Phosphorus-mediated alleviation of aluminum toxicity revealed by the iTRAQ technique in Citrus grandis roots

Abstract

Citrus grandis seedlings were irrigated with nutrient solutions with four Al-P combinations [two Al levels (0 mM and 1.2 mM AlCl3·6H2O) × two P levels (0 μM and 200 μM KH2PO4)] for 18 weeks. Al dramatically inhibited the growth of C. grandis seedlings, as revealed by a decreased dry weight of roots and shoots. Elevating P level could ameliorate the Al-induced growth inhibition and organic acid (malate and citrate) secretion in C. grandis. Using a comparative proteomic approach revealed by the isobaric tags for relative and absolute quantification (iTRAQ) technique, 318 differentially abundant proteins (DAPs) were successfully identified and quantified in this study. The possible mechanisms underlying P-induced alleviation of Al toxicity in C. grandis were proposed. Furthermore, some DAPs, such as GLN phosphoribosyl pyrophosphate amidotransferase 2, ATP-dependent caseinolytic (Clp) protease/crotonase family protein, methionine-S-oxide reductase B2, ABC transporter I family member 17 and pyridoxal phosphate phosphatase, were reported for the first time to respond to Al stress in Citrus plants. Our study provides some proteomic details about the alleviative effects of P on Al toxicity in C. grandis, however, the exact function of the DAPs identified herein in response to Al tolerance in plants must be further investigated.

Fig 1

Effects of P-Al interaction on plant dry weight (DW; shoot, A; root, E), P content (root, B; leaf, C; stem, D) and Al content (root, F; leaf, G; stem, H) in C. grandis seedlings. Bars represent means ± SE (n = 5 for P and Al contents or 10 for plant DW). Differences among the four treatment combinations were analyzed by 2 (B levels) × 2 (Al levels) ANOVA. Different letters indicate a significant difference at p < 0.05.

Fig 2

Effects of the P-Al interaction on root H₂O₂ production (C) and the contents of starch (A), glucose (B) and TBARS (D) in C. grandis seedlings. Bars represent means ± SE (n = 4). Differences among the four treatment combinations were analyzed by 2 (P levels) × 2 (Al levels) ANOVA. Different letters indicate a significant difference at p < 0.05.

Fig 3. Lignin content of C. grandis roots under different P-Al treatments.

Bars represent means ± SE (n = 4). Differences among the four treatment combinations were analyzed by 2 (P levels) × 2 (Al levels) ANOVA. Different letters indicate a significant difference at p < 0.05.

Fig 4. Al-induced secretion of malate and citrate in C. grandis roots.

Malate (A) and citrate (B) secretion from excised roots was measured after 12 or 24 h treatment, respectively, in 0.5 mM CaCl₂ + 0.5 mM AlCl₃·6H₂O or 0.5 mM CaCl₂ solution, pH 4.1–4.2. Bars represent means ± SE (n = 4). Differences among the eight treatments were analyzed by 4 (pretreatments) × 2 (Al levels) ANOVA. Different letters indicate a significant difference at p < 0.05.

Fig 5

Contents of malate (A) and citrate (B) and activity of two enzymes (PEPC, C; GlPX, D) in C. grandis roots. Bars represent means ± SE (n = 4). Differences among the four treatment combinations were analyzed by 2 (P levels) × 2 (Al levels) ANOVA. Different letters indicate a significant difference at p < 0.05.

Fig 6. Principal component analysis (PCA) loading plot of the DAPs in roots under different P-Al treatments.

Three hundred eighteen DAPs from C. grandis roots were transformed for PCA analysis. The first two PCs explained 97.7% of the biological variation in response to P-Al treatments, with PC1 accounting for 92.4% and PC2 5.3%.

Fig 7. Relative expression levels of genes encoding 15 DAPs under P-Al treatments.

A: Ribosomal protein S11-beta (RPS11); B: ribosomal protein L18e/L15 superfamily protein (RPL18); C: UDP-glucose 6-dehydrogenase family protein (UDPGD); D: isocitrate dehydrogenase 1 (ICDH1); E: glyceraldehyde-3-phosphate dehydrogenase-like family protein (G3PDH); F: phosphoenolpyruvate carboxylase family protein (PEPC); G: beta-galactosidase 9 (GAL9); H: glycosyl hydrolase family 38 protein (GH38); I: starch branching enzyme 2.2 (SBE2.2); J: pre-mRNA-processing protein 40B (mRPP); K: tonoplast intrinsic protein 2;3 (TIP2); L: ABC transporter I family member 17(ABCT17); M: glutathione peroxidase 7 (GPX7); N: aluminum sensitive 3 (ALS3); O: lipoxygenase 2 (LOX2). Bars represent means ± SE (n = 5). Differences among the four treatment combinations were analyzed by 2 (P levels) × 2 (Al levels) ANOVA. Different letters indicate a significant difference at p < 0.05.