Genome-Wide Analysis of Light-Regulated Alternative Splicing in Artemisia annua L

Abstract

Artemisinin is currently the most effective ingredient in the treatment of malaria, which is thus of great significance to study the genetic regulation of Artemisia annua. Alternative splicing (AS) is a regulatory process that increases the complexity of transcriptome and proteome. The most common mechanism of alternative splicing (AS) in plant is intron retention (IR). However, little is known about whether the IR isoforms produced by light play roles in regulating biosynthetic pathways. In this work we would explore how the level of AS in A. annua responds to light regulation. We obtained a new dataset of AS by analyzing full-length transcripts using both Illumina- and single molecule real-time (SMRT)-based RNA-seq as well as analyzing AS on various tissues. A total of 5,854 IR isoforms were identified, with IR accounting for the highest proportion (48.48%), affirming that IR is the most common mechanism of AS. We found that the number of up-regulated IR isoforms (1534/1378, blue and red light, respectively) was more than twice that of down-regulated (636/682) after treatment of blue or red light. In the artemisinin biosynthetic pathway, 10 genes produced 16 differentially expressed IR isoforms. This work demonstrated that the differential expression of IR isoforms induced by light has the potential to regulate sesquiterpenoid biosynthesis. This study also provides high accuracy full-length transcripts, which can be a valuable genetic resource for further research of A. annua, including areas of development, breeding, and biosynthesis of active compounds.

Keywords: Artemisia annua; alternative splicing; artemisinin; intron retention; light-regulated; single molecule real-time (SMRT) sequencing.

FIGURE 1

Identification and classification of gene isoforms in Artemisia annua L. (A) The number and proportion of AS and non-AS gene. (B) The number distribution of alternatively spliced isoforms per gene. (C) Types of alternatively spliced isoforms: AA, alternative 3′ splice site; AD, alternative 5′ splice site; ES, exon skipping; IR, intron retention. (D) The number of isoforms produced by one or more splicing mechanisms. Venn diagram shows the combination of the four types of isoforms. (E) The number of new isoforms found in leaf, flower, stem and root. Venn diagram shows the common and specific isoforms in different tissues.

FIGURE 2

Expression of intron retention (IR) isoforms and non-alternatively spliced (non-AS) genes after light treatment. Isoforms with a TPM > 1 were defined as expressed ones. (A,B) Box-plots using log (TPM + 1) as the ordinate show TPM distribution of expressed IR isoforms and non-AS genes under five treatments, respectively. (C,D) the number of expressed IR isoforms and non-AS genes, respectively.

FIGURE 3

Analysis of differential expression IR isoform expression pattern after light treatment. (A,B) Number of up- and down-regulated IR isoforms and non-AS genes after treatment, respectively. (C) Proportion of differentially expressed IR isoforms over all IR isoforms vs. the corresponding profortion of non-AS genes. B, blue light; R, red light; FR, far-red light; WL, white light; D, dark.

FIGURE 4

Expression heatmap of the isoforms involved in artemisinin synthesis. The suffix “ref” indicates the reference isoform. For every gene, the reference isoform present in genome annotation is signed with the suffix “ref” or “rna” plus a series of number, while other isoforms newly identified are signed with pure number in its suffix. For example, CTI12_AA584370.ref61643 is the reference isoform of AACT while CTI12_AA584370.47 is a newly identified AS isoform. AACT, Acetyl-CoA C-acetyltransferase; HMGS, 3-Hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-Hydroxy-3-methylglutaryl-CoA reductase; MK, MVA kinase; PMK, Phospho-MVA kinase; MDC, Diphospho-MVA decarboxylase; DXS, 1-Deoxy-D-xylulose-5-phosphate synthase; DXR, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-(Cytidine-5-diphospho)-2-C-methyl-D-erythritol kinase; MDS, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase; HDS, 4-Hydroxy-3-methylbut-2-enyl-diphosphate synthase; HDR, 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase; GGPPS (E, E, E)-geranylgeranyl diphosphate synthase.