Low pH effects on reactive oxygen species and methylglyoxal metabolisms in Citrus roots and leaves

Abstract

Background: Limited data are available on the responses of reactive oxygen species (ROS) and methylglyoxal (MG) metabolisms to low pH in roots and leaves. In China, quite a few of Citrus are cultivated in acidic soils (pH < 5.0). 'Xuegan' (Citrus sinensis) and 'Sour pummelo' (Citrus grandis) (C. sinensis were more tolerant to low pH than C. grandis) seedlings were irrigated daily with nutrient solution at a pH of 2.5, 3 or 5 for nine months. Thereafter, we examined low pH effects on growth, and superoxide anion production rate (SAP), malondialdehyde (MDA), MG, antioxidants, and enzymes related to ROS and MG detoxification in roots and leaves in order to (a) test the hypothesis that low pH affected ROS and MG metabolisms more in roots than those of leaves, and (b) understand the roles of ROS and MG metabolisms in Citrus low pH-tolerance and -toxicity.

Results: Compared with control, most of the physiological parameters related to ROS and MG metabolisms were greatly altered at pH 2.5, but almost unaffected at pH 3. In addition to decreased root growth, many fibrous roots became rotten and died at pH 2.5. pH 2.5-induced changes in SAP, the levels of MDA, MG and antioxidants, and the activities of most enzymes related to ROS and MG metabolisms were greater in roots than those of leaves. Impairment of root ascorbate metabolism was the most serious, especially in C. grandis roots. pH 2.5-induced increases in MDA and MG levels in roots and leaves, decreases in the ratios of ascorbate/(ascorbate+dehydroascorbate) in roots and leaves and of reduced glutathione/(reduced+oxidized glutathione) in roots were greater in C. grandis than those in C. sinensis.

Conclusions: Low pH affected MG and ROS metabolisms more in roots than those in leaves. The most seriously impaired ascorbate metabolism in roots was suggested to play a role in low pH-induced root death and growth inhibition. Low pH-treated C. sinensis roots and leaves had higher capacity to maintain a balance between ROS and MG production and their removal via detoxification systems than low pH-treated C. grandis ones, thus contribute to the higher acid-tolerance of C. sinensis.

Keywords: Ascorbate metabolism; Citrus; Low pH; Methylglyoxal; Reactive oxygen species.

Fig. 1

Effects of low pH on superoxide anion production rate (SAP; a, d), malondialdehyde (MDA; b, e) and methylglyoxal (MG; c, f) concentrations in Citrus grandis and Citrus sinensis roots (a-c) and leaves (d-f). Bar represent mean ± SE (n = 4 for superoxide anion production and MG or 8 for MDA). Different letters above the bars indicate a significant difference at P < 0.05

Fig. 2

Effects of low pH on ascorbate peroxidase (APX; a, h), monodehydroascorbare reductase (MDHAR; b, i), dehydroascorbate reductase (DHAR; c, j), glutathione reductase (GR; d, k), superoxide dismutase (SOD, e, l), catalase (CAT, f, m) and guaiacol peroxidase (GuPX, g, n) activities in Citrus grandis and Citrus sinensis leaves (a-g) and roots (h-n). Bar represent mean ± SE (n = 4 except for 8 for APX, MDHAR and DHAR). Different letters above the bars indicate a significant difference at P < 0.05

Fig. 3

Effects of low pH on ascorbate oxidase (AO, a, c) and phosphomannose isomerase (PMI, b, d) activities in Citrus grandis and Citrus sinensis leaves (a-b) and roots (c-d). Bar represent mean ± SE (n = 8). Different letters above the bars indicate a significant difference at P < 0.05

Fig. 4

Effects of low pH on ATP sulphurylase (ATPS; a, i), cysteine synthase (CS; b, j), glutathione S-transferase (GST; c, k), glutathione peroxidase (GlPX; d, l), adenosine 5′-phosphosulfate reductase (APR; e, m), sulfite reductase (SiR; f, n), γ-glutamylcysteine synthase (γGCS; g, o) and glutamine synthetase (GS; h, p) activities in Citrus grandis and Citrus sinensis leaves (a-h) and roots (i-p). Bar represent mean ± SE (n = 8 except for 4 for GST and GS). Different letters above the bars indicate a significant difference at P < 0.05. OAS: O-acetyl-l-serine

Fig. 5

Effects of low pH on glyoxalase I (Gly I; a, c) and Gly II (b, d) activities in Citrus grandis and Citrus sinensis leaves (a-b) and roots (c-d). Bar represent mean ± SE (n = 4). Different letters above the bars indicate a significant difference at P < 0.05. SLG: S-D-lactoylglutathione

Fig. 6

Effects of low pH on [ascorbate (ASC) + dehydroascorbate (DHA)] (a, i), ASC (b, j) and DHA (c, k) concentrations, ASC/(ASC + DHA) ratio (d, l), [reduced glutathione (GSH) + oxidized glutathione (GSSG)] (e, m), GSH (f, n) and GSSG (g, o) concentrations, and GSH/(GSH + GSSG) ratio (h, p) in Citrus grandis and Citrus sinensis leaves (a-h) and roots (i-p). Bar represent mean ± SE (n = 8). Different letters above the bars indicate a significant difference at P < 0.05

Fig. 7

Principal component analysis (PCA) loading plots of physiological parameters of Citrus grandis (a) and Citrus sinensis (b) seedlings exposed to different pH levels. TA: ascrobate (ASC) + dehydroascorbate (DHA); TG: reduced glutathione (GSH) + oxidized glutathione (GSSG)

Fig. 8

Principal component analysis (PCA) loading pots of physiological parameters of leaves (a) and roots (b) from Citrus grandis and Citrus sinensis seedlings exposed to different pH levels. TA: ascrobate (ASC) + dehydroascorbate (DHA); TG: reduced glutathione (GSH) + oxidized glutathione (GSSG)

Fig. 9

A diagram showing low pH effects on ROS and MG metabolisms in C. grandis (a) and C. sinensis (b) leaves and roots. In this Figure, we used italics for enzymes and plain format for metabolites. Data from Figs. 1-6 except for H₂O₂ production rate (HP) and electrolyte leakage (EL) from reference [14]. Values in green and blue (black and magenta) were the ratios of pH 3 and pH 2.5 to pH 5 in leaves (roots), respectively. An asterisk indicates a significant difference between pH 2.5 (pH 3) and pH 5 at P < 0.05. An enzyme or metabolite was considered increased or decreased when it had both a relative change of more or less, respectively, than 1 and a P-value of < 0.05. Metabolite concentrations and enzyme activities were determined on a whole tissue extract and not on a subcellular level. GCL: Glutamate-cysteine ligase; NADH-GOGAT: NADH-dependent glutamine-2-oxoglutarate aminotransferase; γGC: γ-glutamylcysteine; γGT: γ-glutamyltransferase; TA: Ascorbate (ASC) + dehydroascorbate (DHA); TG: Reduced GSH + GSSG