Analysis of potential strategies for cadmium stress tolerance revealed by transcriptome analysis of upland cotton

Abstract

In recent years, heavy metal pollution has become a more serious global problem, and all countries are actively engaged in finding methods to remediate heavy metal-contaminated soil. We conducted transcriptome sequencing of the roots of cotton grown under three different cadmium concentrations, and analysed the potential strategies for coping with cadmium stress. Through Gene Ontology analysis, we found that most of the genes differentially regulated under cadmium stress were associated with catalytic activity and binding action, especially metal iron binding, and specific metabolic and cellular processes. The genes responsive to cadmium stress were mainly related to membrane and response to stimulus. The KEGG pathways enriched differentially expressed genes were associated with secondary metabolite production, Starch and sucrose metabolism, flavonoid biosynthesis, phenylalanina metalism and biosynthesis, in order to improve the activity of antioxidant system, repair systems and transport system and reduction of cadmium toxicity. There are three main mechanisms by which cotton responds to cadmium stress: thickening of physical barriers, oxidation resistance and detoxification complexation. Meanwhile, identified a potential cotton-specific stress response pathway involving brassinolide, and ethylene signaling pathways. Further investigation is needed to define the specific molecular mechanisms underlying cotton tolerance to cadmium stress. In this study potential coping strategies of cotton root under cadmium stress were revealed. Our findings can guide the selection of cotton breeds that absorb high levels of cadmium.

Figure 1

RNA-sequencing results and differentially expressed genes. (A) The percentage of RNA-sequencing reads that mapped to the reference. (B) The number of significantly up-regulated (red) and down-regulated (blue) genes between cotton roots treated with three cadmium concentrations (H1, H2 and H3) and the untreated control (H0). (C) Venn diagram illustrating the overlap in transcripts between samples.

Figure 2

Gene ontology classification of cotton root genes differentially expressed under different Cd concentrations. (A) cellular component (B) biological process and (C) molecular function.

Figure 3

Impact of cadmium stress on phenylpropanoid biosynthesis (https://www.kegg.jp/ dbget-bin/www_bget?map00940)under different Cd concentrations.

Figure 4

The physical barrier strategy for cadmium tolerance in cotton. (A) A heat map illustrating the differential expression of genes related to the formation of a physical barrier under Cd stress. x axis is log₂^x value of relative expression level. (B) Lignin biosynthetic pathway genes differentially expressed under cadmium stress. (C) A schematic diagram of the obstruction of cadmium ion transport by the Casparian strip in cotton roots.

Figure 5

The oxidation resistance and complexation detoxification strategies for cadmium tolerance in cotton. (A) A heat map illustrating the differential expression of genes related to oxidation resistance and complexation detoxification under Cd stress. x axis is log₂^x value of relative expression level. (B) A diagram illustrating the functions of differentially expressed genes in oxidation resistance and complexation detoxification

Figure 6

The determination of Superoxide Dismutase (SOD) and thioredoxin peroxidase (TPx) enzyme activity under cadmium stress in cotton roots.

Figure 7

The influence of cadmium stress on cotton root development. (A) The root systems of 30 day old control and Cd stress (5 mg/L) cotton treated seedlings. x axis is log₂^x value of relative expression level. (B) Genes A heat map illustrating the differential expression of genes related to root development under cadmium stress. (C) Diagram illustrating the regulation of root growth under cadmium stress in cotton.

Figure 8

Verification of gene expression in untreated cotton seedlings (H0) and cotton seedlings treated with three different concentrations of cadmium (H1, H2 and H3) by real-time RT-PCR. (A) Metallothionein genes. (B) Facicllin-like arabinogalactan genes. (C) GA synthesis-related genes. (D) Laccase genes. (E) Copper binding protein genes. (F) Superoxide dismutase gene.