Low pH-responsive proteins revealed by a 2-DE based MS approach and related physiological responses in Citrus leaves

Abstract

Background: Rare data are available on the molecular responses of higher plants to low pH. Seedlings of 'Sour pummelo' (Citrus grandis) and 'Xuegan' (Citrus sinensis) were treated daily with nutrient solution at a pH of 2.5, 3, or 6 (control) for nine months. Thereafter, we first used 2-dimensional electrophoresis (2-DE) to investigate low pH-responsive proteins in Citrus leaves. Meanwhile, we examined low pH-effects on leaf gas exchange, carbohydrates, ascorbate, dehydroascorbate and malondialdehyde. The objectives were to understand the adaptive mechanisms of Citrus to low pH and to identify the possible candidate proteins for low pH-tolerance.

Results: Our results demonstrated that Citrus were tolerant to low pH, with a slightly higher low pH-tolerance in the C. sinensis than in the C. grandis. Using 2-DE, we identified more pH 2.5-responsive proteins than pH 3-responsive proteins in leaves. This paper discussed mainly on the pH 2.5-responsive proteins. pH 2.5 decreased the abundances of proteins involved in ribulose bisphosphate carboxylase/oxygenase activation, Calvin cycle, carbon fixation, chlorophyll biosynthesis and electron transport, hence lowering chlorophyll level, electron transport rate and photosynthesis. The higher oxidative damage in the pH 2.5-treated C. grandis leaves might be due to a combination of factors including higher production of reactive oxygen species, more proteins decreased in abundance involved in antioxidation and detoxification, and lower ascorbate level. Protein and amino acid metabolisms were less affected in the C. sinensis leaves than those in the C. grandis leaves when exposed to pH 2.5. The abundances of proteins related to jasmonic acid biosynthesis and signal transduction were increased and decreased in the pH 2.5-treated C. sinensis and C. grandis leaves, respectively.

Conclusions: This is the first report on low pH-responsive proteins in higher plants. Thus, our results provide some novel information on low pH-toxicity and -tolerance in higher plants.

Keywords: 2-DE; Citrus grandis; Citrus sinensis; Leaves; Low pH; Proteomics.

Fig. 1

Leaf gas exchange in response to low pH. a CO₂ assimilation; b stomatal conductance; c intercellular CO₂ concentration. Bars represent means ± SE (n = 5). Different letters above the bars indicate a significant difference at P < 0.05

Fig. 2

Leaf concentrations of nonstructural carbohydrates in response to low pH. a glucose; b fructose; c sucrose; d total soluble sugars (the summation of glucose, fructose and sucrose); e starch; f total nonstructural carbohydrates (TNC; the summation of glucose, fructose, sucrose and starch). Bars represent means ± SE (n = 8). Different letters above the bars indicate a significant difference at P < 0.05

Fig. 3

Leaf ASC + DHA, ASC and DHA concentrations, and ASC/(ASC + DHA) ratio in response to low pH. Bars represent means ± SE (n = 6–8). Different letters above the bars indicate a significant difference at P < 0.05

Fig. 4

Leaf MDA concentration in response to low pH. Bars represent means ± SE (n = 7–8). Different letters above the bars indicate a significant difference at P < 0.05

Fig. 5

Representative 2-DE images of proteins extracted from C. grandis (a-c) and C. sinensis (d-f) leaves

Fig. 6

Classification of low pH-responsive proteins (a-d) and venn diagram analysis of low pH-responsive proteins (e-f). For e, 37 or 43 identified DAPs only presented in the pH 2.5-treated C. grandis or C. sinensis leaves, respectively, and only six identified DAPs with the same accession number were shared by the two. For f, 25 or 9 identified DAPs only presented in the pH 3-treated C. grandis or C. sinensis leaves, respectively, and only two identified DAPs with the same accession number were shared by the two

Fig. 7

qRT-PCR analysis of 26 low-pH responsive protein genes. Relative expression levels of genes encoding 26 low-pH responsive proteins identified in C. grandis (a-b) and C. sinensis (c-d) leaves using actin (a, c) and PRPF31 (b, d) as internal standards, and the correlation analysis of qRT-PCR results and 2-DE data (e-f). For a-d, bars represent means ± SE (n = 3). For the same genes, different letters above the bars indicate a significant difference at P < 0.05. For e and f, 2-DE data from Tables 2 and 3