A CLE–RLK–LBD signaling module promotes de novo shoot regeneration in plants

doi:10.1111/jipb.70300

• Research Article • Next Articles

A CLE–RLK–LBD signaling module promotes de novo shoot regeneration in plants

Ziyao Hu^1†, Langrang Zhang^1†, Siwei Bie^2†, Junpeng Niu¹, Kaiying Chen¹, Xuan Ji¹, Jing Wang¹, Deyan Yang¹, Yanpeng Zhao¹, Yonghong Zhang³, Chen Li³, J. Peter Etchells⁴, Jennifer C. Fletcher^5,6, Chao Zhou^2*, Shinichiro Sawa^7* and Guodong Wang^1*

1. Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry of Ministry of Education, College of Life Sciences, Engineering Research Center of High Value Utilization of Western China Fruit Resources of Ministry of Education, Shaanxi Normal University, Xi'an 710119, China
2. Key Laboratory of Three Gorges Regional Plant Genetics & Germplasm Enhancement (CTGU), Biotechnology Research Center, China Three Gorges University, Yichang 443002, China
3. Laboratory of Medicinal Plant, Hubei Key Laboratory of Embryonic Stem Cell Research, School of Basic Medicine, University of Medicine, Shiyan 442000, China
4. Department of Biosciences, Durham University, Durham DH1 3LE, United Kingdom
5. Plant Gene Expression Center, USDA‐ARS/UC Berkeley, Albany 94710, USA
6. Department of Plant and Microbial Biology, University of California, Berkeley 94720‐3102, USA
7. International Research Center for Agricultural and Environmental Biology (IRCAEB), Kumamoto University, Kumamoto 860‐8555, Japan
^†These authors contributed equally to this work.
^*Correspondences: Guodong Wang (guodong_wang@snnu.edu.cn, Dr. Wang is fully responsible for the distribution of all materials associated with this article); Shinichiro Sawa (sawa@kumamoto-u.ac.jp); Chao Zhou (zhouchao@ctgu.edu.cn)

Received:2025-09-13 Accepted:2026-04-10 Online:2026-05-25
Supported by:
This work was supported by National Natural Science Foundation of China (32370850 to G.W.; 32372147 to C.Z.), KAKENHI (25H02428, 25H00932, and 25K22479 to S.S.), United States Department of Agriculture (CRIS 2030‐21210‐001‐00D to J.C.F), UKRI grant (BB/X000559/1 to J.P.E.), JSPS Fellowship (to G.W.), Leading Talent of Technological Innovation of Shaanxi Special Support Program (20249 to G.W.), Key Project of Natural Science Basic Research Program of Shaanxi (2023‐JC‐ZD‐09 to G.W.), Natural Science Foundation of Shaanxi for Distinguished Young Scholars (2020JC‐29 to G.W.), and Fundamental Research Funds for Central Universities (GK202402003 to G.W.).

Abstract

Abstract: Tissue culture recalcitrance limits plant transformation and genome editing for crop improvement. The development of new, generic factors that enhance plant regeneration efficiency could alleviate this bottleneck. Through precursor gene overexpression, exogenous peptide application, and loss-of-function studies, we show here that CLAVATA3/ESR-RELATED 41 (CLE41), CLE42, CLE44, and CLE46, collectively referred to as TDIF-related genes, function redundantly to promote de novo shoot regeneration. CLE41, CLE42, and CLE44 are abundantly and dynamically expressed in pluripotent callus, whereas CLE46 is shoot-induction medium-activated and specifically expressed at explant wound sites. TDIF-related peptides act through PHLOEM INTERCALATED WITH XYLEM/TDIF RECEPTOR (PXY/TDR)-subfamily members to activate WUSCHEL (WUS)-RELATED HOMEOBOX 14 (WOX14) and enhance shooting capacity. In addition, we find that LATERAL ORGAN BOUNDARIES DOMAIN4 (LBD4) is highly expressed throughout the callus and its expression increases upon TDIF upregulation in a PXY/PXL-dependent manner. Consistently, LBD4 overexpression notably improves regeneration efficiency, whereas its loss decreases regeneration. TDIF-enhanced shoot regeneration is significantly suppressed in lbd4 mutants, indicating that LBD4 is required for TDIF signaling in shoot regeneration enhancement. Furthermore, WOX14 directly activates LBD4 to promote de novo shoot regeneration. Exogenous application of synthetic TDIF-related peptides substantially increases shooting capacity in the recalcitrant crop species Sorghum bicolor L., offering a convenient new avenue to enhance de novo shoot regeneration in crops. In conclusion, we demonstrate that Arabidopsis TDIF-PXY/PXL ligand-receptor pairs markedly boost de novo shoot regeneration through WOX14-potentiated LBD4 induction. Our findings elucidate the molecular basis for a novel function of TDIF-related peptides and their receptors in hormone-driven shoot regeneration and demonstrate their practical application in improving adventitious shoot regeneration in a recalcitrant plant species.

Key words: callus, CLE peptide, shoot regeneration

Ziyao Hu, Langrang Zhang, Siwei Bie, Junpeng Niu, Kaiying Chen, Xuan Ji, Jing Wang, Deyan Yang, Yanpeng Zhao, Yonghong Zhang, Chen Li, J. Peter Etchells, Jennifer C. Fletcher, Chao Zhou, Shinichiro Sawa, Guodong Wang. A CLE–RLK–LBD signaling module promotes de novo shoot regeneration in plants[J]. J Integr Plant Biol., DOI: 10.1111/jipb.70300.

[1]	Guifang Zhang, Ning Zhai, Mulan Zhu, Keyuan Zheng, Yalin Sang, Xiaojuan Li, Lin Xu. Cell wall remodeling during plant regeneration [J]. J Integr Plant Biol., 2025, 67(4): 1060-1076.
[2]	Megan G. Sawchuk and Enrico Scarpella. Polarity, Continuity, and Alignment in Plant Vascular Strands [J]. J Integr Plant Biol., 2013, 55(9): 824-834.
[3]	Yi Qiang, Jinbin Wu, Huibin Han and Guodong Wang. CLE Peptides in Vascular Development [J]. J Integr Plant Biol., 2013, 55(4): 389-394.
[4]	Zhao Zhang, and Bart P.H.J. Thomma. Structure-function Aspects of Extracellular Leucine-rich Repeat-containing Cell Surface Receptors in Plants [J]. J Integr Plant Biol., 2013, 55(12): 1212-1223.
[5]	Hai-Hong Shang, Chuan-Liang Liu, Chao-Jun Zhang, Feng-Lian Li, Wei-Dong Hong and Fu-Guang Li. Histological and Ultrastructural Observation Reveals Significant Cellular Differences between Agrobacterium Transformed Embryogenic and Non-embryogenic Calli of Cotton [J]. J Integr Plant Biol., 2009, 51(5): 456-465.
[6]	Aysel Sivaci and Munevver Sokmen. Effects of Exogenous Indole Butyric Acid and Callus Formation on the Anti-oxidant Activity, Total Phenolic, and Anthocyanin Constituents of Mulberry Cuttings [J]. J Integr Plant Biol., 2006, 48(7): -.
[7]	Lu-Min VAARIO, Kazuo SUZUK. Ectomycorrhizal Synthesis Between Abies firma Roots/Callus and Laccaria bicolor Strain [J]. J Integr Plant Biol., 2004, 46(1): 63-68.
[8]	CHAI Bao-Feng, LIANG Ai-Hua, WANG Wei, Hu Wei. Agrobacterium-mediated Transformation of Kentucky Bluegrass [J]. J Integr Plant Biol., 2003, 45(8): 966-973.
[9]	GANG Yong-Yun, DU Gui-Sen, SHI Ding-Ji , WANG Mei-Zhi , LI Xue-Dong , HUA Zhen-Ling. Establishment of In Vitro Regeneration System of the Atrichum Mosses [J]. J Integr Plant Biol., 2003, 45(12): 1475-1480.
[10]	ZHAO Hu-Cheng, WANG Bo-Chu, CAI Shao-Xi and XI Bao-Shu. Effect of Sound Stimulation on the Lipid Physical States and Metabolism of Plasma Membrane from Chrysanthemum Callus [J]. J Integr Plant Biol., 2002, 44(7): 799-803.
[11]	YAN Ying Cai, LIN Hong Hui, LIANG Hou Guo, DU Lin Fang. A Comparative Study on Detection of the Expression of Alternative Oxidase in Tobacco Callus with the Monoclonal Antibody Against Alternative Oxidase and Antibody Against Synthetic Polypeptide Antibody [J]. J Integr Plant Biol., 2002, 44(10): 1255-1257.
[12]	ZHOU Gong-Ke, LI Hong-Yu, WEN Jiang-Qi, KONG Ying-Zhen, LIANG Hou-Guo. The Cyanide-resistant Respiration in Callus of Nicotiana rustica cv. Gansu Yellow Flower Under Low Temperature [J]. J Integr Plant Biol., 2000, 42(7): 679-683.
[13]	WANG Zhe-Zhi, ZHANG Su-Feng and HU Zheng-Hai. Isolation,Culture and Plant Regeneration of Protoplasts from an Embryogenic Callus Tissue of Gossypium hirsutum [J]. J Integr Plant Biol., 1998, 40(3): -.
[14]	Hai Yan, He Xian-chang, Huang Bing-yan and Wang Jin-lan. Preparation of Gui Culture Medium and Its Application to Wheat Anther Culture [J]. J Integr Plant Biol., 1997, 39(8): -.
[15]	Ma Qing-hu and Song Yan-ru. Expression of Tomato Antisense ACC Synthase Gene in Transgenic Tobacco and Its Role in Shoot Formation [J]. J Integr Plant Biol., 1997, 39(11): -.

A CLE–RLK–LBD signaling module promotes de novo shoot regeneration in plants

HTML

PDF

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments