Constitutive overexpression of the classical arabinogalactan protein gene BcMF18 in Arabidopsis causes defects in pollen intine morphogenesis
- 127 Downloads
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.
KeywordsArabidopsis Brassica campestris Intine Microspore development Arabinogalactan proteins AGP
We thank Prof. Dr Fengming Song for providing pET32a(+) vector and Escherichia coli strain DE3, and Prof. Dr Ying Miao for providing pB7YWG2,0 vector. This research received the support of grants from the National Natural Science Foundation of China [Grant Nos. 31501764, 31471877, and 31572126], the Grand Science and Technology Special Project of Zhejiang Province [Grant No. 2016C02051-6].
SL, LH and JC participated in the design of the experimental plan. SL took charge of plant transformation and phenotype comparative observation. LH performed recombinant vector construction and gene expression analysis. YM took part in western blots and transient expression assays. YY performed bioinformatic analysis and statistical analysis. SL and LH wrote the manuscript, while RP provided commentes on it.
Compliance with ethical standards
Conflict of interest
The authors declare that there is no conflict of interest.
- Driouich A, Zhang GF, Staehelin LA (1993) Effect of brefeldin A on the structure of the Golgi apparatus and on the synthesis and secretion of proteins and polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. Plant Physiol 101:1363–1373CrossRefPubMedPubMedCentralGoogle Scholar
- Fei H, Sawhney VK (2001) Ultrastructural characterization of male sterile33 (ms33) mutant in Arabidopsis affected in pollen desiccation and maturation. Can J Bot 79:118–129Google Scholar
- Gan C (1989) Gene gun accelerates DNA-coated particles to transform intact cells. Scientist 3:25Google Scholar
- Mohebali M, Mirbakhsh M, Keshavarz H (2002) Rapid detection of Pneumocystis Carini in spiratory specimens of rats by calcofluor white staining. Iran J Public Health 31:108–110Google Scholar