Root transcriptome reveals efficient cell signaling and energy conservation key to aluminum toxicity tolerance in acidic soil adapted rice genotype

Abstract

Aluminium (Al) toxicity is the single most important contributing factor constraining crop productivity in acidic soils. Hydroponics based screening of three rice genotypes, a tolerant (ARR09, AR), a susceptible (IR 1552, IR) and an acid soil adapted landrace (Theruvii, TH) revealed that AR accumulates less Al and shows minimum decrease in shoot and root biomass under Al toxicity conditions when compared with IR. Transcriptome data generated on roots (grown in presence or absence of Al) led to identification of ~1500 transcripts per genotype with percentage annotation ranging from 21.94% (AR) to 29.94% (TH). A total of 511, 804 and 912 DEGs were identified in genotypes AR, IR and TH, respectively. IR showed upregulation of transcripts involved in exergonic processes. AR appears to conserve energy by downregulating key genes of glycolysis pathway and maintaining transcript levels of key exergonic step enzymes under Al stress. The tolerance in AR appears to be as a result of novel mechanism as none of the reported Al toxicity genes or QTLs overlap with significant DEGs. Components of signal transduction and regulatory machinery like transcripts encoding zinc finger protein, calcieurin binding protein and cell wall associated transcripts are among the highly upregulated DEGs in AR, suggesting increased and better signal transduction in response to Al stress in tolerant rice. Sequencing of NRAT1 and glycine-rich protein A3 revealed distinct haplotype for indica type AR. The newly identified components of Al tolerance will help in designing molecular breeding tools to enhance rice productivity in acidic soils.

Figure 1

Morphological variation for 10 days old three rice genotypes grown in modified Magnavaca nutrient in presence or absence of Al³⁺ Variation in the root growth pattern (A), aluminium content in root (b) and shoot (C), 2% haematoxylin staining (D–F) of 10 days old rice genotypes grown under control (0 Al³⁺) and treatment (0.54 mM of Al³⁺) for five days (G) root and shoot biomass of treated plants expressed as respective percentage of plants grown under control condition. The encircled portions (D–F) represent the root portion where Al³⁺ gets accumulated Values are means (n = 3), and bars indicate ± standard errors. The significant values are indicated by *Names of the genotypes are given on the X-axis.

Figure 2

Differential gene expression under Al stress condition. (A) The total, unannotated and annotated transcripts identified for the three rice genotypes AR, IR and TH is shown in the bar graph. (B) Bar diagram showing the number of up- and down- regulated genes in response to Al toxicity in the roots of AR, IR and TH. The total number of genes differentially expressed under each condition is given on the top of each bar (C). The correlation of gene expression results obtained from qPCR analysis and RNA-seq for fifteen selected genes.

Figure 3

Top ten GO terms in each of the three categories, molecular function, cellular component and biological process for the three rice genotypes AR, IR and TH. The top GO terms for molecular function (blue), cellular component (green) and biological process (red) categorised for genotypes AR (A), IR (B) and TH (C).

Figure 4

GO enrichment in response to Al stress across three rice genotypes. Significantly, enriched GO categories in the up-regulated genes in tolerant genotype AR (a) and susceptible genotype IR (B) and TH (C) are shown. The genes were analyzed using BiNGO and the biological process terms showing significant enrichment are shown. Node size is proportional to the number of genes in each category and shades represent the significance level (yellow—no significant difference; red −>log₂ fold upregulated; green − <log₂ fold downregulated; scale, P = 005 to P < 00000005).

Figure 5

Heat map representation and K-means clustering of expression profiles of genes differentially expressed under Al stress conditions in AR (A), IR (B) and TH (C). The ion torrent RNA-seq data were re-analyzed, and the FPKM values were log₂ transformed and heat map generated using MeV v4.11 software. Clustering was performed on log₂ fold change for each gene under Al stress conditions when compared with control condition across the three genotypes. Genes exhibiting a similar pattern of expression under Al stress conditions cluster together. Red and green colours denote control (no Al³⁺) and treatment (0.54 mM Al³⁺), respectively. Bar at the bottom represents log₂ fold values.

Figure 6

Mapping statistics of DEGs against already reported (A) genes and (B) QTLs for Al toxicity tolerance in rice. Total number of reported genes mapped- Number; Upregulated DEGs- Up_Regulated, Downregulated DEGs; Down_Regulated; Upregulated DEGs mapped to a reported rice QTL- Up_QTL_mapped; Downregulated DEGs mapped to reported rice QTL- Down_QTL_mapped.

Figure 7

Single nucleotide polymorphism (SNP) and sequence alignment across NRAT 1 (A,C) and glycine-rich A3 (B,D) genes for five rice genotypes. Dark grey and white colours indicate reference and novel allele, respectively. Sequence alignment of NRAT 1 (C) and glycine-rich A3 for five rice genotypes where numbers 1, 2, 3, 4 and 5 indicate reference genotype, AR, AZ, TH and IR, respectively. The encircle portions indicate SNPs unique to AR.

Figure 8

Schematic representation of the probable mechanism of susceptibility, tolerance and adaptation based on Al root transcriptome data for genotypes IR, AR and TH, respectively ↑–upregulation and ↓– downregulation.