PeERF1, a SHINE-Like Transcription Factor, Is Involved in Nanoridge Development on Lip Epidermis of Phalaenopsis Flowers

Abstract

<i>Phalaenopsis</i> orchids have a spectacular floral morphology with a highly evolved lip that offers a landing platform for pollinators. The typical morphological orchid lip features are essential for the special pollination mechanism of <i>Phalaenopsis</i> flowers. Previously, we found that in the lip, a member of the AP2/EREBP protein family was highly expressed. Here, we further confirmed its high expression and characterized its function during lip development. Phylogenetic analysis showed that AP2/EREBP belongs to the Va2 subgroup of ERF transcription factors. We named it PeERF1. We found that <i>PeERF1</i> was only expressed at stage 5, as flowers opened. This coincided with both thickening of the cuticle and development of nanoridges. We performed knockdown expression of <i>PeERF1</i> using CymMV-based virus-induced gene silencing in either the <i>AP2</i> conserved domain, producing <i>PeERF1_AP2</i>-silenced plants, or the <i>SHN</i> specific domain, producing <i>PeERF1_SHN-</i>silenced plants. Using cryo-SEM, we found that the number of nanoridges was reduced only in the <i>PeERF1_AP2</i>-silenced group. This change was found on both the abaxial and adaxial surfaces of the central lip lobe. Expression of <i>PeERF1</i> was reduced significantly in <i>PeERF1_AP2</i>-silenced plants. In cutin biosynthesis genes, expression of both <i>PeCYP86A2</i> and <i>PeDCR</i> was significantly decreased in both groups. The expression of <i>PeCYP77A4</i> was reduced significantly only in the <i>PeERF1_AP2</i>-silenced plants. Although <i>PeGPAT</i> expression was reduced in both silenced plants, but to a lesser degree. The expression of <i>PeERF1</i> was significantly reduced in the petal-like lip of a big-lip variant. <i>PeCYP77A4</i> and <i>PeGPAT</i> in the lip were also reduced, but <i>PeDCR was not</i>. Furthermore, heterologous overexpression of <i>PeERF1</i> in the genus <i>Arabidopsis</i> produced leaves that were shiny on the adaxial surface. Taken together, our results show that in <i>Phalaenopsis</i> orchids PeERF1 plays an important role in formation of nanoridges during lip epidermis development.