MdARF3 switches the lateral root elongation to regulate dwarfing in apple plants

Abstract

Apple rootstock dwarfing and dense planting are common practices in apple farming. However, the dwarfing mechanisms are not understood. In our study, the expression of MdARF3 in the root system of dwarfing rootstock ‘M9’ was lower than in the vigorous rootstock from Malus micromalus due to the deletion of the WUSATAg element in the promoter of the ‘M9’ genotype. Notably, this deletion variation was significantly associated with dwarfing rootstocks. Subsequently, transgenic tobacco ( Nicotiana tabacum ) cv. Xanthi was generated with the ARF3 promoter from ‘M9’ and M. micromalus genotypes. The transgenic apple with 35S::MdARF3 was also obtained. The transgenic tobacco and apple with the highly expressed ARF3 had a longer root system and a higher plant height phenotype. Furthermore, the yeast one-hybrid, luciferase, electrophoretic mobility shift assays, and Chip-qPCR identified MdWOX4-1 in apples that interacted with the pMm-ARF3 promoter but not the pM9-ARF3 promoter. Notably, MdWOX4-1 significantly increased the transcriptional activity of MdARF3 and MdLBD16-2. However, MdARF3 significantly decreased the transcriptional activity of MdLBD16-2. Further analysis revealed that MdARF3 and MdLBD16-2 were temporally expressed during different stages of lateral root development. pMdLBD16-2 was mainly expressed during the early stage of lateral root development, which promoted lateral root production. On the contrary, pMmARF3 was expressed during the late stage of lateral root development to promote elongation. The findings in our study will shed light on the genetic causes of apple plant dwarfism and provide strategies for molecular breeding of dwarfing apple rootstocks.

Acknowledgements

This project was supported by grants from the National Natu-ral Science Foundation of China (32172522 and 31901974), Ear-marked Fund for China Agriculture Research System (CARS-27), and 2115 Talent Development Program of China Agricultural University.

Author contributions

Z.H. and Y.W. conceptualized and designed the study. J.L., Y.F., L.Z., L.J., Y.W., Y.H., and R.Y. conducted the tests. J.L., and Y.F., conducted data analysis. Y.W., Z.H., J.L., and Y.F. wrote the draft manuscript. T.W. and X.Z. edited the draft manuscript. All authors read and approved the final manuscript.

Data availability

The manuscript and supplementary materials provide all the necessary information.

Conflict of interest statement

All authors declare that they have no conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Johnson WC. Methods and results of screening for disease and insect resistant apple rootstocks. Compact Fruit Tree. 2000; 3 :73-6

[2] Webster AD. Dwarfing rootstocks: past, present and future. Compact Fruit Tree. 2002; 35 :67-72

[3] Tworkoski T, Miller S. Rootstock effect on growth of apple scions with different growth habits. Sci Hortic. 2007; 111 :335-43

[4] Cohen S, Naor A, Bennink J. et al. Hydraulic resistance compo- nents of mature apple trees on rootstocks of different vigours. J Exp Bot. 2007; 58 :4213-24

[5] Foster TM, Celton JM, Chagné D. et al. Two quantitative trait loci, Dw1 and Dw2, are primarily responsible for rootstock-induced dwarfing in apple. Hortic Res. 2015; 2 :301-9

[6] Pilcher RLR, Celton JM, Gardiner SE. et al. Genetic markers linked to the dwarfing trait of apple rootstock ‘Malling 9’. J Am Soc Hortic Sci. 2008; 133 :100-6

[7] Li W, Chu C, Li H. et al. Near-gapless and haplotype-resolved apple genomes provide insights into the genetic basis of rootstock-induced dwarfing. Nat Genet. 2024;1-12

[8] Li HL, Zhang H, Yu C. et al. Possible roles of auxin and zeatin for initiating the dwarfing effect of M9 used as apple rootstock or interstock. Acta Physiol Plant. 2012; 34 :235-44

[9] Zhang H, An HS, Wang Y. et al. Low expression of PIN gene family members is involved in triggering the dwarfing effect in M9 interstem but not in M9 rootstock apple tree. Acta Physiol Plant. 2015; 37 :104-22

[10] Guilfoyle TJ, Hagen G. Auxin response factors. Curr Opin Plant Bio. 2007; 10 :453-60

[11] Roosjen M, Paque S, Weijers D. Auxin response factors: output control in auxin biology. J Exp Bot. 2018; 69 :179-88

[12] Kato H, Kouno M, Takeda M. et al. The roles of the sole activator- type auxin response factor in pattern formation of Marchantia polymorpha. Plant Cell Physiol. 2017; 58 :1642-51

[13] Li H, Johnson P, Stepanova A. et al. Convergence of signaling pathways in the control of differential cell growth in Arabidop- sis. Dev Cell. 2004; 7 :193-204

[14] Okushima Y, Overvoorde PJ, Arima K. et al. Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana : unique and overlapping functions of ARF7 and ARF19. Plant Cell. 2005; 17 :444-63

[15] Wang S, Tiwari SB, Hagen G. et al. AUXIN RESPONSE FACTOR7 restores the expression of auxin-responsive genes in mutant Arabidopsis leaf mesophyll protoplasts. Plant Cell. 2005; 17 : 1979-93

[16] Feng ZH, Zhu J, Du X. et al. Effects of three auxin-inducible LBD members on lateral root formation in Arabidopsis thaliana. Planta. 2012; 236 :1227-37

[17] Ren ZX, Liu RY, Gu WT. et al. The Solanum lycopersicum auxin response factor SlARF2 participates in regulating lateral root formation and flower organ senescence. Plant Sci. 2017; 256 : 103-11

[18] Li HF, Ran K, Sun QR. Genome-wide identification and expres- sion analysis of peach auxin response factor gene families. J Plant Biochem Biotechnol. 2016; 25 :349-57

[19] Wu B, Li YH, Wu JY. et al. Over-expression of mango ( Mangifera indica L.) MiARF 2 inhibits root and hypocotyl growth of Arabidop- sis. Mol Biol Rep. 2011; 38 :3189-94

[20] Haecker A, Groß-Hardt R, Geiges B. et al. Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana. Development. 2004; 131 :657-68

[21] Luijten M, Miyashima S, Lenhard M. et al. Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature. 2007; 446 :811-4

[22] Breuninger H, Rikirsch E, Hermann M. et al. Differential expres- sion of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo. Dev Cell. 2008; 14 :867-76

[23] Hirakawa Y, Kondo Y, Fukuda H. TDIF peptide signaling regu- lates vascular stem cell proliferation via theWOX4Homeobox gene in Arabidopsis. Plant Cell. 2010; 22 :2618-29

[24] Suer S, Agusti J, Sanchez P. et al. WOX4 imparts auxin responsive- ness to cambium cells in Arabidopsis. Plant Cell. 2011; 23 :3247-59

[25] Ji J, Strable J, Shimizu R. et al. WOX4 promotes procambial development. Plant Physiol. 2010; 152 :1346-56

[26] Okushima Y, Fukaki H, Onoda M. et al. ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. Plant Cell. 2007; 19 :118-30

[27] Yuan TT, Xu HH, Li J. et al. Auxin abolishes SHI-RELATED SEQUENCE5-mediated inhibition of lateral root development in Arabidopsis. New Phytol. 2020; 225 :297-309

[28] Lee HW, Kim NY, Lee DJ. et al. LBD18/ASL20 regulates lat- eral root formation in combination with LBD16/ASL 18 down- stream of ARF7 and ARF19 in Arabidopsis. Plant Physiol. 2009; 151 : 1377-89

[29] Liu J, Hu X, Qin P. et al. The WOX11-LBD16 pathway promotes pluripotency acquisition in callus cells during de novo shoot regeneration in tissue culture. Plant Cell Physiol. 2018; 59 :734-43

[30] Zhao Y, Hu Y, Dai M. et al. The WUSCHEL-related homeobox gene WOX11 is required to activate shoot-borne crown root development in rice. Plant Cell. 2009; 21 :736-48

[31] Zhao Y, Cheng S, Song Y. et al. The interaction between rice ERF3 and WOX11 promotes crown root development by regu- lating gene expression involved in cytokinin signaling. Plant Cell. 2015; 27 :2469-83

[32] Lv JH, Feng Y, Jiang LZ. et al. Genome-wide identification of WOX family members in nine Rosaceae species and a func- tional analysis of MdWOX13- 1 in drought resistance. Plant Sci. 2023; 328 :111564

[33] Goh T, Joi S, Mimura T. et al. The establishment of asym- metry in Arabidopsis lateral root founder cells is regulated by LBD16/ASL18 and related LBD/ASL proteins. Development. 2012; 139 :883-93

[34] Berckmans B, Vassileva V, Schmid SPC. et al. Auxin-dependent cell cycle reactivation through transcriptional regulation of Ara- bidopsis E2Fa by lateral organ boundary proteins. Plant Cell. 2011; 23 :3671-83

[35] Wang JG, Liu JH, Guo ZL. Natural uORF variation in plants. Trends Plant Sc. 2023; 23 :1360-85

[36] Liang Y, Liu HJ, Yan J. et al. Natural variation in crops: realized understanding, continuing promise. Annu Rev Plant Biol. 2021; 72 : 357-85

[37] Yano K, Yamamoto E, Aya K. et al. Genome-wide association study using whole-genome sequencing rapidly identifies new genes influencing agronomic traits in rice. Nat Genet. 2016; 48 : 927-34

[38] Gaut BS, Seymour DK, Liu QP. et al. Demography and its effects on genomic variation in crop domestication. Nature Plants. 2018; 4 :512-20

[39] Zheng X, Zhao Y, Shan D. et al. MdWRKY 9 overexpression con- fers intensive dwarfing in the m26 rootstock of apple by directly inhibiting brassinosteroid synthetase mddwf 4 expression. New Phytol. 2018; 217 :1086-98

[40] Costes E, Salles J, Garcia G. et al. Growth and branching patterns along the main axis of two apple cultivars grafted on two different rootstocks. Acta Hortic. 2001; 55 :131-8

[41] Seleznyova AN, Thorp TG, White M. et al. Application of archi- tectural analysis and AMAPmod methodology to study dwarfing phenomenon: the branch structure of ’Royal Gala’ apple grafted on dwarfing and non-dwarfing rootstock/interstock combina- tions. Ann Bot. 2003; 91 :665-72

[42] Seleznyova AN, Tustin DS, Thorp TG. Apple dwarfing rootstocks and interstocks affect the type of growth units produced during the annual growth cycle: precocious transition to flowering affects the composition and vigour of annual shoots. Ann Bot. 2008; 101 :679-87

[43] Benková E, Michniewicz M, Sauer M. et al. Efflux-dependent auxin gradients as a common module for plant organ formation. Cell. 2003; 115 :591-602

[44] Himanen K, Boucheron E, Vanneste S. et al. Auxin-mediated cell cycle activation during early lateral root initiation. Plant Cell. 2002; 14 :2339-51

[45] Casimiro I, Beeckman T, Graham N. et al. Dissecting Arabidopsis lateral root development. Trends Plant Sci. 2003; 8 :165-71

[46] Eun KY, Ji HY, Woo SL. Auxin and abscisic acid responses of auxin response factor 3 in Arabidopsis lateral root development. J Plant Biol. 2010; 5 :150-4

[47] Du Y, Scheres B. Lateral root formation and the multiple roles of auxin. J Exp Bot. 2018; 69 :155-67

[48] Lee HW, Cho C, Kim J. Lateral organ boundaries domain16 and 18 act downstream of the AUXIN1 and LIKE-AUXIN3 AUXIN influx carriers to control lateral root development in Arabidopsis. Plant Physiol. 2015; 168 :1792-806

[49] Hu J, Hu X, Yang Y. et al. Strigolactone signaling regulates cam- bial activity through repression of WOX4 by transcription factor BES1. Plant Physiol. 2022; 188 :255-67

[50] Kucukoglu M, Nilsson J, Zheng B. et al. WUSCHEL-RELATED HOMEOBOX4 (WOX4)-like genes regulate cambial cell division activity and secondary growth in Populus trees. New Phytol. 2017; 215 :642-57

[51] Tang XF, Wang CP, Chai GH. et al. Ubiquitinated DA1 negatively regulates vascular cambium activity through modulating the stability of WOX4 in Populus. Plant Cell. 2022; 34 :3364-82

[52] Feng Y, Liu J, Zhai LM. et al. Natural variation in cytokinin maintenance improves salt tolerance in apple rootstocks. Plant Cell Environ. 2019; 42 :424-36

[53] Feng Y, Lv JH, Peng M. et al. Genome-wide identification and characterization of the IPT family members in nine Rosaceae species and a functional analysis of MdIPT5b in cold resistance. Hortic Plant J. 2023; 9 :616-30

[54] Fan XQ, YQ L, Deng CH. et al. Strigolactone regulates adventitious root formation via the MdSMXL7-MdWRKY6- MdBRC1 signaling cascade in apple. Plant J. 2022; 113 : 772-86

[55] Meng D, Yang Q, Dong BY. et al. Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants. Plant Biotechnol J. 2019; 17 : 1804-13

[56] Xie X, Li S, Zhang R. et al. The bHLH transcription fac- tor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant Cell Environ. 2012; 35 :1884-97

[57] Zheng X, Deng W, Luo K. et al. The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep. 2007; 26 :1195-203

[58] Chen J, Li Y, Li Y. et al. AUXIN RESPONSE FACTOR 18-HISTONE DEACETYLASE 6 module regulates floral organ identity in rose ( Rosa hybrida ). Plant Physiol. 2021; 186 :1074-87

[59] Dai H, Li W, Han G. et al. Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple. Sci Hortic. 2013; 164 :202-8

[60] Nowicka A, Tokarz B, Zwyrtková J. et al. Comparative analysis of epigenetic inhibitors reveals different degrees of interference with transcriptional gene silencing and induction of DNA dam- age. Plant J. 2020; 102 :68-84

[61] Zhou Y, Tian X, Yao J. et al. Morphological and photosynthetic responses differ among eight apple scion-rootstock combina- tions. Sci Hortic. 2019; 261 :108981

[62] Wei Q, Ma C, Xu YJ. et al. Control of chrysanthemum flower- ing through integration with an aging pathway. Nat Commun. 2017; 8 :829

[63] Xie Y, Liu Y, Ma M. et al. Arabidopsis FHY3 and FAR1 integrate light and strigolactone signaling to regulate branching. Nat Com- mun. 1955; 2020 :11

[64] Zhai L, Sun Q, Gao M. et al. MxMPK4- 1 phosphorylates NADPH oxidase to trigger the MxMPK6-2-MxbHLH104 pathway medi- ated Fe deficiency responses in apple. Plant Cell Environ. 2022; 45 : 2810-26

[65] Feng Y, Wang Y, Zhang GF. et al. Group-C/S1 bZIP heterodimers regulate MdIPT5b to negatively modulate drought tolerance in apple species. Plant J. 2021; 107 :399-417

[66] Cai XT, Xu P, Zhao PX. et al. Arabidopsis ERF109 mediates cross- talk between jasmonic acid and auxin biosynthesis during lat- eral root formation. Nat Commun. 2014; 5 :5833

[67] Gasic K, Hernandez A, Korban SS. RNA extraction from dif- ferent apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Mol Biol Report. 2004; 22 : 437-8

[68] Edwards K, Johnstone C, Thompson C. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 1991; 19 :1349

[69] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 - ΔΔ CT method. Methods. 2001; 25 :402-8