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<p>Gametophyte-specific promoters are valuable tools for targeted gene expression in breeding, developmental biology, and genome editing. However, the regulatory regions that confer strict gametophyte-specific activity remain poorly characterized in plants. The Plant Intracellular Ras-group Leucine-Rich-Repeat 6 (<i>PIRL6</i>) gene is known to be essential for both male and female gametogenesis, yet the promoter features underlying its specificity were not previously defined. Here, using GUS-based deletion analysis in <i>Arabidopsis thaliana</i>, we identified the minimal <i>PIRL6</i> promoter fragment required for exclusive expression in the male gametophyte. Quantitative assays revealed that this fragment (PA1) drives stronger expression in mature anthers than in younger ones. To understand the broader regulatory context, we identified 50 genes co-expressed with <i>PIRL6</i> and showed through gene ontology analysis that they are mainly involved in cell differentiation. Comparative promoter analysis uncovered three over-represented known motifs (POLLEN1LELAT52, ACGTATERD1, CIACADIANLELHC) and predicted three novel cis-regulatory elements enriched in this gene set. Furthermore, we functionally validated the activity of ABRE and P-box motifs in the <i>PIRL6</i> promoter using a tobacco transient assay. Together, our study defines the functional promoter region of <i>PIRL6</i>, reveals its regulatory architecture, and identifies novel motifs potentially governing male gametophyte-specific gene expression. This provides new insights into the cis-regulatory logic of reproductive development and delivers a characterized promoter resource for gametophyte-targeted applications.</p>

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Functional Profiling and Regulatory Element Analysis of the Male Gametophyte-Specific Promoter Fragment in the Arabidopsis PIRL6 Gene

  • 期刊论文

  • 发表日期：

    2026年1月21日

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  • T. G. Ajith,
  • Jasmine M. Shah

摘要

Gametophyte-specific promoters are valuable tools for targeted gene expression in breeding, developmental biology, and genome editing. However, the regulatory regions that confer strict gametophyte-specific activity remain poorly characterized in plants. The Plant Intracellular Ras-group Leucine-Rich-Repeat 6 (PIRL6) gene is known to be essential for both male and female gametogenesis, yet the promoter features underlying its specificity were not previously defined. Here, using GUS-based deletion analysis in Arabidopsis thaliana, we identified the minimal PIRL6 promoter fragment required for exclusive expression in the male gametophyte. Quantitative assays revealed that this fragment (PA1) drives stronger expression in mature anthers than in younger ones. To understand the broader regulatory context, we identified 50 genes co-expressed with PIRL6 and showed through gene ontology analysis that they are mainly involved in cell differentiation. Comparative promoter analysis uncovered three over-represented known motifs (POLLEN1LELAT52, ACGTATERD1, CIACADIANLELHC) and predicted three novel cis-regulatory elements enriched in this gene set. Furthermore, we functionally validated the activity of ABRE and P-box motifs in the PIRL6 promoter using a tobacco transient assay. Together, our study defines the functional promoter region of PIRL6, reveals its regulatory architecture, and identifies novel motifs potentially governing male gametophyte-specific gene expression. This provides new insights into the cis-regulatory logic of reproductive development and delivers a characterized promoter resource for gametophyte-targeted applications.