Transcriptomic analysis of poco1, a mitochondrial pentatricopeptide repeat protein mutant in Arabidopsis thaliana

Abstract

Background: Flowering is a crucial stage during plant development. Plants may respond to unfavorable conditions by accelerating reproductive processes like flowering. In a recent study, we showed that PRECOCIOUS1 (POCO1) is a mitochondrial pentatricopeptide repeat (PPR) protein involved in flowering time and abscisic acid (ABA) signaling in Arabidopsis thaliana. Here, we use RNA-seq data to investigate global gene expression alteration in the poco1 mutant.

Results: RNA-seq analysis was performed during different developmental stages for wild-type and poco1 plants. The most profound differences in gene expression were found when wild-type and poco1 plants of the same developmental stage were compared. Coverage analysis confirmed the T-DNA insertion in POCO1, which was concomitant with truncated transcripts. Many biological processes were found to be enriched. Several flowering-related genes such as FLOWERING LOCUS T (FT), which may be involved in the early-flowering phenotype of poco1, were differentially regulated. Numerous ABA-associated genes, including the core components of ABA signaling such as ABA receptors, protein phosphatases, protein kinases, and ABA-responsive element (ABRE) binding proteins (AREBs)/ABRE-binding factors (ABFs) as well as important genes for stomatal function, were mostly down-regulated in poco1. Drought and oxidative stress-related genes, including ABA-induced stress genes, were differentially regulated. RNA-seq analysis also uncovered differentially regulated genes encoding various classes of transcription factors and genes involved in cellular signaling. Furthermore, the expression of stress-associated nuclear genes encoding mitochondrial proteins (NGEMPs) was found to be altered in poco1. Redox-related genes were affected, suggesting that the redox state in poco1 might be altered.

Conclusion: The identification of various enriched biological processes indicates that complex regulatory mechanisms underlie poco1 development. Differentially regulated genes associated with flowering may contribute to the early-flowering phenotype of poco1. Our data suggest the involvement of POCO1 in the early ABA signaling process. The down-regulation of many ABA-related genes suggests an association of poco1 mutation with the ABA signaling deficiency. This condition further affects the expression of many stress-related, especially drought-associated genes in poco1, consistent with the drought sensitivity of poco1. poco1 mutation also affects the expression of genes associated with the cellular regulation, redox, and mitochondrial perturbation.

Keywords: A. thaliana; ABA signaling; Flowering time; Mitochondria; POCO1; PPR protein; RNA-seq.

Fig. 1

Experimental setup for RNA-seq and analysis of differentially expressed genes in three comparisons. Overview of the strategy for RNA-seq and analysis of differentially expressed genes. a RNAs were isolated at two time points. Stage 1: Wild-type plants without inflorescence stem and poco1 plants with inflorescence stem (pre-inflorescence-inflorescence). Stage 2: Wild-type plants with inflorescence stem and poco1 plants with flowers (inflorescence-flowering). Three RNA-seq comparisons were performed between wild-type and poco1. Comparisons 1 and 2 are referred to pre-inflorescence-inflorescence and inflorescence-flowering respectively. Comparison 3 is the comparison between wild-type and poco1 plants of the same developmental stage, in which both have inflorescence stem (inflorescence-inflorescence). b The number of up- and down-regulated differentially expressed genes between three comparisons. Differentially expressed genes were defined as those with a fold change either ≥ 2 or ≤ − 2 and an FDR < 0.05. The highest number of differentially expressed genes was observed for inflorescence-inflorescence. Venn diagrams showing unique or common differentially expressed genes in each gene of interest category as c Flowering-related genes, d ABA-related genes, and e Drought and oxidative stress-related genes. Venn diagrams were made by an online tool (http://bioinformatics.psb.ugent.be/webtools/Venn/)

Fig. 2

A summary of the GO enrichment analysis. A summary of the GO analysis in each comparison is represented. The adjusted p-values (Padj) are shown in negative log10 scale (capped at -log10 (Padj) ≤ 16). Biological processes terms in red and blue refer to the up- and down-regulated genes respectively. BP- Biological process

Fig. 3

Analysis of POCO1 transcript in wild-type and mutant. Visual representation of POCO1 reads in RNA samples of wild-type and poco1 mutants. The “Map reads to contigs” tool from the CLC Genomics Workbench 7.5.1 program was used. The total number of reads has been written on the right side for each genotype. poco1 has a T-DNA insertion at position + 318. Transcripts of poco1 show the truncated form and are highly abundant from the + 1 to + 318 position. A red star in poco1 mutants represents the gap in POCO1 sequence, which did not map to any transcript

Fig. 4

Affected genes in core components of ABA signaling and flowering in poco1 in a schematic representation. In Arabidopsis, multiple factors affect flowering time to adapt the unfavorable conditions. In poco1 mutants, the core ABA signaling genes (PYRs/PYLs/RCARs, PP2Cs, SnRK2s and AREBs/ABFs) are down-regulated, which may lead to an impaired ABA and stress response. Down-regulation of ABF3 and ABF4, FRL and up-regulation of GRP7 may have an inhibitory effect on FLC expression. Besides, down-regulation of TEM1 and up-regulation of GI may induce FT expression. Down-regulation of TEM1 also induces the gibberellic acid biosynthesis gene, GA3ox1. Down-regulation of ELF4-L1, ELF4-L2, NAC089 and PHYE and up-regulation of FLP1, PRE1 and FBH2 are consistent with the early-flowering phenotype. Red and blue genes indicate up- and down-regulated genes respectively. Arrows and dashed arrows are indicative of inducing and inhibiting effects respectively

Fig. 5

The possible regulatory network of the gene expression in response to the loss of function of POCO1 in mitochondria. Loss of function of POCO1 is sensed by mitochondria. Subsequently, a retrograde signaling cascade may be activated to launch the gene expression changes. Several transcription factors, which control stress-inducible gene expression are affected. Several processes may be under the influence of defected ABA signaling in poco1. Black lines indicate crosstalk and differential regulation. Dashed arrows are indicative of possible inhibiting effects