Long noncoding RNAs in Brassica rapa L. following vernalization

Abstract

Brassica rapa L. is an important agricultural crop that requires a period of prolonged cold for flowering. This process is known as vernalization. Studies have shown that long noncoding RNAs (lncRNAs) play important roles in abiotic stress responses and several cold-responsive noncoding RNAs have been suggested to be involved in vernalization. We examined the transcriptome of the Chinese cabbage inbred line (B. rapa L. var. pekinensis) RJKB-T24, and identified 1,444 long intergenic noncoding RNAs (lincRNAs), 551 natural antisense transcripts (NATs), and 93 intronic noncoding RNAs (incRNAs); 549 of the 2,088 lncRNAs significantly altered their expression in response to four weeks of cold treatment. Most differentially expressed lncRNAs did not lead to a change of expression levels in mRNAs covering or near lncRNAs, suggesting that the transcriptional responses to four weeks of cold treatment in lncRNA and mRNA are independent. However, some differentially expressed mRNAs had NATs with expression altered in the same direction. These genes were categorized as having an abiotic stress response, suggesting that the paired-expression may play a role in the transcriptional response to vernalization or cold treatment. We also identified short-term cold treatment induced NATs in BrFLC and BrMAF genes, which are involved in vernalization. The lncRNAs we identified differed from those reported in Arabidopsis thaliana, suggesting the role of lncRNAs in vernalization differ between these two species.

Figure 1

Schematic representation of the classification strategy used in this study to identify lncRNAs from RNA-seq data.

Figure 2

Boxplot for the expression of lincRNA, NAT, incRNA, putative mRNA, and mRNA classified transcripts prior to cold treatment (NV) and after 4 weeks of cold treatment (V). Plus (+) symbol is the mean log2 (FPKM) expression for the respective category.

Figure 3

LncRNA expression levels after cold treatment measured by qPCR. Values are means ± s.e. (three biological and technical replicates) of relative expression levels compared with Bractin. NV, without cold treatment; 2wkV, after two weeks of cold treatment; 4wkV, four weeks of cold treatment; 6wkV, six weeks of cold treatment; 6wkV + 7 days, seven days after a return to normal growth conditions after six weeks of cold treatment.

Figure 4

Box plot representing the change of expression levels of lncRNAs and their paired mRNAs by four weeks of cold treatment. In the case of lincRNAs, the genes within 2 kb from lincRNAs were used for comparison. V, with four weeks of cold treatment; NV, without cold treatment.

Figure 5

The relationship of transcriptional changes by four weeks of cold treatment between mRNA and paired incRNAs or NATs. Plot of log2 score of fold change of FPKM in V (four weeks of cold treatment) divided by that in NV (before cold treatment) in NAT and mRNA pairs expression, showing the four possible expression patterns as illustrated by the Venn diagrams on the left. The lower two Venn diagrams show the observed expression patterns for mRNAs containing incRNAs. mRNAs showing shared up- or down-regulation with their respective NAT transcript are shown as triangles. mRNAs showing opposing up- or down-regulation in comparison to their respective NAT transcript are shown as circles.

Figure 6

Strand-specific read coverage plots for BrFLC2, BrFLC1, Bra024350, and Bra024351. Above each coverage plot, the mRNA transcripts are shown in black and the antisense transcripts (NATs) are shown in gray, with exons represented as thick bars and an arrow showing the 5′ to 3′ sense, and thin lines illustrating the introns. For BrFLC1, three splice isoforms are shown relative to the reference genome annotation, illustrating a conservation of mRNA structure for exons 1 through 5, with differential splicing occurring at exons 6 and 7.

Figure 7

Expression levels of three NATs before and after cold treatment. (A) RNA-seq expression (FPKM) for three NATs before (NV) and after (V) four weeks of cold treatment. Differential expression analysis showed that none of the three NATs were differentially expressed at the FDR <0.05 level. (B) qPCR results showing expression ratios normalized to NV for three NATs prior to cold treatment (NV), three, seven, and twelve days into cold treatments (3dV, 7dV, and 12dV, respectively). BrFLC2as (MSTRG.2765), MAF1 homolog Bra024350as (MSTRG.14523), MAF4 homolog Bra024351as (MSTRG.14524).