| Summary: | Successful plant reproduction and fruit formation depend on adequate pollen and pistil development, and pollen–pistil interactions. In <i>Nicotiana tabacum</i>, pollen tubes grow through the intercellular spaces of pistil-specialized tissues, stigmatic secretory zone, and stylar transmitting tissue (STT). These intercellular spaces are supposed to be formed by the modulation of cell wall pectin esterification. Previously we have identified a gene preferentially expressed in pistils encoding a putative pectin acetylesterase (PAE), named <i>NtPAE1</i>. Here, we characterized the <i>NtPAE1</i> gene and performed genome-wide and phylogenetic analyses of PAEs. We identified 30 PAE sequences in the <i>N. tabacum</i> genome, distributed in four clades. The expression of <i>NtPAE1</i> was assessed by RT-qPCR and <i>in situ</i> hybridization. We confirmed <i>NtPAE1</i> preferential expression in stigmas/styles and ovaries and demonstrated its high expression in the STT. Structural predictions and comparisons between NtPAE1 and functional enzymes validated its identity as a PAE. Transgenic plants were produced, overexpressing and silencing the <i>NtPAE1</i> gene. Overexpressed plants displayed smaller flowers while silencing plants exhibited collapsed pollen grains, which hardly germinate. <i>NtPAE1</i> silencing plants do not produce fruits, due to impaired pollen tube growth in their STTs. Thus, NtPAE1 is an essential enzyme regulating pectin modifications in flowers and, ultimately, in plant reproduction.
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