Noncoding RNAs responsive to nitric oxide and their protein-coding gene targets shed light on root hair formation in Arabidopsis thaliana



doi: 10.3389/fgene.2022.958641.


eCollection 2022.

Affiliations

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Camilla Alves Santos et al.


Front Genet.


.

Abstract

An overview of the total Arabidopsis thaliana transcriptome, described previously by our research group, pointed some noncoding RNA (ncRNA) as participants in the restoration of hair-root phenotype in A. thaliana rhd6 mutants, leading us to a deeper investigation. A transcriptional gene expression profiling of seedling roots was performed aiming to identify ncRNA responsive to nitric oxide (GSNO) and auxin (IAA), and their involvement in root hair formation in the rhd6 null mutant. We identified 3,631 ncRNAs, including new ones, in A. thaliana and differential expression (DE) analysis between the following: 1) GSNO-treated rhd6 vs. untreated rhd6, 2) IAA-treated rhd6 vs. untreated rhd6, 3) GSNO-treated rhd6 vs. IAA-treated rhd6, and 4) WS-2 vs. untreated rhd6 detected the greatest number of DE genes in GSNO-treated rhd6. We detected hundreds of in silico interactions among ncRNA and protein-coding genes (PCGs), highlighting MIR5658 and MIR171 precursors highly upregulated in GSNO-treated rhd6 and wild type, respectively. Those ncRNA interact with many DE PCGs involved in hormone signaling, cell wall development, transcription factors, and root hair formation, becoming candidate genes in cell wall modulation and restoration of root hair phenotype by GSNO treatment. Our data shed light on how GSNO modulates ncRNA and their PCG targets in A. thaliana root hair formation.


Keywords:

A. thaliana; auxin; cell wall modulation; lncRNA; miRNA; ncRNA–PCG interaction.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures


FIGURE 1



FIGURE 1

Differentially expressed gene (DEG) distribution of the four independent and paired comparisons performed. (A) Number of upregulated DEGs identified in the comparisons: GSNO-treated rhd6 vs. untreated rhd6, IAA-treated rhd6 vs. untreated rhd6, GSNO-treated rhd6 vs. IAA-treated rhd6, and WS-2 vs. untreated rhd6 (FDR < 0.05). (B) Noncoding RNA type distribution in each comparison. GSNO-treated rhd6 vs. IAA-treated rhd6 comparison is not shown due to the few DEGs found. (C1) Expression values detected for DEGs in IAA-treated rhd6 vs. untreated rhd6 and (C2) GSNO-treated rhd6 vs. untreated rhd6 comparisons. The x-axis shows the log2FC values, and positive values for upregulated and negative values for downregulated for IAA and GSNO treatments, respectively. The y-axis shows the DEGs names. (D) Left: union of DEGs identified downregulated in untreated rhd6 mutants, and upregulated in IAA and GSNO treatments, revealing genes activated by each treatment. Right: union of DEGs identified upregulated in untreated rhd6 mutants, and downregulated in IAA and GSNO treatments, revealing genes deactivated by each treatment.


FIGURE 2



FIGURE 2

Long noncoding RNA (lncRNA) distribution. (A) Number of long noncoding RNAs already known (with AT identifiers) along the five A. thaliana chromosomes. (B) Length of all lncRNA identified, including those with AT identifiers and novel ones (the isoforms were not considered in the count). (C) Distribution of lncRNA and their respective differentially expressed protein-coding gene (PCG) targets. The interacting lncRNA–DEmRNA pairs were located mostly in genic regions (99.8%) and distributed in the following subtypes: containing (lncRNA contains the mRNA partner), nested (lncRNA is contained in the mRNA partner), and overlapping (lncRNA partially overlaps PCG partner). (D) lncRNA–PCG interaction direction and location, being mostly in sense direction and located in exonic regions. The x-axis shows the number of interactions detected and the y-axis shows the interaction direction and location. The number of interactions detected as intronic are very low compared to exonic ones, reason why no yellow bars could be observed for intronic interactions in the figure.


FIGURE 3



FIGURE 3

Results from co-expression analysis between differentially expressed (DE) long noncoding RNAs (DElncRNA) and differentially expressed protein-coding genes (PCGs). (A) Co-expression network generated for DElncRNA and DE PCGs which present similar expression patterns for GSNO treatment. The DElncRNA are represented in yellow and the DE PCG in blue. The lines represent the connections (edges) among DElncRNA and DE PCGs pairs, which may be correlated (green) or anticorrelated (red). (B) Heatmap showing the top 10 most correlated and anticorrelated PCGs and how they vary their expression patterns in the same or opposite direction to DElncRNAs, which are highlighted in yellow. All DElncRNA in the figure are correlated with DE PCG, except for AT1G06777. (C) IAA treatment co-expression network. The DElncRNA are represented in green and the DE PCG are represented in orange. Green lines represent correlated gene pairs, while red lines are anticorrelated gene pairs. (D) Top 10 heatmap with the most correlated and anticorrelated PCGs, and DElncRNAs are highlighted in yellow. AT2G08695 and AT4G08035 DElncRNAs are anticorrelated, while DEmRNA AT3G17185 and AT2G08750 are correlated. In Figures (B,D), the respective color keys in the right side represent the z-scores.


FIGURE 4



FIGURE 4

Small RNA (sRNA) interaction with DElncRNAs and DE PCG. As sRNA, we consider sRNA (could not have their types identified by annotation), nucleolar RNA (snoRNA), and microRNA (miRNA). (A) Top six DElncRNA–sRNA pairs with the strongest interaction energy (negative values). Three of them are differentially expressed in GSNO-treated and WS-2 individuals, potentially acting in the WS-2 root hair formation WS-2 phenotype. The x-axis shows the interaction energy values, whereas the y-axis represents the DElncRNA genes and their differential expression condition. (B) sRNA and DE PCG interaction. Top three DE PCG with the greatest number of Gene Ontology (GO) biological process (BP) terms matches. The BPs are mostly related to nitrogen metabolism and cell wall organization and root development. The x-axis represents the BP matches number and the y-axis represents the most common BP terms identified.


FIGURE 5



FIGURE 5

Differentially expressed genes in GSNO-treated and WS-2 seedlings and their potential participation in the restoration of root hair phenotype in rhd6 mutants. (A) Intersection of differentially expressed genes (DEGs) detected in the comparison between GSNO-treated rhd6 vs. untreated rhd6 and WS-2 vs. rhd6 reveals 25 genes in common for GSNO-treated rhd6 and WS-2 seedlings, of which nine are downregulated and 16 are upregulated. (B) Gene set enrichment analysis (GSEA) performed for GSNO-treated rhd6/WS-2 vs. untreated rhd6 groups. Figure shows DE genes are enriched and contribute with similar expression patterns for the WS-2 root hair formation (RHF) phenotype. (C) GSEA performed for GSNO-treated/IAA-treated/WS-2 vs. rhd6 groups. Figure shows the top 50 genes contributing the most in GSNO- and IAA-treated for the recovery of RHF phenotype observed in WS-2.

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