Proteomic analysis of somatic embryogenesis in Medicago truncatula. Explant cultures grown under 6-benzylaminopurine and 1-naphthaleneacetic acid treatments

Abstract

The Medicago truncatula line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than wild-type Jemalong. We have compared proteomes of tissue cultures from leaf explants of these two lines. Both 2HA and Jemalong explants were grown on media containing the auxin 1-naphthaleneacetic acid and the cytokinin 6-benzylaminopurine. Proteins were extracted from the cultures at different time points (2, 5, and 8 weeks), separated by two-dimensional gel electrophoresis, and detected by silver staining. More than 2,000 proteins could be reproducibly resolved and detected on each gel. Statistical analysis showed that 54 protein spots were significantly (P < 0.05) changed in expression (accumulation) during the 8 weeks of culture, and most of these spots were extracted from colloidal Coomassie-stained two-dimensional gel electrophoresis gels and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry analysis. Using a publicly available expressed sequence tag database and the Mascot search engine, we were able to identify 16 differentially expressed proteins. More than 60% of the differentially expressed protein spots had very different patterns of gene expression between 2HA and Jemalong during the 8 weeks of culture.

Figure 1.

Explant tissue culturing in M. truncatula. A, Schematic diagram of the experimental design for the proteomic analysis of M. truncatula explant cultures grown on auxin and cytokinin. Samples used for proteomic analysis were collected at 2, 5, and 8 weeks. B, Calli from leaf explants of the highly regenerable seed line 2HA and its near isogenic line Jemalong. Line 2HA shows the formation of numerous embryos. Bar = 1 cm. C, Enlargement of 2HA culture showing green embryos. Bar = 5 mm.

Figure 2.

Two-dimensional gel electrophoresis proteome maps of M. truncatula explant cultures. Protein spots were assigned arbitrary identifiers as shown in Tables I and II. First-dimension focusing used 24-cm IPG strips with a linear pH gradient 4 to 7 loaded with 150 μg of total proteins for each strip. In the second dimension, 12–14%T SDS-PAGE gels were used. Proteins were visualized by silver staining. Two regions are enlarged in Figure 3.

Figure 3.

Enlargement of selected regions in Figure 2 to highlight some of the differentially expressed protein spots. Note the differentially expressed protein spot d148 is on the left panel, and spots d74, d139, d140, and d141 are on the right panel. These images were captured from the annotated gels in Melanie 4.