Fig. 7.

Characterization of the acbp4 mutant.

(a) Location of T-DNA insertion within ACBP4 (At3g05420) in the acbp4 mutant (SALK_040164). Primers (P1, ML412; P2, ML418; P3, LBa1) used for genotyping the mutant are indicated.

(b) Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis of ACBP4 expression in wild type and acbp4 mutant using primer pair ML413 and ML416 show the presence of a 0.8-kbp band in wild type that is absent in the mutant. Bottom, ACTIN transcript was used as a control in RT-PCR analysis.

(c) Northern blot analyses of ACBP4 and ACBP5 expression in wild type and the acbp4 mutant. RNAs were extracted from leaves of 4-week-old Arabidopsis germinated and grown on MS medium. DIG-labeled ACBP4- and ACBP5-cDNA probes were used in northern blot analysis and the blots were washed under conditions of high stringency.

(d) Western blot analysis on wild type, acbp4 mutant and two independent acbp4-complemented transgenic lines (cACBP4 #1 and #2) using antibodies against ACBP4. The cross-reacting 73.1-kDa ACBP4 band in wild type and cACBP4 transgenic lines was detected using the Amplified Alkaline Phosphatase Assay Kit. Bottom, gel identically loaded and stained with Coomassie blue.

(e) Thin-layer chromatography of leaf lipids extracted from 4-week-old Arabidopsis of wild type, acbp4 mutant and two independent acbp4-complemented transgenic lines (cACBP4 #1 and #2). The leaf tissues (100 mg) of each genotype were weighed before lipid extraction and the lipids were dissolved in 100 µl of chloroform after drying under nitrogen. Equal volumes (30 µl) of lipids were loaded in each lane. The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are indicated. The experiment was repeated twice with similar results.