Functional profiling of the male gametophyte-specific promoter fragment of Arabidopsis PIRL6 gene and prediction of cis-regulatory elements

Gametophyte-specific promoters drive the expression of genes in male and/or female gametophytes. These have applications in breeding experiments, gene function identification, developmental biology-related studies, and off-lately in genome editing also. The Plant Intracellular Ras-group Leucine-Rich-Repeat 6 (PIRL6) gene is known to be necessary for male and female gametogenesis. Using the GUS- based deletion analysis, we have identified the PIRL6 promoter length that is essential for exclusive expression in the male gametophyte of Arabidopsis thaliana. We studied the strength of various lengths of PIRL6 promoters in different tissues, by GUS expression quantification. The male-gametophyte-specific promoter segment (PA1) exhibited stronger expression in mature anthers than the younger ones. We identified 50 other genes that co-expressed with PIRL6 in Arabidopsis using the Expression Angler tool. Gene ontology (GO) analysis shows that these 51 co-expressing genes were predominantly involved in cell differentiation. By comparing the promoter sequences of these 51 genes, the presence of three over-represented known motifs, POLLEN1LELAT52, ACGTATERD1 and CIACADIANLELHC was identified. We could also predict the presence of three novel cis-regulatory elements in the co-expressing gene network using the MEME suite tool. Additionally, we confirmed the functionality of the ABRE and P-box elements present in PIRL6 promoter using the tobacco leaf transient assay. Thus, we cloned and functionally confirmed the promoter region of PIRL6 required for male gametophyte-specific expression, compared this promoter with those of 50 other co-expressed genes and predicted their functions, and analyzed their cis regulatory regions and predicted three novel motifs also. Key messageA pollen-specific promoter fragment of PIRL6 was functionally characterized using GUS-based deletion analysis. Fifty other co-expressed genes were compared and novel cis-regulatory elements were predicted. Two hormone-responsive elements in the PIRL6 promoter were found to influence the GUS expression. PA1 promoter can be used in experiments that require male gametophyte-specific expression of genes.