Comparative Analysis of WUSCHEL-Related Homeobox Genes Revealed Their Parent-of-Origin and Cell Type-Specific Expression Pattern During Early Embryogenesis in Tobacco

Abstract

WUSCHEL-related homeobox (WOX) gene is a plant-specific clade of homeobox transcription factors. Increasing evidences reveal that WOXs play critical roles in early embryogenesis, which involves zygote development, initiation of zygote division, and apical or basal cell lineage establishment. However, how <i>WOXs</i> regulate these developmental events remains largely unknown, and even detailed expression pattern in gametes and early proembryos is not yet available. Here, 13 <i>WOX</i> family genes were identified in <i>Nicotiana tabacum</i> genome. Comparative analysis of 13 <i>WOX</i> family genes with their homologs in <i>Arabidopsis thaliana</i> reveals relatively conserved expression pattern of <i>WUS</i> and <i>WOX5</i> in shoot/root apical meristem. Whereas variations were also found, e.g., lacking homolog of <i>WOX8</i> (a marker for suspensor cell) in tobacco genome and the expression of <i>WOX2</i>/<i>WOX9</i> in both apical cell and basal cell. Transient transcriptional activity analysis revealed that WOXs in WUS clade have repressive activities for their target's transcription, whereas WOXs in ancient and intermediate clade have activation activities, giving a molecular basis for the phylogenetic classification of tobacco WOXs into three major clades. Expression pattern analysis revealed that some <i>WOXs</i> (e.g., <i>WOX 13a</i>) expressed in both male and female gametes and some <i>WOXs</i> (e.g., <i>WOX 11</i> and <i>WOX 13b</i>) displayed the characteristics of parent-of-origin genes. Interestingly, some <i>WOXs</i> (e.g., <i>WOX2</i> and <i>WOX9</i>), which are essential for early embryo patterning, were <i>de novo</i> transcribed in zygote, indicating relevant mechanism for embryo pattern formation is only established in zygote right after fertilization and not carried in by gametes. We also found that most <i>WOXs</i> displayed a stage-specific and cell type-specific expression pattern. Taken together, this work provides a detailed landscape of <i>WOXs</i> in tobacco during fertilization and early embryogenesis, which will facilitate the understanding of their specific roles in these critical developmental processes of embryogenesis.