Genome-wide analysis of the rice PPR gene family and their expression profiles under different stress treatments

Abstract

Background: Pentatricopeptide-repeat proteins (PPRs) are characterized by tandem arrays of a degenerate 35-amino-acid (PPR motifs), which can bind RNA strands and participate in post-transcription. PPR proteins family is one of the largest families in land plants and play important roles in organelle RNA metabolism and plant development. However, the functions of PPR genes involved in biotic and abiotic stresses of rice (Oryza sativa L.) remain largely unknown.

Results: In the present study, a comprehensive genome-wide analysis of PPR genes was performed. A total of 491 PPR genes were found in the rice genome, of which 246 PPR genes belong to the P subfamily, and 245 genes belong to the PLS subfamily. Gene structure analysis showed that most PPR genes lack intron. Chromosomal location analysis indicated that PPR genes were widely distributed in all 12 rice chromosomes. Phylogenetic relationship analysis revealed the distinct difference between the P and PLS subfamilies. Many PPR proteins are predicted to target chloroplasts or mitochondria, and a PPR protein (LOC_Os10g34310) was verified to localize in mitochondria. Furthermore, three PPR genes (LOC_Os03g17634,LOC_Os07g40820,LOC_Os04g51350) were verified as corresponding miRNA targets. The expression pattern analysis showed that many PPR genes could be induced under biotic and abiotic stresses. Finally, seven PPR genes were confirmed with their expression patterns under salinity or drought stress.

Conclusions: We found 491 PPR genes in the rice genome, and our genes structure analysis and syntenic analysis indicated that PPR genes might be derived from amplification by retro-transposition. The expression pattern present here suggested that PPR proteins have crucial roles in response to different abiotic stresses in rice. Taken together, our study provides a comprehensive analysis of the PPR gene family and will facilitate further studies on their roles in rice growth and development.

Keywords: Expression patterns; Pentatricopeptide-repeat protein; Rice (Oryza sativa L.); Salinity or drought stress; miRNA.

Fig. 1

Numbers of PPR genes and their intron numbers in rice and other species. a PPR proteins in a rice:P subgroup (246), PLS subgroup (24), E subgroup (90) and DYW subgroup (131). b Numbers and subclasses of PPR genes in moss, rice, Arabidopsis, tomato and foxtail millet,. c PPR genes distribution on rice 12 chromosomes. Chromosome 1 contained the most PPR genes (14.6%) and chromosome 9 had the fewest PPR genes (4%). d Number of introns in rice, Arabidopsis, tomato and foxtail millet

Fig. 2

Phylogenetic analysis and synteny analysis of the PPR genes in rice. a Phylogenetic tree of 491 PPR genes in rice. The blue represented the P subfamily and the red represented the PLS subfamily, while the green represented the mixture of PLS proteins and P subfamily genes. Multiple sequence alignment of full-length proteins was performed by ClustalX. The phylogenetic tree was constructed using MEGA7.0 with the neighbor-joining method and 1000 bootstrap replicates. Scale bar represents 0.2 amino acid substitutions per site. b Synteny analysis of PPR genes in rice. Chromosome 1 to 12 are shown with different colors and in a circular form. The different color curves represent the synteny relationship of PPR genes in the rice genome

Fig. 3

Subcellular localization of a PPR gene in rice. a The results of subcellular localization of each subgroup and species using TargetP and Predotar on-line tools. Plastid in purple, mitochondria in green, endoplasmic reticulum (ER) in red, and other locations in blue. b Confocal scanning microscopy showing the subcellular localization of a PPR (LOC_Os10g34310) protein in mitochondria. Cells were treated with MitoTracker Red to detect mitochondria. GFP signals of fusion proteins localized in the chloroplasts of rice protoplasts. Bar = 5 μm

Fig. 4

Validation and expression of selected miRNAs and their target PPR genes. Relative expression levels of three PPR genes as the targets of miRNAs in rice shoot, seeding, P3 and P4, (a, d) osa-miR1862d (a) and its target (LOC_Os03g17634)(d). (b, e) osa-miR396a-5p (b) and its target (LOC_Os07g40820) (e). (c, f) osa-miR444b.2 and its target (LOC_Os04g51350)

Fig. 5

qRT-PCT validation of nine PPR gene expression patterns. a Heatmap and relative expression levels of LOC_Os01g12810, LOC_Os03g19650 and LOC_Os04g49350. b Heatmap and relative expression levels of LOC_Os03g53490, LOC_Os04g14130 and LOC_Os05g28500. c Heatmap and relative expression levels of LOC_Os08g42610, LOC_Os12g01210 and LOC_Os12g44170

Fig. 6

Expression profiles of the PPR genes in response to different stresses. All heat maps were generated using MeV4.9 software with log2-transformed FPKM values. a Expression profiles of PPR genes in rice infected by rice stripe virus (RSV). Mock means control and dpi means days post inoculation. b Expression profiles of PPR genes in rice infected with bacterial blight disease. MDJ8 represents Japonica rice cultivar Mudanjiang8. c Expression profile of PPR genes in rice infected by rice blast disease. VN and GV represent a blast-tolerant cultivar and susceptible cultivar, respectively. d Expression profiles of PPR genes in rice under drought and salt stresses, respectively. N22 is a drought-tolerant cultivar. PK is a salinity-tolerant rice cultivar and IR64 is a susceptible cultivar. e Expression profiles of PPR genes in rice under phosphorus stress. +Pi, -Pi and Pire represent control, phosphate starvation and recovery, respectively. f Expression profiles of PPR genes in rice under cold stress. 9311 and DX (Dongxiang wile rice) are two rice varieties, and CK is the control condition of cold. g Expression profiles of PPR genes in rice under cadmium stress. Rice was treated with cadmium for 1 h and 24 h, and root and shoot were collected for this experiment

Fig. 7

Expression patterns of PPR genes under the salt and drought salt stresses. (a) Heatmap showing different expression profiles of PPR genes in drought-tolerant (Nagina 22, N22) and salinity-tolerant (Pokkali, PK) rice cultivars with IR64 (susceptible cultivar) under the salt stress (SS) and drought stress (DS). (b) Expression levels of 4 genes under salt condition (S) after 12 h, 24 h, 48 h and 72 h. (c) The expression level of 4 genes under drought condition (d)

Fig. 8

The promoter analysis in the seven PPR genes. Different cis-elements were indicated by different color symbols and placed in their relative position on the promoter. Symbols presented above the line indicate the elements at the forward strand, while those below indicate the reverse strand. The ABA-responsive element (ABRE), light response cis-acting element (ACE), light response module (AE-box), anaerobic induction element (ARE), auxin responsive element (AuxRR-core), fungal elicitor responsive element (BOX-W1), gibberellin-responsive element (GARE), heat shock element (HSE), low temperature responsive element (LTR), MYB-binding site (MBS), endosperm expression required element (Skn-1_motif), salicylic acid responsive element (TCA), Methyl jasmonate-responsive element (TGACG motif), defense and stress responsive element (TC-rich repeat), and element conferring high transcription level (5’ UTR Py-rich stretch) were analyzed