Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice (Oryza sativa L.)

Abstract

Autophagy is an intracellular degradation process for recycling macromolecules and organelles. It plays important roles in plant development and in response to nutritional demand, stress, and senescence. Organisms from yeast to plants contain many autophagy-associated genes (ATG). In this study, we found that a total of 33 ATG homologues exist in the rice [Oryza sativa L. (Os)] genome, which were classified into 13 ATG subfamilies. Six of them are alternatively spliced genes. Evolutional analysis showed that expansion of 10 OsATG homologues occurred via segmental duplication events and that the occurrence of these OsATG homologues within each subfamily was asynchronous. The Ka/Ks ratios suggested purifying selection for four duplicated OsATG homologues and positive selection for two. Calculating the dates of the duplication events indicated that all duplication events might have occurred after the origin of the grasses, from 21.43 to 66.77 million years ago. Semi-quantitative RT-PCR analysis and mining the digital expression database of rice showed that all 33 OsATG homologues could be detected in at least one cell type of the various tissues under normal or stress growth conditions, but their expression was tightly regulated. The 10 duplicated genes showed expression divergence. The expression of most OsATG homologues was regulated by at least one treatment, including hormones, abiotic and biotic stresses, and nutrient limitation. The identification of OsATG homologues showing constitutive expression or responses to environmental stimuli provides new insights for in-depth characterization of selected genes of importance in rice.

Figure 1.

Localization of the rice autophagy-associated homologues on rice chromosomes. OsATG homologues classified into different subfamilies are shown in different colours. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Chromosome number is indicated at the bottom of each chromosome. The OsATG homologues present on duplicated chromosomal segments between two chromosomes are connected by lines.

Figure 2.

Phylogenetic relationships of nine ATG subfamilies among rice and other species. The unrooted tree was constructed using the ClustalX program based on the multiple sequence alignment of the ATG protein sequences by the NJ method. The number at each node represents the bootstrap value from 1000 replicates. Accession numbers of OsATG homologues are given in Supplementary Table S1, and gi numbers of ATG proteins from other species are given.

Figure 3.

Expression profiles of OsATG homologues in various rice organs at the booting stage and the seedling stage by semi-quantitative RT–PCR. Total RNAs were from different organs when the rice plants were grown under normal growth conditions. R, roots; C, culms; L, leaf blades; Yp, young panicles; Sh, leaf sheaths. The accession numbers of mRNAs on the right side indicate that the bands from RT–PCR were actually amplified by the primers, and the NA indicates that this gene has no mRNA record in GenBank.

Figure 4.

Semi-quantitative RT–PCR analysis of the relative expression levels of rice OsATG homologues under various hormone treatments. Three-week-old seedlings were irrigated with Hoagland's solution containing 5 μM gibberellic acid (GA₃), 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 μM KT, or 25 μM ABA for 24 h. Expression levels in treated seedlings were normalized to those of the water-treated seedlings, whose expression levels were defined as 1. A gene name with two or more mRNA accession numbers in the parentheses indicates that this set of primers could amplify two or more alternatively spliced mRNA forms; a gene name without an mRNA accession number indicates that this gene has no mRNA record in GenBank; the same applies to Figs 5 and 6.

Figure 5.

Relative expression levels of rice OsATG homologues under abiotic stresses. Three-week-old rice seedlings under normal growth conditions were the control. Expression levels in treated seedlings were normalized to those of the control seedlings, whose expression levels were defined as 1. For salt treatment, the seedlings were kept in a 250 mM NaCl solution for 4 h. For drought treatment, seedlings were dried for 4 h between folds of tissue paper at 28 ± 1°C. For cold treatment, the seedlings were kept at 4 ± 1°C for 4 h. For dark treatment, seedlings were kept in the dark for 48 h.

Figure 6.

Relative expression levels of OsATG homologues in response to ammonium (I) and nitrate (II) treatments. Ten-day-old seedlings were transferred to nitrogen-free medium (−NH₄⁺ or −NO₃⁻) containing modified MS salts for 7 days for nitrogen starvation, then part of these seedlings were transferred to MS medium containing NH₄⁺ or NO₃⁻ (+NH₄⁺ or +NO₃⁻) as the nitrogen source. Total RNAs were extracted from these treated seedlings for RT–PCR. Expression levels in treated seedlings were normalized to those of the control seedlings grown under normal conditions, whose expression levels were defined as 1.

Figure 6.

Relative expression levels of OsATG homologues in response to ammonium (I) and nitrate (II) treatments. Ten-day-old seedlings were transferred to nitrogen-free medium (−NH₄⁺ or −NO₃⁻) containing modified MS salts for 7 days for nitrogen starvation, then part of these seedlings were transferred to MS medium containing NH₄⁺ or NO₃⁻ (+NH₄⁺ or +NO₃⁻) as the nitrogen source. Total RNAs were extracted from these treated seedlings for RT–PCR. Expression levels in treated seedlings were normalized to those of the control seedlings grown under normal conditions, whose expression levels were defined as 1.