Mutations in a plastid-localized elongation factor G alter early stages of plastid development in Arabidopsis thaliana

Abstract

Background: Proper development of plastids in embryo and seedling tissues is critical for plant development. During germination, plastids develop to perform many critical functions that are necessary to establish the seedling for further growth. A growing body of work has demonstrated that components of the plastid transcription and translation machinery must be present and functional to establish the organelle upon germination.

Results: We have identified Arabidopsis thaliana mutants in a gene that encodes a plastid-targeted elongation factor G (SCO1) that is essential for plastid development during embryogenesis since two T-DNA insertion mutations in the coding sequence (sco1-2 and sco1-3) result in an embryo-lethal phenotype. In addition, a point mutation allele (sco1-1) and an allele with a T-DNA insertion in the promoter (sco1-4) of SCO1 display conditional seedling-lethal phenotypes. Seedlings of these alleles exhibit cotyledon and hypocotyl albinism due to improper chloroplast development, and normally die shortly after germination. However, when germinated on media supplemented with sucrose, the mutant plants can produce photosynthetically-active green leaves from the apical meristem.

Conclusion: The developmental stage-specific phenotype of the conditional-lethal sco1 alleles reveals differences in chloroplast formation during seedling germination compared to chloroplast differentiation in cells derived from the shoot apical meristem. Our identification of embryo-lethal mutant alleles in the Arabidopsis elongation factor G indicates that SCO1 is essential for plant growth, consistent with its predicted role in chloroplast protein translation.

Figure 1

The SCO1 mutational map. SCO1 encodes for a predicted protein with a high degree of similarity to an EF-G containing a chloroplast localization signal (At1g62750). The locations of the 4 mutant alleles are indicated. The EMS-mutagenized sco1-1 allele represents a G to A base change within the GTP-binding domain of the gene, which converts a glycine at amino acid 132 to an arginine. The other alleles [sco1-2 (Salk_046154), sco1-3 (Salk_039084), and sco1-4 (Salk_025112)] were isolated from the Salk T-DNA insert collection.

Figure 2

Phenotypes of T-DNA insertion alleles. In 9-d-old siliques, wild-type Arabidopsis ovules were green (A), but in siliques from sco1-2 (B) and sco1-3 (C) heterozygotes, white ovules were found intermixed with normal green ovules, indicating that chloroplast development is disrupted during embryogenesis in these alleles. White ovules accounted for approximately 25% of the total observed (sco1-2, 88 white ovules out of 360; sco1-3, 36 white ovules in 166). The upstream T-DNA insertion in 5-d-old sco1-4 seedlings (D, right) resulted in significantly stunted growth and pale cotyledons when compared to wild-type seedlings of similar age (D, left).

Figure 3

Characterization of the sco1-1 mutant. A-J are from 5-d-old light-grown seedlings, while K and L are from 4-d-old dark-grown seedlings. Upon seedling germination in white-light, the cotyledons of sco1-1 (B) appear colorless compared to wild type (A), but leaves that emerge from the apical meristem are green like wild-type leaves. Chlorophyll autofluorescence and cotyledon cross sections show that sco1-1 cotyledon cells (D and F) are almost completely devoid of chloroplasts except in cells associated with the vasculature, while wild-type (C and E) show a normal complement of chloroplasts in cotyledon cells. The albinism phenotype of sco1-1 is not always complete and green cells can be found in some sco1-1 cotyledons, where they are typically located along the margins of the tissue (G). A cross section of a cotyledon (H) from such a variegated mutant shows cells with a normal complement of chloroplasts adjacent to cells devoid of chloroplasts. Ultrastructural analysis of chloroplasts in these 'sectored' sco1-1 cotyledon mesophyll cells (J) showed that they are similar to chloroplasts in wild-type cotyledons (I). Starch deposition in 4 d-old dark-grown wild-type (K) and sco1-1 hypocotyls (L) appears similar, indicating that amyloplast development is not severely affected in the sco1-1 mutant. Scale bars in I and J are 1 μm.

Figure 4

Expression of SCO1. RTPCR analysis of the EF-G transcript from wild-type, sco1-1, and sco1-4 seedlings demonstrates a lower abundance of transcript level in sco1-4. The TDNA insert in sco1-4 is located 14 base-pairs upstream of the ATG start site, directly affecting its transcription rate. The genetic lesion in sco1-1 does not appear to affect the level of SCO1 mRNA, as its level appears similar to that of wild-type. The level of the loading control UBQ transcript is similar between all three samples.

Figure 5

Development of sco1-1 embryos. Representative embryos were dissected from the middle of siliques on days 8 and 14 after anthesis. With respect to developmental rate and morphology, sco1-1 embryos were similar to wild type. The sco1-1 embryos were green from days 6 to 12, but appeared slightly paler compared to wild-type embryos of similar age. DAF = days old after fertilization.

Figure 6

Gene expression levels of the Arabidopsis nuclear-coded elongation factor Gs. The Arabidopsis genome encodes for a total of three elongation factor Gs, including, SCO1 and two predicted mitochondrial-targeted EF-Gs. Transcript levels of SCO1 are highest in cotyledon tissue, with reduced levels in adult leaves. At1g45332 and At2g45030 transcript levels are highly reduced compared to SCO1 and remain constant between cotyledon and adult tissues. If these two EF-Gs are dual-targeted, they may be able to aid or compensate for the impaired activity of SCO1 in sco1-1 and sco1-4.