BACKGROUND
magnesium , a common constituent in many minerals, comprising 2% of the earth’s crust, is an essential macronutrient used in large amount by plants for their normal growth and development.
mg-deficiency is a widespread nutritional disorder, affecting productivity and quality in agriculture  <cit> .
mg is taken up by plants in the form of divalent mg2+ .
the binding strength of mg2+ to the soil colloids is low, because mg2+ has a large hydrated radius.
therefore, mg is highly prone to leaching, particularly in acidic soils with low cation exchange capacity.
leaching is considered as a key factor affecting mg availability for roots  <cit> .
mg-deficiency can also be induced by high levels of competing elements, such as potassium  and calcium , which strongly inhibit mg uptake by plants  <cit> .
since mg is mobile within the plant, mg-deficiency symptoms first appear on lower and older leaves.
the typical symptom of mg-deficiency is leaf interveinal chlorosis  <cit> .
although mg-deficiency symptoms are well described in plant shoots, the responses of both plant root growth and biomass allocation between roots and shoots to mg-deficiency is more variable.
previous study showed a less severe impact on root growth or shoot growth, depending on the plant species and the system used to create mg-deficiency  <cit> .
mg is the central component of the chlorophyll  molecule and plays a crucial role in photosynthesis such as chl biosynthesis, photochemical reactions, co <dig> fixation and stomata functioning  <cit> .
mg-deficiency-induced inhibition of photosynthesis is a wide phenomenon observed in many plant species  <cit> .
in addition, mg also act as a cofactor and allosteric modulator for more than  <dig> enzymes including ribulose- <dig> -bisphosphate carboxylase , atpase, protein kinases, rna polymerase, phosphatases and glutathione synthase  <cit> .
therefore, mg also functions in many other physiological processes, such as respiration  <cit> , glycolysis, tricarboxylic acid  cycle  <cit> , energy transfer via adenosine triphosphate  <cit> , carbohydrate partitioning between source and sink organs  <cit> , reactive oxygen species  formation and related photooxidative damage  <cit> , protein biosynthesis, and the formation of dna and rna  <cit> .
accordingly, a number of studies have investigated the effects of mg-deficiency on chl synthesis, transport and utilization of photosynthates  <cit> , photochemical reactions, co <dig> fixation  <cit> , respiration, tca cycle  <cit> , and ros metabolism  <cit> .
physiological and molecular biological analyses have allowed us to draw a picture of abiotic stress responses in various plants.
although the physiological targets upon mg-deficiency have been reported by many workers in various plants  <cit> , the knowledge of upstream molecular targets is very limited until recently.
hermans et al.
<cit>  investigated the transcriptomic responses to  <dig>   <dig> and 24 h mg-deficiency, or long-term  mg-deficiency and restoration in arabidopsis thaliana roots and leaves and identified numerous target genes involved in the circadian clock, the redox control of the cell and the protection of the photosynthetic apparatus.
it is worth mentioning that the responses of global transcriptomics to mg-deficiency were asynchronized, with a higher number of differentially expressed genes after  <dig> or 8 h in roots and after 28 h or 1 week in leaves.
while these data are very useful, the abundances of mrnas does not necessarily correspond directly the abundances of their corresponding proteins.
the level of a protein depends not only on transcription rates of the gene, but also on nuclear export and mrna localization, transcript stability, translational regulation and protein degradation.
indeed, there is considerable variability on protein level versus mrna level  <cit> .
since biological processes are ultimately controlled by proteins, a proteomic analysis is necessary to get a better understanding of the plant responses to mg-deficiency.
to our knowledge, little information is available on the changes of protein profile under mg-deficiency in plant roots and leaves  <cit> .
in china, mg-deficiency is frequently observed in citrus plantations and is responsible for the loss of productivity and poor fruit quality  <cit> .
although the effects of mg-deficiency on citrus co <dig> assimilation, photosynthetic electron transport and antioxidant system, carbohydrates and organic acid metabolism have been investigated by a few researchers  <cit> , the knowledge of upstream targets is scarce.
in the present study, we first investigated the proteomic responses of citrus leaves and roots to mg-deficiency using two-dimensional electrophoresis  in order to  enrich our understanding of the molecular mechanisms of plants to deal with mg-deficiency and  understand the molecular mechanisms by which mg-deficiency lead to a decrease in co <dig> assimilation.
RESULTS
seedling growth, leaf, stem and root mg concentration
plant treated with 0 mm mg displayed decreased leaf, stem and root dry weight  and lower concentration of mg in leaves, stems and roots , and leaf mg concentration was much lower than the normal range  <cit> .
based on these results, plants that did not receive mg are considered mg-deficient, and those treated with 1 mm mg are considered mg-sufficient .figure  <dig>
effects of mg-deficiency on growth and mg concentration in leaves, stems and roots.
leaf, stem and root dw.
leaf, stem and root mg concentration.
bars represent means ± se .
different letters above the bars indicate a significant difference at p <  <dig> .
leaf gas exchange, leaf and root respiration and total soluble protein concentration
compared with controls, mg-deficient leaves had lower co <dig> assimilation , stomatal conductance  and transpiration , and higher intercellular co <dig> concentration .figure  <dig>
effects of mg-deficiency on leaf gas exchange, root respiration, and root and leaf total soluble protein.
leaf co <dig> assimilation, stomatal conductance, intercellular co <dig> concentration, transpiration and respiration.
leaf total soluble protein.
root respiration.
root total soluble protein.
bars represent means ± se .
different letters above the bars indicate a significant difference at p <  <dig> .
mg-deficient leaves displayed increased dark respiration  and decreased concentration of total soluble proteins , while mg-deficient roots had decreased respiration  and unchanged concentration of total soluble proteins .
profiles of differentially expressed proteins
2-de was performed to compare the protein profiles between control and mg-deficient roots and leaves.
in order to get credible results, the experiments were performed in  <dig> biological replicates.
after coomassie brilliant blue g- <dig> staining, more than  <dig> clear and reproducible spots were detected on each gel .
comparative analysis of the 2-de maps of control and mg-deficient leaves was performed by pdquest  <dig> .
<dig> software.
a protein spot was considered differentially expressed when the protein had both a fold change of more than  <dig> and a p-value less than  <dig> .
based on the two criteria,  <dig>  differentially expressed protein spots were detected in mg-deficient leaves with high confidence,  <dig> of which displayed increased and  <dig> displayed decreased level under mg-deficient condition; and  <dig>  differentially expressed protein spots were detected in mg-deficient roots with high confidence, including  <dig> protein spots that were upregulated and  <dig> protein spots that were downregulated by mg-deficiency.figure  <dig>
representative gel images of proteins in mg-deficient  and control  leaves.
proteins were separated in the first dimension on an ipg strip ph 3– <dig> and in the second dimension on a 12% slab gel, followed by colloidal coomassie brilliant g- <dig>  an equal amount  of total protein extracts was loaded in each gel.
representative gel images of proteins in mg-deficient  and control  roots.
proteins were separated in the first dimension on an ipg strip ph 3– <dig> and in the second dimension on a 12% slab gel, followed by colloidal coomassie brilliant g- <dig>  an equal amount  of total protein extracts was loaded in each gel.
all these differentially expressed protein spots were excised from the 2-de gels and submitted to maldi-tof/tof-ms and ltq-esi-ms/ms.
in total,  <dig> and  <dig> protein spots were identified in mg-deficient leaves and roots, respectively.
the database searching results are presented in tables  <dig> and  <dig>  according to the biological functional properties, these differentially expressed protein spots in mg-deficient leaves were classified into the following functional categories: carbohydrate and energy metabolism , protein metabolism , stress responses , nucleic acid metabolism , cell wall and cytoskeleton metabolism , cell transport , lipid metabolism , other and unknown biological processes  ; and theses protein spots in mg-deficient roots were involved in carbohydrate and energy metabolism , protein metabolism , stress responses , nucleic acid metabolism , cell wall and cytoskeleton metabolism , lipid metabolism , other and unknown biological processes  .table  <dig>
list of differentially expressed proteins identified using maldi-tof/tof-ms in magnesium -deficient
citrus sinensis
leaves
spot no.
a
accession no.
b
protein identity
organism
mw
mp/sc
score
ratio
c
carbohydrate and energy metabolism
severinia buxifolia
nicotiana tabacum
glycine max
n. tabacum
n. tabacum
n. tabacum
litchi chinensis
panax ginseng
solanum peruvianum
glycine max
nicotiana benthamiana
n. benthamiana
n. tabacum
ricinus communis
gossypium hirsutum
arabidopsis thaliana
a. thaliana
spinacia oleracea
r. communis
citrus clementina
r. communis
citrus trifoliata
a. thaliana
citrus reticulata
perilla frutescens
oryza sativa japonica group
a. thaliana
c. sinensis
populus tremula × populus alba
r. communis
dianthus caryophyllus
protein metabolism
ricinus communis
pisum sativum
plagiomnium affine
trifolium subterraneum
cucumis melo
a. thaliana
o. sativa japonica group
a. thaliana
platycodon grandiflorus
p. grandiflorus
r. communis
a. thaliana
a. thaliana
a. thaliana
brachypodium distachyon
p. sativum
p. sativum
arabidopsis lyrata subsp.
lyrata
allium cepa
stress responses
citrus sinensis
c. sinensis
citrus maxima
c. maxima
hevea brasiliensis
r. communis
r. communis
petunia x hybrida
gossypium hirsutum
lycopersicon peruvianum
prunus salicina
citrus sinensis
citrus sinensis
a. thaliana
nucleic acid metabolism
nepenthes sp.
‘kosobe’
oryza sativa japonica group
a. thaliana
r. communis
cell wall and cytoskeleton metabolism
adiantum capillus-veneris
pueraria montana var.
lobata
olimarabidopsis pumila
cell transport
oryza sativa japonica group
a. lyrata subsp.
lyrata
lipid metabolism
oryza sativa japonica
r. communis
other and unknown biological processes
luziola fluitans
r. communis
pseudotsuga menziesii
c. sinensis
oryza sativa japonica group
vitis viniferai
selaginella moellendorffii
vitis vinifera
hordeum vulgare subsp.
vulgare
v. vinifera
v. vinifera
mp/sc: number of matched peptides/sequence coverage percentage; mw: theoretical molecular weigh;
a: spot number corresponds to the 2-de gel in figure  <dig>
b: gi number is from ncbi database of matched protein.
c: ratio means the ratio of mg-deficiency to control;  <dig> means protein spots were only detected in control roots;  <dig> means protein spots were only detected in the mg-deficient roots.
list of differentially expressed proteins identified using maldi-tof/tof-ms in magnesium -deficient
citrus sinensis
roots
spot no.
a
accession no.
b
protein identity
organism
mw
mp/sc
score
ratio
c
carbohydrate and energy metabolism
ricinus communis
arabidopsis thaliana
a. thaliana
pisum sativum
afrothismia gabonensis
volvox carteri f. nagariensis
dianthus caryophyllus
hordeum vulgare subsp.
vulgare
citrus sinensis
protein metabolism
r. communis
r. communis
r. communis
a. thaliana
a. thaliana
p. sativum
p. sativum
nicotiana benthamiana
stress responses
xerophyta viscosa
brassica rapa subsp.
pekinensis
triticum aestivum
nucleic acid metabolism
beaucarnea recurvata
zea mays
lotus japonicus
cell wall and cytoskeleton metabolism
boehmeria nivea
medicago sativa
vitis vinifera
lipid metabolism
solanum tuberosum
nicotiana tabacum
other and unknown biological processes
oryza sativa japonica grou
vitis vinifera
mp/sc: number of matched peptides/sequence coverage percentage; mw: theoretical molecular weigh;
a: spot number corresponds to the 2-de gel in figure  <dig>
b: gi number is from ncbi database of matched protein.
c: ratio means the ratio of mg-deficiency to control;  <dig> means protein spots were only detected in control roots;  <dig> means protein spots were only detected in the mg-deficient roots.
functional classification of the differentially expressed protein spots in leaves  and roots .
principal component analysis  loading plots and correlation
the majority of differentially expressed proteins involved in protein metabolism and stress responses were highly clustered under mg-deficient leaves.
furthermore, the differentially expressed proteins associated with nucleic acid metabolism, cell transport, lipid metabolism, and cell wall and cytoskeleton metabolism were only plotted in mg-deficient leaves .
however, the differentially expressed proteins in nucleic acid metabolism were clustered in control roots.
the proteins in cell wall and cytoskeleton metabolism were only plotted in mg-deficient roots .figure  <dig>
pca loading plots of differentially expressed proteins in mg-deficient leaves  and roots .
the correlation coefficient matrix presented that the individual differentially expressed proteins were highly correlated within each categorized metabolism, regardless of tissues .
in leaves, the majority of the differentially expressed proteins in carbohydrate and energy metabolism were positively correlated with each other but negatively with the other proteins in the other metabolisms.
the most proteins in the other metabolisms but carbohydrate and energy metabolism were highly positively correlated with each other .
in contrast, the differentially expressed proteins in roots did not show any clear pattern like those in leaves .figure  <dig>
pearson correlation coefficient matrix for the differentially expressed protein spots in mg-deficient leaves  and roots .
red and blue colors indicated positive and negative correlation between the differentially expressed proteins.
transcriptional analysis of genes for some differentially expressed proteins
to verify the changed expression in transcriptional level and evaluate the correlation between mrna and protein levels.
the expression levels of genes for  <dig> differentially expressed proteins from mg-deficient leaves  and roots  were analyzed by qrt-pcr .
of the  <dig> genes, the expression profiles of nine genes  were well correlated with our 2-de data , meaning that the differentially expressed proteins are regulated at the transcriptional level.
however, the transcript level changes of the remaining nine genes  did not match the proteomic observations .
indeed, the transcript levels of genes do not necessarily match the levels of their corresponding proteins, since the abundance of a protein depends not only on transcription rate of the gene expression alone  <cit> .
the discrepancy between the expression levels of the nine genes and the abundance of the corresponding protein  suggests that post-translational modifications  might influence the abundances of these proteins and alter the positions of these proteins on the gel.figure  <dig>
relative expression of  <dig> genes from leaves and of five genes from roots.
relative expression of nine leaf genes encoding cysteine proteinase ; atp synthase cf <dig> alpha subunit, chloroplastic , triosephosphate isomerase , cu/zn superoxide dismutase , phosphoglycerate kinase , ascorbate peroxidase , s-adenosylmethionine synthetase , aconitate hydratase  <dig> , nucleoside diphosphate kinase, putative .
relative expression of four leaf genes encoding atykt <dig> , glyceraldehyde-3-phosphate dehydrogenase b subunit , alcohol dehydrogenase , and ribulose  <dig> -bisphosphate carboxylase, partial , and five root genes encoding proteasome subunit alpha type, putative , cysteine protease, putative , pyruvate decarboxylase, putative , phosphoglycerate kinase , and hexokinase .
bars represent means ± se .
different letters above the bars indicate a significant difference at p <  <dig> .
all the values were expressed relative to the control leaves or roots.
discussion
mg-deficiency triggers different responses in leaves and roots
our results showed that the amounts of differentially expressed proteins were much more in the mg-deficient leaves than in the mg-deficient roots , meaning that the effects of long-term mg-deficiency on protein profiles of leaves were more pronounced than on those of roots.
this agrees with the reports in a. thaliana that the root transcriptome was less severely affected by long-term  mg-deficiency  <cit> , and that the responses of plants to mg-deficiency was asynchronized, with a lower number of differentially regulated genes in leaves  <dig> or 8 h after the removal of mg from the nutrient solution and in roots 28 h after the removal of mg  <cit> .
this is also supported by our results that mg-deficiency decreased leaf concentration of total soluble proteins , but had no influence on root concentration of total soluble proteins .
as shown in tables  <dig> and  <dig>  the majority of the differentially expressed proteins only presented in roots or leaves, only two differentially expressed proteins with the same gi number presented in both roots and leaves .
in leaves, the abundances of putative ferredoxin-nadp reductase  and atp synthase  decreased and the levels of proteins involved in tca cycle  as well as dark respiration were increased in response to mg-deficiency; whereas in roots, the levels of fnr  and atpase α subunit, partial  were upregulated and the abundance of pyruvate decarboxylase, putative  as well as respiration was downregulated by mg-deficiency.
in conclusion, there are many differences in mg-deficiency-induced changes in protein profiles as well as biochemical responses between leaves and roots.
as shown in tables  <dig> and  <dig>  mg-deficiency did not alter the abundances of these proteins potentially involved in mediating mg transport such as the magnesium⁄proton exchanger  <dig>  and the mitochondrial rna splicing  <dig> ⁄magnesium transporter  family  <cit> .
this agrees with the reports that mg-deficiency did not induce the expression of genes associated with the transport of mg, with the only exception of the mrs2- <dig> being downregulated  <cit> .
some differentially expressed proteins  in mg-deficient leaves and chaperonin precursor  in mg-deficient roots were represented by more than one spot .
this might reflect isozymes or ptms of a single protein  <cit> .
proteins involved in carbohydrate and energy metabolism
a crucial role of mg is its involvement in the export of carbohydrates from source to sink sites.
the accumulation of sugars in source leaves, an early symptom of mg-deficiency, usually precedes the decreases in photosynthetic rate and chl concentration  <cit> .
the accumulation of sugars in leaves may repress the expression of genes that encode photosynthetic enzymes, thus decreasing chl concentration and photosynthetic capacity  <cit> .
therefore, these proteins involved in photosynthesis and carbohydrate metabolism and related biological processes might be altered under mg-deficiency.
as expected, many differentially expressed proteins related to carbohydrate and energy metabolism were found in mg-deficient leaves .
our finding that mg-deficiency decreased the abundances of rubisco  and rubisco activase   agrees with the report that mg-deficiency reduced the activities of rubisco and rubisco activase and the expression levels of genes encoding rubisco large subunit , rubisco small subunit , and rubisco activase subunit  in spinach  leaves  <cit> .
mate et al.
<cit>  reported that transgenic tobacco  plants expressing an antisense gene encoding rubisco activase reduced rubisco carbamylation, hence impairing photosynthesis.
two studies with logan   <cit>  revealed that mg-deficiency-induced inhibition of photosynthesis might be associated with both reduced carboxylation efficiency and lower rate of ribulose- <dig> -bisphosphate regeneration.
oxygen evolving enhancer proteins  consist of three subunits, oee  <dig> , oee  <dig>  and oee  <dig> .
these are nuclear-encoded chloroplast proteins, and peripherally bound to photosystem ii  on the luminal side of the thylakoid membrane.
oee <dig> is the most important protein for oxygen evolution and psii stability.
mg-deficiency greatly decreased the level of oee <dig> in leaves .
this means that mg-deficiency might impair the stability of oxygen evolution and psii, as found on mg-deficient c. sinensis and c. grandis  <cit> .
in addition, mg-deficient leaves had lower level of photosynthetic electron transfer-like protein , which agrees with the report that mg-deficiency decreased photosynthetic electron transport by impairing the whole photosynthetic electron transport chain from the psii donor side up to psi, thus decreasing citrus leaf co <dig> assimilation  <cit> .
hajirezaei et al.
<cit>  reported that a small decrease in the activity of fnr by antisense rna led to decreases in photosynthetic rate and nadph level, and increases in the extent of qa reduction and nadp level, and concluded that fnr was one of the rate-limiting steps in photosynthesis and fnr-deficiency-induced inhibition of photosynthesis was caused by impairment of fnr-mediated electron transfer from reduced ferredoxin to nadp.
as shown in table  <dig>  the level of putative fnr  decreased in mg-deficient leaves.
this means that fnr activity might be downregulated by mg-deficiency, thus impairing the photosynthetic electron transfer and increasing the extent of qa reduction, and hence inhibiting photosynthesis.
palatnik et al.
<cit>  demonstrated that fnr-deficient plants were particularly prone to photoinhibitory damage and photooxidative injury.
all these results agree the report that mg-deficiency decreased co <dig> assimilation, and increased the extent of qa reduction and the concentration of malondialdehyde  in citrus leaves  <cit> .
the reduction of 3-phosphoglycerate to triose phosphate, which is reversibly catalyzed by two chloroplast enzymes , is a crucial step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism of the photosynthetic carbon-reduction cycle in the stroma.
price et al.
<cit>  showed that antisense transgenic plants with severely reduced chloroplast gapdh activity were not photoinhibited despite the continuous presence of a large thylakoid proton gradient in the light, and that the electron-transport chain did not become over-reduced due to a shortage of nadp despite the downregulation of photosynthetic electron transport due to the build-up of a large proton gradient.
the observed lower level of gapdh b subunit  might be helpful to lessen photoinhibition and the over-reduction of photosynthetic electron transport, thus lowering photooxidative damage caused by mg-deficiency.
in addition, mg-deficiency decreased leaf abundance of pgk .
therefore, the reduction of 3-phosphoglycerate to triose phosphate might be decreased in mg-deficient leaves due to lower activities of chloroplast gadph and pgk.
mg-deficiency also reduced leaf levels of putative carbonic anhydrase , triosephosphate isomerase , fructose-bisphosphate aldolase, class i  and putative chloroplastic lactoylglutathione lyase  .
carbonic anhydrase, which reversibly catalyzes the interconversion of co <dig> and hco3−, is a major protein constituent of the c <dig> higher plant chloroplast.
stimler et al.
<cit>  showed that stomatal response was mediated by carbonic anhydrase, and that h2s formed in the mesophyll via the reaction of carbonyl sulfide and water with carbonic anhydrase was involved in the stomatal response.
the observed lower abundance of carbonic anhydrase means that its activity might be downregulated in mg-deficient leaves, thus decreasing the stomatal conductance .
however, in addition to stomatal factor, non-stomatal factors such as photosynthetic inhibition also contributed to the lower co <dig> assimilation in mg-deficient leaves, as indicated by the increased intercellular concentration .
ito et al.
<cit>  showed that triosephosphate isomerase and putative plastidic aldolase were inactivated by oxidized glutathione  and reactivated by reduced glutathione .
this agrees with the report that mg-deficient citrus leaves had increased gssg concentration, but decreased gsh level  <cit> .
haake et al.
<cit>  reported that a moderate decrease of plastidic aldolase activity led to a decrease in photosynthesis in antisense transgenic potato plants.
lactoylglutathione lyase  catalyzes the formation of s-d-lactoylglutathione in the presence of glutathione and methylglyoxal, a cytotoxic compound produced spontaneously under physiological conditions from the glycolysis and photosynthesis intermediates glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
besides detoxification of methylglyoxal, the glyoxalase system could also play a role in oxidative stress tolerance by recycling gsh and maintaining glutathione homeostasis  <cit> .
the observed lower level of putative chloroplastic lactoylglutathione lyase  in mg-deficient leaves  agrees with our previous report that mg-deficiency increased gssg concentration and decreased gsh concentration in citrus leaves  <cit> .
to conclude, mg-deficiency decreased the levels of many proteins involved in photosynthesis including rubisco, rubisco activase, oee <dig>  photosynthetic electron transfer-like protein, fnr, aldolase, thus decreasing leaf photosynthesis.
we found that mg-deficiency increased the abundances of nadp-malate dehydrogenase , putative succinate dehydrogenase  and aconitate hydratase   <dig>  in c. sinensis leaves , which agrees with our data that mg-deficient leaves had higher rate of dark respiration  and the report that the activities of enzymes related to glycolysis and tca cycle were enhanced in mg-deficient c. sinensis leaves  <cit> .
nadp-malate dehydrogenase is the key enzyme in the malate/oxalacetate shuttle, which is the major machinery for the transport of excess reducing equivalents generated in chloroplasts to mitochondria  <cit> .
nadp-malate dehydrogenase converts oxalacetate to malate using nadph, facilitating the regeneration of the electron acceptor nadp in the chloroplasts, particularly when co <dig> assimilation is restricted  <cit> .
the observed higher level of nadp-malate dehydrogenase in mg-deficient leaves might contribute to the transport of excess reducing equivalents from chloroplasts to mitochondria and the regeneration of chloroplast nadp, thus preventing oxidative damage and enhancing the tolerance of plant to mg-deficiency.
previous study showed that antisense inhibition of the iron-sulphur subunit of sdh and aco reduced the carbon flow through the tca cycle, and enhanced the photosynthesis and stomatal conductance  <cit> .
this agrees with our data that mg-deficient leaves had increased dark respiration , levels of sdh and aco , and decreased co <dig> assimilation  and stomatal conductance .
also, the abundances of putative dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase  related to tca cycle, and 2-phospho-d-glycerate hydrolase  and  <dig> -bisphosphoglycerate-independent phosphoglycerate mutase  <dig>  involved in glycolysis in leaves increased in response to mg-deficiency .
our finding that mg-deficient leaves had increased abundance of α-amylase , a starch degrading enzyme agrees with the report that the activities of two starch degrading enzymes  increased in mg-deficient spruce needles  <cit> .
thus, starch degradation might be upregulated in mg-deficient c. sinensis leaves.
however, mg-deficiency had little influence on starch concentration in c. sinensis leaves  <cit> .
this might be related to the fact that sucrose biosynthesis and export decreased in response to mg-deficiency  <cit> .
also, the upregulation of starch biosynthesis was not ruled out, because the abundance of adp-glucose pyrophosphorylase catalytic subunit  increased in mg-deficient leaves.
thus, it is reasonable to assume that the rate of starch turnover in mg-deficient leaves might be enhanced  <cit> .
mg-deficient leaves had lower levels of putative atp synthase β subunit , atp syntase γ chain  <dig> , chloroplastic atp synthase cf <dig> α subunit  and chloroplastic atp synthase subunit α  .
this means that atp synthesis catalyzed by atp synthase in mg-deficient leaves might be decreased, which might provide an advantage to prevent energy surplus due to increased dark respiration and the generation of excess reducing equivalents in chloroplasts.
adenosine kinase, which catalyzes the reaction: adenosine + atp → amp + adp, is an essential component for maintaining purine nucleotide pools in arabidopsis.
it also contributes to cytokinin interconversion in arabidopsis  <cit>  and plays a key role in sustaining transmethylation reactions by serving as a coarse metabolic control to reduce the concentration of free adenosine in spinach and sugar beet  cells during salt stress  <cit> .
our finding that the abundance of putative adenosine kinase  in leaves increased in response to mg-deficiency  agrees with the report that adenosine kinase activity, protein and transcripts in spinach and sugar beet leaves were enhanced by salt stress  <cit> .
nucleoside diphosphate kinase  is a ubiquitous housekeeping enzyme that maintains the intracellular levels of all ntps used in biosynthesis except for atp.
in plants, ndpk <dig> is known to regulate the expression of antioxidant genes.
transgenic potato, sweet potato and arabidopsis plants overexpressing arabidopsis ndpk <dig> had increased activities of antioxidant enzymes and enhanced tolerance to methylviologen, salt and temperature stresses  <cit> .
aldehydes, which lead to a rapid and excessive accumulation of ros in plants, may be induced by various abiotic stresses.
alcohol dehydrogenase , which converts aldehydes into alcohols, is essential for plants to survive under anaerobic conditions.
adh gene and/or protein are induced by aba, salt, desiccation, high and low temperatures in various plants.
it has been suggested that the induction of adh by these stresses may trigger a signal transduction cascade, which would lead to a decrease in membrane damage, through adapting plants to oxidative stress  <cit> .
therefore, the antioxidant capacity might be upregulated in mg-deficient leaves due to the increased level of putative ndpk  and adh , hence enhancing the mg-deficiency tolerance.
this agrees with our report that mg-deficient c. sinensis leaves had higher activities of ascorbate peroxidase , superoxide dismutase , glutathione reductase , dehydroascorbate reductase  and guaiacol peroxidase   <cit> .
unlike to leaves, mg-deficiency decreased root respiration  and the activities of enzymes related to glycolysis and tca cycle  <cit> .
as expected, mg-deficiency decreased the abundances of putative pyruvate decarboxylase  and pgk  in roots.
however, root level of pyruvate dehydrogenase e <dig> α subunit  increased in response to mg-deficiency .
in contrast to leaf putative fnr , the level of root fnr  was increased by mg-deficiency.
in higher plants, there are two forms of fnr, a photosynthetic fnr and a heterotrophic fnr.
onda et al.
<cit>  revealed that the interaction of root fnr with ferredoxin  is crucial for an efficient electron flux of nadph-fnr-fd cascade, thus supporting fd-dependent metabolism in non-photosynthetic organs.
xu et al.
<cit>  showed that the abundance of a fnr precursor was reduced by 60% in the roots of null transformant  plants, but maintained in transgenic lines expressing the adenine isopentenyl transferase gene under heat stress, and concluded that the maintenance of fnr level under heat stress could reflect a superior n assimilation capacity of the transgenic lines over the nt line that facilitates their growth under heat stress.
thus, the observed higher level of fnr  in mg-deficient roots  might be an adaptive response.
the biosynthesis of atp in mg-deficient roots might be enhanced due to increased level of mitochondrial atpase α subunit, partial .
adenylate kinase catalyses the reversible formation of adp by the transfer of one phosphate group from atp to amp, thus equilibrating adenylates  <cit> .
thus, the increased abundances of the two proteins might be advantage to maintaining energy balance in mg-deficient roots, when atp production was reduced due to decreased respiration .
like leaves, the level of root adh  increased in response to mg-deficiency.
similar result has been obtained on b-deficient c. sinensis roots  <cit> .
similar to leaf α-amylase, the activity of β-amylase might be upregulated in mg-deficient roots due to enhanced abundance of β-amylase  <dig> , thus increasing the breakdown of starch into sugars in roots.
this agrees with our report that mg-deficiency decreased the concentration of starch in citrus roots  <cit> .
hexokinases  phosphorylate glucose to produce glucose-6-phosphate, the first step of glycolysis.
our finding that mg-deficient roots had higher level of hxk  compared with controls  agrees with the reports that hxks contributed to the survival of acclimated maize root tips via hypoxic pretreatment by allowing the maintenance of a sustained glycolytic rate  <cit> , and that oshxk <dig> , localized at the cytosol, was upregulated under sugar-starvation conditions but downregulated in response to high sugar treatments of the leaf tissues of rice  <cit> , because mg-deficiency decreased or did not affect the concentrations of non-structural carbohydrates in c. sinensis roots  <cit> .
also, hxks might act as a glucose sensor  and, through its catalytic activity, as a crucial regulator of ros levels in plant organelles such as mitochondria and chloroplasts  <cit> .
proteins involved in stress responses
because leaf co <dig> assimilation decreased in response to mg-deficiency , less of the absorbed light energy was utilized in photosynthetic electron transport, particularly under high light.
the excess absorbed photon flux can potentially lead to ros production.
thus, ros production might be enhanced in mg-deficient leaves  <cit> .
to minimize the cellular damage caused by ros, plants have evolved an antioxidant system composed of antioxidants and antioxidant enzymes.
as expected, the leaf levels of antioxidant enzymes  increased in response to mg-deficiency except for chloroplast cu/zn sod  .
this agrees with the report that mg-deficient citrus leaves had higher or similar activities of antioxidant enxymes  except for catalase  <cit> .
aldo-keto reductases , which catalyze the reduction of aldehyde to alcohol, are effective detoxification of peroxidation-derived reactive aldehydes  <cit> .
transgenic tobacco plants overexpressing alfalfa or rice ark had enhanced tolerance against a variety of oxidative damages induced by methylviologen, uv-b irradiation, heavy metals, drought, heat treatment, osmotic and salt stresses  <cit> .
the upregulation of the putative akr  in mg-deficient leaves  might contribute to the oxidative tolerance of leaves.
in addition, mg-deficiency increased the leaf abundances of heat shock proteins , which play a key role in protecting plants against stress by reestablishing normal protein conformation and thus cellular homeostasis, and stress-related protein .
based on these results, we concluded that proteins associated with stress-response functions were upregulated in mg-deficient leaves.
however, the level of late embryogenesis abundant  protein  in leaves decreased in response to mg-deficiency .
like to leaves, the levels of stress-responsive proteins  in roots increased in response to mg-deficiency .
this agrees with the reports that b-deficiency increased the level of nb-arc domain-containing disease resistance protein in c. sinensis roots  <cit> , and that the abundance of grp <dig> in xerophyta viscosa plants increased in response to heat and dehydration  <cit> .
however, the abundances of mitochondrial heat shock 70 kda protein  and heat shock protein hsp <dig>  were decreased in mg-deficient roots , which agrees with the studies that b-deficiency decreased the levels of several hsps  in c. sinensis roots  <cit>  and the abundances of three hsps in lupinus albus roots  <cit> .
proteins involved in protein metabolism
protein synthesis elongation factor tu  plays a central role in the elongation phase of protein synthesis in bacteria and organelles including mitochondria and plastids in plants  <cit> .
we found that the abundance of the putative enongation factor tu  in leaves decreased in response to mg-deficiency , meaning that the biosynthesis of some proteins in plastids and mitochondria might be downregulated.
this agrees with our results that the levels of many chloroplastic  proteins was decreased by mg-deficiency , and that the concentration of total soluble proteins in leaves decreased in response to mg-deficiency .
however, the levels of chloroplastic chaperonin precursor , chloroplastic small ribosomal protein  <dig>  partial  and ribosomal protein s <dig>  related to protein folding and biosynthesis were increased in mg-deficient leaves .
plant proteases play key roles in maintaining strict protein quality control and degrading specific sets of proteins in response to environmental and developmental stimuli.
the majority of the thousand or more proteins that are present in mitochondria are required to be imported from nuclear-encoded cytosolically synthesized precursors, and a number of peptidases are needed to remove the “transient” targeting information present on many, but not all, mitochondrial precursor proteins  <cit> .
the upregulation of mitochondrial processing peptidase β subunit  in mg-deficient leaves  agreed with the increased requirement for removing the “transient” targeting information due to enhanced import resulting from the increased abundance of the putative mitochondrial import inner membrane translocase subunit tim17/tim22/tim <dig> family protein .
ubiquitination, which serves as a versatile ptm, plays a key role in regulating plant response to abiotic stresses.
it has been known that the f-box is a motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction  <cit> , and that the ring zinc-finger domain is essential for the function of ubiquitin e <dig> ligases  <cit> .
the upregulation of f-box domain-containing protein , putative f-box family protein, expressed , putative f-box/kelch-repeat protein  and putative ring zinc finger protein  in mg-deficient leaves  means that leaf ubiquitination might be enhanced in response to mg-deficiency.
also, the abundances of proteins  related to protein degradation increased in mg-deficient leaves .
in conclusion, proteolysis might be enhanced in mg-deficient leaves, thus lowering leaf concentration of total soluble proteins .
however, the abundances of leaf f2e <dig>   and putative skp <dig> protein  decreased in response to mg-deficiency .
amino acids are the structural units that make up proteins and also serve as precursors for many metabolites with multiple functions in plant growth and response to various stresses.
all the four differentially expressed proteins  involved in amino acid metabolism were upregulated in mg-deficient leaves , meaning that the metabolism of amino acids might be upregulated in mg-deficient leaves.
glutathione s-transferases , which catalyze the conjugation of cytotoxic metabolites with the gsh, are mainly involved in detoxifying oxidative-stress metabolites.
glutamine synthetase  catalyses the critical incorporation of inorganic ammonium into the amino acid glutamine.
in plants, two types of gs isozymes, located in the cytosol  and in the chloroplast  have been found.
transgenic plants overexpressing gs <dig> or gs <dig> displayed enhanced tolerance to water or salt stress, respectively  <cit> .
methylation induced by abiotic stress has been supposed to be linked with the numerous biochemical pathways involved in acclimatization and stress response in plants  <cit> .
s-adenosylmethionine is an important methyl donor for numerous transmethylation reactions.
s-adenosylmethionine synthase is a crucial enzyme that directs the flux of methione to s-adenosylmethionine.
these results indicate that the upregulation of amino acid metabolism in mg-deficient leaves might be involved in the adaption of mg-deficient plants.
proteolytic cleavage of proteins by proteases is not limited to the total degradation of mature proteins to free amino acids, but also is relevant for modification and maturation of proteins.
like to leaves, mg-deficiency increased the abundances of root proteins  involved in proteolysis .
however, there was no significant difference in total soluble protein concentration between mg-deficient and control roots .
this implies that the mg-deficiency-induced increase in protease levels may be mainly involved in modification and maturation of proteins or that the biosynthesis is enhanced in mg-deficient roots.
the upregulation of proteases in mg-deficient roots might be an adaptive response of plants to mg-deficiency through maintaining the protein complexes and/or the recycling of n.
the eukaryotic translation initiation factor  <dig>  has multiple roles during the initiation of translation of cytoplasmic mrnas.
the classic functions ascribed to eif <dig> include:  facilitating the charging of the 40s ribosomal subunit with the ternary complex;  bridging between the 40s ribosomal subunit and the eif4g subunit of the cap-binding complex, eif4f; and  inhibiting the association of 40s and 60s ribosomal subunits  <cit> .
the upregulation of eif 3b- <dig>  in mg-deficient roots  might contribute to the translatability of cytoplasmic mrnas, thus maintaining the level of cytoplasmic proteins, which agrees with our result that mg-deficiency did not significantly affect root concentration of total soluble proteins .
however, the levels of root proteins  involved in protein folding and biosynthesis were decreased by mg-deficiency.
proteins involved in nucleic acid metabolism
the downregulation of rna polymerase β chain , spliceosome rna helicase bat <dig>  and transcription factor homolog btf3-like protein  in mg-deficient roots  agrees with our report that all  <dig> differentially expressed proteins related to nucleic acid metabolism were reduced in b-deficient c. sinensis roots except for argonaute family protein  <cit> .
this indicates that the synthesis of rna might be inhibited in mg-deficient roots.
by contrast, the abundances of all the four altered proteins  related to nucleic acid metabolism in leaves increased in response to mg-deficiency , meaning that the metabolism of nucleic acid might be upregulated in mg-deficient leaves.
dead box rna helicases are prominent candidates for rna chaperones because these proteins can use energy derived from atp hydrolysis to actively disrupt misfolded rna structures so that correct folding can occur, and play important roles in plant stress responses  <cit> .
transgenic arabidopsis plants overexpressing a rice gene osbirh <dig>  which encodes a dead-box rna helicase protein, showed enhanced disease resistance against alternaria brassicicola and pseudomonas syringae, and increased tolerance to oxidative stress and elevated expression levels of oxidative defense genes  <cit> .
the upregulation of dead box rna helicases  in mg-deficient leaves  might be an adaptive response of plants to mg-deficiency.
in nonplant systems, the splicing of group-ii introns is mediated by proteins encoded within the introns themselves , whereas only a single maturase orf  has retained in the mitochondrial genomes in plants.
interestingly, higher plant genomes contain four maturase-related genes, which exist in the nucleus as self-standing orfs.
recently, keren et al.
<cit>  showed that atnmat <dig>  a nuclear-encoded maturase, was required for splicing of group-ii introns in arabidopsis mitochondria.
in another study, keren et al.
<cit>  observed that nmat <dig> functions were required for mitochondrial biogenesis and that nmat <dig> mutants displayed growth and developmental defect phenotypes and accumulated high levels of ros, concluding that nmat <dig> was essential for mitochondrial complex i assembly and function.
thus, the observed higher level of maturase, partial  in mg-deficient leaves might be an adaptive response.
proteins involved in cell wall and cytoskeleton metabolism
ca, a constituent of the cell wall, plays a key role in determining the structural rigidity of the cell wall.
also, ca is a regulator that can exert multiple effects on the structure and dynamics of the actin cytoskeleton  <cit> .
it has been well known that high rhizosphere concentration of mg, relative to ca, is inhibitory to the absorption of ca and vice versa.
therefore, the concentration of ca in mg-deficient plant tissues should be enhanced.
indeed, mg-deficiency increased the concentration of ca in rough-lemon  leaves  <cit> .
based on these results, it is reasonable to assume that the mg-deficiency-induced increase in ca concentration might be responsible for the enhanced abundances of myosin class 11– <dig> , ugt <dig>  and pollen coat oleosin-glycine rich protein  involved in cell wall and cytoskeleton metabolism in mg-deficient leaves .
similarly, the abundances of root actin  <dig> , villin  <dig>  and tubulin γ- <dig> chain  were upregulated by mg-deficiency , which is in agreements with the report that mg-deficiency led to enhanced suberization in endodermis and hypodermis of corn roots  <cit> .
the upregulation of proteins related to cell wall and cytoskeleton metabolism in mg-deficient roots and leaves might be an adaptive response of plants to mg-deficiency.
proteins involved in cell transport
proteins that reside in endomembrane organelles, or that are secreted from the cell, reach their sites of function by transport of the endomembrane system  <cit> .
the translocase of the inner membrane  is a complex of proteins found in the inner membrane of the mitochondria and plays a pivotal role in protein import, which is important to cope with stress  <cit> .
the observed higher level of the putative mitochondrial import inner membrane translocase subunit tim17/tim22/tim <dig> family protein  in mg-deficient leaves  can be explained by the fact that mitochondria are the center of oxygen sensing.
under mg-deficiency, high protein import might be required for the increased ros detoxification  <cit> , thus requiring high activity of the tim protein complex  <cit> .
snares are core proteins for vesicle fusion and can be categorized into two types: snares on the vesicle are called v-snares, while snares on the target membrane are called t-snares.
atykt <dig> and atykt <dig>  two t-snares, are essential for membrane fusion mediated by either syp <dig> or stp <dig>  <cit> .
the upregulation of the two proteins means that the transport of proteins might be enhanced in mg-deficient roots.
proteins involved in lipid metabolism
sato-izawa et al.
<cit>  revealed that the inositol- <dig> ,5-trisphosphate 5-phosphatase, an enzyme probably involved in phosphoinositide signaling, was required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes.
perera et al.
<cit>  showed that transgenic arabidopsis plants expressing the type  <dig> inositol- <dig> ,5-trisphosphate 5-phosphatase had enhanced drought tolerance and altered aba signaling.
cytochrome p450s , a superfamily of ubiquitous heme-containing mixed-function monooxygenases proteins, play a key role in biotic and abiotic stresses  <cit> .
in arabidopsis, at least five of the  <dig> cytop450s are induced by abiotic and biotic stresses including alternaria brassicicola or alternaria alternata, paraquat, rose bengal, uv-c stress, heavy metal stress , mechanical wounding, drought, high salinity, low temperature or hormones  <cit> .
transgenic tobacco and potato plants expressing cytp <dig> with increased monooxygenase activity tolerated better oxidative stress after herbicide treatment  <cit> .
therefore, the observed higher level of putative type  <dig> inositol- <dig> ,5-trisphosphate 5-phosphatase cvp <dig>  and putative cytop <dig>  might contribute to mg-deficiency tolerance of plants.
we found that the abundance of root lipoxygenase  increased in response to mg-deficiency , which is in agreement with the report that the level of lox  <dig> was enhanced in b-deficient c. sinensis roots  <cit> .
lox, which catalyses the oxidation of α-linolenic acid into either 9- or 13-hydroperoxy-octadecatrienoic acids, or a mixture of both, is one of the key enzymes responsible for the biosynthesis of jasmonates, which are ubiquitously occurring lipid-derived compounds with signal functions in plant responses to abiotic and biotic stresses  <cit> .
hu et al.
<cit>  demonstrated that tomloxd, a tomato 13-lox gene, was involved in endogenous jasmonate synthesis and tolerance to biotic and abiotic stress.
therefore, both jasmonate biosynthesis and level might be increased in mg-deficient roots, thus enhancing plant mg-deficiency tolerance.
this inference is also supported by the observation that the expression levels of two genes encoding myc <dig>  which is a jasmonate-dependent transcription factor and lox <dig>  which is an enzyme involved in jasmonate biosynthesis  <cit>  in roots increased in response to mg-deficiency .
thus, jasmonate signaling might be involved in the responses of citrus roots to mg-deficiency.
however, the expression of the gene encoding zim/tify-domain , which is the co-repressor of myc <dig> that is ubiquitinilated upon activation of jasmonate signaling cascade  <cit> , did not change in mg-deficient citrus roots .
the plastid acetyl-coenzyme a carboxylase, which catalyzes the first committed step of fatty acid synthesis, is present as a heteromeric complex of at least four different protein subunits: the biotin carboxylase, the biotin carboxyl carrier protein, and the α and β subunits of the carboxyltransferase in most plants.
transgenic tobacco plants with less than 25% of wild-type biotin carboxylase levels displayed severely retarded growth when grown under low-light conditions and a 26% lower leaf fatty acid concentration than wild-type plants  <cit> .
thus, the biosynthesis of fatty acids in mg-deficient roots might be elevated due to enhanced level of biotin carboxylase subunit .
other proteins
ent-kaurene is a tetracyclic hydrocarbon precursor for gibberellins  in plants.
in higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase  and ent-kaurene synthase  from geranylgeranyl diphosphate .
a range of ga-deficient phenotypes of the ga1- <dig> and ga2- <dig> a. thaliana mutants  were restored to wild type, when fungal cps/ks was overexpressed and targeted to plastids.
the over-expressing plants emitted ent-kaurene into the headspace besides its accumulation in the plant body  <cit> .
fleet et al.
<cit>  showed that transgenic arabidopsis plants overexpressing atcps and atks led to increased ent-kaurene production but unchanged concentration of active gas.
therefore, the upregulation of ent-kaurene synthase  in mg-deficient leaves  does not necessarily imply that leaf concentration of gas was enhanced by mg-deficiency.
CONCLUSIONS
we first investigated the proteomic changes induced by long-term mg-deficiency in c. sinensis leaves and roots using 2-de.
in mg-deficient leaves,  <dig> upregulated and  <dig> downregulated proteins were isolated, while only  <dig> upregulated and  <dig> downregulated proteins in mg-deficient roots.
this indicated that proteomes were more affected by long-term mg-deficiency in the leaves than in the roots, which was further supported by our observation that the concentration of total soluble proteins was decreased by mg-deficiency in leaves, but unaffected in roots.
a potential regulatory network of mg-deficiency-induced responses in citrus leaves and roots was proposed through the integration of the present results and available data in the literatures .
mg-deficiency led to decreased abundances of proteins  involved in photosyntheis, thus decreasing leaf co <dig> assimilation.
the adaptive responses of c. sinensis roots and leaves to mg-deficiency might including several aspects:  improving leaf respiration and lowering root respiration, but increasing  the levels of proteins related to atp synthase in roots ;  enhancing the levels of proteins  involved in 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.
therefore, our proteomic analysis provides an integrated view of the adaptive responses occurring in mg-deficient leaves and roots of c. sinensis.
as a first attempt, the present study will be useful for further investigating the roles of mg in higher plants.
it is worth noting that it may provide more data on mg-deficiency in real citrus orchards if we use grafted plants rather than c. sinensis seedlings as experimental materials, but it is difficult for us to compare the present data with the transcriptomic data obtained on arabidopsis roots and leaves  <cit>  and the physiological and biochemical data obtained on c. sinensis roots and leaves  <cit> .
in the further study, we will investigate the effects of rootstocks on mg-deficiency-responsive proteomics using grafted citrus plants from different rootstock-scion combinations including both own-rooted scions and rootstocks as controls to obtain more knowledge on mg-deficiency in real citrus orchards.figure  <dig>
the potential regulatory network of mg-deficiency-induced responses in
citrus
leaves  and roots .
bpg-pgam:  <dig> -bisphosphoglycerate-independent phosphoglycerate mutase 1; cvp2: type i inositol- <dig> ,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/tim <dig> family protein.

 magnesium, a common constituent in many minerals, comprising 2% of the earth’s crust, is an essential macronutrient used in large amount by plants for their normal growth and development. mg-deficiency is a widespread nutritional disorder, affecting productivity and quality in agriculture. mg is taken up by plants in the form of divalent mg2+. the binding strength of mg2+ to the soil colloids is low, because mg2+ has a large hydrated radius. therefore, mg is highly prone to leaching, particularly in acidic soils with low cation exchange capacity. leaching is considered as a key factor affecting mg availability for roots. mg-deficiency can also be induced by high levels of competing elements, such as potassium and calcium, which strongly inhibit mg uptake by plants. since mg is mobile within the plant, mg-deficiency symptoms first appear on lower and older leaves. the typical symptom of mg-deficiency is leaf interveinal chlorosis. although mg-deficiency symptoms are well described in plant shoots, the responses of both plant root growth and biomass allocation between roots and shoots to mg-deficiency is more variable. previous study showed a less severe impact on root growth or shoot growth, depending on the plant species and the system used to create mg-deficiency. mg is the central component of the chlorophyll molecule and plays a crucial role in photosynthesis such as chl biosynthesis, photochemical reactions, co fixation and stomata functioning. mg-deficiency-induced inhibition of photosynthesis is a wide phenomenon observed in many plant species. in addition, mg also act as a cofactor and allosteric modulator for more than enzymes including ribulose- -bisphosphate carboxylase, atpase, protein kinases, rna polymerase, phosphatases and glutathione synthase. therefore, mg also functions in many other physiological processes, such as respiration, glycolysis, tricarboxylic acid cycle, energy transfer via adenosine triphosphate, carbohydrate partitioning between source and sink organs, reactive oxygen species formation and related photooxidative damage, protein biosynthesis, and the formation of dna and rna. accordingly, a number of studies have investigated the effects of mg-deficiency on chl synthesis, transport and utilization of photosynthates, photochemical reactions, co fixation, respiration, tca cycle, and ros metabolism. physiological and molecular biological analyses have allowed us to draw a picture of abiotic stress responses in various plants. although the physiological targets upon mg-deficiency have been reported by many workers in various plants, the knowledge of upstream molecular targets is very limited until recently. hermans et al. investigated the transcriptomic responses to and 24 h mg-deficiency, or long-term mg-deficiency and restoration in arabidopsis thaliana roots and leaves and identified numerous target genes involved in the circadian clock, the redox control of the cell and the protection of the photosynthetic apparatus. it is worth mentioning that the responses of global transcriptomics to mg-deficiency were asynchronized, with a higher number of differentially expressed genes after or 8 h in roots and after 28 h or 1 week in leaves. while these data are very useful, the abundances of mrnas does not necessarily correspond directly the abundances of their corresponding proteins. the level of a protein depends not only on transcription rates of the gene, but also on nuclear export and mrna localization, transcript stability, translational regulation and protein degradation. indeed, there is considerable variability on protein level versus mrna level. since biological processes are ultimately controlled by proteins, a proteomic analysis is necessary to get a better understanding of the plant responses to mg-deficiency. to our knowledge, little information is available on the changes of protein profile under mg-deficiency in plant roots and leaves. in china, mg-deficiency is frequently observed in citrus plantations and is responsible for the loss of productivity and poor fruit quality. although the effects of mg-deficiency on citrus co assimilation, photosynthetic electron transport and antioxidant system, carbohydrates and organic acid metabolism have been investigated by a few researchers, the knowledge of upstream targets is scarce. in the present study, we first investigated the proteomic responses of citrus leaves and roots to mg-deficiency using two-dimensional electrophoresis in order to enrich our understanding of the molecular mechanisms of plants to deal with mg-deficiency and understand the molecular mechanisms by which mg-deficiency lead to a decrease in co assimilation.  seedling growth, leaf, stem and root mg concentration plant treated with 0 mm mg displayed decreased leaf, stem and root dry weight and lower concentration of mg in leaves, stems and roots, and leaf mg concentration was much lower than the normal range. based on these results, plants that did not receive mg are considered mg-deficient, and those treated with 1 mm mg are considered mg-sufficient.figure effects of mg-deficiency on growth and mg concentration in leaves, stems and roots. leaf, stem and root dw. leaf, stem and root mg concentration. bars represent means ± se. different letters above the bars indicate a significant difference at p < . leaf gas exchange, leaf and root respiration and total soluble protein concentration compared with controls, mg-deficient leaves had lower co assimilation, stomatal conductance and transpiration, and higher intercellular co concentration.figure effects of mg-deficiency on leaf gas exchange, root respiration, and root and leaf total soluble protein. leaf co assimilation, stomatal conductance, intercellular co concentration, transpiration and respiration. leaf total soluble protein. root respiration. root total soluble protein. bars represent means ± se. different letters above the bars indicate a significant difference at p < . mg-deficient leaves displayed increased dark respiration and decreased concentration of total soluble proteins, while mg-deficient roots had decreased respiration and unchanged concentration of total soluble proteins. profiles of differentially expressed proteins 2-de was performed to compare the protein profiles between control and mg-deficient roots and leaves. in order to get credible results, the experiments were performed in biological replicates. after coomassie brilliant blue g- staining, more than clear and reproducible spots were detected on each gel. comparative analysis of the 2-de maps of control and mg-deficient leaves was performed by pdquest. software. a protein spot was considered differentially expressed when the protein had both a fold change of more than and a p-value less than. based on the two criteria, differentially expressed protein spots were detected in mg-deficient leaves with high confidence, of which displayed increased and displayed decreased level under mg-deficient condition; and differentially expressed protein spots were detected in mg-deficient roots with high confidence, including protein spots that were upregulated and protein spots that were downregulated by mg-deficiency.figure representative gel images of proteins in mg-deficient and control leaves. proteins were separated in the first dimension on an ipg strip ph 3– and in the second dimension on a 12% slab gel, followed by colloidal coomassie brilliant g- an equal amount of total protein extracts was loaded in each gel. representative gel images of proteins in mg-deficient and control roots. proteins were separated in the first dimension on an ipg strip ph 3– and in the second dimension on a 12% slab gel, followed by colloidal coomassie brilliant g- an equal amount of total protein extracts was loaded in each gel. all these differentially expressed protein spots were excised from the 2-de gels and submitted to maldi-tof/tof-ms and ltq-esi-ms/ms. in total, and protein spots were identified in mg-deficient leaves and roots, respectively. the database searching results are presented in tables  and according to the biological functional properties, these differentially expressed protein spots in mg-deficient leaves were classified into the following functional categories: carbohydrate and energy metabolism, protein metabolism, stress responses, nucleic acid metabolism, cell wall and cytoskeleton metabolism, cell transport, lipid metabolism, other and unknown biological processes; and theses protein spots in mg-deficient roots were involved in carbohydrate and energy metabolism, protein metabolism, stress responses, nucleic acid metabolism, cell wall and cytoskeleton metabolism, lipid metabolism, other and unknown biological processes.table list of differentially expressed proteins identified using maldi-tof/tof-ms in magnesium -deficient citrus sinensis leaves spot no. a accession no. b protein identity organism mw mp/sc score ratio c carbohydrate and energy metabolism severinia buxifolia nicotiana tabacum glycine max n. tabacum n. tabacum n. tabacum litchi chinensis panax ginseng solanum peruvianum glycine max nicotiana benthamiana n. benthamiana n. tabacum ricinus communis gossypium hirsutum arabidopsis thaliana a. thaliana spinacia oleracea r. communis citrus clementina r. communis citrus trifoliata a. thaliana citrus reticulata perilla frutescens oryza sativa japonica group a. thaliana c. sinensis populus tremula × populus alba r. communis dianthus caryophyllus protein metabolism ricinus communis pisum sativum plagiomnium affine trifolium subterraneum cucumis melo a. thaliana o. sativa japonica group a. thaliana platycodon grandiflorus p. grandiflorus r. communis a. thaliana a. thaliana a. thaliana brachypodium distachyon p. sativum p. sativum arabidopsis lyrata subsp. lyrata allium cepa stress responses citrus sinensis c. sinensis citrus maxima c. maxima hevea brasiliensis r. communis r. communis petunia x hybrida gossypium hirsutum lycopersicon peruvianum prunus salicina citrus sinensis citrus sinensis a. thaliana nucleic acid metabolism nepenthes sp. ‘kosobe’ oryza sativa japonica group a. thaliana r. communis cell wall and cytoskeleton metabolism adiantum capillus-veneris pueraria montana var. lobata olimarabidopsis pumila cell transport oryza sativa japonica group a. lyrata subsp. lyrata lipid metabolism oryza sativa japonica r. communis other and unknown biological processes luziola fluitans r. communis pseudotsuga menziesii c. sinensis oryza sativa japonica group vitis viniferai selaginella moellendorffii vitis vinifera hordeum vulgare subsp. vulgare v. vinifera v. vinifera mp/sc: number of matched peptides/sequence coverage percentage; mw: theoretical molecular weigh; a: spot number corresponds to the 2-de gel in figure  b: gi number is from ncbi database of matched protein. c: ratio means the ratio of mg-deficiency to control; means protein spots were only detected in control roots; means protein spots were only detected in the mg-deficient roots. list of differentially expressed proteins identified using maldi-tof/tof-ms in magnesium -deficient citrus sinensis roots spot no. a accession no. b protein identity organism mw mp/sc score ratio c carbohydrate and energy metabolism ricinus communis arabidopsis thaliana a. thaliana pisum sativum afrothismia gabonensis volvox carteri f. nagariensis dianthus caryophyllus hordeum vulgare subsp. vulgare citrus sinensis protein metabolism r. communis r. communis r. communis a. thaliana a. thaliana p. sativum p. sativum nicotiana benthamiana stress responses xerophyta viscosa brassica rapa subsp. pekinensis triticum aestivum nucleic acid metabolism beaucarnea recurvata zea mays lotus japonicus cell wall and cytoskeleton metabolism boehmeria nivea medicago sativa vitis vinifera lipid metabolism solanum tuberosum nicotiana tabacum other and unknown biological processes oryza sativa japonica grou vitis vinifera mp/sc: number of matched peptides/sequence coverage percentage; mw: theoretical molecular weigh; a: spot number corresponds to the 2-de gel in figure  b: gi number is from ncbi database of matched protein. c: ratio means the ratio of mg-deficiency to control; means protein spots were only detected in control roots; means protein spots were only detected in the mg-deficient roots. functional classification of the differentially expressed protein spots in leaves and roots. principal component analysis loading plots and correlation the majority of differentially expressed proteins involved in protein metabolism and stress responses were highly clustered under mg-deficient leaves. furthermore, the differentially expressed proteins associated with nucleic acid metabolism, cell transport, lipid metabolism, and cell wall and cytoskeleton metabolism were only plotted in mg-deficient leaves. however, the differentially expressed proteins in nucleic acid metabolism were clustered in control roots. the proteins in cell wall and cytoskeleton metabolism were only plotted in mg-deficient roots.figure pca loading plots of differentially expressed proteins in mg-deficient leaves and roots. the correlation coefficient matrix presented that the individual differentially expressed proteins were highly correlated within each categorized metabolism, regardless of tissues. in leaves, the majority of the differentially expressed proteins in carbohydrate and energy metabolism were positively correlated with each other but negatively with the other proteins in the other metabolisms. the most proteins in the other metabolisms but carbohydrate and energy metabolism were highly positively correlated with each other. in contrast, the differentially expressed proteins in roots did not show any clear pattern like those in leaves.figure pearson correlation coefficient matrix for the differentially expressed protein spots in mg-deficient leaves and roots. red and blue colors indicated positive and negative correlation between the differentially expressed proteins. transcriptional analysis of genes for some differentially expressed proteins to verify the changed expression in transcriptional level and evaluate the correlation between mrna and protein levels. the expression levels of genes for differentially expressed proteins from mg-deficient leaves and roots were analyzed by qrt-pcr. of the genes, the expression profiles of nine genes were well correlated with our 2-de data, meaning that the differentially expressed proteins are regulated at the transcriptional level. however, the transcript level changes of the remaining nine genes did not match the proteomic observations. indeed, the transcript levels of genes do not necessarily match the levels of their corresponding proteins, since the abundance of a protein depends not only on transcription rate of the gene expression alone. the discrepancy between the expression levels of the nine genes and the abundance of the corresponding protein suggests that post-translational modifications might influence the abundances of these proteins and alter the positions of these proteins on the gel.figure relative expression of genes from leaves and of five genes from roots. relative expression of nine leaf genes encoding cysteine proteinase; atp synthase cf alpha subunit, chloroplastic, triosephosphate isomerase, cu/zn superoxide dismutase, phosphoglycerate kinase, ascorbate peroxidase, s-adenosylmethionine synthetase, aconitate hydratase, nucleoside diphosphate kinase, putative. relative expression of four leaf genes encoding atykt, glyceraldehyde-3-phosphate dehydrogenase b subunit, alcohol dehydrogenase, and ribulose -bisphosphate carboxylase, partial, and five root genes encoding proteasome subunit alpha type, putative, cysteine protease, putative, pyruvate decarboxylase, putative, phosphoglycerate kinase, and hexokinase. bars represent means ± se. different letters above the bars indicate a significant difference at p < . all the values were expressed relative to the control leaves or roots. discussion mg-deficiency triggers different responses in leaves and roots our results showed that the amounts of differentially expressed proteins were much more in the mg-deficient leaves than in the mg-deficient roots, meaning that the effects of long-term mg-deficiency on protein profiles of leaves were more pronounced than on those of roots. this agrees with the reports in a. thaliana that the root transcriptome was less severely affected by long-term mg-deficiency, and that the responses of plants to mg-deficiency was asynchronized, with a lower number of differentially regulated genes in leaves or 8 h after the removal of mg from the nutrient solution and in roots 28 h after the removal of mg. this is also supported by our results that mg-deficiency decreased leaf concentration of total soluble proteins, but had no influence on root concentration of total soluble proteins. as shown in tables  and the majority of the differentially expressed proteins only presented in roots or leaves, only two differentially expressed proteins with the same gi number presented in both roots and leaves. in leaves, the abundances of putative ferredoxin-nadp reductase and atp synthase decreased and the levels of proteins involved in tca cycle as well as dark respiration were increased in response to mg-deficiency; whereas in roots, the levels of fnr and atpase α subunit, partial were upregulated and the abundance of pyruvate decarboxylase, putative as well as respiration was downregulated by mg-deficiency. in conclusion, there are many differences in mg-deficiency-induced changes in protein profiles as well as biochemical responses between leaves and roots. as shown in tables  and mg-deficiency did not alter the abundances of these proteins potentially involved in mediating mg transport such as the magnesium⁄proton exchanger and the mitochondrial rna splicing ⁄magnesium transporter family. this agrees with the reports that mg-deficiency did not induce the expression of genes associated with the transport of mg, with the only exception of the mrs2- being downregulated. some differentially expressed proteins in mg-deficient leaves and chaperonin precursor in mg-deficient roots were represented by more than one spot. this might reflect isozymes or ptms of a single protein. proteins involved in carbohydrate and energy metabolism a crucial role of mg is its involvement in the export of carbohydrates from source to sink sites. the accumulation of sugars in source leaves, an early symptom of mg-deficiency, usually precedes the decreases in photosynthetic rate and chl concentration. the accumulation of sugars in leaves may repress the expression of genes that encode photosynthetic enzymes, thus decreasing chl concentration and photosynthetic capacity. therefore, these proteins involved in photosynthesis and carbohydrate metabolism and related biological processes might be altered under mg-deficiency. as expected, many differentially expressed proteins related to carbohydrate and energy metabolism were found in mg-deficient leaves. our finding that mg-deficiency decreased the abundances of rubisco and rubisco activase agrees with the report that mg-deficiency reduced the activities of rubisco and rubisco activase and the expression levels of genes encoding rubisco large subunit, rubisco small subunit, and rubisco activase subunit in spinach leaves. mate et al. reported that transgenic tobacco plants expressing an antisense gene encoding rubisco activase reduced rubisco carbamylation, hence impairing photosynthesis. two studies with logan revealed that mg-deficiency-induced inhibition of photosynthesis might be associated with both reduced carboxylation efficiency and lower rate of ribulose- -bisphosphate regeneration. oxygen evolving enhancer proteins consist of three subunits, oee, oee and oee. these are nuclear-encoded chloroplast proteins, and peripherally bound to photosystem ii on the luminal side of the thylakoid membrane. oee is the most important protein for oxygen evolution and psii stability. mg-deficiency greatly decreased the level of oee in leaves. this means that mg-deficiency might impair the stability of oxygen evolution and psii, as found on mg-deficient c. sinensis and c. grandis. in addition, mg-deficient leaves had lower level of photosynthetic electron transfer-like protein, which agrees with the report that mg-deficiency decreased photosynthetic electron transport by impairing the whole photosynthetic electron transport chain from the psii donor side up to psi, thus decreasing citrus leaf co assimilation. hajirezaei et al. reported that a small decrease in the activity of fnr by antisense rna led to decreases in photosynthetic rate and nadph level, and increases in the extent of qa reduction and nadp level, and concluded that fnr was one of the rate-limiting steps in photosynthesis and fnr-deficiency-induced inhibition of photosynthesis was caused by impairment of fnr-mediated electron transfer from reduced ferredoxin to nadp. as shown in table  the level of putative fnr decreased in mg-deficient leaves. this means that fnr activity might be downregulated by mg-deficiency, thus impairing the photosynthetic electron transfer and increasing the extent of qa reduction, and hence inhibiting photosynthesis. palatnik et al. demonstrated that fnr-deficient plants were particularly prone to photoinhibitory damage and photooxidative injury. all these results agree the report that mg-deficiency decreased co assimilation, and increased the extent of qa reduction and the concentration of malondialdehyde in citrus leaves. the reduction of 3-phosphoglycerate to triose phosphate, which is reversibly catalyzed by two chloroplast enzymes, is a crucial step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism of the photosynthetic carbon-reduction cycle in the stroma. price et al. showed that antisense transgenic plants with severely reduced chloroplast gapdh activity were not photoinhibited despite the continuous presence of a large thylakoid proton gradient in the light, and that the electron-transport chain did not become over-reduced due to a shortage of nadp despite the downregulation of photosynthetic electron transport due to the build-up of a large proton gradient. the observed lower level of gapdh b subunit might be helpful to lessen photoinhibition and the over-reduction of photosynthetic electron transport, thus lowering photooxidative damage caused by mg-deficiency. in addition, mg-deficiency decreased leaf abundance of pgk. therefore, the reduction of 3-phosphoglycerate to triose phosphate might be decreased in mg-deficient leaves due to lower activities of chloroplast gadph and pgk. mg-deficiency also reduced leaf levels of putative carbonic anhydrase, triosephosphate isomerase, fructose-bisphosphate aldolase, class i and putative chloroplastic lactoylglutathione lyase. carbonic anhydrase, which reversibly catalyzes the interconversion of co and hco3−, is a major protein constituent of the c higher plant chloroplast. stimler et al. showed that stomatal response was mediated by carbonic anhydrase, and that h2s formed in the mesophyll via the reaction of carbonyl sulfide and water with carbonic anhydrase was involved in the stomatal response. the observed lower abundance of carbonic anhydrase means that its activity might be downregulated in mg-deficient leaves, thus decreasing the stomatal conductance. however, in addition to stomatal factor, non-stomatal factors such as photosynthetic inhibition also contributed to the lower co assimilation in mg-deficient leaves, as indicated by the increased intercellular concentration. ito et al. showed that triosephosphate isomerase and putative plastidic aldolase were inactivated by oxidized glutathione and reactivated by reduced glutathione. this agrees with the report that mg-deficient citrus leaves had increased gssg concentration, but decreased gsh level. haake et al. reported that a moderate decrease of plastidic aldolase activity led to a decrease in photosynthesis in antisense transgenic potato plants. lactoylglutathione lyase catalyzes the formation of s-d-lactoylglutathione in the presence of glutathione and methylglyoxal, a cytotoxic compound produced spontaneously under physiological conditions from the glycolysis and photosynthesis intermediates glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. besides detoxification of methylglyoxal, the glyoxalase system could also play a role in oxidative stress tolerance by recycling gsh and maintaining glutathione homeostasis. the observed lower level of putative chloroplastic lactoylglutathione lyase in mg-deficient leaves agrees with our previous report that mg-deficiency increased gssg concentration and decreased gsh concentration in citrus leaves. to conclude, mg-deficiency decreased the levels of many proteins involved in photosynthesis including rubisco, rubisco activase, oee photosynthetic electron transfer-like protein, fnr, aldolase, thus decreasing leaf photosynthesis. we found that mg-deficiency increased the abundances of nadp-malate dehydrogenase, putative succinate dehydrogenase and aconitate hydratase in c. sinensis leaves, which agrees with our data that mg-deficient leaves had higher rate of dark respiration and the report that the activities of enzymes related to glycolysis and tca cycle were enhanced in mg-deficient c. sinensis leaves. nadp-malate dehydrogenase is the key enzyme in the malate/oxalacetate shuttle, which is the major machinery for the transport of excess reducing equivalents generated in chloroplasts to mitochondria. nadp-malate dehydrogenase converts oxalacetate to malate using nadph, facilitating the regeneration of the electron acceptor nadp in the chloroplasts, particularly when co assimilation is restricted. the observed higher level of nadp-malate dehydrogenase in mg-deficient leaves might contribute to the transport of excess reducing equivalents from chloroplasts to mitochondria and the regeneration of chloroplast nadp, thus preventing oxidative damage and enhancing the tolerance of plant to mg-deficiency. previous study showed that antisense inhibition of the iron-sulphur subunit of sdh and aco reduced the carbon flow through the tca cycle, and enhanced the photosynthesis and stomatal conductance. this agrees with our data that mg-deficient leaves had increased dark respiration, levels of sdh and aco, and decreased co assimilation and stomatal conductance. also, the abundances of putative dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase related to tca cycle, and 2-phospho-d-glycerate hydrolase and -bisphosphoglycerate-independent phosphoglycerate mutase involved in glycolysis in leaves increased in response to mg-deficiency. our finding that mg-deficient leaves had increased abundance of α-amylase, a starch degrading enzyme agrees with the report that the activities of two starch degrading enzymes increased in mg-deficient spruce needles. thus, starch degradation might be upregulated in mg-deficient c. sinensis leaves. however, mg-deficiency had little influence on starch concentration in c. sinensis leaves. this might be related to the fact that sucrose biosynthesis and export decreased in response to mg-deficiency. also, the upregulation of starch biosynthesis was not ruled out, because the abundance of adp-glucose pyrophosphorylase catalytic subunit increased in mg-deficient leaves. thus, it is reasonable to assume that the rate of starch turnover in mg-deficient leaves might be enhanced. mg-deficient leaves had lower levels of putative atp synthase β subunit, atp syntase γ chain, chloroplastic atp synthase cf α subunit and chloroplastic atp synthase subunit α. this means that atp synthesis catalyzed by atp synthase in mg-deficient leaves might be decreased, which might provide an advantage to prevent energy surplus due to increased dark respiration and the generation of excess reducing equivalents in chloroplasts. adenosine kinase, which catalyzes the reaction: adenosine + atp → amp + adp, is an essential component for maintaining purine nucleotide pools in arabidopsis. it also contributes to cytokinin interconversion in arabidopsis and plays a key role in sustaining transmethylation reactions by serving as a coarse metabolic control to reduce the concentration of free adenosine in spinach and sugar beet cells during salt stress. our finding that the abundance of putative adenosine kinase in leaves increased in response to mg-deficiency agrees with the report that adenosine kinase activity, protein and transcripts in spinach and sugar beet leaves were enhanced by salt stress. nucleoside diphosphate kinase is a ubiquitous housekeeping enzyme that maintains the intracellular levels of all ntps used in biosynthesis except for atp. in plants, ndpk is known to regulate the expression of antioxidant genes. transgenic potato, sweet potato and arabidopsis plants overexpressing arabidopsis ndpk had increased activities of antioxidant enzymes and enhanced tolerance to methylviologen, salt and temperature stresses. aldehydes, which lead to a rapid and excessive accumulation of ros in plants, may be induced by various abiotic stresses. alcohol dehydrogenase, which converts aldehydes into alcohols, is essential for plants to survive under anaerobic conditions. adh gene and/or protein are induced by aba, salt, desiccation, high and low temperatures in various plants. it has been suggested that the induction of adh by these stresses may trigger a signal transduction cascade, which would lead to a decrease in membrane damage, through adapting plants to oxidative stress. therefore, the antioxidant capacity might be upregulated in mg-deficient leaves due to the increased level of putative ndpk and adh, hence enhancing the mg-deficiency tolerance. this agrees with our report that mg-deficient c. sinensis leaves had higher activities of ascorbate peroxidase, superoxide dismutase, glutathione reductase, dehydroascorbate reductase and guaiacol peroxidase. unlike to leaves, mg-deficiency decreased root respiration and the activities of enzymes related to glycolysis and tca cycle. as expected, mg-deficiency decreased the abundances of putative pyruvate decarboxylase and pgk in roots. however, root level of pyruvate dehydrogenase e α subunit increased in response to mg-deficiency. in contrast to leaf putative fnr, the level of root fnr was increased by mg-deficiency. in higher plants, there are two forms of fnr, a photosynthetic fnr and a heterotrophic fnr. onda et al. revealed that the interaction of root fnr with ferredoxin is crucial for an efficient electron flux of nadph-fnr-fd cascade, thus supporting fd-dependent metabolism in non-photosynthetic organs. xu et al. showed that the abundance of a fnr precursor was reduced by 60% in the roots of null transformant plants, but maintained in transgenic lines expressing the adenine isopentenyl transferase gene under heat stress, and concluded that the maintenance of fnr level under heat stress could reflect a superior n assimilation capacity of the transgenic lines over the nt line that facilitates their growth under heat stress. thus, the observed higher level of fnr in mg-deficient roots might be an adaptive response. the biosynthesis of atp in mg-deficient roots might be enhanced due to increased level of mitochondrial atpase α subunit, partial. adenylate kinase catalyses the reversible formation of adp by the transfer of one phosphate group from atp to amp, thus equilibrating adenylates. thus, the increased abundances of the two proteins might be advantage to maintaining energy balance in mg-deficient roots, when atp production was reduced due to decreased respiration. like leaves, the level of root adh increased in response to mg-deficiency. similar result has been obtained on b-deficient c. sinensis roots. similar to leaf α-amylase, the activity of β-amylase might be upregulated in mg-deficient roots due to enhanced abundance of β-amylase, thus increasing the breakdown of starch into sugars in roots. this agrees with our report that mg-deficiency decreased the concentration of starch in citrus roots. hexokinases phosphorylate glucose to produce glucose-6-phosphate, the first step of glycolysis. our finding that mg-deficient roots had higher level of hxk compared with controls agrees with the reports that hxks contributed to the survival of acclimated maize root tips via hypoxic pretreatment by allowing the maintenance of a sustained glycolytic rate, and that oshxk, localized at the cytosol, was upregulated under sugar-starvation conditions but downregulated in response to high sugar treatments of the leaf tissues of rice, because mg-deficiency decreased or did not affect the concentrations of non-structural carbohydrates in c. sinensis roots. also, hxks might act as a glucose sensor and, through its catalytic activity, as a crucial regulator of ros levels in plant organelles such as mitochondria and chloroplasts. proteins involved in stress responses because leaf co assimilation decreased in response to mg-deficiency, less of the absorbed light energy was utilized in photosynthetic electron transport, particularly under high light. the excess absorbed photon flux can potentially lead to ros production. thus, ros production might be enhanced in mg-deficient leaves. to minimize the cellular damage caused by ros, plants have evolved an antioxidant system composed of antioxidants and antioxidant enzymes. as expected, the leaf levels of antioxidant enzymes increased in response to mg-deficiency except for chloroplast cu/zn sod. this agrees with the report that mg-deficient citrus leaves had higher or similar activities of antioxidant enxymes except for catalase. aldo-keto reductases, which catalyze the reduction of aldehyde to alcohol, are effective detoxification of peroxidation-derived reactive aldehydes. transgenic tobacco plants overexpressing alfalfa or rice ark had enhanced tolerance against a variety of oxidative damages induced by methylviologen, uv-b irradiation, heavy metals, drought, heat treatment, osmotic and salt stresses. the upregulation of the putative akr in mg-deficient leaves might contribute to the oxidative tolerance of leaves. in addition, mg-deficiency increased the leaf abundances of heat shock proteins, which play a key role in protecting plants against stress by reestablishing normal protein conformation and thus cellular homeostasis, and stress-related protein. based on these results, we concluded that proteins associated with stress-response functions were upregulated in mg-deficient leaves. however, the level of late embryogenesis abundant protein in leaves decreased in response to mg-deficiency. like to leaves, the levels of stress-responsive proteins in roots increased in response to mg-deficiency. this agrees with the reports that b-deficiency increased the level of nb-arc domain-containing disease resistance protein in c. sinensis roots, and that the abundance of grp in xerophyta viscosa plants increased in response to heat and dehydration. however, the abundances of mitochondrial heat shock 70 kda protein and heat shock protein hsp were decreased in mg-deficient roots, which agrees with the studies that b-deficiency decreased the levels of several hsps in c. sinensis roots and the abundances of three hsps in lupinus albus roots. proteins involved in protein metabolism protein synthesis elongation factor tu plays a central role in the elongation phase of protein synthesis in bacteria and organelles including mitochondria and plastids in plants. we found that the abundance of the putative enongation factor tu in leaves decreased in response to mg-deficiency, meaning that the biosynthesis of some proteins in plastids and mitochondria might be downregulated. this agrees with our results that the levels of many chloroplastic proteins was decreased by mg-deficiency, and that the concentration of total soluble proteins in leaves decreased in response to mg-deficiency. however, the levels of chloroplastic chaperonin precursor, chloroplastic small ribosomal protein partial and ribosomal protein s related to protein folding and biosynthesis were increased in mg-deficient leaves. plant proteases play key roles in maintaining strict protein quality control and degrading specific sets of proteins in response to environmental and developmental stimuli. the majority of the thousand or more proteins that are present in mitochondria are required to be imported from nuclear-encoded cytosolically synthesized precursors, and a number of peptidases are needed to remove the “transient” targeting information present on many, but not all, mitochondrial precursor proteins. the upregulation of mitochondrial processing peptidase β subunit in mg-deficient leaves agreed with the increased requirement for removing the “transient” targeting information due to enhanced import resulting from the increased abundance of the putative mitochondrial import inner membrane translocase subunit tim17/tim22/tim family protein. ubiquitination, which serves as a versatile ptm, plays a key role in regulating plant response to abiotic stresses. it has been known that the f-box is a motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction, and that the ring zinc-finger domain is essential for the function of ubiquitin e ligases. the upregulation of f-box domain-containing protein, putative f-box family protein, expressed, putative f-box/kelch-repeat protein and putative ring zinc finger protein in mg-deficient leaves means that leaf ubiquitination might be enhanced in response to mg-deficiency. also, the abundances of proteins related to protein degradation increased in mg-deficient leaves. in conclusion, proteolysis might be enhanced in mg-deficient leaves, thus lowering leaf concentration of total soluble proteins. however, the abundances of leaf f2e and putative skp protein decreased in response to mg-deficiency. amino acids are the structural units that make up proteins and also serve as precursors for many metabolites with multiple functions in plant growth and response to various stresses. all the four differentially expressed proteins involved in amino acid metabolism were upregulated in mg-deficient leaves, meaning that the metabolism of amino acids might be upregulated in mg-deficient leaves. glutathione s-transferases, which catalyze the conjugation of cytotoxic metabolites with the gsh, are mainly involved in detoxifying oxidative-stress metabolites. glutamine synthetase catalyses the critical incorporation of inorganic ammonium into the amino acid glutamine. in plants, two types of gs isozymes, located in the cytosol and in the chloroplast have been found. transgenic plants overexpressing gs or gs displayed enhanced tolerance to water or salt stress, respectively. methylation induced by abiotic stress has been supposed to be linked with the numerous biochemical pathways involved in acclimatization and stress response in plants. s-adenosylmethionine is an important methyl donor for numerous transmethylation reactions. s-adenosylmethionine synthase is a crucial enzyme that directs the flux of methione to s-adenosylmethionine. these results indicate that the upregulation of amino acid metabolism in mg-deficient leaves might be involved in the adaption of mg-deficient plants. proteolytic cleavage of proteins by proteases is not limited to the total degradation of mature proteins to free amino acids, but also is relevant for modification and maturation of proteins. like to leaves, mg-deficiency increased the abundances of root proteins involved in proteolysis. however, there was no significant difference in total soluble protein concentration between mg-deficient and control roots. this implies that the mg-deficiency-induced increase in protease levels may be mainly involved in modification and maturation of proteins or that the biosynthesis is enhanced in mg-deficient roots. the upregulation of proteases in mg-deficient roots might be an adaptive response of plants to mg-deficiency through maintaining the protein complexes and/or the recycling of n. the eukaryotic translation initiation factor has multiple roles during the initiation of translation of cytoplasmic mrnas. the classic functions ascribed to eif include: facilitating the charging of the 40s ribosomal subunit with the ternary complex; bridging between the 40s ribosomal subunit and the eif4g subunit of the cap-binding complex, eif4f; and inhibiting the association of 40s and 60s ribosomal subunits. the upregulation of eif 3b- in mg-deficient roots might contribute to the translatability of cytoplasmic mrnas, thus maintaining the level of cytoplasmic proteins, which agrees with our result that mg-deficiency did not significantly affect root concentration of total soluble proteins. however, the levels of root proteins involved in protein folding and biosynthesis were decreased by mg-deficiency. proteins involved in nucleic acid metabolism the downregulation of rna polymerase β chain, spliceosome rna helicase bat and transcription factor homolog btf3-like protein in mg-deficient roots agrees with our report that all differentially expressed proteins related to nucleic acid metabolism were reduced in b-deficient c. sinensis roots except for argonaute family protein. this indicates that the synthesis of rna might be inhibited in mg-deficient roots. by contrast, the abundances of all the four altered proteins related to nucleic acid metabolism in leaves increased in response to mg-deficiency, meaning that the metabolism of nucleic acid might be upregulated in mg-deficient leaves. dead box rna helicases are prominent candidates for rna chaperones because these proteins can use energy derived from atp hydrolysis to actively disrupt misfolded rna structures so that correct folding can occur, and play important roles in plant stress responses. transgenic arabidopsis plants overexpressing a rice gene osbirh which encodes a dead-box rna helicase protein, showed enhanced disease resistance against alternaria brassicicola and pseudomonas syringae, and increased tolerance to oxidative stress and elevated expression levels of oxidative defense genes. the upregulation of dead box rna helicases in mg-deficient leaves might be an adaptive response of plants to mg-deficiency. in nonplant systems, the splicing of group-ii introns is mediated by proteins encoded within the introns themselves, whereas only a single maturase orf has retained in the mitochondrial genomes in plants. interestingly, higher plant genomes contain four maturase-related genes, which exist in the nucleus as self-standing orfs. recently, keren et al. showed that atnmat a nuclear-encoded maturase, was required for splicing of group-ii introns in arabidopsis mitochondria. in another study, keren et al. observed that nmat functions were required for mitochondrial biogenesis and that nmat mutants displayed growth and developmental defect phenotypes and accumulated high levels of ros, concluding that nmat was essential for mitochondrial complex i assembly and function. thus, the observed higher level of maturase, partial in mg-deficient leaves might be an adaptive response. proteins involved in cell wall and cytoskeleton metabolism ca, a constituent of the cell wall, plays a key role in determining the structural rigidity of the cell wall. also, ca is a regulator that can exert multiple effects on the structure and dynamics of the actin cytoskeleton. it has been well known that high rhizosphere concentration of mg, relative to ca, is inhibitory to the absorption of ca and vice versa. therefore, the concentration of ca in mg-deficient plant tissues should be enhanced. indeed, mg-deficiency increased the concentration of ca in rough-lemon leaves. based on these results, it is reasonable to assume that the mg-deficiency-induced increase in ca concentration might be responsible for the enhanced abundances of myosin class 11–, ugt and pollen coat oleosin-glycine rich protein involved in cell wall and cytoskeleton metabolism in mg-deficient leaves. similarly, the abundances of root actin, villin and tubulin γ- chain were upregulated by mg-deficiency, which is in agreements with the report that mg-deficiency led to enhanced suberization in endodermis and hypodermis of corn roots. the upregulation of proteins related to cell wall and cytoskeleton metabolism in mg-deficient roots and leaves might be an adaptive response of plants to mg-deficiency. proteins involved in cell transport proteins that reside in endomembrane organelles, or that are secreted from the cell, reach their sites of function by transport of the endomembrane system. the translocase of the inner membrane is a complex of proteins found in the inner membrane of the mitochondria and plays a pivotal role in protein import, which is important to cope with stress. the observed higher level of the putative mitochondrial import inner membrane translocase subunit tim17/tim22/tim family protein in mg-deficient leaves can be explained by the fact that mitochondria are the center of oxygen sensing. under mg-deficiency, high protein import might be required for the increased ros detoxification, thus requiring high activity of the tim protein complex. snares are core proteins for vesicle fusion and can be categorized into two types: snares on the vesicle are called v-snares, while snares on the target membrane are called t-snares. atykt and atykt two t-snares, are essential for membrane fusion mediated by either syp or stp. the upregulation of the two proteins means that the transport of proteins might be enhanced in mg-deficient roots. proteins involved in lipid metabolism sato-izawa et al. revealed that the inositol-,5-trisphosphate 5-phosphatase, an enzyme probably involved in phosphoinositide signaling, was required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. perera et al. showed that transgenic arabidopsis plants expressing the type inositol-,5-trisphosphate 5-phosphatase had enhanced drought tolerance and altered aba signaling. cytochrome p450s, a superfamily of ubiquitous heme-containing mixed-function monooxygenases proteins, play a key role in biotic and abiotic stresses. in arabidopsis, at least five of the cytop450s are induced by abiotic and biotic stresses including alternaria brassicicola or alternaria alternata, paraquat, rose bengal, uv-c stress, heavy metal stress, mechanical wounding, drought, high salinity, low temperature or hormones. transgenic tobacco and potato plants expressing cytp with increased monooxygenase activity tolerated better oxidative stress after herbicide treatment. therefore, the observed higher level of putative type inositol-,5-trisphosphate 5-phosphatase cvp and putative cytop might contribute to mg-deficiency tolerance of plants. we found that the abundance of root lipoxygenase increased in response to mg-deficiency, which is in agreement with the report that the level of lox was enhanced in b-deficient c. sinensis roots. lox, which catalyses the oxidation of α-linolenic acid into either 9- or 13-hydroperoxy-octadecatrienoic acids, or a mixture of both, is one of the key enzymes responsible for the biosynthesis of jasmonates, which are ubiquitously occurring lipid-derived compounds with signal functions in plant responses to abiotic and biotic stresses. hu et al. demonstrated that tomloxd, a tomato 13-lox gene, was involved in endogenous jasmonate synthesis and tolerance to biotic and abiotic stress. therefore, both jasmonate biosynthesis and level might be increased in mg-deficient roots, thus enhancing plant mg-deficiency tolerance. this inference is also supported by the observation that the expression levels of two genes encoding myc which is a jasmonate-dependent transcription factor and lox which is an enzyme involved in jasmonate biosynthesis in roots increased in response to mg-deficiency. thus, jasmonate signaling might be involved in the responses of citrus roots to mg-deficiency. however, the expression of the gene encoding zim/tify-domain, which is the co-repressor of myc that is ubiquitinilated upon activation of jasmonate signaling cascade, did not change in mg-deficient citrus roots. the plastid acetyl-coenzyme a carboxylase, which catalyzes the first committed step of fatty acid synthesis, is present as a heteromeric complex of at least four different protein subunits: the biotin carboxylase, the biotin carboxyl carrier protein, and the α and β subunits of the carboxyltransferase in most plants. transgenic tobacco plants with less than 25% of wild-type biotin carboxylase levels displayed severely retarded growth when grown under low-light conditions and a 26% lower leaf fatty acid concentration than wild-type plants. thus, the biosynthesis of fatty acids in mg-deficient roots might be elevated due to enhanced level of biotin carboxylase subunit. other proteins ent-kaurene is a tetracyclic hydrocarbon precursor for gibberellins in plants. in higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase and ent-kaurene synthase from geranylgeranyl diphosphate. a range of ga-deficient phenotypes of the ga1- and ga2- a. thaliana mutants were restored to wild type, when fungal cps/ks was overexpressed and targeted to plastids. the over-expressing plants emitted ent-kaurene into the headspace besides its accumulation in the plant body. fleet et al. showed that transgenic arabidopsis plants overexpressing atcps and atks led to increased ent-kaurene production but unchanged concentration of active gas. therefore, the upregulation of ent-kaurene synthase in mg-deficient leaves does not necessarily imply that leaf concentration of gas was enhanced by mg-deficiency.  we first investigated the proteomic changes induced by long-term mg-deficiency in c. sinensis leaves and roots using 2-de. in mg-deficient leaves, upregulated and downregulated proteins were isolated, while only upregulated and downregulated proteins in mg-deficient roots. this indicated that proteomes were more affected by long-term mg-deficiency in the leaves than in the roots, which was further supported by our observation that the concentration of total soluble proteins was decreased by mg-deficiency in leaves, but unaffected in roots. a potential regulatory network of mg-deficiency-induced responses in citrus leaves and roots was proposed through the integration of the present results and available data in the literatures. mg-deficiency led to decreased abundances of proteins involved in photosyntheis, thus decreasing leaf co assimilation. the adaptive responses of c. sinensis roots and leaves to mg-deficiency might including several aspects: improving leaf respiration and lowering root respiration, but increasing the levels of proteins related to atp synthase in roots; enhancing the levels of proteins involved in 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. therefore, our proteomic analysis provides an integrated view of the adaptive responses occurring in mg-deficient leaves and roots of c. sinensis. as a first attempt, the present study will be useful for further investigating the roles of mg in higher plants. it is worth noting that it may provide more data on mg-deficiency in real citrus orchards if we use grafted plants rather than c. sinensis seedlings as experimental materials, but it is difficult for us to compare the present data with the transcriptomic data obtained on arabidopsis roots and leaves and the physiological and biochemical data obtained on c. sinensis roots and leaves. in the further study, we will investigate the effects of rootstocks on mg-deficiency-responsive proteomics using grafted citrus plants from different rootstock-scion combinations including both own-rooted scions and rootstocks as controls to obtain more knowledge on mg-deficiency in real citrus orchards.figure the potential regulatory network of mg-deficiency-induced responses in citrus leaves and roots. bpg-pgam: -bisphosphoglycerate-independent phosphoglycerate mutase 1; cvp2: type i inositol-,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/tim family protein. 
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. 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 the levels of proteins related to atp synthase in roots; enhancing the levels of proteins involved in reactive oxygen species 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. 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. magnesium -deficiency is frequently observed in citrus plantations and is responsible for the loss of productivity and poor fruit quality. fifty-nine upregulated and downregulated protein spots were isolated in mg-deficient leaves, while only upregulated and downregulated protein spots in mg-deficient roots. mg-deficiency had decreased levels of proteins involved in photosynthesis, thus decreasing leaf photosynthesis. mg-deficiency decreased the levels of many proteins involved in photosynthesis, thus decreasing leaf photosynthesis. thereafter, we first investigated the proteomic responses of c. sinensis roots and leaves to mg-deficiency using two-dimensional electrophoresis in order to enrich our understanding of the molecular mechanisms of plants to deal with mg-deficiency and understand the molecular mechanisms by which mg-deficiency lead to a decrease in photosynthesis. 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. knowledge on the effects of mg-deficiency on upstream targets is scarce.
 magnesium -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’ were irrigated with mg-deficient or mg-sufficient 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 in order to enrich our understanding of the molecular mechanisms of plants to deal with mg-deficiency and understand the molecular mechanisms by which mg-deficiency lead to a decrease in photosynthesis.  fifty-nine upregulated and downregulated protein spots were isolated in mg-deficient leaves, while only upregulated and 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 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 the levels of proteins related to atp synthase in roots; enhancing the levels of proteins involved in reactive oxygen species 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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords citrus sinensismagnesium -deficiencyphotosynthesisproteomicsrespirationreactive oxygen speciesissue-copyright-statement© the author 2015 