The pan genome analysis of WOX gene family in apple and the two sides of MdWUS-1 in promoting leaf-borne shoot

Abstract

Unlike animals, plants are sessile organisms that cannot move freely in response to fluctuating and complex environments. As a result, plant development follows post-embryonic processes, enabling flexible developmental strategies to adapt to changing environment. The WUSCHEL-related homeobox (WOX) gene family plays a crucial role in regulating these post-embryonic processes in plants. In this study, we performed an evolutionary analysis of the WOX gene family across 29 plant species, isolating a total of 330 WOX family genes. Our study identified a fern protein with similar length and conserved motifs to WUS gene of spermatophyte, suggesting that the modern clade of the WOX family may have already diverged in ferns. Furthermore, we conducted a pan-genome analysis of the WOX family in Malus, examining the number and gene characteristics of WOX family members across eight varieties. The promoter elements of WUS-1, WUS-2, WOX5-1, and WOX5-2 in different Malus varieties were analyzed further. Additionally, we examined the expression patterns of modern clade WOX family members in developing tissues and during leaf-borne shoot regeneration of Malus. We developed the transgenic lines with inducible overexpression of MdWUS-1 or MdWOX5-1, which revealed that mild upregulation of MdWUS-1 significantly promoted leaf-borne shoot formation, while strong upregulation of MdWUS-1 led to browning and death of explants, likely due to oxidative stress. These findings provide new insights into the evolution of the WOX gene family from ferns into seed plants and lay the foundation for further studies on the spatiotemporal regulation of gene expression during shoot regeneration.

Acknowledgments

This research was financially supported by the National Key Research and Development Program of China 2023YFD2301000, the Taishan Program of Shandong Province (grants tsqn202408127), and the National Natural Science Foundation of China (32372659 and 31972357)。.

Author contributions

L. L. and Y. F. S. performed research and wrote the paper. M.Y. L., S. X. X., X. F. L., Y. Z., and C. A. W. contributed new reagents/analytic tools. Y. W., L.Y. Y., Z. T., J. L. W., B. K. G., and P. Z. C conducted database collation and bioinformatics analysis. J. G. H., K. Y., and C. C. Z., provided the critical discussion. G. D. Y., X. T., and S. Z. Z. designed research.

Data availability statement

Supplementary figures and supplementary data are available at Horticulture Research online.

Conflict of interests

The authors declare no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Beckers A, Mamiya A, Furutani M. et al. Multiple layers of regu-lators emerge in the network controlling lateral root organogen-esis. Trends Plant Sci. 2024; 30:499-514

[2] Laux T, Mayer KF, Berger J. et al. The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis. Develop-men. 1996; 122:87-96

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

[4] Mayer KF, Schoof H, Haecker A. et al. Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell. 1998; 95:805-15

[5] Stahl Y, Wink RH, Ingram GC. et al. A signaling module control-ling the stem cell niche in Arabidopsis root meristems. Curr Biol. 2009; 19:909-14

[6] Nardmann J, Reisewitz P, Werr W. Discrete shoot and root stem cell-promoting WUS/WOX5 functions are an evolutionary inno-vation of angiosperms. MolBiolEvol. 2009; 26:1745-55

[7] van der Graaff E, Laux T, Rensing SA. The WUS homeobox-containing (WOX) protein family. Genome Biol. 2009; 10:248

[8] Zhang Y, Jiao Y, Jiao H. et al. Two-step functional innovation of the stem-cell factors WUS/WOX5 during plant evolution. Mol Biol Evol. 2017c; 34:640-53

[9] Pagel M. Natural selection 150 years on. Nature. 2009; 457: 808-11

[10] Marchant DB, Chen G, Cai S. et al. Dynamic genome evolution in a model fern. Nat Plants. 2022; 8:1038-51

[11] Fang Y, Qin X, Liao Q. et al. The genome of homosporous maidenhair fern sheds light on the euphyllophyte evolution and defences. Nat Plants. 2022; 8:1024-37

[12] Li F-W, Brouwer P, Carretero-Paulet L. et al. Fern genomes elu-cidate land plant evolution and cyanobacterial symbioses. Nat Plants. 2018; 4:460-72

[13] Huang X, Wang W, Gong T. et al. The flying spider-monkey tree fern genome provides insights into fern evolution and arbores-cence. Nat Plants. 2022; 8:500-12

[14] Meng WJ, Cheng ZJ, Sang YL. et al. Type-B ARABIDOP-SIS RESPONSE REGULATORs specify the shoot stem cell niche by dual regulation of WUSCHEL. Plant Cell. 2017; 29: 1357-72

[15] Zhai N, Xu L. Pluripotency acquisition in the middle cell layer of callus is required for organ regeneration. Nat Plants. 2021; 7: 1453-60

[16] Zhang TQ, Lian H, Zhou CM. et al. A two-step model for de novo activation of WUSCHEL during plant shoot regeneration. Plant Cell. 2017b; 29:1073-87

[17] Wang K, Shi L, Liang X. et al. The gene TaWOX5 overcomes geno-type dependency in wheat genetic transformation. Nat Plants. 2022; 8:110-7

[18] Zhang F, May A, Irish VF. Type-B ARABIDOPSIS RESPONSE REG-ULATORs directly activate WUSCHEL. Trends Plant Sci. 2017a; 22: 815-7

[19] Maher MF, Nasti RA, Vollbrecht M. et al. Plant gene editing through de novo induction of meristems. Nat Biotechnol. 2020; 38: 84-9

[20] Wang N, Ryan L, Sardesai N. et al. Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum. Nat Plants. 2023; 9:255-70

[21] Lowe K, Wu E, Wang N. et al. Morphogenic regulators baby boom and Wuschel improve monocot transformation. Plant Cell. 2016; 28:1998-2015

[22] Schena M, Davis RW. HD-zip proteins: members of an Arabidop-sis homeodomain protein superfamily. Proc Natl Acad Sci USA. 1992; 89:3894-8

[23] Jiang W, Wang Z, Li Y. et al. FERONIA regulates salt tolerance in Arabidopsis by controlling photorespiratory flux. Plant Cell. 2024; 36:4732-51

[24] Smetana O, Mäkilä R, Lyu M. et al. High levels of auxin signalling define the stem-cell organizer of the vascular cambium. Nature. 2019; 565:485-9

[25] Liu W, Xu L. Recruitment of IC-WOX genes in root evolution. Trends Plant Sci. 2018; 23:490-6

[26] Hernández-Coronado M, Dias Araujo PC, Ip PL. et al. Plant glutamate receptors mediate a bet-hedging strategy between regeneration and defense. Dev Cell. 2022; 57:451-465.e6

[27] Yang W, Zhai H, Wu F. et al. Peptide REF1 is a local wound signal promoting plant regeneration. Cell. 2024; 187:3024-3038.e14

[28] Wu H, Qu X, Dong Z. et al. WUSCHEL triggers innate antiviral immunity in plant stem cells. Science. 2020; 370:227-31

[29] Feng S, Liu Z, Chen H. et al. PHGD: an integrative and user-friendly database for plant hormone-related genes. iMeta. 2024; 3:e164

[30] Finn RD, Coggill P, Eberhardt RY. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 2016;44:D279-85

[31] Goodstein DM, Shu S, Howson R. et al. Phytozome: a com-parative platform for green plant genomics. Nucleic Acids Res. 2012;40:D1178-86

[32] Liu Z, Zhang C, He J. et al. plantGIR: a genomic database of plants. Hortic Res. 2024b;11:uhae342

[33] Lu S, Wang J, Chitsaz F. et al.CDD/SPARCLE:the conserved domain database in 2020. Nucleic Acids Res.2020;48:D265-d268

[34] Wu T,LiuZ,YuT. et al.Flowering genes identification, network analysis, and database construction for 837 plants. Hortic Res. 2024;11:uhae013

[35] Chen C, Wu Y, Li J. et al. TBtools-II: a ‘one for all, all for one’ bioinformatics platform for biological big-data mining. Mol Plant. 2023; 16:1733-42

[36] LiuL, QuJ, WangC. et al. An efficient genetic transformation system mediated by rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion. Plant Biotechnol J. 2024a; 22:2093-103