BACKGROUND
in many acidic soils through the tropics and subtropics, aluminum -toxicity is a major factor limiting crop productivity.
approximately 30 % of the world’s total land area is acidic  <cit> .
furthermore, the acidity of the soils is increasing due to the environmental problems including some farming practices and acid rain  <cit> .
when soil ph is less than  <dig> , toxic forms of al  are solubilized into the soil solution and accumulate to high concentration that rapidly inhibits plant root growth by damaging the roots functionally and structurally, subsequently decreasing nutrient and water uptake, eventually resulting in poor crop growth and productivity  <cit> .
a traditional strategy is to raise soil ph by application of lime, thus alleviating al-toxicity; however, the approach is both costly and ecologically unsound from the long-term point of view.
a more efficient strategy is to breed al-tolerant crop cultivars.
fortunately, plants display wide variation in their ability to deal with al-toxicity.
variation in al-tolerance makes it possible to breed tolerant cultivars.
the success of breeding programs relies on an understanding of the physiological, biochemical and molecular mechanisms that plants tolerate al-toxicity.
since biological processes are ultimately controlled by proteins, identification and characterization of al-tolerant proteins will not only increase our understanding of the molecular mechanisms on plant al-tolerance, but also will provide new information that researchers will use to screen and breed crop cultivars suited for acidic soils with higher active al.
during the long-term evolutionary process, higher plants have evolved two main strategies  that enable them to tolerate high level of soil active al  <cit> .
although several mechanisms for the external detoxification  have been suggested, the al-induced secretion of organic acid anions such as citrate, malate and oxalate from roots is the most-studied mechanism of al-tolerance in higher plants  <cit> .
genes involved in al-induced root secretion of citrate and malate have been isolated from wheat , sorghum  and barley .
transgenic plants over-expressing these genes displayed increased al-activated root secretion of malate and/or citrate and enhanced al-tolerance  <cit> .
internal detoxification is mainly reached by both complexation and sequestration of al  <cit> .
gene expression networks revealed by transcriptomics offer us the opportunity to understand the molecular mechanisms of plant al-toxicity and -tolerance.
although al-induced changes in gene expressions have been examined in some detail , limited data are available on al-toxicity-responsive proteins in higher plants.
alteration of plant proteins is an inevitable process to deal with environmental stresses including al-toxicity  <cit> .
proteomics is a very powerful tool for analyzing the functions of the plant proteins.
abundance of a protein is not only regulated at transcriptional, but also at translational and post-translational levels.
therefore, proteomic analysis may provide more accurate and comprehensive data than what genomic studies can provide.
recently, there were several studies reporting al-induced changes in plant proteins using two-dimensional gel electrophoresis  or isobaric tags for relative and absolute quantification  technique.
study with al-tolerant rice  cultivar showed that root abundances of copper/zinc superoxide dismutase , s-adenosylmethionine synthetase, cysteine  synthase, 1-aminocydopropane-1-carboxylate oxidase , glutathione s-transferase  increased in response to al-toxicity, and that cys synthase might play a key role in al-tolerance  <cit> .
in al-tolerant soybean cultivar roots, al induced the production of heat shock proteins , gst, chalcone-related synthetase, gtp-binding protein, and atp binding cassette  transporter atp-binding protein  <cit> .
duressa et al.
<cit>  observed that al resulted in a distinct protein profile changes in al-tolerant and al-sensitive soybean genotypes.
in tomato roots, dehydroascorbate reductase , glutathione reductase , catalase , mitochondrial aldehyde dehydrogenase , catechol oxidase, quinone reductase, and lactoylglutathione lyase involved in antioxidation and detoxification were induced by al  <cit> .
dai et al.
<cit>  isolated  <dig> proteins associated with al-tolerance in al-tolerant wild barley xz <dig>  there were  <dig> proteins, which were up-regulated in xz <dig> roots, but down-regulated or unaltered in both al-tolerant barley cultivar dayton and al-sensitive wild barley xz <dig>  oh et al.
<cit>  identified  <dig> up-regulated protein spots such as s-adenosylmethionine, oxalate oxidase , malate dehydrogenase , cys synthase, ascorbate peroxidase  and  <dig> down-regulated protein spots such as hsp  <dig>  o-methytransferase  <dig>  enolase and amylogenin in + al wheat roots.
wang et al.
<cit>  identified  <dig> al-toxicity-responsive proteins from al-sensitive and -tolerant rice roots.
they observed that glycolysis/gluconeogenesis was the most significantly up-regulated biochemical process in al-toxic roots.
to our knowledge, data available on the effects of al-toxicity on root proteomics of woody plants are very limited.
citrus spp.
are cultivated mainly in acidic and strong acidic soils such as red soil, yellow soil or lateritic red soil.
in  <dig>  li et al.
<cit>  investigated the ph of  <dig> ‘guanximiyou’ pummelo  orchard soils from pinghe county, zhangzhou, china.
up to 90 % of soils had a lower ph than  <dig> .
low ph and high al are the factors contributing to poor citrus growth and shortened lifespan of trees  <cit> .
in this study, we compared al-induced quantitative and qualitative changes in proteomes that occurred in roots of al-tolerant citrus sinensis and al-sensitive c. grandis seedlings  <cit>  using itraq technique in order to identify differentially expressed proteins involved in al-tolerance and to understasnd the molecular mechanisms of plants to deal with al-toxicity.
RESULTS
plant growth and al concentration in roots
as shown in fig.
1a-c,  <dig>  mm al-treated  c. grandis seedings displayed decreased whole plant and shoot dry weights  and unchanged root dw, compared with controls, while al-toxicity did not significantly affect c. sinensis whole plant, shoot and root dws.
there was no significant difference in whole plant, shoot and root dws between the two species at each given al treatment.
root concentration of al was higher in + al seedlings than controls, while did not significantly differ between the two species at each given al treatment .fig.
<dig> effects of al-toxicity on plant growth and root al concentration in c. sinensis and c. grandis seedlings.
a-c whole plant, shoot and root dry weights .
d root al concentration.
bar represents the mean ± se .
differences among four treatment combinations were analyzed by  <dig>  ×  <dig>  anova.
different letters above the bars indicate a significant difference at p <  <dig>
it is worth mentioning that  <dig>  mm al treatment decreased c. grandis root dw, but had no influence on c. sinensis root dw and that al-toxicity-induced decreases in whole plant and shoot dws were more severe in c. grandis than in c. sinensis seedlings, when 20 μm h3bo <dig> in nutrient solution was replaced by  <dig>  μm h3bo <dig>  in addition, al concentration was not lower in c. sinensis than in c. grandis roots at each given al level .
primary data analysis and protein identification
a total of  <dig>  <dig> spectra were produced from the itraq experiment using control and al-toxic c. sinensis and c. grandis roots as materials.
by analyzing these spectra, we identified  <dig>  <dig> known spectra,  <dig>  <dig> unique spectra,  <dig>  <dig> peptides,  <dig>  <dig> unique peptides and  <dig> proteins, respectively .
the number of proteins in the genome is  <dig> , due to the reasons of samples and technical restrain,  <dig> identified proteins in current proteomic research is normal in plants.
protein number decreased with increased number of peptides that matched to proteins, but over 67 % of the proteins  included at least two peptides .fig.
<dig> spectra, peptides and proteins identified from itraq proteomics by searching against c. sinensis database , number of peptides that match to proteins using mascot , protein mass distribution , distribution of protein sequence coverage , and distribution of peptide length
both protein mass distribution and distribution of sequence coverage were summarized in fig.
2c and d. proteins with 20–30 kda and 30–40 kda were at the most abundant, followed by proteins with 40–50 kda and 50–60 kda.
protein number decreased with increased sequence coverage.
figure 2e shows the distribution of peptide length in citrus roots.
less than  <dig>  % of all peptides are ≥  <dig> amino acid residues and  <dig>  % are between  <dig> and  <dig> residues.
differentially expressed proteins by itraq
in this study, a protein was considered differentially expressed when the protein had both a log <dig> fold-change of more than  <dig>  and a p-value of less than  <dig> .
based on the two criteria,  <dig> differentially expressed proteins were detected in + al c. sinensis roots,  <dig>  of which displayed increased and  <dig>  displayed decreased abundance.
these proteins were related to sulfur  metabolism, stress and defense response, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, cell transport, biological regulation and signal transduction, cell wall and cytoskeleton metabolism, jasmonic acid  biosynthesis and others .
in + al c. grandis roots, we isolated  <dig>  up- and  <dig>  down-regulated proteins, which were grouped into following functional categories: s metabolism, stress and defense response, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, cell transport, biological regulation and signal transduction, cell wall and cytoskeleton metabolism and others .
figures  <dig> and  <dig> showed al-induced alterations of proteins involved in s metabolism in c. sinensis and c. grandis roots and ja biosynthesis in c. sinensis roots, respectively.fig.
<dig> classification of al-induced differentially expressed proteins in c. sinensis  and c. grandis  roots and venn diagram analysis of differentially expressed proteins in citrus roots .
among these overlapping proteins,  <dig>  proteins were regulated in the same  direction upon al exposure
fig.
<dig> al-induced changes in s metabolism in c. sinensis and c. grandis roots.
up-regulated proteins presented in + al c. sinensis and c. grandis  roots were labeled in red .
abbreviations: aps, adenosine phosphosulphate; gsh, reduced glutathione; gssg, oxidized glutathione
fig.
<dig> metabolic scheme of ja biosynthesis in citrus sinensis roots.
up-regulated proteins in + al roots were labeled in red.
abbreviations: lox, lipoxygenase; 13-hpot, -hydroperoxyoctadecatrienoic acid; aos, allene oxide synthase;  <dig> -eot,  <dig> -epoxyoctadecatrienoic acid; aoc, allene oxide cyclase; cis--opda; cis--12-oxophytodienoic acid; opr, oxophytodienoic acid reductase; opc- <dig>  3-oxo-2--pentenyl)-cyclopentane-1-octanoic acid; acx, acyl-coa-oxidase
as shown in fig.
3c, a total of  <dig> differentially expressed proteins were detected from + al c. grandis and c. sinensis roots.
among these proteins,  <dig>  proteins only presented in c. sinensis  and  <dig> proteins were shared by the two species.
of the  <dig> overlapping proteins,  <dig> proteins were regulated in the same direction upon al exposure in the both species, the remaining four proteins were regulated in the opposite direction.
qrt-pcr analysis of genes for some differentially expressed proteins
to valuate the correlation between mrna and protein levels, we assayed the expression levels of genes for  <dig> differentially expressed proteins  in c. sinensis and c. grandis roots .
for c. grandis roots, the expression levels of nine genes matched well with our itraq data, while only four genes matched well with the itraq data for c. sinensis roots .
six genes [i.e., gi| <dig>  gi| <dig>  gi| <dig>  gi| <dig>  gi| <dig> and gi|3913733) were regulated differentially at transcript and protein levels in the both species.
thus, it is reasonable to assume that the discrepancy between the expression levels of genes and the abundances of the corresponding protein might be caused by post-translational modifications .fig.
<dig> relative expression levels of genes from c. sinensis  and c. grandis  roots.
relative abundances of genes encoding atp sulfurylase  <dig> , probable glutathione peroxidase  <dig> , oxalate oxidase  <dig> , o-acetylserine lyase , partial , catalase , blue copper protein , glyoxylase i, partial , glutathione transferase , partial , peroxidase  and hydroxyacylglutathione hydrolase  in al-toxic and control roots revealed by qrt-pcr.
bar represents the mean ± se .
unpaired t-test was applied for comparision between means.
different letters above the bars indicate a significant difference at p <  <dig> .
all the values were expressed relative to the control roots
discussion
c. sinensis is more tolerant to al-toxicity than c. grandis
our results showed that al-toxicity inhibited c. grandis shoot growth, but had no significant influence on c. sinensis growth, and that root al concentration did not differ between the two species .
when 20 μm h3bo <dig> in nutrient solution was replaced by  <dig>  μm h3bo <dig>   <dig>  mm al inhibited c. grandis root and shoot growth, but only significantly inhibited c. sinensis shoot growth even though al concentration was higher in al-toxic c. sinensis roots than in al-toxic c. grandis ones.
futhermore, al-toxicity-induced inhibition of shoot growth was less severe in c. sinensis seedlings than in c. grandis ones .
to conclude, the present work, like that of previous workers  <cit> , demonstrates that c. sinensis is more tolerant to al-toxicity than c. grandis.
as shown in additional file  <dig>  we identified  <dig> differentially expressed proteins from + al c. sinensis roots, while only  <dig> differentially expressed proteins were identified from + al c. grandis roots.
this demonstrated that the metabolic flexibility was more remarkable in c. sinensis roots than in c. grandis ones in response to al, which may contribute to the higher al-tolerance of c. sinensis.
s metabolism was up-regulated in al-treated roots
s metabolism is a core pathway for the synthesis of molecules required for some environmental stresses including al  <cit> .
transgenic plants over-expressing s metabolism-related genes such as gst and glutathione peroxidase  displayed enhanced tolerance to al-toxicity and/or to oxidative stress .
the up-regulation of atp sulfurylase  <dig>  and probable gpx  <dig> in c. sinensis roots and cys synthase and gst in c. sinensis and c. grandis roots in response to al  indicated that s metabolism was enhanced in al-treated roots.
this agrees with the reports that s metabolism-related enzymes  were induced by various stresses including al  <cit> , and that al-toxicity increased rice root concentrations of reduced glutathione  + oxidized glutathione  and gsh  <cit>  and citrus leaf concentrations of gsh and gssg  <cit> .
thus, s metabolism may play a role in response to al-toxicity by the synthesis of molecules required for al detoxification and confer al-tolerance in both c. grandis and c. sinensis, particularly in the latter.
proteins involved in stress and defense response
increased reactive oxygen species  level is commonly presumed to be a major component of al-toxicity.
here, the abundances of six antioxidant enzymes  increased in + al c. sinensis roots .
al-induced up-regulation of antioxidant enzymes has also been observed in al-toxic roots of tomato  <cit>  and rice  <cit> .
however, we only identified one up-regulated  and two down-regulated  antioxidant enzymes from + al c. grandis ones .
germin and germin-like proteins , which have different enzyme functions such as oxo and sod, play a role in various abiotic stresses  <cit> .
study showed that al-induced oxo expression in wheat roots could be involved in detoxification mechanism  <cit> .
thus, al-induced increase in the levels of oxo  <dig> and seven glps in c. sinensis roots might play a role in al-tolerance of c. sinensis.
however, we only isolated an up-regulated oxo  <dig> in + al c. grandis roots .
also, we isolated four up-regulated proteins  involved in ros scavenging from + al c. sinensis roots, while only one up-regulated bcp  and one down-regulated bcp  were isolated from + al c. grandis roots .
to sum up, the abundances of proteins involved in ros detoxification were greatly enhanced in + al c. sinensis roots, but were less affected in + al c.grandis ones.
in addition to ros formation, al-toxicity also results in overproduction of aldehydes and methylglyoxal  through the activation of various enzymatic and non-enzymatic reactions.
as expected, the abundances of aldh 7b involved in the detoxification of the aldehydes, alcohol dehydrogenase -like  <dig>  which catalyzes the inter-conversions between alcohols and aldehydes, and glyoxalase  i and gly ii involved in the detoxification of mg were enhanced in al-toxic c. sinensis roots, while the abundance of the probable aldo-keto reductase   <dig> involved in the detoxification of lipid peroxidation- and/or glycolysis-derived reactive carbonyls  was down-regulated in + al c. grandis roots .
thus, the ability of aldehydes and mg detoxification might be enhanced in + al c. sinensis roots, thus contributing its higher al-tolerance, but down-regulated in + al c. grandis roots.
the up-regulation of two hsps  in + al c. sinensis roots  agrees with the report that two protein spots of dnak-type molecular chaperone hsc <dig>   in al-tolerant arabidopsis ecotype increased in response to al-toxicity  <cit> , and that the abundance of hsp  increased only in al-tolerant rice roots  <cit> .
dehydrins, or group  <dig> lea  proteins, can function as antioxidants, ion sequestrants, or metal ion transporters in plant phloem sap  <cit> .
al-induced up-regulation of desiccation-related protein pcc13- <dig> in + al c. sinensis roots  agrees with the report that root expression level of gene encoding desiccated-related protein under al-stress was higher in al-tolerant soybean genotype than in al-sensitive one  <cit> .
however, al-toxicity decreased the levels of lea14-a in c. sinensis roots .
we isolated seven up-regulated  and one down-regulated  pathogenesis-related  proteins from + al c. sinensis roots, four down-regulated  and one up-regulated  pr proteins from + al c. grandis ones.
al-induced increase in s-norcoclaurine synthase  was higher in c. sinensis than in c. grandis roots.
generally speaking, al-toxicity increased the abundances of pr proteins in c. sinensis roots and decreased their levels in c. grandis roots .
this agrees with the reports that pr- <dig> protein was highly up-regulated at the transcriptional and translational levels in al-tolerant soybean cultivar roots  <cit> , that root pr bet v i family protein  were expressed at higher level in the tolerant rice cultivar than in the sensitive one  <cit> , and that the expression levels of several pr genes were higher in al-tolerant soybean genotype roots than in al-sensitive ones  <cit> .
thus, the up-regulation of pr proteins in + al c. sinensis roots might contribute to its al-tolerance.
harpin proteins can lead to multiple responses in plants, such as systemic acquired resistance, hypersensitive response, and enhancement of growth and drought tolerance.
over-expression of a harpin-encoding gene in rice increased drought tolerance through aba signaling  <cit> .
activated harpin binding protein- <dig>  can act upstream of the salicylic acid , ja, and ethylene signaling pathways in plant cells  <cit> .
the up-regulation of hrbp <dig> in + al c. sinensis roots might play a role in al-tolerance through acting those signaling pathways.
to conclude, the total ability of antioxidation and detoxification was greatly enhanced in + al c. sinensis roots, thus improving the al-tolerance of c. sinensis.
proteins involved in carbohydrate and energy metabolism
rapid turnover rate of fine roots requires systematic rebuilding of tissues, which is associated with a high reduced carbon cost.
building new root biomass not only requires carbon skeletons to produce cellulose, lignin and other structural compounds, it also requires metabolic energy .
under stress conditions, the demand may increase owing to initiation of response mechanisms and secondary metabolism  <cit> .
thus, carbohydrate and energy metabolism might be enhanced in al-treated roots to maintain the primary metabolic pathways and the carbohydrate balance.
as expected, all the  <dig> differentially expressed proteins involved in carbohydrate metabolism were up-regulated in + al c. sinensis roots except for two proteins .
wang et al.
<cit>  observed that glycolysis/gluconeogenesis was the most significantly up-regulated biochemical process in rice roots in response to al, suggesting that the enhancement of root glycolytic/gluconeogenetic pathway might promote al-tolerance by balancing the level of available energy to prevent intracellular shortage.
based on these results, we concluded that the up-regulation of carbohydrate and energy metabolism in + al c. sinensis roots was an adaptive response to meet the increased requirement for carbon skeletons and energy.
by contrast, we identified four down- and nine up-regulated proteins involved in carbohydrate and energy metabolism from + al c. grandis roots .
obviously, al-induced adaptive responses in carbohydrate and energy metabolism were less pronounced in c. grandis than in c. sinensis roots.
al-induced inhibition of nucleic acid biosynthesis
we found that all these differentially expressed proteins related to nucleic acid metabolism in c. sinensis and c. grandis roots except for protein rad-like  <dig> involved in regulation of transcription in the two species and ribonuclease, which catalyzes the degradation of rna into smaller components in c. grandis, were down-regulated in response to al-toxicity , demonstrating that al-toxicity might impair nucleic acid biosynthesis in c. sinensis and c. grandis roots and increased rna degradation in c. grandis roots.
this agrees with the report that al-toxicity decreased the concentrations of rna and dna, and increased the activities of deoxyribonuclease and ribonuclease in longan  leaves  <cit> .
al-induced alteration in protein metabolism
as shown in additional file  <dig>  all the  <dig> differentially expressed ribosomal proteins involved in mature ribosome assembly and translation processes were down-regulated in + al c. sinensis  and c. grandis  roots.
in addition, the abundances of two proteins  involved in translation processes, eukaryotic translation initiation factor 5b-like involved in the initiation phase of eukaryotic translation, and peptidyl-prolyl cis-trans isomerase fkbp15- <dig> involved in protein folding in c. sinensis roots, and of two proteins  involved in protein folding decreased in response to al-stress except for peptidyl-prolyl cis-trans isomerase in c. sinensis roots.
therefore, we concluded that al-toxicity impaired protein biosynthesis in roots.
similar results have been obtained on al-stressed rice roots  <cit> .
the al-induced down-regulation of many proteins involved in protein biosynthesis and processing, which are the major consumers of atp and nutrients, indicated that the translation was reasonably regulated to save energy and nutrients in roots, especially in the al-tolerant species.
similar results have been obtained on al-stressed arabidopsis  <cit>  and rice  <cit>  roots.
in addition, the widespread down-regulation of root ribosomal proteins, especially in c. sinensis roots, might indicate a redeployment of resources to meet the greater demands for amino acids in non-ribosomal peptide synthesis of glutathione and phytochelation for al complexation.
this agrees with the above inference that s metabolism was up-regulated in al-treated roots, especially in c. sinensis roots, and with the reports that al-toxicity increased rice root concentrations of gsh + gssg and gsh  <cit>  and citrus leaf concentrations of gsh and gssg  <cit> .
protein degradation not only provides molecular substrates for plant respiration but also initiates adaptive responses to biotic stresses by reallocating nutrients from non-essential areas of metabolism to vital cellular activities  <cit> .
as expected, we isolated  <dig> up-regulated  and one down-regulated  proteins in proteiolysis from + al c. sinensis roots, five upregulated  and two down-regulated  proteins in proteolysis from + al in c. grandis roots .
wang et al.
<cit>  found that the abundance of cys protease  and mitochondrial-processing peptidase subunit ] were increased in + al rice roots, suggesting that cys protease might play a role in resisting against toxic al because root cys protease was expressed at a higher level in the tolerant than in the sensitive cultivar when exposed to al-toxicity.
the more widespread up-regulation of protein in proteolysis in c. sinensis than in c. grandis roots might contribute to higher al-tolerance of c. sinensis.
inactive proteins  and proteins, which are no longer required for cell, are tagged by ubiquitin for proteolysis.
unlike the above protease, the abundances of all the  <dig> differentially expressed proteins involved in ubiquitination in c. sinensis  and c. grandis  roots decreased in response to al-toxicity , meaning that the ubiquitination of some proteins might be impaired in al-stressed roots.
six proteins related to amino acid metabolism were altered in + al c. sinensis and c. grandis roots .
al-induced decrease in the abundance of aspartate transaminase in c. sinensis roots implied that al-toxicity might decrease n metabolism flux, because it is the major enzyme controlling aspartate that is used to transport n from sources to sinks  <cit> .
this disagrees with the report that al-toxicity increased aspartate transaminase level in rice roots, especially in al-tolerant cultivar  <cit> .
in plants, sarcorsine oxidase metabolizes both sarcosine and pipecolate with preferential utilization of the latter as an endogenous substrate  <cit> .
here, we first isolated up-regulated sacrosine oxidase in + al c. sinensis roots.
nitrite reductase  is considered to be a controlling enzyme in plant no <dig> assimilation.
the up-regulation of nir in + al c. sinensis roots agrees with the report that combined treatment of acidity and pb2+ led to an increase in nir activity of soybean roots  <cit> .
phenylalanine ammonia-lyase  catalyzes the nonoxidative deamination of l-phenylalanine to form trans-cinnamic acid and a free ammonium ion.
we observed that al-toxicity only affected pal level in al-intolerant c. grandis roots .
however, wang et al.
<cit>  showed that al-toxicity decreased pal abundance in rice roots, especially in al-tolerant cultivar.
glutamate decarboxylase  catalyzes the conversion of glutamate to γ-aminobutyrate .
root-specific gad  is essential for sustaining gaba level in arabidopsis  <cit> .
therefore, gaba level might be elevated in al-treated c. grandis roots due to enhanced level of putative gad .
the observed higher level of putative gad also agrees with the report that rice root gad was expressed primarily under phosphate deprivation  <cit> .
proteins involved in cell transport
as shown in additional file  <dig>  we obtained seven up- and three down-regulated proteins in cell transport from + al c. sinensis roots, and three up- and three down-regulated proteins from + al c. grandis roots.
abc transporter facilitates the movement of s-glutathionylated toxins formed in gst-catalyzed conjugation of gsh to toxins and other substrates across biological membranes  <cit> .
the up-regulation of abc transporter i family member  <dig> in + al c. sinensis roots agrees with the increased reguirement for the transport of s-glutathionylated toxins due to increased gst level.
al-induced increase in abc transporter has been observed in soybean roots  <cit> .
in arabidopsis, a putative abc transporter-like protein is required for al-tolerance  <cit> .
the induction of aquaporin in + al c. sinensis roots might be involved in al-tolerance of c. sinensis by regulating the transport of water or micronutrients across cell membranes  <cit> .
however, wang et al.
<cit>  reported that the abundance of aquaporin pip <dig> was down-regulated by ca.
50 % only in al-tolerant rice cultivar, and that nip <dig> in al-tolerant and -sensitive roots was up-regulated to a similar extent by al.
the up-regulation of porin/voltage-dependent anion-selective channel protein in + al c. sinensis agrees with the report that the abundance of root mitochondrial outer membrane porin  <dig>  increased in al-tolerant rice cultivar when exposed to al-toxicity, and decreased in al-sensitive cultivar  <cit> .
plasma membrane h+-atpase has an important role in plant response to nutrient and environmental stresses, such as salt stress, al-stress, p and k deficiencies  <cit> .
mitochondrial adp/atp carrier plays a central role in aerobic cell energetics by providing the atp generated by oxidative phosphorylation to the cytosol  <cit> .
sec <dig>  an endoplasmic reticulum  membrane protein translocator, participates in the translocation of newly-synthesized proteins into the lumen of the er.
the hydrolysis of the signal peptide for newly-synthesized proteins is carried out by specific proteases called signal peptidases located on the luminal side of er.
the removal of the signal peptide is crucial for the maintenance of er homeostasis  <cit> .
the induction of protein transport protein sec <dig> subunit β in + al c. sinensis roots might provide an adaptive response by maintaining er homeostasis.
however, its abundance decreased in + al c. grandis roots.
by contrast, three proteins  in protein transport decreased in + al c. sinensis roots.
besides their role in fe storage, ferritins also play a role in preventing oxidative damage by storing free fe in a safe form  <cit> .
our finding that + al c. sinensis roots had higher abundance of ferritin  <dig> agrees with the report that there was an increase in the expression of the ferritin fe storage gene, atfer <dig> in p-deficient arabidopsis leaves  <cit> .
nucleobase-ascorbate transporters  have been identified in prokaryotes, fungi, plants and mammals.
in plants, maize leaf permease <dig>  has been characterized by functional complementation of a purine transport-deficient aspergillus nidulans strain and is necessary for proper chloroplast development in maize.
interestingly, leaf permease <dig> protein does not contain a plastidic transit peptide and is expressed only in non-photosynthetic tissues such as roots and etiolated leaves  <cit> .
the up-regulation of putative permease  <dig> in + al c. sinensis roots might be involved in al-tolerance throught maintaing chloroplast morphology.
plant lipid transfer proteins  are responsible for the shuttling of phospholipids and other fatty acid groups between cell membranes.
some ltps with broad specificity are termed non-specific ltps .
we found that the abundances of nsltp <dig> and nsltp-like protein decreased in + al c. grandis roots.
this agrees with the reports that nsltp  was up-regulated in al-tolerant cultivar rice roots, and down-regulated in al-sensitive ones  <cit> , that the expression levels of two genes encoding ltp-like proteins  in wheat roots were higher in al-tolerant than in al-sensitive near isogenic-line  <cit> , and that mrna levels of ltps were higher in al-tolerant than in al-sensitive soybean genotype  <cit> .
similarly, putative phosphatidylglycerol/phosphatidylinositol transfer protein ddb_g0282179-like involved in lipid transport was increased in + al c. sinensis roots.
however, wang et al.
<cit>  showed that al-toxicity decreased the abundance of ltp in al-tolerance rice roots, but did not signifaicantly affect its level in al-sensitive ones.
taken all together, the ability of cell transport was enhanced in + al roots, especially in c. sinensis, which might contribute to its higher al-tolerance.
proteins involved in biological regulation and signal transduction
perception and transmission of stress signals by the cell play crucial roles in plant response to abiotic stresses.
protein kinases and phosphatases are the key players in cell signals.
here, we isolated five up-regulated proteins in phosphorylation  and dephosphorylation  in + al c. sinensis roots , demonstrating that al-toxicity triggered phosphorylation-dependent signal transduction pathway in c. sinensis roots, which might be involved in its al-tolerance.
by contrast, two down-regulated  and two up-regulated  proteins in phosphorylation and one up-regulated protein in dephosphorylation  were detected in + al c. grandis roots .
this suggested that the balance between phosphorylation and dephosphorylation might be upset and phosphorylation of some proteins might be impaired in + al c. grandis roots.
obviously, c. sinensis roots had a higher capacity to keep a better balance between phosphorylation and dephosphorylation than c. grandis ones under al-toxicity, which might contribute to c. sinensis al-tolerance.
purple acid phosphatases  play important roles in p acquisition and recycling in plants  <cit> .
wang et al.
<cit>  observed that pap <dig> expressed at a higher level in al-tolerant than in al-sensitive rice root, suggesting that al-tolerant cultivar might relieve the al-toxicity through improving the p acquisition.
the higher up-regulation of pap <dig> in + al c. sinensis than in c. grandis roots agrees with our report that the former might more effectively acquire p than the latter with or without al-stress  <cit> .
plasma membrane-associated cation-binding protein  <dig>  is involved in intracellular signals through interaction with phosphatidylinositol phosphates and calmodulin  <cit> .
our finding that + al c. sinensis roots displayed higher abundance of pcap <dig>  agrees with the report that cu-stress indced the expression of pcap <dig> in arabidopsis  <cit> .
our finding that the level of putative g3bp-like protein-like, a protein involved in the ras signal transduction pathway, was decreased only in + al c. grandis roots .
this agrees with the report that the abundance of g3bp was up-regulated in wild al-tolerant barley  and unchanged in al-tolerant barley cultivar , and down-regulated in al-sensitive wild barley  when exposed to al-toxicity  <cit> .
these results indicated that the down-regulation of g3bp might be associated with the lower al-tolerance of c. grandis.
proteins involved in cell wall and cytoskeleton metabolism
plant cytoskeleton is highly dynamic networks of proteinaceous components consisting mainly of microtubules and microfilaments.
al-induced growth inhibition and swelling of roots demonstrated that plant cytoskeleton could be a target of al-toxicity  <cit> .
studies showed that root microtubules and microfilaments were altered by al-toxicity  <cit> .
in wheat, al led to disorganization of actin filaments and formation of actin deposits  <cit> .
as expected, we identified five down-regulated  and two down-regulated ] proteins related to cytoskeleton metabolism in c. sinensis and c. grandis roots, respectively.
apart from cytoskeleton proteins, we isolated two down-regulated proteins  in cell wall metabolism in + al c. sinensis roots .
to conclude, al-induced alterations in root cell wall and cytoskeleton metabolism differed between the both species.
ja biosynthesis
phytohormones play a key role in plant response to biotic stresses.
among these, one of the most important signal molecules is ja  <cit> .
here, we obtained four up-regulated proteins  involved in ja biosynthesis .
lox is one of the key enzymes responsible for the biosynthesis of ja.
al-induced up-regulation of lox has also been reported in soybean  <cit>  and cassia tora  <cit>  roots.
in arabidopsis and rice, opr <dig> and opr  <dig> are responsible for ja production, respectively  <cit> .
acx catalyze the first step in the peroxisomal β-oxidation stage of ja biosynthesis.
wound-induced ja accumulation was reduced in a mutant that was defective in acx <dig> and was abolished in a mutant that was impaired in both acx <dig> and its closely related paralog, acx <dig>  <cit> .
thus, both ja biosynthesis and level might be increased in + al c. sinensis roots, thus enhancing plant al-tolerance.
because lox may increase the formation of oxidation products, al-induced up-regulation of lox has been suggested to be a response to al-toxicity  <cit> .
CONCLUSIONS
our results demonstrate that c. sinensis is more tolerant to al-toxicity than c. grandis.
using itraq technique, we isolated  <dig> differentially expressed proteins from + al c. sinensis and c. grandis roots.
among these proteins,  <dig> proteins only presented in c. sinensis,  <dig> proteins only presented in c. grandis, and  <dig> proteins were shared by the two species.
in the  <dig> overlapping proteins,  <dig>  proteins were regulated in the same  direction upon al exposure.
this indicated more remarkable metabolic flexibility in c. sinenis than in c. grandis roots, thus improving the al-tolerance of c. sinensis.
as shown in fig.
<dig>  the higher al-tolerance of c. sinensis might be related to following several factors, including:  activation of s metabolism;  greatly improving the total ability of antioxidation and detoxification;  up-regulation of carbohydrate and energy metabolism;  enhancing cell transport;  decreasing  proteins related to protein synthesis ;  maintaining a better balance between protein phosphorylation and dephosphorylation; and  increasing ja biosynthesis.
to sum up, we identified more differentially expressed proteins than those of previous studies in other plant roots and provided the most integrated view of the adaptive responses occurring in + al roots.
therefore, our analysis of root al-toxicity-responsive proteins will increase our understanding of al-tolerant mechanisms in higher plants.fig.
<dig> a potential model for the adaptive responses of c. sinensis and c. grandis roots to al-toxicity.
cgr: c. grandis roots; csr: c. sinensis roots; gly: glyoxalase; misap: mitochondrial intermembrane space import and assembly protein; pepc: phosphoenolpyruvate carboxylase; pm atpase: plasma membrane h+-atpase; ppiase: peptidyl-prolyl cis-trans isomerase; plap: plastid-lipid-associated protein; r-protein: ribosomal protein; ss: sucrose synthase; trx m: thioredoxin m; ptp: phosphatidylglycerol/phosphatidylinositol transfer protein; vap: vesicle-associated protein

 in many acidic soils through the tropics and subtropics, aluminum -toxicity is a major factor limiting crop productivity. approximately 30 % of the world’s total land area is acidic. furthermore, the acidity of the soils is increasing due to the environmental problems including some farming practices and acid rain. when soil ph is less than, toxic forms of al are solubilized into the soil solution and accumulate to high concentration that rapidly inhibits plant root growth by damaging the roots functionally and structurally, subsequently decreasing nutrient and water uptake, eventually resulting in poor crop growth and productivity. a traditional strategy is to raise soil ph by application of lime, thus alleviating al-toxicity; however, the approach is both costly and ecologically unsound from the long-term point of view. a more efficient strategy is to breed al-tolerant crop cultivars. fortunately, plants display wide variation in their ability to deal with al-toxicity. variation in al-tolerance makes it possible to breed tolerant cultivars. the success of breeding programs relies on an understanding of the physiological, biochemical and molecular mechanisms that plants tolerate al-toxicity. since biological processes are ultimately controlled by proteins, identification and characterization of al-tolerant proteins will not only increase our understanding of the molecular mechanisms on plant al-tolerance, but also will provide new information that researchers will use to screen and breed crop cultivars suited for acidic soils with higher active al. during the long-term evolutionary process, higher plants have evolved two main strategies that enable them to tolerate high level of soil active al. although several mechanisms for the external detoxification have been suggested, the al-induced secretion of organic acid anions such as citrate, malate and oxalate from roots is the most-studied mechanism of al-tolerance in higher plants. genes involved in al-induced root secretion of citrate and malate have been isolated from wheat, sorghum and barley. transgenic plants over-expressing these genes displayed increased al-activated root secretion of malate and/or citrate and enhanced al-tolerance. internal detoxification is mainly reached by both complexation and sequestration of al. gene expression networks revealed by transcriptomics offer us the opportunity to understand the molecular mechanisms of plant al-toxicity and -tolerance. although al-induced changes in gene expressions have been examined in some detail, limited data are available on al-toxicity-responsive proteins in higher plants. alteration of plant proteins is an inevitable process to deal with environmental stresses including al-toxicity. proteomics is a very powerful tool for analyzing the functions of the plant proteins. abundance of a protein is not only regulated at transcriptional, but also at translational and post-translational levels. therefore, proteomic analysis may provide more accurate and comprehensive data than what genomic studies can provide. recently, there were several studies reporting al-induced changes in plant proteins using two-dimensional gel electrophoresis or isobaric tags for relative and absolute quantification technique. study with al-tolerant rice cultivar showed that root abundances of copper/zinc superoxide dismutase, s-adenosylmethionine synthetase, cysteine synthase, 1-aminocydopropane-1-carboxylate oxidase, glutathione s-transferase increased in response to al-toxicity, and that cys synthase might play a key role in al-tolerance. in al-tolerant soybean cultivar roots, al induced the production of heat shock proteins, gst, chalcone-related synthetase, gtp-binding protein, and atp binding cassette transporter atp-binding protein. duressa et al. observed that al resulted in a distinct protein profile changes in al-tolerant and al-sensitive soybean genotypes. in tomato roots, dehydroascorbate reductase, glutathione reductase, catalase, mitochondrial aldehyde dehydrogenase, catechol oxidase, quinone reductase, and lactoylglutathione lyase involved in antioxidation and detoxification were induced by al. dai et al. isolated proteins associated with al-tolerance in al-tolerant wild barley xz there were proteins, which were up-regulated in xz roots, but down-regulated or unaltered in both al-tolerant barley cultivar dayton and al-sensitive wild barley xz oh et al. identified up-regulated protein spots such as s-adenosylmethionine, oxalate oxidase, malate dehydrogenase, cys synthase, ascorbate peroxidase and down-regulated protein spots such as hsp o-methytransferase enolase and amylogenin in + al wheat roots. wang et al. identified al-toxicity-responsive proteins from al-sensitive and -tolerant rice roots. they observed that glycolysis/gluconeogenesis was the most significantly up-regulated biochemical process in al-toxic roots. to our knowledge, data available on the effects of al-toxicity on root proteomics of woody plants are very limited. citrus spp. are cultivated mainly in acidic and strong acidic soils such as red soil, yellow soil or lateritic red soil. in li et al. investigated the ph of ‘guanximiyou’ pummelo orchard soils from pinghe county, zhangzhou, china. up to 90 % of soils had a lower ph than. low ph and high al are the factors contributing to poor citrus growth and shortened lifespan of trees. in this study, we compared al-induced quantitative and qualitative changes in proteomes that occurred in roots of al-tolerant citrus sinensis and al-sensitive c. grandis seedlings using itraq technique in order to identify differentially expressed proteins involved in al-tolerance and to understasnd the molecular mechanisms of plants to deal with al-toxicity.  plant growth and al concentration in roots as shown in fig. 1a-c,  mm al-treated c. grandis seedings displayed decreased whole plant and shoot dry weights and unchanged root dw, compared with controls, while al-toxicity did not significantly affect c. sinensis whole plant, shoot and root dws. there was no significant difference in whole plant, shoot and root dws between the two species at each given al treatment. root concentration of al was higher in + al seedlings than controls, while did not significantly differ between the two species at each given al treatment.fig. effects of al-toxicity on plant growth and root al concentration in c. sinensis and c. grandis seedlings. a-c whole plant, shoot and root dry weights. d root al concentration. bar represents the mean ± se. differences among four treatment combinations were analyzed by  ×  anova. different letters above the bars indicate a significant difference at p <  it is worth mentioning that  mm al treatment decreased c. grandis root dw, but had no influence on c. sinensis root dw and that al-toxicity-induced decreases in whole plant and shoot dws were more severe in c. grandis than in c. sinensis seedlings, when 20 μm h3bo in nutrient solution was replaced by  μm h3bo in addition, al concentration was not lower in c. sinensis than in c. grandis roots at each given al level. primary data analysis and protein identification a total of spectra were produced from the itraq experiment using control and al-toxic c. sinensis and c. grandis roots as materials. by analyzing these spectra, we identified known spectra, unique spectra, peptides, unique peptides and proteins, respectively. the number of proteins in the genome is, due to the reasons of samples and technical restrain, identified proteins in current proteomic research is normal in plants. protein number decreased with increased number of peptides that matched to proteins, but over 67 % of the proteins included at least two peptides.fig. spectra, peptides and proteins identified from itraq proteomics by searching against c. sinensis database, number of peptides that match to proteins using mascot, protein mass distribution, distribution of protein sequence coverage, and distribution of peptide length both protein mass distribution and distribution of sequence coverage were summarized in fig. 2c and d. proteins with 20–30 kda and 30–40 kda were at the most abundant, followed by proteins with 40–50 kda and 50–60 kda. protein number decreased with increased sequence coverage. figure 2e shows the distribution of peptide length in citrus roots. less than  % of all peptides are ≥  amino acid residues and  % are between and residues. differentially expressed proteins by itraq in this study, a protein was considered differentially expressed when the protein had both a log fold-change of more than and a p-value of less than. based on the two criteria, differentially expressed proteins were detected in + al c. sinensis roots, of which displayed increased and displayed decreased abundance. these proteins were related to sulfur metabolism, stress and defense response, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, cell transport, biological regulation and signal transduction, cell wall and cytoskeleton metabolism, jasmonic acid biosynthesis and others. in + al c. grandis roots, we isolated up- and down-regulated proteins, which were grouped into following functional categories: s metabolism, stress and defense response, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, cell transport, biological regulation and signal transduction, cell wall and cytoskeleton metabolism and others. figures  and showed al-induced alterations of proteins involved in s metabolism in c. sinensis and c. grandis roots and ja biosynthesis in c. sinensis roots, respectively.fig. classification of al-induced differentially expressed proteins in c. sinensis and c. grandis roots and venn diagram analysis of differentially expressed proteins in citrus roots. among these overlapping proteins, proteins were regulated in the same direction upon al exposure fig. al-induced changes in s metabolism in c. sinensis and c. grandis roots. up-regulated proteins presented in + al c. sinensis and c. grandis roots were labeled in red. abbreviations: aps, adenosine phosphosulphate; gsh, reduced glutathione; gssg, oxidized glutathione fig. metabolic scheme of ja biosynthesis in citrus sinensis roots. up-regulated proteins in + al roots were labeled in red. abbreviations: lox, lipoxygenase; 13-hpot, -hydroperoxyoctadecatrienoic acid; aos, allene oxide synthase; -eot, -epoxyoctadecatrienoic acid; aoc, allene oxide cyclase; cis--opda; cis--12-oxophytodienoic acid; opr, oxophytodienoic acid reductase; opc- 3-oxo-2--pentenyl)-cyclopentane-1-octanoic acid; acx, acyl-coa-oxidase as shown in fig. 3c, a total of differentially expressed proteins were detected from + al c. grandis and c. sinensis roots. among these proteins, proteins only presented in c. sinensis and proteins were shared by the two species. of the overlapping proteins, proteins were regulated in the same direction upon al exposure in the both species, the remaining four proteins were regulated in the opposite direction. qrt-pcr analysis of genes for some differentially expressed proteins to valuate the correlation between mrna and protein levels, we assayed the expression levels of genes for differentially expressed proteins in c. sinensis and c. grandis roots. for c. grandis roots, the expression levels of nine genes matched well with our itraq data, while only four genes matched well with the itraq data for c. sinensis roots. six genes [i.e., gi| gi| gi| gi| gi| and gi|3913733) were regulated differentially at transcript and protein levels in the both species. thus, it is reasonable to assume that the discrepancy between the expression levels of genes and the abundances of the corresponding protein might be caused by post-translational modifications.fig. relative expression levels of genes from c. sinensis and c. grandis roots. relative abundances of genes encoding atp sulfurylase, probable glutathione peroxidase, oxalate oxidase, o-acetylserine lyase, partial, catalase, blue copper protein, glyoxylase i, partial, glutathione transferase, partial, peroxidase and hydroxyacylglutathione hydrolase in al-toxic and control roots revealed by qrt-pcr. bar represents the mean ± se. unpaired t-test was applied for comparision between means. different letters above the bars indicate a significant difference at p < . all the values were expressed relative to the control roots discussion c. sinensis is more tolerant to al-toxicity than c. grandis our results showed that al-toxicity inhibited c. grandis shoot growth, but had no significant influence on c. sinensis growth, and that root al concentration did not differ between the two species. when 20 μm h3bo in nutrient solution was replaced by  μm h3bo  mm al inhibited c. grandis root and shoot growth, but only significantly inhibited c. sinensis shoot growth even though al concentration was higher in al-toxic c. sinensis roots than in al-toxic c. grandis ones. futhermore, al-toxicity-induced inhibition of shoot growth was less severe in c. sinensis seedlings than in c. grandis ones. to conclude, the present work, like that of previous workers, demonstrates that c. sinensis is more tolerant to al-toxicity than c. grandis. as shown in additional file we identified differentially expressed proteins from + al c. sinensis roots, while only differentially expressed proteins were identified from + al c. grandis roots. this demonstrated that the metabolic flexibility was more remarkable in c. sinensis roots than in c. grandis ones in response to al, which may contribute to the higher al-tolerance of c. sinensis. s metabolism was up-regulated in al-treated roots s metabolism is a core pathway for the synthesis of molecules required for some environmental stresses including al. transgenic plants over-expressing s metabolism-related genes such as gst and glutathione peroxidase displayed enhanced tolerance to al-toxicity and/or to oxidative stress. the up-regulation of atp sulfurylase and probable gpx in c. sinensis roots and cys synthase and gst in c. sinensis and c. grandis roots in response to al indicated that s metabolism was enhanced in al-treated roots. this agrees with the reports that s metabolism-related enzymes were induced by various stresses including al, and that al-toxicity increased rice root concentrations of reduced glutathione  + oxidized glutathione and gsh and citrus leaf concentrations of gsh and gssg. thus, s metabolism may play a role in response to al-toxicity by the synthesis of molecules required for al detoxification and confer al-tolerance in both c. grandis and c. sinensis, particularly in the latter. proteins involved in stress and defense response increased reactive oxygen species level is commonly presumed to be a major component of al-toxicity. here, the abundances of six antioxidant enzymes increased in + al c. sinensis roots. al-induced up-regulation of antioxidant enzymes has also been observed in al-toxic roots of tomato and rice. however, we only identified one up-regulated and two down-regulated antioxidant enzymes from + al c. grandis ones. germin and germin-like proteins, which have different enzyme functions such as oxo and sod, play a role in various abiotic stresses. study showed that al-induced oxo expression in wheat roots could be involved in detoxification mechanism. thus, al-induced increase in the levels of oxo and seven glps in c. sinensis roots might play a role in al-tolerance of c. sinensis. however, we only isolated an up-regulated oxo in + al c. grandis roots. also, we isolated four up-regulated proteins involved in ros scavenging from + al c. sinensis roots, while only one up-regulated bcp and one down-regulated bcp were isolated from + al c. grandis roots. to sum up, the abundances of proteins involved in ros detoxification were greatly enhanced in + al c. sinensis roots, but were less affected in + al c.grandis ones. in addition to ros formation, al-toxicity also results in overproduction of aldehydes and methylglyoxal through the activation of various enzymatic and non-enzymatic reactions. as expected, the abundances of aldh 7b involved in the detoxification of the aldehydes, alcohol dehydrogenase -like which catalyzes the inter-conversions between alcohols and aldehydes, and glyoxalase i and gly ii involved in the detoxification of mg were enhanced in al-toxic c. sinensis roots, while the abundance of the probable aldo-keto reductase involved in the detoxification of lipid peroxidation- and/or glycolysis-derived reactive carbonyls was down-regulated in + al c. grandis roots. thus, the ability of aldehydes and mg detoxification might be enhanced in + al c. sinensis roots, thus contributing its higher al-tolerance, but down-regulated in + al c. grandis roots. the up-regulation of two hsps in + al c. sinensis roots agrees with the report that two protein spots of dnak-type molecular chaperone hsc in al-tolerant arabidopsis ecotype increased in response to al-toxicity, and that the abundance of hsp increased only in al-tolerant rice roots. dehydrins, or group lea proteins, can function as antioxidants, ion sequestrants, or metal ion transporters in plant phloem sap. al-induced up-regulation of desiccation-related protein pcc13- in + al c. sinensis roots agrees with the report that root expression level of gene encoding desiccated-related protein under al-stress was higher in al-tolerant soybean genotype than in al-sensitive one. however, al-toxicity decreased the levels of lea14-a in c. sinensis roots. we isolated seven up-regulated and one down-regulated pathogenesis-related proteins from + al c. sinensis roots, four down-regulated and one up-regulated pr proteins from + al c. grandis ones. al-induced increase in s-norcoclaurine synthase was higher in c. sinensis than in c. grandis roots. generally speaking, al-toxicity increased the abundances of pr proteins in c. sinensis roots and decreased their levels in c. grandis roots. this agrees with the reports that pr- protein was highly up-regulated at the transcriptional and translational levels in al-tolerant soybean cultivar roots, that root pr bet v i family protein were expressed at higher level in the tolerant rice cultivar than in the sensitive one, and that the expression levels of several pr genes were higher in al-tolerant soybean genotype roots than in al-sensitive ones. thus, the up-regulation of pr proteins in + al c. sinensis roots might contribute to its al-tolerance. harpin proteins can lead to multiple responses in plants, such as systemic acquired resistance, hypersensitive response, and enhancement of growth and drought tolerance. over-expression of a harpin-encoding gene in rice increased drought tolerance through aba signaling. activated harpin binding protein- can act upstream of the salicylic acid, ja, and ethylene signaling pathways in plant cells. the up-regulation of hrbp in + al c. sinensis roots might play a role in al-tolerance through acting those signaling pathways. to conclude, the total ability of antioxidation and detoxification was greatly enhanced in + al c. sinensis roots, thus improving the al-tolerance of c. sinensis. proteins involved in carbohydrate and energy metabolism rapid turnover rate of fine roots requires systematic rebuilding of tissues, which is associated with a high reduced carbon cost. building new root biomass not only requires carbon skeletons to produce cellulose, lignin and other structural compounds, it also requires metabolic energy. under stress conditions, the demand may increase owing to initiation of response mechanisms and secondary metabolism. thus, carbohydrate and energy metabolism might be enhanced in al-treated roots to maintain the primary metabolic pathways and the carbohydrate balance. as expected, all the differentially expressed proteins involved in carbohydrate metabolism were up-regulated in + al c. sinensis roots except for two proteins. wang et al. observed that glycolysis/gluconeogenesis was the most significantly up-regulated biochemical process in rice roots in response to al, suggesting that the enhancement of root glycolytic/gluconeogenetic pathway might promote al-tolerance by balancing the level of available energy to prevent intracellular shortage. based on these results, we concluded that the up-regulation of carbohydrate and energy metabolism in + al c. sinensis roots was an adaptive response to meet the increased requirement for carbon skeletons and energy. by contrast, we identified four down- and nine up-regulated proteins involved in carbohydrate and energy metabolism from + al c. grandis roots. obviously, al-induced adaptive responses in carbohydrate and energy metabolism were less pronounced in c. grandis than in c. sinensis roots. al-induced inhibition of nucleic acid biosynthesis we found that all these differentially expressed proteins related to nucleic acid metabolism in c. sinensis and c. grandis roots except for protein rad-like involved in regulation of transcription in the two species and ribonuclease, which catalyzes the degradation of rna into smaller components in c. grandis, were down-regulated in response to al-toxicity, demonstrating that al-toxicity might impair nucleic acid biosynthesis in c. sinensis and c. grandis roots and increased rna degradation in c. grandis roots. this agrees with the report that al-toxicity decreased the concentrations of rna and dna, and increased the activities of deoxyribonuclease and ribonuclease in longan leaves. al-induced alteration in protein metabolism as shown in additional file all the differentially expressed ribosomal proteins involved in mature ribosome assembly and translation processes were down-regulated in + al c. sinensis and c. grandis roots. in addition, the abundances of two proteins involved in translation processes, eukaryotic translation initiation factor 5b-like involved in the initiation phase of eukaryotic translation, and peptidyl-prolyl cis-trans isomerase fkbp15- involved in protein folding in c. sinensis roots, and of two proteins involved in protein folding decreased in response to al-stress except for peptidyl-prolyl cis-trans isomerase in c. sinensis roots. therefore, we concluded that al-toxicity impaired protein biosynthesis in roots. similar results have been obtained on al-stressed rice roots. the al-induced down-regulation of many proteins involved in protein biosynthesis and processing, which are the major consumers of atp and nutrients, indicated that the translation was reasonably regulated to save energy and nutrients in roots, especially in the al-tolerant species. similar results have been obtained on al-stressed arabidopsis and rice roots. in addition, the widespread down-regulation of root ribosomal proteins, especially in c. sinensis roots, might indicate a redeployment of resources to meet the greater demands for amino acids in non-ribosomal peptide synthesis of glutathione and phytochelation for al complexation. this agrees with the above inference that s metabolism was up-regulated in al-treated roots, especially in c. sinensis roots, and with the reports that al-toxicity increased rice root concentrations of gsh + gssg and gsh and citrus leaf concentrations of gsh and gssg. protein degradation not only provides molecular substrates for plant respiration but also initiates adaptive responses to biotic stresses by reallocating nutrients from non-essential areas of metabolism to vital cellular activities. as expected, we isolated up-regulated and one down-regulated proteins in proteiolysis from + al c. sinensis roots, five upregulated and two down-regulated proteins in proteolysis from + al in c. grandis roots. wang et al. found that the abundance of cys protease and mitochondrial-processing peptidase subunit ] were increased in + al rice roots, suggesting that cys protease might play a role in resisting against toxic al because root cys protease was expressed at a higher level in the tolerant than in the sensitive cultivar when exposed to al-toxicity. the more widespread up-regulation of protein in proteolysis in c. sinensis than in c. grandis roots might contribute to higher al-tolerance of c. sinensis. inactive proteins and proteins, which are no longer required for cell, are tagged by ubiquitin for proteolysis. unlike the above protease, the abundances of all the differentially expressed proteins involved in ubiquitination in c. sinensis and c. grandis roots decreased in response to al-toxicity, meaning that the ubiquitination of some proteins might be impaired in al-stressed roots. six proteins related to amino acid metabolism were altered in + al c. sinensis and c. grandis roots. al-induced decrease in the abundance of aspartate transaminase in c. sinensis roots implied that al-toxicity might decrease n metabolism flux, because it is the major enzyme controlling aspartate that is used to transport n from sources to sinks. this disagrees with the report that al-toxicity increased aspartate transaminase level in rice roots, especially in al-tolerant cultivar. in plants, sarcorsine oxidase metabolizes both sarcosine and pipecolate with preferential utilization of the latter as an endogenous substrate. here, we first isolated up-regulated sacrosine oxidase in + al c. sinensis roots. nitrite reductase is considered to be a controlling enzyme in plant no assimilation. the up-regulation of nir in + al c. sinensis roots agrees with the report that combined treatment of acidity and pb2+ led to an increase in nir activity of soybean roots. phenylalanine ammonia-lyase catalyzes the nonoxidative deamination of l-phenylalanine to form trans-cinnamic acid and a free ammonium ion. we observed that al-toxicity only affected pal level in al-intolerant c. grandis roots. however, wang et al. showed that al-toxicity decreased pal abundance in rice roots, especially in al-tolerant cultivar. glutamate decarboxylase catalyzes the conversion of glutamate to γ-aminobutyrate. root-specific gad is essential for sustaining gaba level in arabidopsis. therefore, gaba level might be elevated in al-treated c. grandis roots due to enhanced level of putative gad. the observed higher level of putative gad also agrees with the report that rice root gad was expressed primarily under phosphate deprivation. proteins involved in cell transport as shown in additional file we obtained seven up- and three down-regulated proteins in cell transport from + al c. sinensis roots, and three up- and three down-regulated proteins from + al c. grandis roots. abc transporter facilitates the movement of s-glutathionylated toxins formed in gst-catalyzed conjugation of gsh to toxins and other substrates across biological membranes. the up-regulation of abc transporter i family member in + al c. sinensis roots agrees with the increased reguirement for the transport of s-glutathionylated toxins due to increased gst level. al-induced increase in abc transporter has been observed in soybean roots. in arabidopsis, a putative abc transporter-like protein is required for al-tolerance. the induction of aquaporin in + al c. sinensis roots might be involved in al-tolerance of c. sinensis by regulating the transport of water or micronutrients across cell membranes. however, wang et al. reported that the abundance of aquaporin pip was down-regulated by ca. 50 % only in al-tolerant rice cultivar, and that nip in al-tolerant and -sensitive roots was up-regulated to a similar extent by al. the up-regulation of porin/voltage-dependent anion-selective channel protein in + al c. sinensis agrees with the report that the abundance of root mitochondrial outer membrane porin increased in al-tolerant rice cultivar when exposed to al-toxicity, and decreased in al-sensitive cultivar. plasma membrane h+-atpase has an important role in plant response to nutrient and environmental stresses, such as salt stress, al-stress, p and k deficiencies. mitochondrial adp/atp carrier plays a central role in aerobic cell energetics by providing the atp generated by oxidative phosphorylation to the cytosol. sec an endoplasmic reticulum membrane protein translocator, participates in the translocation of newly-synthesized proteins into the lumen of the er. the hydrolysis of the signal peptide for newly-synthesized proteins is carried out by specific proteases called signal peptidases located on the luminal side of er. the removal of the signal peptide is crucial for the maintenance of er homeostasis. the induction of protein transport protein sec subunit β in + al c. sinensis roots might provide an adaptive response by maintaining er homeostasis. however, its abundance decreased in + al c. grandis roots. by contrast, three proteins in protein transport decreased in + al c. sinensis roots. besides their role in fe storage, ferritins also play a role in preventing oxidative damage by storing free fe in a safe form. our finding that + al c. sinensis roots had higher abundance of ferritin agrees with the report that there was an increase in the expression of the ferritin fe storage gene, atfer in p-deficient arabidopsis leaves. nucleobase-ascorbate transporters have been identified in prokaryotes, fungi, plants and mammals. in plants, maize leaf permease has been characterized by functional complementation of a purine transport-deficient aspergillus nidulans strain and is necessary for proper chloroplast development in maize. interestingly, leaf permease protein does not contain a plastidic transit peptide and is expressed only in non-photosynthetic tissues such as roots and etiolated leaves. the up-regulation of putative permease in + al c. sinensis roots might be involved in al-tolerance throught maintaing chloroplast morphology. plant lipid transfer proteins are responsible for the shuttling of phospholipids and other fatty acid groups between cell membranes. some ltps with broad specificity are termed non-specific ltps. we found that the abundances of nsltp and nsltp-like protein decreased in + al c. grandis roots. this agrees with the reports that nsltp was up-regulated in al-tolerant cultivar rice roots, and down-regulated in al-sensitive ones, that the expression levels of two genes encoding ltp-like proteins in wheat roots were higher in al-tolerant than in al-sensitive near isogenic-line, and that mrna levels of ltps were higher in al-tolerant than in al-sensitive soybean genotype. similarly, putative phosphatidylglycerol/phosphatidylinositol transfer protein ddb_g0282179-like involved in lipid transport was increased in + al c. sinensis roots. however, wang et al. showed that al-toxicity decreased the abundance of ltp in al-tolerance rice roots, but did not signifaicantly affect its level in al-sensitive ones. taken all together, the ability of cell transport was enhanced in + al roots, especially in c. sinensis, which might contribute to its higher al-tolerance. proteins involved in biological regulation and signal transduction perception and transmission of stress signals by the cell play crucial roles in plant response to abiotic stresses. protein kinases and phosphatases are the key players in cell signals. here, we isolated five up-regulated proteins in phosphorylation and dephosphorylation in + al c. sinensis roots, demonstrating that al-toxicity triggered phosphorylation-dependent signal transduction pathway in c. sinensis roots, which might be involved in its al-tolerance. by contrast, two down-regulated and two up-regulated proteins in phosphorylation and one up-regulated protein in dephosphorylation were detected in + al c. grandis roots. this suggested that the balance between phosphorylation and dephosphorylation might be upset and phosphorylation of some proteins might be impaired in + al c. grandis roots. obviously, c. sinensis roots had a higher capacity to keep a better balance between phosphorylation and dephosphorylation than c. grandis ones under al-toxicity, which might contribute to c. sinensis al-tolerance. purple acid phosphatases play important roles in p acquisition and recycling in plants. wang et al. observed that pap expressed at a higher level in al-tolerant than in al-sensitive rice root, suggesting that al-tolerant cultivar might relieve the al-toxicity through improving the p acquisition. the higher up-regulation of pap in + al c. sinensis than in c. grandis roots agrees with our report that the former might more effectively acquire p than the latter with or without al-stress. plasma membrane-associated cation-binding protein is involved in intracellular signals through interaction with phosphatidylinositol phosphates and calmodulin. our finding that + al c. sinensis roots displayed higher abundance of pcap agrees with the report that cu-stress indced the expression of pcap in arabidopsis. our finding that the level of putative g3bp-like protein-like, a protein involved in the ras signal transduction pathway, was decreased only in + al c. grandis roots. this agrees with the report that the abundance of g3bp was up-regulated in wild al-tolerant barley and unchanged in al-tolerant barley cultivar, and down-regulated in al-sensitive wild barley when exposed to al-toxicity. these results indicated that the down-regulation of g3bp might be associated with the lower al-tolerance of c. grandis. proteins involved in cell wall and cytoskeleton metabolism plant cytoskeleton is highly dynamic networks of proteinaceous components consisting mainly of microtubules and microfilaments. al-induced growth inhibition and swelling of roots demonstrated that plant cytoskeleton could be a target of al-toxicity. studies showed that root microtubules and microfilaments were altered by al-toxicity. in wheat, al led to disorganization of actin filaments and formation of actin deposits. as expected, we identified five down-regulated and two down-regulated ] proteins related to cytoskeleton metabolism in c. sinensis and c. grandis roots, respectively. apart from cytoskeleton proteins, we isolated two down-regulated proteins in cell wall metabolism in + al c. sinensis roots. to conclude, al-induced alterations in root cell wall and cytoskeleton metabolism differed between the both species. ja biosynthesis phytohormones play a key role in plant response to biotic stresses. among these, one of the most important signal molecules is ja. here, we obtained four up-regulated proteins involved in ja biosynthesis. lox is one of the key enzymes responsible for the biosynthesis of ja. al-induced up-regulation of lox has also been reported in soybean and cassia tora roots. in arabidopsis and rice, opr and opr are responsible for ja production, respectively. acx catalyze the first step in the peroxisomal β-oxidation stage of ja biosynthesis. wound-induced ja accumulation was reduced in a mutant that was defective in acx and was abolished in a mutant that was impaired in both acx and its closely related paralog, acx. thus, both ja biosynthesis and level might be increased in + al c. sinensis roots, thus enhancing plant al-tolerance. because lox may increase the formation of oxidation products, al-induced up-regulation of lox has been suggested to be a response to al-toxicity.  our results demonstrate that c. sinensis is more tolerant to al-toxicity than c. grandis. using itraq technique, we isolated differentially expressed proteins from + al c. sinensis and c. grandis roots. among these proteins, proteins only presented in c. sinensis, proteins only presented in c. grandis, and proteins were shared by the two species. in the overlapping proteins, proteins were regulated in the same direction upon al exposure. this indicated more remarkable metabolic flexibility in c. sinenis than in c. grandis roots, thus improving the al-tolerance of c. sinensis. as shown in fig.  the higher al-tolerance of c. sinensis might be related to following several factors, including: activation of s metabolism; greatly improving the total ability of antioxidation and detoxification; up-regulation of carbohydrate and energy metabolism; enhancing cell transport; decreasing proteins related to protein synthesis; maintaining a better balance between protein phosphorylation and dephosphorylation; and increasing ja biosynthesis. to sum up, we identified more differentially expressed proteins than those of previous studies in other plant roots and provided the most integrated view of the adaptive responses occurring in + al roots. therefore, our analysis of root al-toxicity-responsive proteins will increase our understanding of al-tolerant mechanisms in higher plants.fig. a potential model for the adaptive responses of c. sinensis and c. grandis roots to al-toxicity. cgr: c. grandis roots; csr: c. sinensis roots; gly: glyoxalase; misap: mitochondrial intermembrane space import and assembly protein; pepc: phosphoenolpyruvate carboxylase; pm atpase: plasma membrane h+-atpase; ppiase: peptidyl-prolyl cis-trans isomerase; plap: plastid-lipid-associated protein; r-protein: ribosomal protein; ss: sucrose synthase; trx m: thioredoxin m; ptp: phosphatidylglycerol/phosphatidylinositol transfer protein; vap: vesicle-associated protein 
these proteins were classified into following categories: sulfur metabolism, stress and defense response, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, cell transport, biological regulation and signal transduction, cell wall and cytoskeleton metabolism, and jasmonic acid biosynthesis. our results demonstrated that metabolic flexibility was more remarkable in c. sinenis than in c. grandis roots, thus improving the al-tolerance of c. sinensis. the aims of this work were to determine the molecular mechanisms of plants to deal with al-toxicity and to identify differentially expressed proteins involved in al-tolerance. the higher al-tolerance of c. sinensis may be related to several factors, including: activation of sulfur metabolism; greatly improving the total ability of antioxidation and detoxification; up-regulation of carbohydrate and energy metabolism; enhancing cell transport; decreased abundances of proteins involved in protein synthesis; keeping a better balance between protein phosphorylation and dephosphorylation; and increasing ja biosynthesis. this provided the most integrated view of the adaptive responses occurring in al-toxicity roots. we isolated differentially expressed proteins from + al citrus roots. c. sinensis was more tolerant to al-toxicity than c. grandis. among these proteins, proteins only presented in c. sinensis, and proteins were shared by the two species. thereafter, we investigated citrus root protein profiles using isobaric tags for relative and absolute quantification. seedlings of ‘xuegan’ and ‘sour pummelo’ were treated for 18 weeks with nutrient solution containing or  mm alcl3 · 6h2o.
 limited information is available on aluminum -toxicity-responsive proteins in woody plant roots. seedlings of ‘xuegan’ and ‘sour pummelo’ were treated for 18 weeks with nutrient solution containing or  mm alcl3 · 6h2o. thereafter, we investigated citrus root protein profiles using isobaric tags for relative and absolute quantification. the aims of this work were to determine the molecular mechanisms of plants to deal with al-toxicity and to identify differentially expressed proteins involved in al-tolerance.  c. sinensis was more tolerant to al-toxicity than c. grandis. we isolated differentially expressed proteins from + al citrus roots. among these proteins, proteins only presented in c. sinensis, and proteins were shared by the two species. of the overlapping proteins, proteins were regulated in the same direction upon al exposure in the both species. these proteins were classified into following categories: sulfur metabolism, stress and defense response, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, cell transport, biological regulation and signal transduction, cell wall and cytoskeleton metabolism, and jasmonic acid biosynthesis. the higher al-tolerance of c. sinensis may be related to several factors, including: activation of sulfur metabolism; greatly improving the total ability of antioxidation and detoxification; up-regulation of carbohydrate and energy metabolism; enhancing cell transport; decreased abundances of proteins involved in protein synthesis; keeping a better balance between protein phosphorylation and dephosphorylation; and increasing ja biosynthesis.  our results demonstrated that metabolic flexibility was more remarkable in c. sinenis than in c. grandis roots, thus improving the al-tolerance of c. sinensis. this provided the most integrated view of the adaptive responses occurring in al-toxicity roots. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords aluminum-toxicitycitrusitraqproteomicsrootsulfur metabolismissue-copyright-statement© the author 2015 