SPL13 controls tomato lateral branch outgrowth by regulating brassinosteroid biosynthesis and signal transduction

Abstract

Plant architecture can directly impact on tomato (Solanum lycopersicum) fruit production. Our previous work has demonstrated an important role of microRNA156a (miR156a) in determining the lateral branches mainly by regulating SQUAMOSA PROMOTER BINDING PROTEIN LIKE 13 (SPL13) expression in tomato. However, the regulatory pathway by which the miR156a-SPL13 module regulates branching remains obscure. In this study, we examined the relationship between SPL13 and two other genes, BRANCHED1b (BRC1b) and DWARF (DWF), which have previously been known to regulate lateral branch development in tomato. Our findings indicate that SPL13 directly interacts with the promoters of BRC1b and DWF, enhancing BRC1b expression while inhibiting DWF expression, thereby finely regulating lateral branch development in tomatoes. Additionally, overexpression of BRC1b or knockout of DWF were found to rescue the increased lateral branch outgrowth phenotype of the spl13 mutant plants. Furthermore, brassinosteroid (BR) content was high in spl13 mutant plants, supporting the notion that SPL13 negatively regulates BR biosynthesis. These findings suggest that SPL13 serves as an activator of the BR signaling downstream gene BRC1b and a repressor of the BR biosynthesis gene DWF to regulate lateral branch outgrowth in tomato. One-sentence summary: The miR156a-targeted transcription factor SlSPL13 regulates plant architecture in tomato.

Acknowledgements

We thank C. Xu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China) and R. Yin (Shanghai Jiao Tong University, China) for suggestions and for critically revising our manuscript. This research received funding from the National Natural Science Foundation of China (grant nos 32202512, 32360766, and 32072595), the Natural Science Foundation of Jiangxi Province (grants 20242BAB25377 and 20224BAB215026), and the China Agriculture Research System (CARS-23-A13).

Author contributions

Z.Y. and J.Z. conceived and structured the study. L.C., F.Z., L.J., F.H., G.A., J.Y., T.W., and Z.W. conducted experiments and fieldwork, analyzed data, and drew conclusions. L.C., Z.H., J.Z., and R.M.L. wrote the manuscript.

Data availability statement

The manuscript and online Supporting Information contain all data underpinning this study’s findings.

Conflicts of interest statement

The authors report no financial conflicts of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Zhang X , Zou Z , Zhang J . et al. Over-expression of sly-miR156a in tomato results in multiple vegetative and reproductive trait alterations and partial phenocopy of the sft mutant . FEBS Lett. 2011; 585: 435-9

[2] Cui LG , Shan JX , Shi M . et al. The miR156-SPL9-DFR pathway coordinates the relationship between development and abiotic stress tolerance in plants. Plant J. 2014; 80: 1108-17

[3] Poethig RS . The past, present, and future of vegetative phase change. Plant Physiol . 2010; 154: 541-4

[4] Wang H , Wang H . The miR156/SPL module, a regulatory hub and versatile toolbox, gears up crops for enhanced agronomic traits. Mol Plant. 2015; 8: 677-88

[5] Yu S , Lian H , Wang JW . Plant developmental transitions: the role of microRNAs and sugars. Curr Opin Plant Biol. 2015; 27: 1-7

[6] Yu ZX , Wang LJ , Zhao B . et al. Progressive regulation of sesquiterpene biosynthesis in Arabidopsis and patchouli (Pogostemon cablin) by the miR156-targeted SPL transcription factors. Mol Plant. 2015; 8: 98-110

[7] Guo C , Xu Y , Shi M . et al. Repression of miR156 by miR159 regulates the timing of the juvenile-to-adult transition in Arabidopsis. Plant Cell . 2017; 29: 1293-304

[8] Cui L , Zheng F , Wang J . et al. miR156a-targeted SBP-box transcription factor SlSPL13 regulates inflorescence morphogenesis by directly activating SFT in tomato . Plant Biotechnol J. 2020; 18: 1670-82

[9] Xie K , Wu C , Xiong L . Genomic organization, differential expression, and interaction of SQUAMOSA promoter-binding-like transcription factors and microRNA156 in rice. Plant Physiol . 2006; 142: 280-93

[10] Wu G , Park MY , Conway SR . et al. The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell. 2009; 138: 750-9

[11] Jiao Y , Wang Y , Xue D . et al. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nat Genet. 2010; 42: 541-4

[12] Miura K , Ikeda M , Matsubara A . et al. OsSPL14 promotes panicle branching and higher grain productivity in rice. Nat Genet. 2010; 42: 545-9

[13] Wang S , Wu K , Yuan Q . et al. Control of grain size, shape and quality by OsSPL16 in rice. Nat Genet. 2012; 44: 950-4

[14] Wang S , Li S , Liu Q . et al. The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality. Nat Genet. 2015; 47: 949-54

[15] Si L , Chen J , Huang X . et al. OsSPL13 controls grain size in cultivated rice. Nat Genet. 2016; 48: 447-56

[16] Wang J , Zhou L , Shi H . et al. A single transcription factor promotes both yield and immunity in rice. Science. 2018; 361: 1026-8

[17] Wu G , Poethig RS . Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3. Development. 2006; 133: 3539-47

[18] Gandikota M , Birkenbihl RP , Hohmann S . et al. The miRNA156/157 recognition element in the 3’ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. Plant J. 2007; 49: 683-93

[19] Schwarz S , Grande AV , Bujdoso N . et al. The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis. Plant Mol Biol. 2008; 67: 183-95

[20] Wang JW , Schwab R , Czech B . et al. Dual effects of miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana . Plant Cell. 2008; 20: 1231-43

[21] Wang JW , Czech B , Weigel D . miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana . Cell. 2009; 138: 738-49

[22] Usami T , Horiguchi G , Yano S . et al. The more and smaller cells mutants of Arabidopsis thaliana identify novel roles for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes in the control of heteroblasty. Development. 2009; 136: 955-64

[23] Yu N , Cai WJ , Wang SC . et al. Temporal control of trichome distribution by microRNA156-targeted SPL genes in Arabidopsis thaliana . Plant Cell. 2010; 22: 2322-35

[24] Hyun Y , Richter R , Vincent C . et al. Multi-layered regulation of SPL15 and cooperation with SOC1 integrate endogenous flowering pathways at the Arabidopsis shoot meristem. Dev Cell. 2016; 37: 254-66

[25] Jiao Z , Shi X , Xu R . et al. HOS1 ubiquitinates SPL9 for degradation to modulate salinity-delayed flowering. J Integr Plant Biol. 2024; 66: 2600-12

[26] Manning K , Tor M , Poole M . et al. A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nat Genet. 2006; 38: 948-52

[27] Otsuga D , DeGuzman B , Prigge MJ . et al. REVOLUTA regulates meristem initiation at lateral positions. Plant J. 2001; 25: 223-36

[28] Gallavotti A , Zhao Q , Kyozuka J . et al. The role of barren stalk1 in the architecture of maize . Nature. 2004; 432: 630-5

[29] Martin-Trillo M , Grandio EG , Serra F . et al. Role of tomato BRANCHED1-like genes in the control of shoot branching. Plant J. 2011; 67: 701-14

[30] Du Y , Liu L , Li M . et al. UNBRANCHED3 regulates branching by modulating cytokinin biosynthesis and signaling in maize and rice. New Phytol. 2017; 214: 721-33

[31] Liu J , Cheng X , Liu P . et al. miR156-targeted SBP-box transcription factors interact with DWARF53 to regulate TEOSINTE BRANCHED1 and BARREN STALK1 expression in bread wheat. Plant Physiol. 2017; 174: 1931-48

[32] Song X , Lu Z , Yu H . et al. IPA1 functions as a downstream transcription factor repressed by D53 in strigolactone signaling in rice. Cell Res. 2017; 27: 1128-41

[33] Wang L , Zhang Q . Boosting rice yield by fine-tuning SPL gene expression. Trends Plant Sci. 2017; 22: 643-6

[34] Lu ZF , Yu H , Xiong GS . et al. Genome-wide binding analysis of the transcription activator IDEAL PLANT ARCHITECTURE1 reveals a complex network regulating rice plant ARCHITECTURE. Plant Cell. 2013; 25: 3743-59

[35] Yang B , Sun Y , Minne M . et al. SPL13 controls a root apical meristem phase change by triggering oriented cell divisions. Science. 2024; 386: eado4298

[36] Chen S , Song X , Zheng Q . et al. The transcription factor SPL13 mediates strigolactone suppression of shoot branching by inhibiting cytokinin synthesis in Solanum lycopersicum. J Exp Bot. 2023; 74: 5722-35

[37] Singh AP , Savaldi-Goldstein S . Growth control: brassinosteroid activity gets context. J Exp Bot. 2015; 66: 1123-32

[38] Xia X , Dong H , Yin Y . et al. Brassinosteroid signaling integrates multiple pathways to release apical dominance in tomato. Proc Natl Acad Sci USA. 2021; 118: e2004384118

[39] Wu CY , Trieu A , Radhakrishnan P . et al. Brassinosteroids regulate grain filling in rice. Plant Cell . 2008; 20: 2130-45

[40] Takeda T , Suwa Y , Suzuki M . et al. The OsTB1 gene negatively regulates lateral branching in rice. Plant J . 2003; 33: 513-20

[41] Aguilar-Martinez JA , Poza-Carrion C , Cubas P . Arabidopsis BRANCHED1 acts as an integrator of branching signals within axillary buds. Plant Cell. 2007; 19: 458-72

[42] Studer A , Zhao Q , Ross-Ibarra J . et al. Identification of a functional transposon insertion in the maize domestication gene tb1. Nat Genet. 2011; 43: 1160-3

[43] Shen J , Zhang Y , Ge D . et al. CsBRC1 inhibits axillary bud outgrowth by directly repressing the auxin efflux carrier CsPIN3 in cucumber. Proc Natl Acad Sci USA. 2019; 116: 17105-14

[44] Klein J , Saedler H , Huijser P . A new family of DNA binding proteins includes putative transcriptional regulators of the Antirrhinum majus floral meristem identity gene SQUAMOSA. Mol Gen Genet. 1996; 250: 7-16

[45] Axtell MJ , Bartel DP . Antiquity of microRNAs and their targets in land plants. Plant Cell . 2005; 17: 1658-73

[46] Chuck G , Cigan AM , Saeteurn K . et al. The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA . Nat Genet. 2007; 39: 544-9

[47] Feng S , Xu Y , Guo C . et al. Modulation of miR156 to identify traits associated with vegetative phase change in tobacco (Nicotiana tabacum) . J Exp Bot. 2016; 67: 1493-504

[48] Aung B , Gao R , Gruber MY . et al. MsmiR156 affects global gene expression and promotes root regenerative capacity and nitrogen fixation activity in alfalfa. Transgenic Res. 2017; 26: 541-57

[49] Massoumi M , Krens FA , Visser RG . et al. Azacytidine and miR156 promote rooting in adult but not in juvenile Arabidopsis tissues. J Plant Physiol. 2017; 208: 52-60

[50] Xu XZ , Li X , Hu XW . et al. High miR156 expression is required for auxin-induced adventitious root formation via MxSPL26 independent of PINs and ARFs in malus xiaojinensis. Front Plant Sci. 2017; 8: 1509

[51] Yang J , Wei H , Hou M . et al. ZmSPL13 and ZmSPL29 act together to promote vegetative and reproductive transition in maize. New Phytol . 2023; 239: 1505-20

[52] Feyissa BA , Arshad M , Gruber MY . et al. The interplay between miR156/SPL13 and DFR/WD40-1 regulate drought tolerance in alfalfa. BMC Plant Biol. 2019; 19: 434

[53] Ma Y , Xue H , Zhang F . et al. The miR156/SPL module regulates apple salt stress tolerance by activating MdWRKY100 expression . Plant Biotechnol J. 2021; 19: 311-23

[54] Wang DR , Zhang CL , Wang X . et al. MicroRNA156-SPL13B module induces parthenocarpy through the gibberellin pathway. Plant Biotechnol J. 2025; 23: 5536-49

[55] Jing X , Wang D , Liu X . et al. SBP transcription factor MdSPL13B positively regulates salt tolerance in apple. Hortic Plant J . 2025;

[56] Yao Y , Wu X , Yang Y . et al. SPL transcription factors in plants: a potential stress regulator. Hortic Plant J. 2025;

[57] Wang B , Smith SM , Li J . Genetic regulation of shoot architecture. Annu Rev Plant Biol. 2018; 69: 437-68

[58] Wakabayashi T , Hamana M , Mori A . et al. Direct conversion of carlactonoic acid to orobanchol by cytochrome P450 CYP722C in strigolactone biosynthesis. Sci Adv. 2019; 5: eaax9067

[59] Wang L , Wang B , Yu H . et al. Transcriptional regulation of strigolactone signalling in Arabidopsis. Nature . 2020; 583: 277-81

[60] Gou JY , Felippes FF , Liu CJ . et al. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell. 2011; 23: 1512-22

[61] Jones B , Frasse P , Olmos E . et al. Down-regulation of DR12, an auxin-response-factor homolog, in the tomato results in a pleiotropic phenotype including dark green and blotchy ripening fruit. Plant J. 2002; 32: 603-13

[62] Hellens RP , Allan AC , Friel EN . et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods . 2005; 1: 13

[63] Stone SL , Hauksdottir H , Troy A . et al. Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol . 2005; 137: 13-30

[64] Xin P , Yan J , Fan J . et al. An improved simplified high-sensitivity quantification method for determining brassinosteroids in different tissues of rice and Arabidopsis. Plant Physiol. 2013; 162: 2056-66