Model for PINNA1, PALM1 and SGL1 action in the pattern formation of compound leaf in M. truncatula.

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Data availability

M. truncatula sequence data from this article can be found in the M. truncatula genome version Mt4.0 under the following accession numbers: Medtr3g112290 (PINNA1), Medtr5g014400 (PALM1), Medtr3g098560 (SGL1). The relevant accession number for PINNA1 in GenBank is MN265866. Other data for the current study are available from the paper and its Supplementary Information, the indicated public databases, or from the corresponding authors upon request.

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Acknowledgements

We thank Z. Gu for assistance with SEM; Y. Hu for providing the plasmid pGREEN800IILUC; members of the J.C. and Z. Xu laboratory for their valuable input; and S. Khederzadeh for reading this manuscript. These studies were founded by National Natural Science Foundation of China grants U1702234, 31471171 and XDB27030106 (to J.C.), and the Yunnan Applied Basic Research Project 2016FB041. J.C. is also supported by the One Hundred Talent Project of the Chinese Academy of Sciences, High-end Scientific and Technological Talents in Yunnan Province (2015HA031, 2015HA032), the CAS 135 program and Core Botanical Gardens, Chinese Academy of Sciences.

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Author notes

  1. These authors contributed equally: Liangliang He, Yu Liu.

Authors and Affiliations

  1. CAS Key Laboratory of Tropical Plant Resources and Sustainable Use, CAS Center for Excellence for Molecular Plant Sciences, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, China

    Liangliang He, Yu Liu, Hua He, Xiaojia Zhang, Youhan Li, Yawen Mao, Shaoli Zhou, Xiaoling Zheng, Quanzi Bai, Baolin Zhao, Yongmei Xia & Jianghua Chen

  2. Center of Economic Botany, Core Botanical Gardens, Chinese Academy of Sciences, Menglun, Mengla, China

    Liangliang He & Yu Liu

  3. University of Chinese Academy of Sciences, Beijing, China

    Liangliang He, Xiaojia Zhang, Yawen Mao, Shaoli Zhou, Xiaoling Zheng, Quanzi Bai & Jianghua Chen

  4. School of Life Sciences, University of Science and Technology of China, Hefei, China

    Ye Liu, Jinfeng Qi, Dongfa Wang & Jianghua Chen

  5. Noble Research Institute, Ardmore, OK, USA

    Jiangqi Wen & Kirankumar S. Mysore

  6. Department of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, OK, USA

    Million Tadege

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Contributions

J.C. designed research; L.H. and Yu L. performed most of the experiments; J.Q., Y.X. and Ye L. participated in RNA in situ hybridization; X. Zhang, Y. Li and X. Zheng participated in genetic transformation; H.H., Y.M., S.Z. and Q.B. participated in EMSA, yeast two-hybrid, BiFC, pull-down and ChIP assays; J.W., K.S.M., M.T. and Y.X. contributed new reagent and analytic tools; L.H. and J.C. analysed data; L.H. and J.C. wrote the manuscript with input from M.T., Ye L., J.Q., B.Z. and D.W.

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Correspondence to Jianghua Chen.

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Peer review information Nature Plants thanks Yuling Jiao, Miltos Tsiantis, Hirokazu Tsukaya and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phenotypic analysis of WT and the pinna1-1 mutant.

a,b, Images of 3-week-old (a) and 7-week-old (b) WT and pinna1-1 plants. These experiments were repeated independently three times with similar results. Scale bars, 4 cm. c, Phenotypic variation of pinna1-1 leaves with a pie chart showing the percentage distribution. This experiment was repeated independently three times with similar results. Scale bar, 1 cm. d, SEM images of shoot apices with P1 to P3 leaf primordia. Yellow dotted circles indicate no difference in proximal regions of the P3 TL primordia between WT and pinna1-1. This experiment was repeated independently three times with similar results. Scare bares, 20 μm.

Extended Data Fig. 2 Molecular cloning of PINNA1 gene and cosegregation analysis.

a, Simplified schema of the Tnt1 insertion site-based mapping strategy. Red and blue short-lines represent the recovered Tnt1 FSTs of two different mutant lines. b, Images of representative WT, pinna1-1, -2, -3, -4 and -5 leaves. This experiment was repeated independently three times with similar results. Scale bar, 1 cm. c, The detailed positions (red numbers indicate position from the first ATG codon) of Tnt1 insertion sites in pinna1 alleles. Yellow shaded letters indicate the exon sequences. The consensus GT/AG splice sequences are boxed. d, PINNA1 gene structure, the Tnt1 insertion site of pinna1-1 and the primers (arrowheads) used for genotyping. e, Cosegregation analysis of a F2 population derived from a cross of pinna1-1 x WT (R108). 41 out of 172 F2 individuals showing mutant phenotype were homozygous for Tnt1 insertion. The primer pair Fw100-RvTAG100 was used for detecting intact PINNA1 genomic fragments (upper); the primer pair Fw394-TntR2 was used for verifying the existence of Tnt1 insertion within the PINNA1 genomic region (lower). This experiment was repeated independently three times with similar results.

Extended Data Fig. 3 Genetic complementation of pinna1.

a, 3-week-old plants of pinna1-2 and two 35S::PINNA1-GFP/pinna1-2 transgenic lines with partially rescued (#2-3) and completely rescued (#2-1) phenotypes. This experiment was repeated independently three times with similar results. Scare bar, 2 cm. b,c, Normal RT-PCR (b) and RT-qPCR (c) analysis of PINNA1 expression level in young leaves of seven T0 independent lines (35S::PINNA1-GFP) compared to pinna1-1 and pinna1-2. The normal RT-PCR experiment was repeated independently two times with similar results. The RT-qPCR experiment was repeated three times using independent biological replicates with similar results, and one representative result is shown (mean ± SD, n = 3). P, 35S::PINNA1-GFP plasmid. M, molecular weight markers. PINNA1 gene structure, the positions of Tnt1 insertions in pinna1-1 and pinna1-2, and the RT-qPCR primer-pair are shown in the top panel. d, A 7-week-old 35S::PINNA1-GFP/pinna1-2 transgenic plant (#2-12) with a completely rescued phenotype. This experiment was repeated independently three times with similar results. Scare bar, 5 cm. e, Western blot analysis of T1 progeny from the 35S:PINNA1-GFP/pinna1-2 transgenic plant (#2-12) using an anti-GFP antibody. A DR5::GFP transgenic plant used as positive control. 3 to 10, independent T1 plants. This experiment was repeated independently three times with similar results. f, A 7-week-old pinna1-2/pPINNA1:GFP-PINNA1 transgenic line (#G1) with a completely rescued phenotype. This experiment was repeated independently three times with similar results. Scare bar, 5 cm. g, Normal RT-PCR analysis of PINNA1 expression level in shoot buds of two independent pinna1-2/pPINNA1::GFP-PINNA1 transgenic lines. P, pPINNA1:GFP-PINNA1 plasmid. This experiment was repeated independently three times with similar results.

Source data

Extended Data Fig. 4 Maximum likelihood tree of TALE-superfamily homeodomain proteins from several angiosperms.

Legume species (M. truncatula, pea, soybean and L. japonicas), related core eudicots (S. lycopersicum, C. hirsute and A. thaliana), basal eudicots (Aquilegia coerulea), monocots (Oryza sativa and Zea mays), and basal angiosperms (Amborella trichopoad) are chosen for analysis. This is a detailed representation of the tree shown in Fig. 2e. Bootstrap values are out of 2,000 replicate.

Extended Data Fig. 5 Expression pattern of PINNA1 during leaf development.

Longitudinal serial sections of WT apex showing PINNA1 expression in leaf primordium but not in the shoot apical meristem. This experiment was repeated independently three times with similar results. TL, terminal leaflet; LL, lateral leaflet. Scale bars, 50 μm.

Extended Data Fig. 6 Genetic interaction between pinna1-1 and sgl1-2 in the elp1-3 genetic background.

elp1-3 developed normal trifoliate form leaves a, while pinna1-1 elp1-3 developed typical pinnate form leaves (indicated as “Pinnate”) b. sgl1-2 elp1-3 developed simple-like leaves having a single leaflet c, and pinna1-1 sgl1-2 elp1-3 strongly resembled the sgl1-2 elp1-3 by mostly developing simple-like leaves of single leaflet (indicated as “SL”) (except a few dissected leaves having two or three clustered leaflets that indicated as “DL”) d. SL, single leaflet; DL, dissected leaf. Proportions of leaf types in each genotype are shown in pie charts. Blue, trifoliate; red, pinnate; green, single; orange, dissected leaves with clustered leaflets. These experiments were repeated independently three times with similar results. Scale bars, 1 cm.

Extended Data Fig. 7 RNA in situ hybridization of SGL1 in palm1 elp1 and pinna1 palm1 elp1.

a, Serial longitudinal sections of the palm1-3 elp1-3 P4 leaf primordium. b,c, Serial longitudinal sections of the pinna1-1 palm1-3 elp1-3 leaf primordia at P4 (b) and at P5 (c) stages. LP, leaf primordia. These experiments were repeated independently three times with similar results. Scale bars, 50 μm.

Extended Data Fig. 8 BiFC analysis of the in vivo interaction between PINNA1 and PALM1.

Epidermal cells of the N. benthamiana leaf were co-transformed with GFPn-PINNA1/GFPc-PALM1, GFPn-PINNA1/GFPc, or GFPn/GFPc-PALM1. GFPn, N-terminal portion of GFP; GFPc, C-terminal portion of GFP. When GFPn- and GFPc-tagged proteins are in close proximity, the GFPn and GFPc portions interact and produce GFP fluorescence. This experiment was repeated independently three times with similar results. Scale bars, 10 μm.

Extended Data Fig. 9 Quantitative analysis of PALM1 and PINNA1 expression level in pinna1 and palm1 respectively.

a, RT-qPCR of PALM1 expression in the lateral leaflet parts of the first emerged young leaves (P7) from WT and pinna1-1 shoots. Data shown as mean ± SD (n = 3 biologically independent replicates). b, RT-qPCR of PINNA1 expression in different parts of the first emerged young leaves (P7) from WT and palm1-3 shoots. Data shown as mean ± SD (n = 3 biologically independent replicates).

Source data

Extended Data Fig. 10 RNA in situ hybridization of SGL1 in WT, palm1 and pinna1.

Shown are serial longitudinal sections of the P3 leaf primordium. An upregulated SGL1 transcription was found in palm1-3 P3 LL primordia (cyan arrows). This experiment was repeated independently three times with similar results.

Supplementary information

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He, L., Liu, Y., He, H. et al. A molecular framework underlying the compound leaf pattern of Medicago truncatula. Nat. Plants 6, 511–521 (2020). https://doi.org/10.1038/s41477-020-0642-2

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