Mitochondrial aldehyde dehydrogenase activity is required for male fertility in maize

Abstract

Some plant cytoplasms express novel mitochondrial genes that cause male sterility. Nuclear genes that disrupt the accumulation of the corresponding mitochondrial gene products can restore fertility to such plants. The Texas (T) cytoplasm mitochondrial genome of maize expresses a novel protein, URF13, which is necessary for T cytoplasm-induced male sterility. Working in concert, functional alleles of two nuclear genes, rf1 and rf2, can restore fertility to T cytoplasm plants. Rf1 alleles, but not Rf2 alleles, reduce the accumulation of URF13. Hence, Rf2 differs from typical nuclear restorers in that it does not alter the accumulation of the mitochondrial protein necessary for T cytoplasm-induced male sterility. This study established that the rf2 gene encodes a soluble protein that accumulates in the mitochondrial matrix. Three independent lines of evidence establish that the RF2 protein is an aldehyde dehydrogenase (ALDH). The finding that T cytoplasm plants that are homozygous for the rf2-R213 allele are male sterile but accumulate normal amounts of RF2 protein that lacks normal mitochondrial (mt) ALDH activity provides strong evidence that rf2-encoded mtALDH activity is required to restore male fertility to T cytoplasm maize. Detailed genetic analyses have established that the rf2 gene also is required for anther development in normal cytoplasm maize. Hence, it appears that the rf2 gene was recruited recently to function as a nuclear restorer. ALDHs typically have very broad substrate specificities. Indeed, the RF2 protein is capable of oxidizing at least three aldehydes. Hence, the specific metabolic pathway(s) within which the rf2-encoded mtALDH acts remains to be discovered.

Figure 1.

Immunoblot Analysis of RF2 Protein Accumulation. (A) Different organs from mutant and wild-type maize plants. w, Rf2-Ky21/Rf2-Ky21; m, rf2-m8904/rf2-m8904. Both maize strains carried T cytoplasm and had been backcrossed to the inbred line Ky21 for three generations. The lower panel is a duplicated gel stained with Coomassie Blue and serves as a protein loading control. Ear, a young unpollinated ear ∼10 cm long; young tassel, premeiotic stage of microsporogenesis; older tassel, early microspore stage of microsporogenesis; mesocotyl and root, both from 7-day-old seedlings. (B) Seedlings of different cytoplasmic and nuclear backgrounds. Total protein extracts were prepared from 7-day-old seedlings homozygous for the indicated alleles in the indicated cytoplasmic background. Rf2-Ky21 and rf2-m8904 are in a Ky21 genetic background, and the rf2-R213 allele is carried by the inbred line R213 with the indicated cytoplasm. The lower panel is a duplicated gel stained with Coomassie blue and serves as a protein loading control.

Figure 2.

RF2 Protein Accumulates in Mitochondria. (A) Homogenate (Hg), purified chloroplastic (Ch), microsomal (Ms), cytosolic (Cy), and mitochondrial (Mt) fractions of Ky21 seedlings were subjected to SDS-PAGE and stained with Coomassie blue. (B) A duplicate gel was transferred to a nitrocellulose membrane and reacted with affinity-purified anti-RF2 antibodies. (C) Protein extracts from isolated mitochondria of the inbred line Ky21 were treated with Triton X-100 and papain as indicated, subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and reacted with affinity-purified anti-RF2 antibodies. (D) Purified mitochondria from Ky21 seedlings were subjected to sonication and centrifugation to isolate the soluble and membrane fractions and then subjected to immunoblot analysis. Mt, purified mitochondria; Inf, insoluble fraction; Sf, soluble fraction.

Figure 3.

Immunolocalization of the RF2 Protein. (A) Diagram of a cross-section of one locule of an anther (from Kiesselbach, 1980). The approximate position of the areas shown in (B) and (C) is indicated by the box. (B) Cross-section of a T cytoplasm anther of the inbred line Ky21 that is homozygous for the Rf2-Ky21 allele incubated with affinity-purified rabbit anti-RF2 antibodies, followed by gold-labeled goat anti-rabbit IgG antibodies and silver enhancement. (C) Cross-section of an anther from T cytoplasm rf2-m8904/rf2-m8904 treated as described for (B). E, epidermis; EL, endothecium; ML, middle layer; PM, microspore pollen mother cell; T, tapetum. .

Figure 4.

ALDH Assay of E. coli–Expressed Recombinant RF2. Crude extracts of E. coli expressing the RF2 protein were assayed for ALDH activity. RFU 460, relative fluorescence units at 460 nm.

Figure 5.

The rf2 Gene Can Complement an E. coli ald-Deficient Mutant. (A) Basal medium plus glucose. (B) Basal medium plus 1,2-propanediol. −1, JA111; −2, JA111(DE3); (+), JA111(DE3)pALD9; Vector, JA111 (DE3)pET-17b; RF2-1, JA111(DE3)pMAP11-1; RF2-2, JA111(DE3) pMAP11-2.

Figure 6.

Mitochondrial ALDH Assay. ALDH activity was measured in extracts from mitochondria purified from unpollinated ears produced by T cytoplasm (A) or N cytoplasm (B) plants or from etiolated seedlings (C) of the indicated genotypes. Results from one typical experiment are shown. Data from (A) and (B) are summarized in Table 2. RFU, relative fluorescence units.

Figure 7.

Structure of the RF2 Protein. This structure was predicted by SWISS-MODEL (Guex and Peitsch, 1997) using bovine mtALDH (Protein Data Bank number 1AG8) as a template. The stereo images were prepared using MOLMOL (Koradi et al., 1996). Residues Glu³¹⁷, Pro³²³, Cys³⁵¹, Asn⁴⁰⁰, and Ile⁵²² in the RF2 sequence are equivalent to amino acid residues Glu²⁶⁷, Pro²⁷³, Cys³⁰¹, Lys³⁵⁰, and Ser⁴⁷² in Protein Data Bank number 1AG8, respectively.

Figure 8.

Anther Arrest in rf2 Plants Carrying N or T Cytoplasm. (A) Normal anther development on a tassel branch from N cytoplasm Ky21. (B) to (E) Various degrees of anther arrest on tassel branches from different plants in a segregating population resulting from the cross (N) rf2-m9323/rf2-m9323 × rf2-m9323/Rf2-Ky21. Arrows identify arrested anthers. (F) Normal anther development on a tassel branch from a fully suppressed T cytoplasm plant homozygous for rf2-m9390. (G) to (I) Various degrees of anther arrest on tassel branches from partially suppressed T cytoplasm plants homozygous for rf2-m9437. (J) Completely sterile tassel branch from an unsuppressed T cytoplasm plant homozygous for rf2-m9437.

Figure 9.

Microscopic Observation of Anther Arrest. (A) Illustration of a normal maize spikelet. (B) A spikelet from an N cytoplasm Ky21 plant. (C) A spikelet from an N cytoplasm plant homozygous for rf2-m9323 and near-isogenic with Ky21. (D) and (E) Anthers from the lower florets of N cytoplasm plants homozygous for rf2-m8904. (F) and (G) Anthers from the lower florets of N cytoplasm plants homozygous for rf2-m9323.