Proteomic analysis of Citrus sinensis roots and leaves in response to long-term magnesium-deficiency

Abstract

Background: Magnesium (Mg)-deficiency is frequently observed in Citrus plantations and is responsible for the loss of productivity and poor fruit quality. Knowledge on the effects of Mg-deficiency on upstream targets is scarce. Seedlings of 'Xuegan' [Citrus sinensis (L.) Osbeck] were irrigated with Mg-deficient (0 mM MgSO4) or Mg-sufficient (1 mM MgSO4) nutrient solution for 16 weeks. Thereafter, we first investigated the proteomic responses of C. sinensis roots and leaves to Mg-deficiency using two-dimensional electrophoresis (2-DE) in order to (a) enrich our understanding of the molecular mechanisms of plants to deal with Mg-deficiency and (b) understand the molecular mechanisms by which Mg-deficiency lead to a decrease in photosynthesis.

Results: Fifty-nine upregulated and 31 downregulated protein spots were isolated in Mg-deficient leaves, while only 19 upregulated and 12 downregulated protein spots in Mg-deficient roots. Many Mg-deficiency-responsive proteins were involved in carbohydrate and energy metabolism, followed by protein metabolism, stress responses, nucleic acid metabolism, cell wall and cytoskeleton metabolism, lipid metabolism and cell transport. The larger changes in leaf proteome versus root one in response to Mg-deficiency was further supported by our observation that total soluble protein concentration was decreased by Mg-deficiency in leaves, but unaffected in roots. Mg-deficiency had decreased levels of proteins [i.e. ribulose-1,5-bisphosphate carboxylase (Rubisco), rubisco activase, oxygen evolving enhancer protein 1, photosynthetic electron transfer-like protein, ferredoxin-NADP reductase (FNR), aldolase] involved in photosynthesis, thus decreasing leaf photosynthesis. To cope with Mg-deficiency, C. sinensis leaves and roots might respond adaptively to Mg-deficiency through: improving leaf respiration and lowering root respiration, but increasing (decreasing) the levels of proteins related to ATP synthase in roots (leaves); enhancing the levels of proteins involved in reactive oxygen species (ROS) scavenging and other stress-responsive proteins; accelerating proteolytic cleavage of proteins by proteases, protein transport and amino acid metabolism; and upregulating the levels of proteins involved in cell wall and cytoskeleton metabolism.

Conclusions: Our results demonstrated that proteomics were more affected by long-term Mg-deficiency in leaves than in roots, and that the adaptive responses differed between roots and leaves when exposed to long-term Mg-deficiency. Mg-deficiency decreased the levels of many proteins involved in photosynthesis, thus decreasing leaf photosynthesis.

Figure 1

Effects of Mg-deficiency on growth and Mg concentration in leaves, stems and roots. (A-C) Leaf, stem and root DW. (D-F) Leaf, stem and root Mg concentration. Bars represent means ± SE (n = 10 except for 9 for leaf, stem and root DW of Mg-deficient seedlings). Different letters above the bars indicate a significant difference at P < 0.05.

Figure 2

Effects of Mg-deficiency on leaf gas exchange, root respiration, and root and leaf total soluble protein. (A-E) Leaf CO₂ assimilation, stomatal conductance, intercellular CO₂ concentration, transpiration and respiration. (F) Leaf total soluble protein. (G) Root respiration. (H) Root total soluble protein. Bars represent means ± SE (n = 5 except for 8 for leaf CO₂ assimilation, stomatal conductance, intercellular CO₂ concentration and transpiration of control and Mg-deficient seedlings, respectively). Different letters above the bars indicate a significant difference at P < 0.05.

Figure 3

Representative gel images of proteins in Mg-deficient (A) and control (B) leaves. Proteins were separated in the first dimension on an IPG strip pH 3–7 and in the second dimension on a 12% slab gel, followed by colloidal Coomassie Brilliant G-250. An equal amount (1.5 mg) of total protein extracts was loaded in each gel.

Figure 4

Representative gel images of proteins in Mg-deficient (A) and control (B) roots. Proteins were separated in the first dimension on an IPG strip pH 3–7 and in the second dimension on a 12% slab gel, followed by colloidal Coomassie Brilliant G-250. An equal amount (1.5 mg) of total protein extracts was loaded in each gel.

Figure 5

Functional classification of the differentially expressed protein spots in leaves (A) and roots (B).

Figure 6

PCA loading plots of differentially expressed proteins in Mg-deficient leaves (A) and roots (B).

Figure 7

Pearson correlation coefficient matrix for the differentially expressed protein spots in Mg-deficient leaves (A) and roots (B). Red and blue colors indicated positive and negative correlation between the differentially expressed proteins.

Figure 8

Relative expression of 13 genes from leaves and of five genes from roots. (A) Relative expression of nine leaf genes encoding cysteine proteinase (L4); ATP synthase CF1 alpha subunit, chloroplastic (L33), triosephosphate isomerase (L44), Cu/Zn superoxide dismutase (L78), phosphoglycerate kinase (L80), ascorbate peroxidase (L96), S-adenosylmethionine synthetase (L102), aconitate hydratase 3 (L104), nucleoside diphosphate kinase, putative (L105). (B) Relative expression of four leaf genes encoding ATYKT62 (L110), glyceraldehyde-3-phosphate dehydrogenase B subunit (L114), alcohol dehydrogenase (L117), and ribulose 1,5-bisphosphate carboxylase, partial (L118), and five root genes encoding proteasome subunit alpha type, putative (R1), cysteine protease, putative (R13), pyruvate decarboxylase, putative (R85), phosphoglycerate kinase (R95), and hexokinase (R121). Bars represent means ± SE (n = 3). Different letters above the bars indicate a significant difference at P < 0.05. All the values were expressed relative to the control leaves or roots.

Figure 9

The potential regulatory network of Mg-deficiency-induced responses in Citrus leaves (A) and roots (B). BPG-PGAM: 2,3-Bisphosphoglycerate-independent phosphoglycerate mutase 1; CVP2: Type I inositol-1,4,5-trisphosphate 5-phosphatase CVP2; DBA- RNA helicase: Dead box ATP-dependent RNA helicase; DLST: Dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase; DRP: Disease resistance protein; Fru: Fructose; Glu: Glucose; Gs: Stomatal conductance; LOS2: 2-Phospho-D-glycerate hydrolase; NADP-MDH: NADP-malate dehydrogenase; PCOGRP: Pollen coat oleosin-glycine rich protein; PDC: Pyruvate decarboxylase; PETLP: Photosynthetic electron transfer-like protein; Pn: Photosynthesis; Suc: Sucrose; Tim17/Tim22/Tim23: Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein.