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Molecular Cloning and Functional Characterization of FOUR LIPS in Pomegranate, a Protein Involved in Regulating the Gravitropic Set-Point Angle of Adventitious Roots

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Abstract

Self-rooted pomegranate seedlings are widely used in the horticultural industry to cut costs and time. However, these seedlings produce shallow roots that exhibit poor cold resistance. Thus, deeper adventitious roots generated through gravitropism are imperative for seedling survival, and understanding the molecular mechanisms of gravitropism can facilitate improved breeding techniques. We hypothesized that gravitropism in pomegranate is partially controlled by pomegranate FOUR LIPS (PgFLP), an R2R3-MYB protein that interacts with and controls the transcriptional expression of PgPIN10, which facilitates transmembrane auxin signaling. We studied subcellular localization of PgFLP, quantified auxin levels, and measured gravitropic set-point angle (GSA) to investigate the underlying mechanisms regulating PgPIN10 expression during the formation of GSA in pomegranate adventitious roots. We found that PgFLP was localized to the nucleus based on use of green florescent proteins, suggesting that this protein is a transcription factor. When using the tractable 35S::PgFLP, we observed stronger gravitational response in overexpression lines leading to a narrower GSA than in the wild-type Arabidopsis, and the expression of PgFLP and PgPIN10 in ‘Lanbaoshi’ (LBS; a deep-rooted cultivar) was higher than that in ‘Taishanhong’ (TSH; a shallow-rooted cultivar), which indicates that PgFLP may participate in regulating the GSA of adventitious roots via PgPIN10 in pomegranate. Taken together, our results indicate that the pomegranate R2R3-MYB transcription factor, PgFLP, plays a vital role in setting the GSA of adventitious roots in this crop species.

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Data Availability and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

FLP:

FOUR LIPS

PIN:

Pin-formed

IAA:

Indole-3-acetic acid

GSA:

Gravitropic set-point angle

TFs:

Transcription factors

TSH:

Taishanhong

LBS:

Lanbaoshi

GFP:

Green fluorescent protein

MS:

Murashige and Skoog

WT:

Wild-type

GUS:

β-Glucuronidase

qRT-PCRL:

Quantitative reverse transcription-PCR

Y1H:

Yeast one-hybrid

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Acknowledgements

We thank Dr. **ang Shen for pCAMBIA1300 vector and the seeds of Arabidopsis thaliana (ecotype ‘Columbia’).

Funding

This work was supported by the National Natural Science Foundation of China (32102310), the Shandong Key Research and Development Program, China (2018GNC111009 and 2021LZGC007), the Shandong Agricultural Improvement Seed Projects, China (2017LZN023), the Agricultural Science and Technology Innovation Engineering Discipline Team of Shandong Academy of Agricultural Sciences (CXGC2022D02). The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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Authors and Affiliations

  1. Shandong Institute of Pomology, Tai’an, 271000, Shandong, China

    Zenghui Wang, Chuanzeng Wang, Lijuan Feng & Yanlei Yin

  2. School of Biological Science and Technology, University of **an, **an, 250022, China

    Jialin Li

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  1. Zenghui Wang
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  3. Chuanzeng Wang
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  4. Lijuan Feng
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Contributions

YLY and ZHW conceived the project. YLY and ZHW designed the experiments. ZHW performed most of the experiments, and ZHW wrote the paper. JLL helped with the transient expression assays; JLL, CZW and LJF helped with the phenotypic observations and the paper revision. All authors read and approved the final manuscript.

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Correspondence to Yanlei Yin.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Wang, Z., Li, J., Wang, C. et al. Molecular Cloning and Functional Characterization of FOUR LIPS in Pomegranate, a Protein Involved in Regulating the Gravitropic Set-Point Angle of Adventitious Roots. J. Plant Biol. 65, 377–387 (2022). https://doi.org/10.1007/s12374-022-09360-7

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