Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei

doi:10.1186/s12870-020-2303-z

. 2020 Mar 6;20(1):105.

doi: 10.1186/s12870-020-2303-z.

Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei

Zhi Wang¹, Tianqing Zhu¹, Wenjun Ma¹, Erqin Fan^{1

2}, Nan Lu¹, Fangqun Ouyang¹, Nan Wang¹, Guijuan Yang¹, Lisheng Kong³, Guanzheng Qu², Shougong Zhang¹, Junhui Wang⁴

Affiliations

¹ Present address: State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, People's Republic of China.
² Present address: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, 26 Hexing Road, Harbin, 150040, People's Republic of China.
³ Present address: Department of Biology Centre for Forest Biology, University of Victoria, Victoria 11, BC, Canada.
⁴ Present address: State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, People's Republic of China. wangjh808@sina.com.

PMID: 32143577
PMCID: PMC7060540
DOI: 10.1186/s12870-020-2303-z

Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei

Zhi Wang et al. BMC Plant Biol. 2020.

. 2020 Mar 6;20(1):105.

doi: 10.1186/s12870-020-2303-z.

Authors

Zhi Wang¹, Tianqing Zhu¹, Wenjun Ma¹, Erqin Fan^{1

2}, Nan Lu¹, Fangqun Ouyang¹, Nan Wang¹, Guijuan Yang¹, Lisheng Kong³, Guanzheng Qu², Shougong Zhang¹, Junhui Wang⁴

Affiliations

¹ Present address: State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, People's Republic of China.
² Present address: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, 26 Hexing Road, Harbin, 150040, People's Republic of China.
³ Present address: Department of Biology Centre for Forest Biology, University of Victoria, Victoria 11, BC, Canada.
⁴ Present address: State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, People's Republic of China. wangjh808@sina.com.

PMID: 32143577
PMCID: PMC7060540
DOI: 10.1186/s12870-020-2303-z

Abstract

Background: "Bairihua", a variety of the Catalpa bungei, has a large amount of flowers and a long flowering period which make it an excellent material for flowering researches in trees. SPL is one of the hub genes that regulate both flowering transition and development.

Results: SPL homologues CbuSPL9 was cloned using degenerate primers with RACE. Expression studies during flowering transition in "Bairihua" and ectopic expression in Arabidopsis showed that CbuSPL9 was functional similarly with its Arabidopsis homologues. In the next step, we used Y2H to identify the proteins that could interact with CbuSPL9. HMGA, an architectural transcriptional factor, was identified and cloned for further research. BiFC and BLI showed that CbuSPL9 could form a heterodimer with CbuHMGA in the nucleus. The expression analysis showed that CbuHMGA had a similar expression trend to that of CbuSPL9 during flowering in "Bairihua". Intriguingly, ectopic expression of CbuHMGA in Arabidopsis would lead to aberrant flowers, but did not effect flowering time.

Conclusions: Our results implied a novel pathway that CbuSPL9 regulated flowering development, but not flowering transition, with the participation of CbuHMGA. Further investments need to be done to verify the details of this pathway.

Keywords: Architectural transcriptional factor; Catalpa bungei; Flowering; HMGA; SPL.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

**Fig. 1**
The phylogenetic relationship and motif composition analysis of the CbuSPL9 gene in *C. bungei*. a Multiple sequence alignment and sequence logo of the *C. bungei* SBP-box domain. Sequence alignment was performed with DNAMAN. The two conserved zinc fingers and NLS are indicated. The sequence logo was obtained from MEME online software. The overall height of the stack indicates the sequence conservation at that position. b The phylogenetic tree was constructed with MEGA 6.0 by the neighbor-joining (NJ) method with 1000 bootstrap replicates. Bootstrap support is indicated at each node. *A. thaliana* (At), *C. bungei* (Cbu)

**Fig. 2**
Expression profile of *CbuSPL9* in the flower buds and leaf buds during the developmental periods of *C. bungei*. T1-T3 represents the flowering buds and leaf buds collected in the dormant period; T4-T5 represents the flowering buds and leaf buds collected in the germination period; T6-T9 represents the flowering buds and leaf buds collected in the floral transition period; and T10-T12 represents the flowering buds and leaf buds collected in the reproductive period. D1: Image of flowering buds in the dormant period; D2: Image of leaf buds in the dormant period; G1: Image of flowering buds in the germination period; G2: Image of leaf buds in the germination period; F1: Section of flowering buds in the floral transition period; note the flat generative apex (Ga); F2: Section of leaf buds in floral transition period, note the bulged vegetative apex (V); R1: Image of flowering buds in the reproductive period; and R2: Image of leaf buds in the reproductive period. Notably, even though floral transition was never observed in the leaf buds, the leaf buds collected in the period corresponding to floral transition are henceforth called F2 and R2 for convenience. Error bars indicate SD from three independent biological replicates. *Difference between flowering buds (black) and leaf buds (gray) is significant (Student’s test; p < 0.05). **Difference between flowering buds (black) and leaf buds (gray) is highly significant (Student’s test; p < 0.01)

**Fig. 3**
Phenotype of overexpression mutants of *CbuSPL9*. a The change in floral organs occurred in the CbuSPL9-overexpressing transgenic Arabidopsis lines. Image of a normal flower from col. as the control (a/I); and the floral organ mutant from oespl9 (a/II-VI). a/II showing shrunken petals (White arrow) and increased stamens (Right arrow); a/III showing loss petal (White arrow); a/IV showing loss petal (White arrow); a/V showing increased petal (White arrow); a/VI showing overlapping petals (White arrow) and loss stamens (Right arrow); b Comparison of the flowering phenotype in both the col. and oespl9 transgenic plants. From top to bottom: col. (control) and oespl9 (transgenic plants). Bars = 2 mm in (a/II-VI), 1.5 cm in (b). c and d are the statistical data of the time of flower bud emergence and the number of rosette leaves. The oespl9 transgenic plants (black), the oe-hmga transgenic plants (gray) and the col. plants (white). A total of 30 plants were averaged to obtain the mean. Error bars indicate SD

**Fig. 4**
The sequence and expression profile analysis of *CbuHMGA*. a Sequence analysis of the CbuHMGA protein by SMART. b The expression analysis of *CbuHMGA* between flowering buds and leaf buds during the development periods. T1-T3 represents the flowering buds and leaf buds collected in the dormant period; T4-T5 represents the flowering buds and leaf buds collected in the germination period; T6-T9 represents the flowering buds and leaf buds collected in the floral transition period; and T10-T12 represents the flowering buds and leaf buds collected in the reproductive period. Notably, even though floral transition was never observed in the leaf buds, the leaf buds collected in the period corresponding to floral transition are henceforth called the floral transition period and reproductive period for convenience. Error bars indicate SD from three independent experiments. *Difference between flowering buds (black) and leaf buds (gray) is significant (Student’s test; p < 0.05). **Difference between flowering buds (black) and leaf buds (gray) is highly significant (Student’s test; p < 0.01)

**Fig. 5**
Interaction between the two proteins CbuSPL9 and CbuHMGA. a Nuclear localization of the CbuSPL9 protein and CbuHMGA protein. The GFP (control) gene, CbuSPL9-GFP fusion gene and CbuHMGA-GFP fusion gene were expressed transiently in *Nicotiana benthamiana* leaf epidermal cells and observed with confocal microscopy. DAPI, DAPI for nuclear staining image; GFP, GFP green fluorescence image; Merge, the merged images of bright-field, GFP and DAPI staining. b Binding of GST-CbuSPL9 to HIS-CbuHMGA, with GST-CbuSPL9 concentrations of 2381.00 nm, 1191.0 nm, 595.30 nm, 297.60 nm, 148.80 nm assessed by real-time biolayer interferometry. c Yeast two-hybrid assays for the interactions between CbuSPL9 and CbuHMGA. CbuHMGA (as prey) was fused with the GAL4 activation domain (AD) in pGADT7, while CbuSPL9 (as bait) was fused with the GAL DNA-binding domain (BD) in pGBKT7. The positive control was as follows: pGADT7-T and pGBK-53. The negative control was as follows: pGADT7-T and pGBK-Lam. Interactions are indicated by the blue color on SD/−Trp/−Leu/−His/−Ade/X-α-gal medium. d Confocal images of the BiFC analysis in protoplasts from *P. trichocarpa*. CbuSPL9 was fused with EYFP^C, and CbuHMGA was fused with EYFP^N. EYFP signal was detected in the nucleus of the S1–21 protoplasts transfected by H2A:mCherry and CbuSPL9:EYFP^C with (A) CbuHMGA:EYFP^N. Bars = 50 μm

**Fig. 6**
Phenotype of overexpression mutants of *CbuHMGA*. a The change in floral organs occurred in the overexpression of the CbuHMGA transgenic Arabidopsis lines. The images are the floral organ mutant from oe-hmga transgenic Arabidopsis lines (a/I-VI). a/I showing shrunken petals (White arrow); a/II showing dislocation petals (White arrow) and separating stamens (Red arrow); a/III showing dislocation petals (White arrow); a/IV showing overlapping petals (White arrow); a/V showing increased petal (White arrow) and loss stamens (Red arrow); a/VI showing increased petals (White arrow); Bars = 2 mm in (a/I-VI). b Expression profiles of atSPL9 homologous genes in the oespl9 transgenic plants, oe-hmga transgenic plants and col. represents the PCR results in oe-hmga; represents the PCR results in oespl9; and represents the PCR results in col. Three independent biological replicates were performed, and each replicate was measured in triplicate. The error bars show the standard deviation of the results of three technical replicates

formula image — **Fig. 6**
Phenotype of overexpression mutants of *CbuHMGA*. a The change in floral organs occurred in the overexpression of the CbuHMGA transgenic Arabidopsis lines. The images are the floral organ mutant from oe-hmga transgenic Arabidopsis lines (a/I-VI). a/I showing shrunken petals (White arrow); a/II showing dislocation petals (White arrow) and separating stamens (Red arrow); a/III showing dislocation petals (White arrow); a/IV showing overlapping petals (White arrow); a/V showing increased petal (White arrow) and loss stamens (Red arrow); a/VI showing increased petals (White arrow); Bars = 2 mm in (a/I-VI). b Expression profiles of atSPL9 homologous genes in the oespl9 transgenic plants, oe-hmga transgenic plants and col. represents the PCR results in oe-hmga; represents the PCR results in oespl9; and represents the PCR results in col. Three independent biological replicates were performed, and each replicate was measured in triplicate. The error bars show the standard deviation of the results of three technical replicates

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References

1. Jing D, Xia Y, Chen F, Wang Z, Zhang S, Wang J. Ectopic expression of a Catalpa bungei (Bignoniaceae) PISTILLATA homologue rescues the petal and stamen identities in Arabidopsis pi-1 mutant. Plant Sci. 2015;231:40–51. doi: 10.1016/j.plantsci.2014.11.004. - DOI - PubMed
1. Mouradov A, Cremer F, Coupland G. Control of flowering time: interacting pathways as a basis for diversity. Plant Cell. 2002;14(Suppl):S111–S130. doi: 10.1105/tpc.001362. - DOI - PMC - PubMed
1. Flowers JM, Hanzawa Y, Hall MC, Moore RC, Purugganan MD. Population genomics of the arabidopsis thaliana flowering time gene network. Mol Biol Evol. 2009;26:2475–2486. doi: 10.1093/molbev/msp161. - DOI - PubMed
1. Fornara F, de Montaigu A, Coupland G. SnapShot: control of flowering in arabidopsis. Cell. 2010;141:3–5. doi: 10.1016/j.cell.2010.04.024. - DOI - PubMed
1. Bouché F, Lobet G, Tocquin P, Périlleux C. FLOR-ID: An interactive database of flowering-time gene networks in Arabidopsis thaliana. Nucleic Acids Res. 2016;44:D1167–D1171. doi: 10.1093/nar/gkv1054. - DOI - PMC - PubMed

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[1] Jing D, Xia Y, Chen F, Wang Z, Zhang S, Wang J. Ectopic expression of a Catalpa bungei (Bignoniaceae) PISTILLATA homologue rescues the petal and stamen identities in Arabidopsis pi-1 mutant. Plant Sci. 2015;231:40–51. doi: 10.1016/j.plantsci.2014.11.004. - DOI - PubMed

[2] Jing D, Xia Y, Chen F, Wang Z, Zhang S, Wang J. Ectopic expression of a Catalpa bungei (Bignoniaceae) PISTILLATA homologue rescues the petal and stamen identities in Arabidopsis pi-1 mutant. Plant Sci. 2015;231:40–51. doi: 10.1016/j.plantsci.2014.11.004. - DOI - PubMed

[3] Mouradov A, Cremer F, Coupland G. Control of flowering time: interacting pathways as a basis for diversity. Plant Cell. 2002;14(Suppl):S111–S130. doi: 10.1105/tpc.001362. - DOI - PMC - PubMed

[4] Mouradov A, Cremer F, Coupland G. Control of flowering time: interacting pathways as a basis for diversity. Plant Cell. 2002;14(Suppl):S111–S130. doi: 10.1105/tpc.001362. - DOI - PMC - PubMed

[5] Flowers JM, Hanzawa Y, Hall MC, Moore RC, Purugganan MD. Population genomics of the arabidopsis thaliana flowering time gene network. Mol Biol Evol. 2009;26:2475–2486. doi: 10.1093/molbev/msp161. - DOI - PubMed

[6] Flowers JM, Hanzawa Y, Hall MC, Moore RC, Purugganan MD. Population genomics of the arabidopsis thaliana flowering time gene network. Mol Biol Evol. 2009;26:2475–2486. doi: 10.1093/molbev/msp161. - DOI - PubMed

[7] Fornara F, de Montaigu A, Coupland G. SnapShot: control of flowering in arabidopsis. Cell. 2010;141:3–5. doi: 10.1016/j.cell.2010.04.024. - DOI - PubMed

[8] Fornara F, de Montaigu A, Coupland G. SnapShot: control of flowering in arabidopsis. Cell. 2010;141:3–5. doi: 10.1016/j.cell.2010.04.024. - DOI - PubMed

[9] Bouché F, Lobet G, Tocquin P, Périlleux C. FLOR-ID: An interactive database of flowering-time gene networks in Arabidopsis thaliana. Nucleic Acids Res. 2016;44:D1167–D1171. doi: 10.1093/nar/gkv1054. - DOI - PMC - PubMed

[10] Bouché F, Lobet G, Tocquin P, Périlleux C. FLOR-ID: An interactive database of flowering-time gene networks in Arabidopsis thaliana. Nucleic Acids Res. 2016;44:D1167–D1171. doi: 10.1093/nar/gkv1054. - DOI - PMC - PubMed

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Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei

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Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei

Authors

Affiliations

Abstract

Conflict of interest statement

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