# Comparative Genomic Analysis and Functional Identification of CER1 and CER3 Homologs in Rice Wax Synthesis

## TL;DR

This paper identifies key genes involved in wax production in rice, which could help improve stress tolerance in crops.

## Contribution

The study systematically compares CER1 and CER3 homologs in rice and identifies OsCER3a as a major contributor to wax synthesis.

## Key findings

- OsCER3a mutation significantly reduces wax production in rice.
- CER1 and CER3 genes form complexes during wax synthesis.
- Promoter analysis reveals stress-responsive elements in CER genes.

## Abstract

This study provides a comprehensive characterization of CER1 and CER3 gene families in rice, revealing their evolutionary divergence into two distinct subgroups despite originating from a common ancestral lineage. These genes display different tissue-specific expression patterns. Promoter analysis identified an abundance of cis-regulatory elements responsive to light and drought, suggesting potential involvement in abiotic stress adaptation. Subcellular localization confirmed endoplasmic reticulum (ER) targeting, consistent with their enzymatic functions in wax biosynthetic pathways. Protein–protein interaction assays further revealed that OsCER1 proteins interact with OsCER3 homologs, mirroring conserved interactions observed in dicot species such as Arabidopsis thaliana. Functional analysis of transgenic and knockout lines highlighted OsCER3a as a key regulator of wax accumulation, while other homologs exhibited partial or redundant activity. Collectively, these findings enhance our understanding of CER1-CER3 module functionality in rice and offer promising targets for genetic engineering aimed at improving cuticular wax deposition and abiotic stress resilience in cereal crops.

Alkane is a predominant wax component, whose production requires the aids of CER1 and CER3. In rice, OsCER1 and OsCER3 are present in multiple copies. Until now, the roles of these genes have been studied individually; however, a systematic comparison of their relative contributions to cuticular wax biosynthesis has not yet been carried out. Phylogenetic tree analysis revealed that CER1s and CER3s from different plants are classified into two subgroups. RT-qPCR analysis showed that these genes display distinct expression patterns, revealing their specific roles in wax production. Promoter prediction analysis showed that cis-elements responding to light, phytohormones and stress are enriched in the promoter region of OsCER1s and OsCER3s. These proteins are all localized in the endoplasmic reticulum. Further study showed that OsCER1s and OsCER3s are inclined to form a complex during the wax synthesis. Finally, the wax analysis of single mutants showed that among the examined genes, OsCER3a mutation greatly reduced the total wax amounts to 19.6% of wild-type plant with a decrease in most of wax components, whereas mutation of other genes including OsCER3b, OsCER3c, OsCER1a and OsCER1c slightly or barely affect wax production, suggesting that OsCER3a plays major roles in rice wax production whereas other proteins redundantly participate in the wax synthesis. Additionally, the wax increasing rates of Arabidopsis expressing OSCER1 are lower than those of overexpressing AtCER1. Taken together, our study identified the predominant genes involved in wax production, which will be useful for genetically engineering rice with enhanced stress tolerance.