# Genome-Wide Identification of the PAL Gene Family in Camellia nitidissima and Functional Characterization of CnPAL1 Gene by In Vitro Expression

## TL;DR

This study identifies and characterizes the PAL gene family in Camellia nitidissima, focusing on the CnPAL1 gene's function and enzyme activity.

## Contribution

The study provides the first genome-wide analysis of the PAL gene family in Camellia nitidissima and experimentally validates the function of CnPAL1.

## Key findings

- Six CnPAL genes were identified and analyzed for their structure, function, and expression patterns.
- CnPAL1 showed high expression and confirmed enzyme activity through prokaryotic expression and UPLC-MS analysis.
- Collinearity analysis revealed conserved relationships between CnPALs and PAL genes in other Camellia species.

## Abstract

Background: PAL genes are crucial for plant growth and stress response, yet studies on the PAL gene family in Camellia nitidissima are sparse. Methods: The PAL gene family was screened from the entire genome of C. nitidissima, and their physicochemical properties, chromosomal locations, intraspecific and interspecific collinearity, conserved motifs, phylogenetic trees, cis-acting elements, and gene structures were analyzed. The expression patterns of the CnPAL genes were compared across different tissues, and the highly expressed CnPAL1 gene was expressed in prokaryotes, and its enzyme activity was validated using UPLC-MS technology. Results: The results revealed that six CnPALs were identified in the C. nitidissima genome, distributed unevenly across six chromosomes. The CnPAL proteins shared similar physicochemical properties, with highly conserved motifs and gene structures. Promoter analysis showed multiple cis-acting elements in the CnPALs genes. Intra-species collinearity analysis revealed that all CnPALs were collinear with multiple PAL genes in C. nitidissima, while inter-species collinearity analysis indicated that CnPALs were collinear with the PAL genes in Camellia oleifera and Camellia sinensis. Furthermore, the transcriptomic data of C. nitidissima demonstrated tissue-specific expression of the CnPALs, although qRT-PCR validation showed some discrepancies with the sequencing result. The qRT-PCR revealed varied expression patterns among the six CnPALs, with the CnPAL1 gene showing relatively higher expression levels. Subsequently, cloning, prokaryotic expression, and enzyme activity analysis confirmed the effective catalytic activity of the CnPAL1 protein. Conclusions: This study lays the foundation for understanding the functions of CnPAL genes and offers insights for genetic improvement of C. nitidissima.