Root iTRAQ protein profile analysis of two Citrus species differing in aluminum-tolerance in response to long-term aluminum-toxicity

Abstract

Background: Limited information is available on aluminum (Al)-toxicity-responsive proteins in woody plant roots. Seedlings of 'Xuegan' (Citrus sinensis) and 'Sour pummelo' (Citrus grandis) were treated for 18 weeks with nutrient solution containing 0 (control) or 1.2 mM AlCl3 · 6H2O (+Al). Thereafter, we investigated Citrus root protein profiles using isobaric tags for relative and absolute quantification (iTRAQ). The aims of this work were to determine the molecular mechanisms of plants to deal with Al-toxicity and to identify differentially expressed proteins involved in Al-tolerance.

Results: C. sinensis was more tolerant to Al-toxicity than C. grandis. We isolated 347 differentially expressed proteins from + Al Citrus roots. Among these proteins, 202 (96) proteins only presented in C. sinensis (C. grandis), and 49 proteins were shared by the two species. Of the 49 overlapping proteins, 45 proteins were regulated in the same direction upon Al exposure in the both species. These proteins were classified into following categories: sulfur metabolism, stress and defense response, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, cell transport, biological regulation and signal transduction, cell wall and cytoskeleton metabolism, and jasmonic acid (JA) biosynthesis. The higher Al-tolerance of C. sinensis may be related to several factors, including: (a) activation of sulfur metabolism; (b) greatly improving the total ability of antioxidation and detoxification; (c) up-regulation of carbohydrate and energy metabolism; (d) enhancing cell transport; (e) decreased (increased) abundances of proteins involved in protein synthesis (proteiolysis); (f) keeping a better balance between protein phosphorylation and dephosphorylation; and (g) increasing JA biosynthesis.

Conclusions: Our results demonstrated that metabolic flexibility was more remarkable in C. sinenis than in C. grandis roots, thus improving the Al-tolerance of C. sinensis. This provided the most integrated view of the adaptive responses occurring in Al-toxicity roots.

Fig. 1

Effects of Al-toxicity on plant growth and root Al concentration in C. sinensis and C. grandis seedlings. a-c Whole plant, shoot and root dry weights (DWs). d Root Al concentration. Bar represents the mean ± SE (n = 10 for plant DW or 4 for Al concentration). Differences among four treatment combinations were analyzed by 2 (species) × 2 (Al levels) ANOVA. Different letters above the bars indicate a significant difference at P < 0.05

Fig. 2

Spectra, peptides and proteins identified from iTRAQ proteomics by searching against C. sinensis database (a), number of peptides that match to proteins using MASCOT (b), protein mass distribution (c), distribution of protein sequence coverage (d), and distribution of peptide length (e)

Fig. 3

Classification of Al-induced differentially expressed proteins in C. sinensis (a) and C. grandis (b) roots and venn diagram analysis of differentially expressed proteins in Citrus roots (c). Among these overlapping proteins, 45 (4) proteins were regulated in the same (opposite) direction upon Al exposure

Fig. 4

Al-induced changes in S metabolism in C. sinensis and C. grandis roots. Up-regulated proteins presented in + Al C. sinensis and C. grandis (only in C. sinensis) roots were labeled in red (blue). Abbreviations: APS, adenosine phosphosulphate; GSH, reduced glutathione; GSSG, oxidized glutathione

Fig. 5

Metabolic scheme of JA biosynthesis in Citrus sinensis roots. Up-regulated proteins in + Al roots were labeled in red. Abbreviations: LOX, lipoxygenase; 13-HPOT, (13S)-hydroperoxyoctadecatrienoic acid; AOS, allene oxide synthase; 12,13-EOT, 12,13-epoxyoctadecatrienoic acid; AOC, allene oxide cyclase; cis-(+)-OPDA; cis-(+)-12-oxophytodienoic acid; OPR, oxophytodienoic acid reductase; OPC-8, 3-oxo-2-(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid; ACX, acyl-CoA-oxidase

Fig. 6

Relative expression levels of genes from C. sinensis (a-j) and C. grandis (k-t) roots. Relative abundances of genes encoding ATP sulfurylase 1 (ATPS1, gi|281426908; a and k), probable glutathione peroxidase 4 (GPX 4, gi|75154467; b and l), oxalate oxidase 2 (OXO 2, gi|1171937; c and m), O-acetylserine (thiol)lyase (cysteine synthase), partial (gi|34099833; d and n), catalase (gi|378724814; CAT, e and o), blue copper protein (gi|21264375; f and p), glyoxylase I, partial (gi|301341860; g and q), glutathione transferase (GST), partial (gi|380863042; h and r), peroxidase (POD, gi|110007377; i and s) and hydroxyacylglutathione hydrolase (Glyoxylase II, gi|3913733; j and t) in Al-toxic and control roots revealed by qRT-PCR. Bar represents the mean ± SE (n = 3). Unpaired t-test was applied for comparision between means. Different letters above the bars indicate a significant difference at P < 0.05. All the values were expressed relative to the control roots

Fig. 7

A potential model for the adaptive responses of C. sinensis and C. grandis roots to Al-toxicity. CGR: C. grandis roots; CSR: C. sinensis roots; Gly: Glyoxalase; MISAP: Mitochondrial intermembrane space import and assembly protein; PEPC: Phosphoenolpyruvate carboxylase; PM ATPase: Plasma membrane H⁺-ATPase; PPIase: Peptidyl-prolyl cis-trans isomerase; PLAP: Plastid-lipid-associated protein; R-protein: Ribosomal protein; SS: Sucrose synthase; Trx m: Thioredoxin m; PTP: Phosphatidylglycerol/phosphatidylinositol transfer protein; VAP: Vesicle-associated protein