Arabidopsis EMB1990 Encoding a Plastid-Targeted YlmG Protein Is Required for Chloroplast Biogenesis and Embryo Development

Abstract

In higher plants, embryo development originated from fertilized egg cell is the first step of the life cycle. The chloroplast participates in many essential metabolic pathways, and its function is highly associated with embryo development. However, the mechanisms and relevant genetic components by which the chloroplast functions in embryogenesis are largely uncharacterized. In this paper, we describe the Arabidopsis EMB1990 gene, encoding a plastid-targeted YlmG protein which is required for chloroplast biogenesis and embryo development. Loss of the EMB1990/YLMG1-1 resulted in albino seeds containing abortive embryos, and the morphological development of homozygous emb1990 embryos was disrupted after the globular stage. Our results showed that EMB1990/YLMG1-1 was expressed in the primordia and adaxial region of cotyledon during embryogenesis, and the encoded protein was targeted to the chloroplast. TEM observation of cellular ultrastructure showed that chloroplast biogenesis was impaired in emb1990 embryo cells. Expression of certain plastid genes was also affected in the loss-of-function mutants, including genes encoding core protein complex subunits located in the thylakoid membrane. Moreover, the tissue-specific genes of embryo development were misexpressed in emb1990 mutant, including genes known to delineate cell fate decisions in the SAM (shoot apical meristem), cotyledon and hypophysis. Taken together, we propose that the nuclear-encoded YLMG1-1 is targeted to the chloroplast and required for normal plastid gene expression. Hence, YLMG1-1 plays a critical role in Arabidopsis embryogenesis through participating in chloroplast biogenesis.

Keywords: Arabidopsis; YLMG; chloroplast; embryo development; plastid gene expression.

FIGURE 1

Characterization and complementation of Arabidopsis emb1990 mutants. (A) Schematic diagrams of EMB1990/YLMG1-1 gene. The positions of T-DNA insertions in emb1990-1 and emb1990-2 mutants are shown in the schematic diagrams. Arrowheads indicate the positions of primers used for genotyping. (B–F) Seed development in siliques of wild-type, mutants, and complemented plants. White arrows highlight the aborted white ovules, and the siliques were placed as morphological apical to basal from left to right. Bars = 1 mm.

FIGURE 2

Embryo development in emb1990-1 and emb1990-2 mutants. (A–F) Embryos from late globular stage to bent cotyledon stage in wild-type ovules. (G–L) emb1990-1 and (M–R) emb1990-2 embryos from siliques at different development stage as similar as wild-type embryos showed in (A–F). Scale bars = 20 μm.

FIGURE 3

Expression pattern of EMB1990/YLMG1-1 gene in Arabidopsis tissues and organs. (A) Expression levels of EMB1990/YLMG1-1genes in different tissues by qPCR assay. R, root; S, stem; L, leave; Sl, seedling; In, inflorescence; F, flower; 1Si, 1 DAP silique; 2Si, 2 DAP silique; 3Si, 3 DAP silique; 4Si, 4 DAP silique; 5Si, 5 DAP silique; 6Si, 6 DAP silique; 7Si, 7 DAP silique. (B–F) GUS activity in pYLMG1-1::GUS transgenic plants. (B) Flower; (C) inflorescence; (D) 7 DAG seedling; (E) 14 DAG seedling; (F) rosette leave. Scale bars = 2mm. (G–J) Fluorescence analysis of embryos at different stage from pYLMG1-1::YLMG1-1-Venus transgenic plants. (G) Globular stage; (H) Heart stage; (G) torpedo stage; (G) bent cotyledon stage. Scale bars = 20 μm.

FIGURE 4

The regulation of YLMG1-1 expression by dark and light. (A) Quantitative PCR analysis of YLMG1-1 expression in seedlings. CK-7 DAG, control check of 7 DAG seedlings; Dark, 24 h dark treatment; CK-8 DAG, control check of 8 DAG seedlings; Recovery, 24 h normal light cycle after 24 h dark treatment. The vertical axis shows the relative expression levels, and the asterisk indicates a significant difference (Student’s t-test,^∗P < 0.05, ^∗∗P < 0.01). (B–E) GUS staining signals in seedlings from pYLMG1-1::GUS transgenic plants. (B) 7 DAG seedling in normal light cycles; (C) 7 DAG seedling with 6 days normal light cycles and 24 h dark treatment; (D) 8 DAG seedling in normal light cycles; (E) 8 DAG seedling under normal light cycle after 24 h dark treatment.

FIGURE 5

Ultrastructure observation of chloroplast in wild-type and emb1990-1 embryo cells. (A,B) Cell and chloroplast in WT embryo at 5 DAP. (C,D) Cell and chloroplast in emb1990-1 embryos at 5 DAP. (B,D) Are enlarged images of the small black boxes in (A,C), respectively. N, nucleolus; Nu, nucleus; CW, cell wall; Gr, grana; St, stroma; Ve, vesicles. Scale bars = 1 μm.

FIGURE 6

Expression analysis of plastid genes involved in thylakoid formation between WT and emb1990-1 embryos by qRT-PCR. (A–L) Relative transcription level of plastid genes in WT and emb1990-1 embryos. The RNA was isolated from normal ovules of 5 DAP in wild-type and white ovules of 5 DAP in emb1990-1 plants. The vertical axis shows the relative expression levels, and the asterisk indicates a significant difference (Student’s t-test, ^∗P < 0.05, ^∗∗P < 0.01).

FIGURE 7

Expression analysis of nuclear genes known to delineate the embryo cell fate decisions between WT and emb1990-1 embryos by qRT-PCR. (A–T) Relative transcription level of nuclear genes in WT and emb1990-1 embryos. The RNA was isolated from normal ovules of 5 DAP in wild-type and white ovules of 5 DAP in emb1990-1 plants. The vertical axis shows the relative expression levels, and the asterisk indicates a significant difference (Student’s t-test, ^∗P < 0.05, ^∗∗P < 0.01).