Figure 4.

Localization of At5g62080-GFP on microspores at a midstage (stage 10) of anther development in Arabidopsis. A, Presence of At5g62080-LTP-GFP (encoded by GFP attached to full-length LTP) and absence of At5g62080-SP-GFP (GFP attached to the LTP region encoding only the N-terminal signal peptide) on microspores of plants transformed with the respective genes. In the third row, microspores were stained with tinopal for the cellulosic cell wall. GFP fluorescence is shown in green, and exine autofluorescence (or tinopal stain) is shown in magenta. Nontransformed plants (wild-type) were used as a control. Scale bars are in μm. B, Localization of At5g62080 in flowers and other Arabidopsis organs with immunoblotting and specificity of anti-At5g62080 antibodies (Ab). At left, total proteins of equal amounts from the indicated organs were subjected to immunoblotting after SDS-PAGE with antibodies against a synthetic fragment of a unique sequence of At5g62080. Immunodetection of tubulin was used as a control. Young flower buds were stages 1 to 10; late flower buds were stages 11 to 13. At right, His-tagged recombinant protein (1 ng each) of the indicated LTPs, whose transcripts were present mostly in flowers among all organs (Table I), were subjected to the same immunodetection. The antibodies were highly specific to At5g62080 and barely recognized the closest-related At5g07230 (Supplemental Fig. S2). The antibodies recognized only one band on the SDS-PAGE blot; only this portion of the immunoblot is shown. The molecular masses of the endogenous and recombinant At5g62080 were 6.6 and 7.9 kD, respectively. C, Immuno-TEM of the outermost portion of microspores (MSP) probed with antibodies against At5g62080. The right panel is an enlarged image of the left panel. Immunogold particles were present in the locule and exine (both nexine and sexine). Quantification of the gold particles is shown in Supplemental Table S1. Scale bar is in μm.