Natural antisense transcripts are significantly involved in regulation of drought stress in maize

Abstract

Natural antisense transcripts (NATs) are a prominent and complex class of regulatory RNAs. Using strand-specific RNA sequencing, we identified 1769 sense and antisense transcript pairs (NAT pairs) in two maize inbreds with different sensitivity to drought, as well as in two derivative recombination inbred lines (RILs). A significantly higher proportion of NATs relative to non-NATs are specifically expressed under water stress (WS). Surprisingly, expression of sense and antisense transcripts produced by NAT pairs is significantly correlated, particularly under WS. We found an unexpected large proportion of NATs with protein coding potential, as estimated by ribosome release scores. Small RNAs significantly accumulate within NAT pairs, with 21 nt smRNA particularly enriched in overlapping regions of these pairs of genes. The abundance of these smRNAs is significantly altered in the leafbladeless1 mutant, suggesting that these genes may be regulated by the tasiRNA pathway. Further, NATs are significantly hypomethylated and include fewer transposable element sequences relative to non-NAT genes. NAT gene regions also exhibit higher levels of H3K36me3, H3K9ac, and H3K4me3, but lower levels of H3K27me3, indicating that NAT gene pairs generally exhibit an open chromatin configuration. Finally, NAT pairs in 368 diverse maize inbreds and 19 segregating populations were specifically enriched for polymorphisms associated with drought tolerance. Taken together, the data highlight the potential impact of that small RNAs and histone modifications have in regulation of NAT expression, and the significance of NATs in response to WS.

Figure 1.

Response of natural antisense transcript (NAT) pairs to drought stress. (A) Differentially expressed (DE) NATs among the tested lines with different levels of drought sensitivity. The colored rings represent the numbers and percentages (%) of DE NATs between different samples. C1: Up- or down-regulated DE NATs in both the two drought tolerant lines and the two drought sensitive lines. C2: Up-regulated DE NATs in the two drought tolerant lines, but down-regulated in the two drought sensitive lines, or vice versa. C3: Up- or down-regulated DE NATs in one of the drought tolerant lines and also in one of the drought sensitive lines. C4: Up- or down-regulated DE NATs in one of the drought tolerant lines, but down- or up-regulated in one of the drought sensitive lines. C5: No significant expression changes. The Venn diagram of the DE NATs is in the center of the circle. P1: AC7643. P2: AC7729/TZSRW. R1: RIL208. R2: RIL64. The up- and down-regulated NATs are presented by red and blue colors, respectively. (B) Cumulative frequency distribution of gene expression fold change under water stress. Different colors indicate different groups of genes. Antisense transcript (n = 3948), sense transcript (n = 3884), housekeeping genes (n = 2328), lncRNAs of maize (n = 1550) and non-NATs in the maize transcriptome (n = 112 636) are depicted in dark pink, vivid red, dark blue, dark grey and dark cyan, respectively. The color code is displayed in left corner. (C) Standard deviation of fold change variation in gene expression. (D) Expression correlation of sense and antisense transcription in NAT pairs in response to water stress. Portrayed are the 516 NAT pair genes with at least a twofold expression increase or decrease under water stress. For each NAT pair, expression fold change under water stress (WS) and well water (WW) for the sense transcript was plotted against the antisense transcript. (E and F) Concordant and discordant NAT pairs with respect to response to drought stress, respectively.

Figure 2.

Expression and inheritance patterns of NAT pairs. (A) Density distribution for expression levels of sense and antisense transcripts, as well as WW-specific and WS-specific sense and antisense transcripts. Kinds of transcripts are as indicated. WW: well water; WS: water stress. (B) Cumulative frequency distribution of sense and antisense transcripts expression ratios. (C) Density distribution of Shannon entropy estimates of NATs, sense transcripts, housekeeping genes, lncRNAs and non-NATs, as well as antisense and sense transcripts whose response to drought was significant (sig.) or nonsignificant (ns). A color code is displayed in the top right corner and bottom right corner, respectively. (D–H) Two-dimensional density estimation of gene expression patterns in two recombinant inbred lines compared with two parental lines for housekeeping genes, NAT pair sense transcripts, non-NATs (non-housekeeping genes), NATs and lncRNAs, respectively. The x-axis and y-axis represent the gene expression-level deviation in recombination inbred lines relative to their parental lines, AC7643 and AC7729/TZSRW, respectively. (I) Distribution of expression-level deviations in recombination inbred lines relative to their parental lines, AC7643 and AC7729/TZSRW.

Figure 3.

Correlation of gene expression and translational efficiency. A scatterplot of gene translational efficiency (log scale, y-axis) and expression level (log scale, x-axis) for sense and antisense transcripts, housekeeping genes and lncRNAs. Along each axis, all points are summarized using an overlaid density plot. The color code is displayed in top right corner.

Figure 4.

(A-D) The degree of enrichment of smRNAs, DNA methylation, H3K36me3 and TEs. The average enrichment levels are plotted with respect to transcription start sites (TSS) and transcription termination sites (TTS). The figure legend is in the top right corner. On the x-axis, kilobytes from the start and end of transcription are shown. In the y-axis, the number of reads was averaged in a 100 bp sliding window moving in 10 bp increments.

Figure 5.

NAT pairs associated with drought-related traits in different populations. The average number of SNPs (per kb) associated with survival rate under water stress (WS) condition, kernel oil content and days to tassel under water well (WW) condition in maize association population for (A) NAT pair genes and non-NAT genes, (B) NAT pair genes that significantly responded to drought stress (NAT.sig), showed no response to drought stress (NAT.ns), only expressed under WS condition (NAT.WS.special) and only expressed under WW condition (NAT.WW.special). The average number of SNPs (per kb) associated with anthesis date, plant height and grain yield under WW in 19 bi-parental populations for (C) NAT pairs genes and non-NAT genes, (D) NAT pairs genes that significantly responded to drought stress (NAT.sig), showed no response to drought stress (NAT.ns), only expressed under WS condition (NAT.WS.special) and only expressed under WW condition (NAT.WW.special).