Transcriptomic Analysis of Distal Parts of Roots Reveals Potentially Important Mechanisms Contributing to Limited Flooding Tolerance of Canola (Brassica napus) Plants

Abstract

Since most of the root metabolic activities as well as root elongation and the uptake of water and mineral nutrients take place in the distal parts of roots, we aimed to gain insight into the physiological and transcriptional changes induced by root hypoxia in the distal parts of roots in canola (Brassica napus) plants, which are relatively sensitive to flooding conditions. Plants were subject to three days of root hypoxia via lowering oxygen content in hydroponic medium, and various physiological and anatomical features were examined to characterize plant responses. Untargeted transcriptomic profiling approaches were also applied to investigate changes in gene expression that took place in the distal root tissues in response to hypoxia. Plants responded to three days of root hypoxia by reducing growth and gas exchange rates. These changes were accompanied by decreases in leaf water potential (Ψ_leaf) and root hydraulic conductivity (L_pr). Increased deposition of lignin and suberin was also observed in the root tissues of hypoxic plants. The transcriptomic data demonstrated that the effect of hypoxia on plant water relations involved downregulation of most BnPIPs in the root tissues with the exception of BnPIP1;3 and BnPIP2;7, which were upregulated. Since some members of the PIP1 subfamily of aquaporins are known to transport oxygen, the increase in BnPIP1;3 may represent an important hypoxia tolerance strategy in plants. The results also demonstrated substantial rearrangements of different signaling pathways and transcription factors (TFs), which resulted in alterations of genes involved in the regulation of L_pr, TCA (tricarboxylic acid) cycle-related enzymes, antioxidant enzymes, and cell wall modifications. An integration of these data enabled us to draft a comprehensive model of the molecular pathways involved in the responses of distal parts of roots in B. napus. The model highlights systematic transcriptomic reprogramming aimed at explaining the relative sensitivity of Brassica napus to root hypoxia.

Keywords: aquaporins; hypoxia; roots; transcription factors.

Figure 1

Total plant dry weight change of different hypoxia treatment durations (A), dry weight of different part of plants (B) and root length (C) of control plants and in plants subjected to hypoxia for three days. Means ± SE are shown (n = 6–8). Asterisks indicate a difference between aerated and hypoxia (p ≤ 0.05 denoted by *; t-test).

Figure 2

Net photosynthesis (P_n, (A)), stomatal conductance (g_s, (B_), and transpiration rate (E, (C)) in control plants and in plants subjected to hypoxia for three days. Means ± SE are shown (n = 6–8). Asterisks indicate statistically significant differences between aerated and hypoxia (p ≤ 0.05 denoted by *; t-test).

Figure 3

Leaf water potential (Ψ_leaf, (A)),root hydraulic conductivity (L_pr, (B)), L_pr in plants treated with AgNO₃ (% control) (C), and percentage of the apoplastic light green SF yellowish dye concentration of the concentration applied to roots (D) in aerated plants and in plants subjected to hypoxia for three days. Means ± SE are shown (n = 6–8). Asterisks indicate a difference between aerated and hypoxia (p ≤ 0.05 denoted by *; t-test).

Figure 4

Cross sections of distal root segments in control B. napus plants (A,C) and in plants treated for three days with root hypoxia (B,D). Lignin autofluorescence was visualized following UV irradiation (A,B). Suberin deposition stained with Sudan 7B was visualized with light microscopy (C,D).

Figure 5

Expression analysis of the differentially expressed genes (DEGs) in distal root tissues of three samples of aerated control plants (c1, c2, c3) and in three samples from plants subjected to three days of root hypoxia (hy1, hy2, hy3). (A) Hierarchical cluster analysis with different columns in the figure representing different samples, and different rows representing different genes. The colors from blue to yellow indicate gene expression from low to high respectively. (B) Bland–Altman (MA) plot. Each point in the MA plot represents a gene. The blue points represent downregulated genes, the red points represent upregulated genes, and the grey points represent unchanged genes. (C) Fragments per kilobase of transcript per million mapped reads (FPKM) boxplot. (D) Venn diagram representing the specific and common DEGs across the control vs. hypoxia.

Figure 6

Classification of B. napus hypoxia stress responsive genes for each KEGG category. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of DEGs in B. napus. The x-axis shows the number of genes. (B) Gene ontology (GO) analyses of the DEGs. The y-axis shows the names of the GO terms, and the x-axis presents the number of genes.

Figure 7

Validation of RNA-seq results through qRT-PCR analysis. (A,B) Fold change of differentially expressed genes through qPCR method. (C) Correlation analysis of differentially expressed genes between qPCR analysis and RNA-seq experiment. Different letters indicate statistically significant differences between different genes (Tukey’s test; p ≤ 0.05; one-way ANOVA).

Figure 8

The analysis of differentially expressed transcription factors (TFs) in 3 days of hypoxia-treated roots. (A) The pie chart presents the percentage of 15 different families’ categories of transcription factors. TFs less than 1% of the total are not marked. Differentially expressed TFs in drought stress. (B) The distribution of transcription factors in upregulated and downregulated TF families. (C) The heatmap transcript profiles of selected transcription factor genes related to hypoxia response in B. napus roots. Columns and rows in the heatmap represent samples and genes, respectively. Sample names are displayed below the heatmaps. The color bar is the scale for the expression levels of each gene.

Figure 9

A model of hypoxia-response mechanisms in B. napus. (A) Hypoxia stress in roots is rapidly recognized through ABA, ROS, and ethylene signaling pathway. The signaling pathways stimulate a transcriptional cascade. The physiological-responsive genes are associated with a range of metabolic activities including cell wall composition adjustments, AQP alterations, antioxidants production, and TCA related enzymes. (B) Hypoxia-induced genes involved in respiration processes. Genes were labeled using individual heatmaps. The color bar is the scale for the expression levels of each gene on the basis of FPKM (fragments per kilobase million) value.

Figure 10

A model based on transcriptomic analysis for the responses of distal root tissues in canola to three days of root hypoxia. Hypoxia stress triggers alterations of cytosolic Ca²⁺, ABA, ethylene, and reactive oxygen species (ROS). Hypoxia-responsive signaling substances activated expression alteration of TF regulators including bHLH, AP2-ERF, WRKY, and NAC. The AP2-ERF expression was linked to downregulation of the TCA cycle, which resulted in ATP depletion leading to the energy crisis and affecting gas exchange and protein phosphorylation. As aerobic respiration was inhibited under hypoxia stress, fermentation activated NIP2;1 and PIP1;2 and helped with the efflux of lactic acid and CO₂ respectively. The NAC TFs were associated with generation of ROS, and ROS activated antioxidant systems involving SOD, APX, and POD. PIP1;3 was upregulated, possibly to enhance the oxygen influx into the cytoplasm. The PIP2;7 was upregulated to keep water homeostasis. Decreased pH and phosphorylation led to closure of water-transporting aquaporins, including PIP2;2, and led to decreases in L_pr and, consequently, Ψ_leaf. Increased ROS were associated with the cell wall modifications of root distal segments, gas exchange, and water relations of hypoxic canola plants.