Constitutive overexpression of the classical arabinogalactan protein gene BcMF18 in Arabidopsis causes defects in pollen intine morphogenesis

Arabinogalactan proteins (AGPs) are essential members of hyperglycosylated glycoproteins and are critical for the sexual reproduction of plants. To our knowledge, currently Arabodopsis FLA3 and Brassica BcMF8 are the only two members of AGPs reported concerning pollen intine formation. Previously, the orthologue of AtAGP6 in B. campestris, BcMF18, was isolated and proved to affect microspore development. In this study, western blot assay and subcellular localization showed that BcMF18 encoded an approximately 19.5 kDa secretory AGP protein which anchored to extracellular matrices. Ectopic overexpression of BcMF18 (OE) in Arabodopsis caused reduced male fertility and resulted in short siliques with low seed set. Approximately 46% of OE pollen grains were aborted and lacked viability, without all cytoplasmic materials and nuclei. Pollen abortion which led to defective microspore production with no cellulose was observed to start at the bicellular stage. At this point, intine layer displayed abnormalities and degraded from the bicellular stage, which led to the failure in keeping the internal environment stability and resulted in hollow remnants of microspores. However, the basic structure and patterning of the exine layer of collapsed pollen grains seemed unaffected, completed with intact nexine, baculum, and tectum to constitute a pronounced reticulate structure filled with typhine. Based on these results presented here, it is proposed that BcMF18 functions in Arabodopsis and is associated with pollen intine formation, possibly through participating in the formation of proteoglycan structures, which provide strong evidence for subfunctionalization from AtAGP6 to BcMF18.