Transcript levels of orf288 are associated with the hau cytoplasmic male sterility system and altered nuclear gene expression in Brassica juncea

Abstract

Cytoplasmic male sterility (CMS) is primarily caused by chimeric genes located in the mitochondrial genomes. In Brassica juncea, orf288 has been identified as a CMS-associated gene in the hau CMS line; however, neither the specific abortive stage nor the molecular function of the gene have been determined. We therefore characterized the hau CMS line, and found that defective mitochondria affect the development of archesporial cells during the L2 stage, leading to male sterility. The expression level of the orf288 transcript was higher in the male-sterility line than in the fertility-restorer line, although no significant differences were apparent at the protein level. The toxicity region of ORF288 was found to be located near the N-terminus and repressed growth of Escherichia coli. However, transgenic expression of different portions of ORF288 indicated that the region that causes male sterility resides between amino acids 73 and 288, the expression of which in E. coli did not result in growth inhibition. Transcriptome analysis revealed a wide range of genes involved in anther development and mitochondrial function that were differentially expressed in the hau CMS line. This study provides new insights into the hau CMS mechanism by which orf288 affects the fertility of Brassica juncea.

Keywords: Anther development; Brassica; CMS; cytoplasmic male sterility; hau; orf288; plant mitochondria.

Fig. 1.

Anther phenotypes in the hau CMS lines and the maintainer line in B. juncea. (A, B) A flower from the hau CMS maintainer line (6-102B). (D, E) A flower from the hau CMS line (6-102A). Scale bars =20 mm. (C, F) A mature anther from the hau CMS maintainer line (C) and the hau CMS line (F). Scale bars =200 µm. (This figure is available in colour at JXB online.)

Fig. 2.

Anther development defects in the hau CMS line in B. juncea. The images are semi-thin sections from (A–E) the hau CMS maintainer line (6-102B) and (F–J) the hau CMS line (6-102A) showing anther development from stages 1–5. Abbreviations: Ar, archesporial cell; E, epidermis; En, endothecium; L1, L2, and L3, the three cell layers of the stamen primordia; L2-d and L3-d, the L2- and L3-derived cells; ML, middle layer; MMC, microspore mother cell; Sp, sporogenous cells; StR, stomium region; T, tapetum; V, vascular tissue; 1°P, primary parietal layer; 1°Sp, primary sporogenous layer; and 2°P, secondary parietal cell layers. Scale bar =25 µm. (This figure is available in colour at JXB online.)

Fig. 3.

TEM analysis of anthers from the hau CMS maintainer line (6-102B) and the hau CMS line (6-102A) at different stages. Anther development from stages 1–5 in 6-102B (A–E) and in 6-102A (F–J). The mitochondrial structures of the cells from (A–E) and (F–J) are shown in (K–O) and (P–T), respectively. Abbreviations: M, mitochondria; P, plastid; N, nucleus; and Va, vacuole. Scale bars: (D) 0.2 µm, (B, F, H, L, P, R) 0.5 µm, (C, E, G, J, N, Q, T) 1 µm, (A, K, M, O, S) 2 µm, and (I) 5 µm.

Fig. 4.

orf288 and atp6 expression patterns in the hau CMS lines. RNA gel-blotting analysis for (A) the orf288 and (B) the atp6 gene regions in the male-fertile, male-sterile, and nuclear-restorer floral buds: BjA, floral buds (<0.5 mm) from the hau CMS line (6-102A) in B. juncea; BjB, floral buds (<0.5 mm) from the hau CMS maintainer line (6-102B) in B. juncea; BnA, the male-sterile line in B. napus; and BnB, the maintainer line in B. napus. BnR1, BnR2 and BnR3 are different-sized floral buds (<0.5 mm; 0.5–2 mm; 2–4 mm) from the fertility-restorer line in B. napus.

Fig. 5.

Western blotting analysis of ORF288 between the hau CMS lines and the fertility-restorer lines. BjA, floral buds (<0.5 mm) from the hau CMS line in B. juncea; BnA, floral buds (<0.5 mm) from the male-sterile line in B. napus; BnB, floral buds (<0.5 mm) from the maintainer line in B. napus; and BnR, floral buds (<0.5 mm) from the fertility restorer line in B. napus.

Fig. 6.

The cytotoxic region of the CMS-associated gene orf288. (A) Truncated orf288 with different transmembrane domains: (a) full-length DNA sequence with three transmembrane domains; (b) truncated orf288 without the first transmembrane domain; (c) truncated orf288 without any transmembrane domains; (d) truncated orf288 that does not include 105-aa at the C-terminus; (e) truncated orf288 with only the third transmembrane domain; and (f) truncated orf288 with only part of the third transmembrane domain. (B) Effect of the overexpression of different truncated orf288 fragments on the growth of E. coli cells: (a–f) indicate the different truncated fragments shown in (A). IPTG was added when cell growth reached an OD₅₅₀ of 0.6. PET indicates the control expression vector induced by IPTG.

Fig. 7.

Fertility associated with truncated ORF288 expression in Arabidopsis. (A) Anthers from transgenic truncated orf288 fragments (a, c) and the wild-type (b, d). (B) The siliques from transgenic truncated orf288 fragments (e, f) and the wild-type (j). (C, D) qPCR and RT-PCR analysis of the expression levels of the truncated orf288 fragments in different tissues from transgenic plants. L1 and B1, leaves and floral buds from a transgenic male sterility T₃ plants; A2/3, anthers from two transgenic T₃ male sterility plants; NS2/3, floral buds without anthers from two transgenic T₃ male sterility plants; L4 and B4, leaves and floral buds from a transgenic semi-male sterility T₃ plants; A5/6, anthers from two transgenic T₃ semi-male sterility plants; and NS5/6, the floral buds without anthers from another two transgenic T₃ semi-male sterility plants. (This figure is available in colour at JXB online.)

Fig. 8.

qRT-PCR analysis of the expression profile of homologous genes involved in early anther development in B. juncea for the hau CMS line (6-102A) and its maintainer line (6-102B). Actin was used as an internal control to normalize the transcript levels.