Analysis of the rice mitochondrial carrier family reveals anaerobic accumulation of a basic amino acid carrier involved in arginine metabolism during seed germination

Abstract

Given the substantial changes in mitochondrial gene expression, the mitochondrial proteome, and respiratory function during rice (Oryza sativa) germination under anaerobic and aerobic conditions, we have attempted to identify changes in mitochondrial membrane transport capacity during these processes. We have assembled a preliminary rice mitochondrial carrier gene family of 50 members, defined its orthology to carriers of known function, and observed significant changes in microarray expression data for these rice genes during germination under aerobic and anaerobic conditions and across rice development. To determine if these transcript changes reflect alteration of the carrier profile itself and to determine which members of the family encode the major mitochondrial carrier proteins, we analyzed mitochondrial integral membrane protein preparations using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass spectrometry, identifying seven distinct carrier proteins. We have used mass spectrometry-based quantitative approaches to compare the abundance of these carriers between mitochondria from dry seeds and those from aerobic- or anaerobic-germinated seeds. We highlight an anaerobic-enhanced basic amino acid carrier and show concomitant increases in mitochondrial arginase and the abundance of arginine and ornithine in anaerobic-germinated seeds, consistent with an anaerobic role of this mitochondria carrier. The potential role of this carrier in facilitating mitochondrial involvement in arginine metabolism and the plant urea cycle during the growth of rice coleoptiles and early seed nitrate assimilation under anaerobic conditions are discussed.

Figure 1.

Phylogenetic unrooted tree of rice and the Arabidopsis MC family proteins. In total, 58 Arabidopsis (Atxgxxxxx) and 50 rice (Osxxgxxxxx) protein sequences were used in the analysis. Asterisks indicate greater than 70% branch confidence (10,000 reiterations of bootstrapping). Naming of carrier function is based on the Arabidopsis carriers in each case. Cpt, Chloroplastic; Mt, mitochondrial; Prx, peroxisomal.

Figure 2.

Hierarchical clustering of transcripts for genes encoding MC proteins from rice. The Affymetrix rice genome microarray data were derived from the Gene Expression Omnibus within the National Center of Biotechnology Information database. The normalized data were hierarchically clustered using average linkage based on Euclidean distance. Genes annotated with carets indicate that these genes were absent in dry seeds, during early germination, and in more than four other tissues/stresses. A, Air; N, nitrogen.

Figure 3.

Respiratory function of mitochondria during rice embryo germination. Whole embryo and mitochondrial respiration rates were obtained from dry seeds, 48-h-imbibed anaerobic-treated seeds, and 48-h-imbibed aerobic-treated seeds. Data presented are averages ± se (n = 3). FW, Fresh weight.

Figure 4.

Isolation of integral membrane proteins from rice mitochondria. A, SDS-PAGE analysis of whole mitochondria (Mt), carbonated extracted soluble fraction (Sol), peripheral membrane proteins (PM), and integral membrane proteins (IM) stained with colloidal Coomassie Brilliant Blue G250. B, Western blot of a matrix protein (E1α), peripheral membrane protein (F₁α), and integral membrane protein (UCP). Numbers on the left indicate apparent molecular mass.

Figure 5.

Integral membrane proteins from mitochondria isolated from rice embryos dissected from dry seeds and germinated under aerobic and anaerobic conditions. Representative SDS-PAGE gels are shown from carbonate-extracted mitochondria isolated from embryos of dry seeds, 48-h-imbibed anaerobic-treated seeds (48 N), and 48-h-imbibed aerobic-treated seeds (48A) revealing the integral membrane proteome. Numbers on the left indicate apparent molecular mass, and alphanumerics on the right indicate the protein bands extracted for analysis by MS (Table I).

Figure 6.

The relative abundance of members of the MC family in rice. Samples were prepared from carbonate-extracted mitochondrial membranes isolated from embryos of dry seeds (white bars), 48-h-imbibed anaerobic-treated seeds (gray bars), and 48-h-imbibed aerobic-treated seeds (black bars). A, Average spectral counts for each OsMC member. B, Analysis of spectral counts by Poisson regression analysis displayed as relative Poisson coefficients ± se (n = 3). The statistical analysis results can be seen in Supplemental Table S1. * P < 0.05.

Figure 7.

Changes in BAC, arginase, Arg, and Orn levels from dry, anoxically imbibed, and aerobically imbibed embryos. A, The relative abundance of BAC1 (Os10g42299) assessed by QqQ SRM MS. Mitochondria isolated from embryos of dry seeds (white bar), 48-h-imbibed anaerobic-treated seeds (gray bar), and 48-h-imbibed aerobic-treated seeds (dark gray bar) were carbonate extracted. The abundance from SRM transitions (862.90/769.39, 862.90/1,113.59, 862.90/1,184.59, 849.40/1,031.59, 849.40/735.40, 849.40/1,130.59) was then obtained. These abundance changes were then combined for each treatment triplicate, and the results are shown in relative abundance ± se (n = 3; * P < 0.05). B, The relative abundance of arginase assessed by western blot. Mitochondrial proteins isolated from embryos of dry seeds (D), 48-h-imbibed anaerobic-treated seeds (N), and 48-h-imbibed aerobic-treated seeds (A) were separated by SDS-PAGE. Numbers below represent relative band intensity. C and D, Ratio of metabolite abundance in embryos relative to dry seed embryos. Dry seeds (white bars), 48-h-imbibed anaerobic-treated seeds (gray bars), and 48-h-imbibed aerobic-treated seeds (dark gray bars) were used for Arg (C) and Orn (D). * P < 0.05.

Figure 8.

Quantitative transcript analysis of PiC and BAC in rice embryos. Gene-specific quantitative RT-PCR was used to determine transcript levels over the first 48 h of germination under aerobic (black circles) and anaerobic (gray circles) conditions. Data are given relative to total RNA, with average values ± se shown (n = 3).

Figure 9.

The BAC and the Orn-urea cycle in animals (A) and plants (B). ARG-Mn²⁺, Arginase; ArgSuc, arginosuccinate; ASL, arginosuccinate lyase; ASS, arginosuccinate synthase; CarbP, carbamoyl phosphate; CPS, carbamoyl phosphate synthase; FUM, fumarate; OCT, Orn carbamoyltransferase; ORC1/2, human mitochondrial Orn carrier; OsmBAC1/2, rice mitochondrial BAC carrier. Question marks indicate currently unknown carriers for Arg and Orn in plastids.