BcMF8, a putative arabinogalactan protein-encoding gene, contributes to pollen wall development, aperture formation and pollen tube growth in Brassica campestris.
OBJECTIVE
The arabinogalactan protein (AGP) gene family is involved in plant reproduction. However, little is known about the function of individual AGP genes in pollen development and pollen tube growth. In this study, Brassica campestris male fertility 8 (BcMF8), a putative AGP-encoding gene previously found to be pollen specific in Chinese cabbage (B. campestris ssp. chinensis), was investigated.


METHODS
Real-time reverse transcription-PCR and in situ hybridization were used to analyse the expression pattern of BcMF8 in pistils. Prokaryotic expression and western blots were used to ensure that BcMF8 could encode a protein. Antisense RNA technology was applied to silence gene expression, and morphological and cytological approaches (e.g. scanning electron microscopy and transmission electron microscopy) were used to reveal abnormal phenotypes caused by gene silencing.


RESULTS
The BcMF8 gene encoded a putative AGP protein that was located in the cell wall, and was expressed in pollen grains and pollen tubes. The functional interruption of BcMF8 by antisense RNA technology resulted in slipper-shaped and bilaterally sunken pollen with abnormal intine development and aperture formation. The inhibition of BcMF8 led to a decrease in the percentage of in vitro pollen germination. In pollen that did germinate, the pollen tubes were unstable, abnormally shaped and burst more frequently relative to controls, which corresponded to an in vivo arrest of pollen germination at the stigma surface and retarded pollen tube growth in the stylar transmitting tissues.


CONCLUSIONS
The phenotypic defects of antisense BcMF8 RNA lines (bcmf8) suggest a crucial function of BcMF8 in modulating the physical nature of the pollen wall and in helping in maintaining the integrity of the pollen tube wall matrix.
Laboratory of Cell & Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou 310058, China.
Sue
S
Lin
Heng
H
Dong
Fang
F
Zhang
Lin
L
Qiu
Fangzhan
F
Wang
Jiashu
J
Cao
Li
L
Huang
D015153:Blotting, Western; D001937:Brassica / Q000235:genetics* / Q000254:growth & development; D018506:Gene Expression Regulation, Plant; D017403:In Situ Hybridization; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D009088:Mucoproteins / Q000235:genetics* / Q000378:metabolism; D051479:Plant Infertility; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011058:Pollen / Q000235:genetics / Q000254:growth & development / Q000648:ultrastructure; D053205:Pollen Tube / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D016372:RNA, Antisense / Q000235:genetics
D009088:Mucoproteins; D010940:Plant Proteins; D016372:RNA, Antisense; C006619:arabinogalactan proteins
AGP; Brassica campestris; Chinese cabbage; aperture; aperture formation; arabinogalactan proteins; intine; pollen tube; pollen wall development