Analysis of the molecular and biochemical mechanisms involved in the symbiotic relationship between Arbuscular mycorrhiza fungi and Manihot esculenta Crantz

Abstract

Introduction: Plants and arbuscular mycorrhizal fungi (AMF) mutualistic interactions are essential for sustainable agriculture production. Although it is shown that AMF inoculation improves cassava physiological performances and yield traits, the molecular mechanisms involved in AM symbiosis remain largely unknown. Herein, we integrated metabolomics and transcriptomics analyses of symbiotic (Ri) and asymbiotic (CK) cassava roots and explored AM-induced biochemical and transcriptional changes.

Results: Three weeks (3w) after AMF inoculations, proliferating fungal hyphae were observable, and plant height and root length were significantly increased. In total, we identified 1,016 metabolites, of which 25 were differentially accumulated (DAMs) at 3w. The most highly induced metabolites were 5-aminolevulinic acid, L-glutamic acid, and lysoPC 18:2. Transcriptome analysis identified 693 and 6,481 differentially expressed genes (DEGs) in the comparison between CK (3w) against Ri at 3w and 6w, respectively. Functional enrichment analyses of DAMs and DEGs unveiled transport, amino acids and sugar metabolisms, biosynthesis of secondary metabolites, plant hormone signal transduction, phenylpropanoid biosynthesis, and plant-pathogen interactions as the most differentially regulated pathways. Potential candidate genes, including nitrogen and phosphate transporters, transcription factors, phytohormone, sugar metabolism-related, and SYM (symbiosis) signaling pathway-related, were identified for future functional studies.

Discussion: Our results provide molecular insights into AM symbiosis and valuable resources for improving cassava production.

Keywords: arbuscular mycorrhiza; candidate gene; cassava; fungi; metabolome; symbiosis; transcriptome.

Figure 1

Microscopic observation and impacts of AM symbiosis on some physiological traits after three (3w) and six (6w) weeks of AMF inoculation. (A, B) Microscopic image of symbiotic cells at 3w of control (CK) and inoculated plants (Ri), respectively. (C, D) Microscopic images of symbiotic cells at 6w of control (CK) and inoculated plants (Ri), respectively. The green organelles in D and F indicate proliferating fungal hyphae. Pictures were taken at 10x, and scale bars indicate 50 µm. (E–J) Evaluation of some physiological traits, including plant height, stem thickness, root length, root dry weight, and leaf chlorophyll content, respectively. The data are presented as mean ± SD of three replicates. Comparisons were evaluated between CK and Ri at each time point using t-test at P < 0.05. * and ** indicate significant differences at P < 0.05 and P < 0.01, respectively. “ns” indicate not significantly different.

Figure 2

Principal component (A) and hierarchical clustering analyses (B) of asymbiotic (CK) and symbiotic (Ri) cassava roots based on their respective metabolite profiles. 3w and 6w indicate three and six weeks after AMF inoculation, respectively.

Figure 3

Differentially accumulated metabolites (DAMs) in pairwise comparison between groups and functional analysis. (A) Number of up- and down-regulated DAMs between groups. (B) Venn diagram showing the number of common DAMs shared by groups. (C) Heatmap of DAMs in between CK and Ri-3w. (D) Classification of DAMs between CK and Ri-6w. (E) KEGG annotation and enrichment result of DAMs between CK and Ri-3w.

Figure 4

Differentially expressed genes (DEGs) and functional analyses. (A) Number of up- and down-regulated DEGs in pairwise comparison between groups. (B) Venn diagram showing the number of common DEGs shared by groups. (C, D) GO and KEGG annotation and enrichment analyses of DEGs between CK and Ri-3w, respectively.

Figure 5

Transcription levels of DEGs related to nitrogen and phosphorus metabolisms. (A) Phosphorus and nitrogen transporters. (B) Other DEGs related to nitrogen metabolism. FPKM, Fragments Per Kilobase of transcript per Million mapped reads. PHT, inorganic phosphate transporter; AMT, ammonium transporter; NRT, high-affinity nitrate transporter; NPF, protein NRT1/PTR family-like. Genes’ annotation is presented in Tables S9A, B .

Figure 6

Transcription levels of DEGs related to sugar metabolism (A) and phenylpropanoid pathway (B). FPKM, Fragments Per Kilobase of transcript per Million mapped reads. Genes’ annotation is presented in Tables S9C, D .

Figure 7

Transcription levels of DEGs belonging to transcription factor (A) and phytohormones (B) families. ABA, abscisic acid; AUX, auxin; GA, gibberellic acid; SL, strigolactones. FPKM, Fragments Per Kilobase of transcript per Million mapped reads. Genes’ annotation is presented in Tables S9F, G .

Figure 8

qRT-PCR validation of the RNA-seq data. Twelve DEGs were randomly selected for the qRT-PCR analysis. (A) Expression patterns of selected genes by RNA-seq and qRT-PCR. (B) Linear regression analysis of RNA-seq and qRT-PCR data. Relative expression indicates FPKM values for RNA-seq and the quantified expression levels by the 2¯^ΔΔCT method for qRT-PCR.