BACKGROUND
manganese , which is the twelfth abundant element and the third most common element in the earth’s crust, is absorbed mainly as mn2+ by plant roots  <cit> .
mn, an essential trace element for the normal growth and development of higher plant, is involved in many biochemical processes.
with decreasing ph, the amount of exchangeable mn  increases in the soil solution  <cit> .
like other heavy metals, however, mn is harmful to most of the plants when present in excess  <cit> .
after aluminum , mn-toxicity is probably the most important factor limiting plant productivity in acidic soils, which comprise up to 50% of the world’s potentially arable lands  <cit> .
furthermore, the acidity of the soils is gradually increasing due to rapid industrialization, the emission of acidic gases and consequently acid deposition  <cit> .
disturbance of plant metabolism by mn-toxicity happens in multiple ways.
toxic effects of mn on plants include inhibition of growth, transpiration and photosynthesis  <cit> , apoplastic deposition of oxidized mn and phenolics  <cit> , induction of oxidative stress through direct generation of reactive oxygen species   <cit> , interfering with the absorption, translocation, and use of other mineral elements  <cit> , impairment of leaf structure and chloroplast ultrastructure  <cit> , alteration of hormone balances  <cit> , modification of enzyme  activities, and affecting carbohydrate, amino acid, protein and nucleic acid metabolisms  <cit> .
to deal with heavy metal stresses, plants have evolved a considerable degree of developmental plasticity, including adaptive responses via cascades of molecular networks  <cit> .
increasing evidence shows that plant responses to heavy metal stresses is associated with changes in the expression profiles of genes involved in a broad spectrum of physiological, biochemical and cellular processes including carbohydrate and energy metabolism, photosynthesis, protein biosynthesis and degradation, nucleic acid metabolism, signal transduction, transcriptional regulation, cell transport and stress responses  <cit> .
however, limited data are available on the differential expression of genes in response to mn-toxicity in plants.
techniques for gene expression analyses in plants have been widely explored.
cdna-amplified fragment length polymorphism , which does not require prior sequence information, is an efficient, sensitive, and reproducible technology for the discovery and identification of genes based on their polymorphism or differential expression patterns  <cit> .
this technique is a robust and high-throughput tool for analysis of genome-wide gene expression fluctuation induced by a specific stress and is also a useful tool for the isolation of novel genes  <cit> .
citrus belongs to evergreen subtropical fruit trees and is cultivated in humid and subhumid of tropical, subtropical, and temperate regions of the world mainly on acidic soils.
although the effects of mn-toxicity on citrus chloroplast ultrastructure, co <dig> assimilation, carbohydrates, photosynthetic electron transport and antioxidant systems have been investigated  <cit> , very little is known about mn-toxicity-responsive genes in citrus plants.
in this study, we investigated the effects of mn-toxicity on growth, leaf co <dig> assimilation, leaf concentrations of malondialdehyde , chlorophyll  and total soluble protein, root, stem and leaf concentration of mn, leaf phosphorus  and magnesium  concentrations, and expression of leaf genes revealed by cdna-aflp in citrus grandis and citrus sinensis seedlings having different mn-tolerance.
the objectives of this study were to understand the mechanisms of citrus mn-tolerance and to identify differentially expressed genes, which might be involved in mn-tolerance.
RESULTS
plant growth, root, stem and leaf mn concentration, and leaf mg and p concentrations
for c. grandis, mn-toxicity decreased root, shoot and whole plant  dry weight , and increased the ratio of root dw to shoot dw.
however, mn-toxicity did not significantly affect root, shoot and whole plant dw in c. sinensis seedlings except for increased ratio of root dw to shoot dw.
root dw, whole plant dw and the ratio of root dw to shoot dw were higher in c. sinensis seedlings than in c. grandis ones or similar between two species, except that shoot dw was lower in the former at the  <dig> μm mn treatment .
in addition, a few c. grandis leaves from the minority of mn-toxicity plants became interveinal chlorosis or necrotic blotching of foliage, while no visible symptoms occurred in mn-toxicity c. sinensis leaves .
as shown in figure  <dig>  mn-toxicity increased root, stem and leaf mn concentration, mn distribution in roots, mn uptake per plant and per root dw, and decreased mn distribution in stems and leaves.
under control condition, all these parameters did not significantly differ between c. grandis and c. sinensis seedlings.
when exposed to mn-toxicity, stem mn concentration, mn uptake per plant and mn distribution in roots were higher in c. sinensis seedlings than in c. grandis ones, while leaf mn concentration and mn distribution in leaves were lower in the former than in the latter.
mn-toxicity decreased p and mg concentrations in c. grandis leaves, but did not significantly affect them in c. sinensis leaves .
leaf total soluble protein, mda and chl concentrations and gas exchange
total soluble protein concentration was decreased by mn-toxicity in c. grandis leaves, but was not significantly affected in c. sinensis ones .
as shown in figure 4b, mn-toxicity did not significantly affect leaf concentration of mda.
mn-toxicity decreased co <dig> assimilation , transpiration  and stomatal conductance  in leaves of c. grandis and c. sinensis, especially in the former.
mn-toxicity increased intercellular co <dig> concentration in c. grandis leaves, but did not significantly affect it in c. sinensis leaves .
mn-toxicity did not significantly affect leaf concentrations of chl a+b, chl a and chl b, and the ratio of chl a to chl b in the two citrus species .
identification of differentially expressed genes by cdna-aflp
a total of  <dig> selective primer combinations were used for the cdna-aflp analysis in order to identify the genes responsive to mn-toxicity in the leaves of two citrus species differing in mn-tolerance .
for c. grandis leaves, a total of 16– <dig>  clear and unambiguous transcript-derived fragments  were detected with each premier combination, which resulted in approximately  <dig> tdfs, ranging from 100– <dig> bp.
a total of  <dig> differentially expressed and reproducible tdfs were recovered from the silver-stained cdna-aflp gels based on their presence, absence or difference in the levels of expression.
all these tdfs were re-amplified, ligated and sequenced, and  <dig> cdna fragments produced useable sequence data.
tdf sequences were compared with those present in the genbank database .
of the  <dig> tdf sequences,  <dig> tdfs showed significant homology to genes encoding known or putative proteins, and  <dig> tdfs were homologous to genes encoding uncharacterized proteins, hypothetical proteins or unknown proteins.
the remaining  <dig> tdfs did not show homology to any nucleotide or amino sequence in the public databases.
of these  <dig> matched tdfs,  <dig>  tdfs increased and  <dig>  decreased in response to mn-toxicity.
according to the biological functional properties, these tdfs were classified into the following functional categories: biological regulation and signal transduction , carbohydrate and energy metabolism , nucleic acid metabolism , protein metabolism , lipid metabolism , cell wall metabolism , stress responses , cell transport , other and unknown biological processes  .
for c. sinensis leaves, a total of 16– <dig>  clear and unambiguous tdfs were obtained with each premier pair, which yield approximately  <dig> tdfs, ranging from 100– <dig> bp.
a total of  <dig> differentially expressed and reproducible tdfs were isolated from the silver-stained cdna-aflp gels.
all these tdfs were sequenced, and produced readable sequences .
of the  <dig> tdfs,  <dig> tdfs were homologous to genes encoding known or putative proteins, and three tdfs belonged to genes encoding hypothetical proteins.
the remaining five tdfs had no database matches.
among the  <dig> matched tdfs, seven  tdfs were up-regulated and eight  was down-regulated by mn-toxicity.
these tdfs were involved in biological regulation and signal transduction , carbohydrate and energy metabolism , nucleic acid metabolism , protein metabolism , cell transport , other and unknown biological processes  .
qrt-pcr analysis of some mn-toxicity-responsive genes
to validate the cdna-aflp expression patterns,  <dig> tdfs from c. grandis leaves and one tdf from c. sinensis ones were selected for qrt-pcr analysis.
the expression levels of all these tdfs except for two tdfs  matched well with the expression profiles observed with cdna-aflp .
the discrepancy in expression patterns for two tdfs between qrt-pcr and cdna-aflp analysis might be due to gene family complexity.
nevertheless, the cdna-aflp technique allowed us to isolate the differentially expressed genes under mn-toxicity.
discussion
c. sinensis is more tolerant to mn-toxicity than c. grandis
as shown in figures  <dig> and 2a-c,  <dig> μm mn treatment greatly inhibited c. grandis plant growth, especially the shoots and increased the concentration of mn in roots, stems and leaves, and foliar mn concentration for  <dig> μm mn treatment was far more than the sufficiency range of 25– <dig> mg kg- <dig> dw for sweet orange  leaves  <cit> .
based on these results, plants that received  <dig> μm mn are considered mn-excess .
mn-toxicity-induced decrease in plant dw and increase in root dw/shoot dw ratio  agree with the previous results obtained on c. grandis <cit>  and with the view that mn-toxicity affects plant tops more than root systems  <cit> .
mn-toxicity, however, had no influence on the ratio of root dw to shoot dw in lucerne  plants, despite mn-toxicity depressed growth of shoots and roots  <cit> .
our results showed that mn-toxicity c. grandis plants had decreased root, shoot and root + shoot dw, and increased ratio of root dw to shoot dw, while mn-toxicity did not significantly affect c. sinensis growth except for increased root dw/shoot dw ratio , meaning that c. sinensis is more tolerant to mn-toxicity than c. grandis.
this is also supported by our data that the gas exchange in c. grandis leaves was affected by mn-toxicity far more than in c. sinensis ones , and that mn-toxicity decreased total soluble protein, p and mg concentrations only in c. grandis leaves .
like that of previous workers  <cit> , the observed lower co <dig> assimilation in mn-toxicity leaves of c. grandis and c. sinensis was primarily caused by non-stomatal factors because the decreases in both co <dig> assimilation and stomatal conductance was accompanied by unchanged or increased intercellular co <dig> assimilation .
it is noteworthy that the reduction in co <dig> assimilation in leaves of mn-toxicity plants could not attributed to photo-oxidative damage and decreased chl, because there were no significant differences in leaf concentrations of mda , chl a+b, chl a and chl b  between control and mn-toxicity leaves.
similar results have been obtained on c. grandis <cit> .
under mn-toxicity, the majority of mn in c. sinensis and c. grandis plants was retained in the roots , as previously found for c. grandis <cit> , lucerne  <cit>  and douglas fir  <cit> .
however, in rice exposed to mn-toxicity, mn was predominantly accumulated in leaves compared with roots  <cit> .
the tolerance of plants to mn is associated not only with low mn uptake, but also with relatively little mn translocation from roots to shoots  <cit> .
our results showed that under mn-toxicity, c. sinensis plants accumulated more mn in roots and less mn in shoots than c. grandis ones, and that the concentration of mn was lower in mn-toxicity c. sinensis leaves than in c. grandis ones .
this might contribute to the mn-tolerance of c. sinensis.
we isolated  <dig> differentially expressed tdfs from mn-toxicity c. grandis leaves, which belong to different functional categories , meaning that mn-toxicity affected different physiological and biochemical pathways.
in contrast, we only identified  <dig> differentially expressed tdfs from mn-toxicity c. sinensis leaves .
obviously, the transcript profile was less affected by mn-toxicity in c. sinensis leaves compared to c. grandis ones, which may be associated with the less mn accumulation in mn-toxicity c. sinensis leaves .
these data also support above inference that c. sinensis is more tolerant to mn-toxicity than c. grandis.
genes involved in biological regulation and signal transduction
protein phosphorylation, a versatile post-translational modification , is involved in almost all plant signal pathways and plays important roles in regulation of abiotic stress responses  <cit> .
phosphorylation and dephosphorylation of a protein often serve as an “on-and-off” switch in the regulation of cellular activities.
for optimal regulation, kinases and phosphatases must strike a balance in any given cell  <cit> .
as shown in additional file  <dig>  mn-toxicity decreased the expression levels of genes involved in phosphorylation  and dephosphorylation  except for increased expression of a mitogen-activated protein kinase  <dig>  gene in c. grandis leaves.
this means that the balance between phosphorylation and dephosphorylation was upset and phosphorylation of some proteins might be impaired in mn-toxicity c. grandis ones.
mapk cascades are important signal modules that convert signals generated at the receptors/sensors to appropriate cellular responses  <cit> .
increasing evidence demonstrates the role of mapk signal in different heavy metal stresses for different plant species  <cit> .
the observed higher expression level of mapk  <dig>  in mn-toxicity leaves agrees with the previous reports that excess copper , cadmium  and mercury  led to the activation of a novel mapk gene osmsrmk <dig> from japonica-type rice  <cit> , and that mapk pathways were activated by excess cu and cd in m. sativa seedlings  <cit> .
thus, mapk cascade might play a role in the responses of plants to mn-toxicity.
in contrast to c. grandis, vh1-interacting kinase  gene in c. sinensis leaves was induced by mn-toxicity .
mapks are able to phosphorylate different substrates in different cellular compartments, including transcription factors  in the nucleus  <cit> .
thus, genes related to tfs might be affected in mn-toxicity c. grandis leaves due to altered expression level of mapk  <dig>  gene .
as expected, mn-toxicity increased the expression levels of genes encoding tf jumonji domain-containing protein  and myb family transcription factor , and decreased the expression levels of genes encoding tf ilr <dig> , c3h <dig> type zinc finger protein , putative tf  and dna-binding storekeeper protein-related transcriptional regulator  in c. grandis leaves .
jumonji c  domain-containing proteins have been shown to function as demethylases and to involve in chromatin structure and gene expression  <cit> .
recently, govind et al.
<cit>  showed that two jumonji tfs  and other genes  were induced in drought-stressed peanut  plants, which agrees with our results that mn-toxicity c. grandis leaves had higher mrna levels of gene encoding tf jumonji domain-containing protein .
they also found that silencing of jumonji  made the transgenic tobacco  plants more tolerant to drought, while down-regulation of hsp <dig> resulted in susceptibility  <cit> .
rampey et al.
<cit>  reported that ilr3- <dig> arabidopsis seedlings were less sensitive than wild type to mn-toxicity.
ilr <dig> is a basic helix–loop–helix type tf, which seems to regulate metal homeostasis in part through the action of putative fe/mn ccc1-like  transporters.
therefore, the down-regulation of ilr <dig> in mn-toxicity c. grandis leaves might be an adaptive response.
like c. grandis, the expression of dna-binding storekeeper protein-related transcriptional regulator  was inhibited in c. sinensis leaves.
ilr <dig> , however, was up-regulated in mn-toxicity c. sinensis leaves .
calcium  is a secondary messenger and has been suggested to participate in heavy metal signal  <cit> .
indeed, ca concentration in runner bean  cells greatly increased in response to cd stress  <cit> .
ca may interact with calmodulin to propagate the signal and ultimately to regulate downstream genes involved in heavy metal transport, metabolism, and tolerance  <cit> .
in this study, mn-toxicity decreased the expression levels of ca-dependent protein kinase 1-like , calmodulin-binding transcription activator  <dig>  and ca2+-binding protein  genes in c. grandis leaves, and increased the expression levels of ca-binding ef-hand domain-containing protein  gene in c. grandis leaves and calmodulin-binding transcription activator  <dig>  in c. sinensis ones .
thus, genes related to ca2+ signal might be involved in response to mn-toxicity.
busov et al.
<cit>  characterized a 5ng <dig> gene from juvenile loblolly pine shoots, which was highly and specifically induced by auxin prior to adventitious root formation.
toxicity of mn, on the other hand, led to auxin deficiency caused by activation of indole-acetic acid oxidase under excess mn  <cit> .
this supports our data that auxin-induced protein 5ng <dig> gene  was down-regulated in c. sinensis leaves .
similarly, the transcript abundance of auxin-response factor  in c. grandis leaves also decreased in response to mn toxicity .
however, mn-toxicity increased 5ng <dig> mrna level in c. grandis ones .
transducin/wd- <dig> repeat-containing protein is a g protein involved in a wide range of functions that include signal transduction, transcription and stress-tolerant function.
it was up-regulated by excess cu in germinating rice seeds  <cit> , but was down-regulated by mn-toxicity in c. grandis leaves .
vq motif-containing proteins play important roles in plant growth, development and defense responses.
loss-of function mutants and/or overexpression lines for the vq genes are altered in seed size, tolerance to abiotic stress, or resistance to pathogen infection  <cit> .
liu et al.
<cit>  reported that transgenic arabidopsis plants overexpressing the atarvq <dig>  a gene encoding a plant-specific vq motif-containing protein, were more resistant to arsenate stress.
recently, cheng et al.
<cit>  showed that plant vq motif-containing proteins played critical roles in the network of wrky-mediated gene expression.
we found that under mn-toxicity, the expression level of vq genes  was decreased in c. grandis, which is in agreement with the previous report that atarvq <dig> expression was strongly down-regulated by arsenate stress  <cit> .
zhang et al.
<cit>  observed that a. thaliana fes1a could prevent cytosolic hsp <dig> degradation.
thus, the observed lower mrna level of hsp <dig> nucleotide exchange factor fes <dig>  in mn-toxicity c. grandis leaves agrees with the results that the expression levels of hsp <dig>  and stromal  <dig> kda heat shock-related protein, chloroplastic-like gene  in c. grandis ones decreased in response to mn-toxicity .
similar result has been obtained on b-deficient c. sinensis roots  <cit> .
phosphatidylinositol  4-kinase catalyzes the phosphorylation of ptdins in the d- <dig> position of the inositol ring.
this is the committed step in the synthetic pathway leading to ptdins  <dig> -bisphosphate .
apart from this function in signal transduction, both ptdins 4-p and ptdins  <dig> -p <dig> appear to have other cellular functions  in c. grandis leaves decreased in response to mn-toxicity , which disagrees with mn-stimulated ptdins 4-kinase from dunaliella parva <cit> .
based on these results above mentioned, we conclude that the responses of citrus plants to mn-toxicity might be regulated in multiple signal pathways.
genes involved in carbohydrate and energy metabolism
thirteen genes in c. grandis leaves and two genes in c. sinensis ones related to carbohydrate and energy metabolism were altered under mn-toxicity .
atp synthase is an important enzyme that provides energy for the cell to use through the synthesis of atp.
hamilton et al.
<cit>  showed that exposure of wheat to al increased mitochondrial f1f0-atpase activity only in the al-tolerant wheat variety, and concluded that the al-induced increase in atp synthase activity was an adaptive response involved in al tolerance.
similar to al-toxicity, mn-toxicity increased the expression levels of synthase subunit alpha  in c. grandis  and c. sinensis  leaves and atp synthase subunit alpha  in c. grandis leaves .
aconitate hydrolyase  involved in glycolysis catalyses the interconversion of citrate and isocitrate, via a cis-aconitate.
our finding that the expression of ah  <dig>  was induced in mn-toxicity c. grandis leaves means that its activity was enhanced by mn-toxicity, which is in agreement with the previous report that ah was up-regulated by cd stress  <cit> .
in addition, mn-toxicity also increased the expression levels of nadph-ferrihemoprotein reductase  and cytochrome  c biogenesis orf <dig>  genes related to respiration in c. grandis leaves .
overall, the metabolic pathways related to atp synthesis and reducing power production might be activated in mn-stressed leaves to yield more energy needed to meet the high-energy demand of stressed cell.
by contrast, the mrna level of gene encoding 2-phospho-d-glycerate hydrolase , an essential and ubiquitous glycolytic enzyme responsible for the catalysis of the conversion of 2-phosphoglycerate  to phosphoenolpyruvate , was down-regulated in mn-toxicity c. grandis leaves .
this agrees with the previous report that the abundance of enolase in agrostis stolonifera leaves was decreased after  <dig> d of heat stress  <cit> .
ferreira et al.
in populus euphratica leaves observed that the level of enolase was transiently increased after  <dig> h of heat stress, followed by a decrease after  <dig> h of heat stress  <cit> .
however, other studies showed that leaf enolase was induced in various plants by different abiotic stresses, such as water stress in maize  <cit> , cold stress in tobacco  <cit> , salt stress in ice plant   <cit>  and cd-stress in phytolacca americana <cit> .
like to enolase, the expression level of gene encoding cyt p <dig> , an enzyme typically catalyzing the reaction: rh + o <dig> + nadph + h+ → roh + h2o + nadp+, was reduced in mn-toxicity c. grandis leaves , which might contribute to maintaining nadph homeostasis by decreasing its utilization.
however, the expression of cyt p <dig>  family  <dig>  subfamily a, polypeptide  <dig>  gene in c. sinsnsis leaves was induced by mn-toxicity , which is in agreement with the report that at least  <dig> of the  <dig> cyt p450s were induced in arabidopsis by abiotic and biotic stress 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 cyt p <dig> tolerated better oxidative stress after herbicide treatment  <cit> .
therefore, the observed higher mrna level of cyt p <dig>  family  <dig>  subfamily a, polypeptide  <dig> might be advantegous to mn-toxicity tolerance of c. sinensis plants.
thioredoxins  are small proteins, which are involved in the cell redox regulation.
trx m, a chloroplastic protein, preferentially activates nadp-malate dehydrogenase , a key enzyme involved in carbon fixation and sugar biosynthesis during photosynthesis.
historically the m-type trxs were so named because they were able to activate nadp-mdh  <cit> .
the activity of nadp-mdh might be down-regulated in mn-toxicity c. grandis leaves due to lower mrna levels of trx m  and trx m <dig>  .
evidence shows that trx m <dig>  m <dig>  and m <dig> could act as antioxidants, i.e.
possibly by serving as hydrogen donors for a trx-dependent peroxidase  <cit> .
trehalose-6-phosphate synthase  is a key enzyme for synthesizing trehalose, which plays an important role in abiotic stress tolerance of plants  <cit> .
the observed lower level of tps  in mn-xicity c. grandis leaves  indicates that mn-toxicity might decrease leaf concentration of trehalose, thus lowering the stress tolerance.
atp sulfurylase  activity were enhanced by sulphur  deprivation and reduced after resupply of so42– <cit> .
heiss et al.
<cit>  reported that cd stress strongly increased the expression levels of atps and 5’-adenylysulfate reductase in roots and leaves of 6-week-old brassica juncea, accompanied by enhanced cysteine concentration in roots and leaves, and concluded that roots and leaves responded to cd stress with a coordinative up-regulation of several s assimilation enzymes to meet an increased demand for cysteine during phytochelatin synthesis.
however, the expression of atps  was down-regulated in mn-toxicity c. grandis leaves .
udp-glycosyltransferases , which glycosylate a broad array of aglycones, including plant hormones, all major classes of plant secondary metabolites, and xenobiotics such as herbicides, play an important role for the stabilization, enhancement of water solubility and detoxification of natural products  <cit> .
cytokinins can be glucosylated to form o-glucosides and n-glucosides.
the glycoconjugates are inactive and are considered to play a role in hormonal homeostasis.
hou et al.
<cit>  showed that ugt76c <dig> and ugt76c <dig>  recognized all cytokinins and glucosylated them at the n <dig> and n <dig> positions.
veselov et al.
<cit>  reported that cd stress sharply lowered the concentration of cytokinin in wheat roots and shoots caused by elevated activity of cytokinin oxidase.
obviously, the down-regulation of udp-glycosyltransferase 76f1-like  gene in mn-toxicity c. grandis leaves  is advantageous to cytokinin homeostasis under mn-toxicity if leaf concentration of cytokinin decreased in response to mn-toxicity.
genes involved in nucleic acid metabolism
evidence shows that heavy metals inhibit plant nucleic acid metabolism  <cit> .
as expected, the expression levels of genes encoding tho complex, subunit  <dig> , dna polymerase phi subunit , histone h <dig> , dna - methyltransferase drm2-like  and luc7-like protein 3-like  in c. grandis leaves  and genes encoding tho complex, subunit  <dig>  and histone h <dig>  in c. sinensis ones  decreased in response to mn-toxicity, indicating that leaf nucleic acid metabolism might be impaired by mn-toxicity.
genes involved in protein metabolism
mn-toxicity has been demonstrated to affect protein metabolism in plants  <cit> .
we found that the expression levels of three ribosomal genes encoding ribosomal protein s <dig> , ribosomal protein s <dig>  and 60s ribosomal protein l <dig>  mitochondrial-like , which are involved in mature ribosome assembly and translation processes, were down-regulated in mn-toxicity c. grandis leaves , while only one ribosomal gene encoding 60s ribosomal protein l <dig>  mitochondrial-like  in c. sinensis leaves was down-regulated by mn-toxicity .
this indicates that the biosynthesis of protein in mn-toxicity c. grandis leaves might be impaired more severe than in mn-toxicity c. sinensis ones, which is in agreement with our data that mn-toxicity decreased the concentration of total soluble protein in c. grandis leaves, but did not affect its concentration in c. sinensis ones .
however, the expression of 30s ribosomal protein s <dig> gene  was induced in mn-toxicity c. grandis leaves .
translational control of protein synthesis depends on numerous eukaryotic initiation factors .
our finding that the expression of gene encoding similar to translation initiation factor if <dig>  was down-regulated in mn-toxicity c. grandis leaves  agrees with our previous report that the abundances of eif4b <dig>  eif <dig> subunit and eif3g <dig> in c. sinensis roots decreased in response to b-deficiency  <cit> .
however, salt and heavy metal induced the accumulation of rice eif5a- <dig> and eif5a- <dig> mrnas in rice cells  <cit> .
elongation factors whose expressions have been demonstrated to correlate to the high rate of protein synthesis in developing plant tissues  <cit>  facilitate translational elongation, from the formation of the first peptide bond to the formation of the last one in the ribosome.
guo et al.
<cit>  isolated a translation elongation factor 2-like protein gene from a cold defective arabidopsis mutant, los 1– <dig>  the los1- <dig> mutant plants were impaired in protein synthesis under cold stress.
we found that the expression level of one gene encoding translation elongation factor- <dig> alpha, partial  in c. grandis leaves decreased in response to mn-toxicity , which agrees with the previous results obtained on drought-stressed soybean nodules  <cit> , anoxia maize roots  <cit> , and b-deficient roots of c. sinensis <cit> , brassica napus <cit>  and lupinus albus <cit> .
however, the abundances of chloroplast translational elongation factor tu and elongation factor p in rice leaves increased in response to cd stress  <cit> .
thus, it appears that the influence of abiotic stress on translation elongation factors depends on plant species and kinds of stresses.
in addition, the expression level of gene encoding tetratricopeptide repeat-containing protein  involved in protein synthesis was down-regulated in mn-toxicity c. grandis leaves .
these results further demonstrate that protein biosynthesis is impaired in mn-toxicity c. grandis leaves.
mn-toxicity increased the expression levels of genes encoding atp-dependent clp protease , cysteine proteinase  and xylem cysteine proteinase  <dig>  in c. grandis leaves .
this indicates that the hydrolysis of some proteins might be up-regulated in mn-toxicity leaves, thus decreasing leaf concentration of total soluble protein.
this is also supported by our data that mn-toxicity leaves had lower concentration of total soluble protein .
however, the expression levels of genes encoding carboxyl-terminal peptidase , papain family cysteine protease , cathepsin b-like cysteine proteinase like protein  and α/β-hydrolase-like protein  decreased in response to mn-toxicity .
like other ptms , ubiquitination, which serves as a versatile ptm, plays a key role in regulating plant response to abiotic stresses  <cit> .
the up-regulation of genes encoding mnd1-interacting protein  <dig>  and ubiquitin-conjugating enzyme e <dig>  <dig>  in mn-toxicity c. grandis leaves  might be an adaptive response to mn-toxicity.
however, the expression levels of ubiquitin-correlative genes such as ubiquitin-protein ligase  and btb and math domain-containing protein  in c. grandis leaves decreased in response to mn-toxicity , meaning that the ubiquitination of some proteins might be impaired in mn-toxicity c. grandis leaves.
three genes  involved in amino acid metabolism was down-regulated in mn-toxicity c. grandis leaves .
this means that the biosynthesis of amino acids might be impaired in mn-toxicity c. grandis leaves.
chorismate synthase catalyzes the last of the seven steps in the shikimate pathway which is used in prokaryotes, fungi and plants for the biosynthesis of aromatic amino acids  <cit> .
cbs catalyzes the first step of the transsulfuration pathway from homocysteine to cystathionine.
jung et al.
<cit>  observed that cbsx <dig> directly modulated trx in chloroplasts, which affected the level of h2o <dig> and, consequently, the expression of the genes involved in secondary cell-wall thickening, concluding that cbsx <dig> protein played a critical role in thickening of the secondary cell walls of the endothecium during anther dehiscence in arabidopsis.
transgenic tobacco plants overexpressing oscbsx <dig> isolated from rice displayed enhanced tolerance to salinity, heavy metal, and oxidative stress  <cit> .
this observed lower expression level of gene encoding cbs domain-containing protein  in c. grandis leaves  disagrees with our previous data that the abundance of cbs family protein in c. sinensis roots increased in response to b-deficiency  <cit> .
genes involved in lipid metabolism
lecithin-cholesterol acyltransferase  catalyzes the transacylation of acyl groups from phospholipids to sterols in mammals and yeast.
sterol acylation is an essential process of sterol homeostasis in eukaryotic cells.
in arabidopsis, phospholipid sterol acyltransferase  <dig> , which displays homology with the mammalian lcat, catalyzes a phospholipid-dependent  formation of sterol esters  <cit> .
recent work with arabidopsis showed that sterol ester concentration decreased in leaves of psat1- <dig> or psat1- <dig> mutants accompanied by an early leaf senescence phenotype, suggesting a major contribution of the psat <dig> in maintaining both free sterol homeostasis in plant cell membranes and leaf viability during developmental aging  <cit> .
the up-regulation of lecithin-cholesterol acyltransferase-like  <dig>  in mn-toxicity c. grandis leaves might enhance the leaf concentration of sterol ester, thus preventing leaf senescence.
genes involved in cell wall metabolism
our results showed that the expression levels of genes  involved in cell wall biosynthesis were down-regulated in mn-toxicity c. grandis leaves .
in addition, the mrna levels of gene encoding glycoside hydrolase family  <dig> protein , which catalyze the hydrolytic cleavage of the pectin and gene encoding cell wall-associated hydrolase  were up-regulated in mn-toxicity c. grandis leaves .
therefore, the formation of cell wall might be impaired in mn-toxicity leaves.
however, the expression of gene encoding protein trichome birefringence-like  <dig>  was induced in mn-toxicity c. grandis leaves .
genes involved in stress responses
mn-toxicity has been demonstrated to stimulate ros production in plants  <cit> .
since mn-toxicity did not affect the concentration of mda in citrus leaves , some protective antioxidant enzymes should be up-regulated to meet the increased requirement for scavenging ros.
our results showed that the expression of gene encoding catalase , an enzyme involved in scavenging h2o <dig>  was induced in mn-toxicity c. grandis leaves , which agrees with the previous report that mn-toxicity c. grandis leaves had increased specific activity of cat and similar leaf area-based activity of cat  <cit> .
besides ros scavenger enzymes, this detoxification mechanism also involves “house-keeping” enzymes.
the family of nudix hydrolases , which catalyze the hydrolytic breakdown of nucleoside diphosphates linked to some other moieties such as a phosphate, sugar or nucleoside, are one of these “house-keeping” enzyme families  <cit> .
ogawa et al.
<cit>  showed that atnudx <dig>  a chloroplastic atnudx, played an important role in modulation of the nadh and/or nadph pools through the hydrolysis of nadh to reduced nicotinamide mononucleotide  in arabidopsis chloroplasts.
transgenic arabidopsis plants overexpressing atnudx <dig> and atnudx <dig> displayed enhanced tolerance to oxidative stress, resulting from the maintenance of nad+ and atp levels by nucleotide recycling of free adp-ribose molecules  <cit> .
the higher expression level of nudx <dig>  in c. grandis leaves  agrees with the previous reports that the abundance of nudx homolog  <dig> in c. sinensis roots increased in response to b-deficiency  <cit>  and chrysanthemum lavandulifolium clnudx <dig>  clnudx <dig>  clnudx <dig> and clnudx <dig> were induced by salt, drought, heat, and cold stresses  <cit> .
however, the expression levels of genes encoding monodehydroascorbate reductase , peroxidase  <dig> , glutathione s-transferase  tau <dig> , nadp-dependent alkenal double bond reductase p <dig> , trx m , trx m <dig>  chloroplastic-like , and cbs domain-containing protein  related to oxidative defense in c. grandis leaves decreased in response to mn-toxicity .
it is noteworthy that mn-toxicity c. grandis leaves had higher specific activity of mdar and leaf area-based activity of mdar compared with controls  <cit> .
the discrepancy between the expression level of mdar and the activity of the corresponding enzyme indicates that ptms might affect mdar activity.
senescence is a genetically programmed decline in various cellular processes and involves in the hydrolysis of macromolecules such as proteins and lipids.
it is governed by the developmental age and is induced or enhanced by biotic and abiotic stresses.
generally in plants the term senescence and programmed cell death  denote the processes that initiate the programmed death of individual cell.
the senescence can be considered as one of the examples for pcd.
as expected, the expression level of one gene encoding for putative senescence-associated protein  was up-regulated by mn-toxicity in the less mn-tolerant c. grandis leaves .
this agrees with the previous report that the gene was highly differentially expressed only in the heat-sensitive fescue  genotype at 44°c  <cit> .
in addition, the expression levels of apoptosis linked genes  were enhanced in mn-toxicity c. grandis leaves .
thus, it is reasonable to assume that the mn-toxicity sensitive c. grandis plants were under great stress and leaf senescence was accelerated, which might contribute to plant survival by using more metabolites through glycolysis, and protein and lipid degradation.
as shown in figure  <dig>  the mn-toxicity-induced senescence in c. grandis leaves was a very complicated process.
hsps/chaperones have been demonstrated to play a key role in protecting plants against heavy metal stress  <cit> .
the observed lower expression levels of genes encoding heat shock protein-related, partial , heat shock protein 60-3a , hsp <dig>  and stromal  <dig> kda heat shock-related protein, chloroplastic-likes  in mn-toxicity c. grandis leaves  indicate decreased protein synthesis in these leaves, as indicated by decreased leaf concentration of total soluble proteins .
this is similar to the previous report that the abundance of hsp <dig> was enhanced in leaves of high cd-accumulating soybean cultivar, but was lowered in low cd-accumulating one  <cit> .
one crucial adaptive mechanism of plants to p-deficiency is the immediate cleavage of phosphate  from phosphorylated substrates.
phosphoethanolamine/phosphocholine phosphatase  may release pi from organic to maintain pi homeostasis of plant cells  <cit> .
the down-regulation of ppspase, putative gene  in c. grandis leaves  might decrease the release of pi from phosphorylated substrates, thus lowering leaf p concentration .
this agrees with the previous report that mn-toxicity decreased p concentration in tea leaves  <cit> .
genes involved in cell transport
eight genes in c. grandis leaves and one gene in c. sinensis ones were regulated by mn-toxicity .
we found that the expression of citrus sucrose transporter  <dig>  was induced in mn-toxicity c. grandis leaves, which is in agreement with the previous report that the gene strongly expressed in source, sugar exporting organs  <cit> , because mn-toxicity increased or did not affect the concentrations of non-structural carbohydrates in c. grandis leaves  <cit> .
the up-regulation of citrus sucrose transporter <dig> might be helpful to decrease sugar accumulation in mn-toxicity leaves, which might in turn lead to feedback suppression of co <dig> assimilation  <cit> .
one of strategies of plants to deal with heavy metals is to transport them out of the cells, thereby removing them from the cytosol.
kim et al.
<cit>  observed that transgenic a. thaliana plants overexpressing the abc transporter atpdr <dig> were more resistant to cd or lead  and displayed lower cd concentration in roots and shoots than wild-type plants, while atpdr <dig> rnai transgenic plants and t-dna insertion lines were more sensitive to cd or pb and had higher cd concentration, concluding that the abc transporter atpdr <dig> is a cd extrusion pump conferring heavy metal resistance.
the down-regulation of genes encoding abc-transporter-like protein  and abc transporter family protein  in c. grandis leaves  indicates that the extrusion of mn from leaves might be lessened in response to mn-toxicity, which might be one of the causes that mn-toxicity c. grandis leaves accumulated more mn than c. sinensis ones .
evidence has shown that cora-like proteins may represent the major transport systems in eukaryotes such as yeast, animals, and plants  <cit> .
the down-regulation of mg transporter cora-like protein gene in mn-toxicity c. grandis  and c. sinensis  leaves might reduce mg transport, hence decreasing leaf mg concentration.
this agrees with the previous report that mn-toxicity decreased leaf concentration of mg  <cit> .
interestingly, mg concentration was decreased by mn-toxicity only in c. grandis leaves, but was not significantly affected in c. sinensis leaves .
this means that leaf mg concentration is also regulated by other factors.
plant cyclic nucleotide gated channels , which comprise a large gene family in arabidopsis, have been proposed to be involved in multiple plant physiological processes including plant growth and heavy metal toxicity tolerance  <cit> .
evidence suggests that cngcs paly a role in heavy metal homeostasis, for example, in tobacco, overexpression of tobacco cngc  led to hypersensitivity to pb  <cit> .
however, sunkar et al.
<cit>  showed that transgenic tobacco plants overexpressing a truncated ntcbp <dig> exhibited improved tolerance to pb2+ and decreased uptake of this metal ion.
we found that the expression of cyclic nucleotide gated channel  <dig> gene  was down-regulated in mn-toxicity c. grandis leaves , suggesting that the gene might be involved in the response to mn-toxicity.
our results showed that the expression of gene encoding protease inhibitor/seed storage/lipid transfer protein  family protein  was down-regulated in mn-toxicity c. grandis leaves , indicating that the exchange of lipids between membranes might be decreased.
CONCLUSIONS
our results clearly demonstrated that c. sinensis was more tolerant to mn-toxicity than c. grandis.
under mn-toxicity, c. sinensis plants accumulated more mn in roots and less mn in shoots  than c. grandis ones, and that the leaf concentration of mn was lower in the former.
this might contribute to the mn-tolerance of c. sinensis.
in this study, we first used the cdna-aflp technique to compare the mrna levels of genes from control and mn-toxicity leaves of two citrus species differing in mn-tolerance.
in mn-toxicity c. grandis leaves,  <dig> up-regulated and  <dig> down-regulated genes were isolated, while only seven up-regulated and eight down-regulated genes were identified in mn-toxicity c. sinensis ones.
obviously, mn-toxicity affected gene expression far less in c. sinensis leaves than in c. grandis ones, which might be associated with less leaf mn concentration in mn-toxicity c. grandis leaves.
cdna-aflp analysis suggests that the responses of c. grandis leaves to mn-toxicity might include following several aspects:  accelerating leaf senescence;  activating the metabolic pathway related to atpase synthesis and reducing power production;  decreasing cell transport;  inhibiting protein and nucleic acid metabolisms;  impairing the formation of cell wall; and  triggering multiple signal transduction pathways.
we also identified many new mn-toxicity-responsive genes involved in biological and signal transduction , carbohydrate metabolism , protein metabolism , stress responses  and cell transport .
further studies will elucidate the roles of these genes in response to mn-toxicity, which will help me to design mn-tolerant transgenic crops.

 manganese, which is the twelfth abundant element and the third most common element in the earth’s crust, is absorbed mainly as mn2+ by plant roots. mn, an essential trace element for the normal growth and development of higher plant, is involved in many biochemical processes. with decreasing ph, the amount of exchangeable mn increases in the soil solution. like other heavy metals, however, mn is harmful to most of the plants when present in excess. after aluminum, mn-toxicity is probably the most important factor limiting plant productivity in acidic soils, which comprise up to 50% of the world’s potentially arable lands. furthermore, the acidity of the soils is gradually increasing due to rapid industrialization, the emission of acidic gases and consequently acid deposition. disturbance of plant metabolism by mn-toxicity happens in multiple ways. toxic effects of mn on plants include inhibition of growth, transpiration and photosynthesis, apoplastic deposition of oxidized mn and phenolics, induction of oxidative stress through direct generation of reactive oxygen species, interfering with the absorption, translocation, and use of other mineral elements, impairment of leaf structure and chloroplast ultrastructure, alteration of hormone balances, modification of enzyme activities, and affecting carbohydrate, amino acid, protein and nucleic acid metabolisms. to deal with heavy metal stresses, plants have evolved a considerable degree of developmental plasticity, including adaptive responses via cascades of molecular networks. increasing evidence shows that plant responses to heavy metal stresses is associated with changes in the expression profiles of genes involved in a broad spectrum of physiological, biochemical and cellular processes including carbohydrate and energy metabolism, photosynthesis, protein biosynthesis and degradation, nucleic acid metabolism, signal transduction, transcriptional regulation, cell transport and stress responses. however, limited data are available on the differential expression of genes in response to mn-toxicity in plants. techniques for gene expression analyses in plants have been widely explored. cdna-amplified fragment length polymorphism, which does not require prior sequence information, is an efficient, sensitive, and reproducible technology for the discovery and identification of genes based on their polymorphism or differential expression patterns. this technique is a robust and high-throughput tool for analysis of genome-wide gene expression fluctuation induced by a specific stress and is also a useful tool for the isolation of novel genes. citrus belongs to evergreen subtropical fruit trees and is cultivated in humid and subhumid of tropical, subtropical, and temperate regions of the world mainly on acidic soils. although the effects of mn-toxicity on citrus chloroplast ultrastructure, co assimilation, carbohydrates, photosynthetic electron transport and antioxidant systems have been investigated, very little is known about mn-toxicity-responsive genes in citrus plants. in this study, we investigated the effects of mn-toxicity on growth, leaf co assimilation, leaf concentrations of malondialdehyde, chlorophyll and total soluble protein, root, stem and leaf concentration of mn, leaf phosphorus and magnesium concentrations, and expression of leaf genes revealed by cdna-aflp in citrus grandis and citrus sinensis seedlings having different mn-tolerance. the objectives of this study were to understand the mechanisms of citrus mn-tolerance and to identify differentially expressed genes, which might be involved in mn-tolerance.  plant growth, root, stem and leaf mn concentration, and leaf mg and p concentrations for c. grandis, mn-toxicity decreased root, shoot and whole plant dry weight, and increased the ratio of root dw to shoot dw. however, mn-toxicity did not significantly affect root, shoot and whole plant dw in c. sinensis seedlings except for increased ratio of root dw to shoot dw. root dw, whole plant dw and the ratio of root dw to shoot dw were higher in c. sinensis seedlings than in c. grandis ones or similar between two species, except that shoot dw was lower in the former at the μm mn treatment. in addition, a few c. grandis leaves from the minority of mn-toxicity plants became interveinal chlorosis or necrotic blotching of foliage, while no visible symptoms occurred in mn-toxicity c. sinensis leaves. as shown in figure  mn-toxicity increased root, stem and leaf mn concentration, mn distribution in roots, mn uptake per plant and per root dw, and decreased mn distribution in stems and leaves. under control condition, all these parameters did not significantly differ between c. grandis and c. sinensis seedlings. when exposed to mn-toxicity, stem mn concentration, mn uptake per plant and mn distribution in roots were higher in c. sinensis seedlings than in c. grandis ones, while leaf mn concentration and mn distribution in leaves were lower in the former than in the latter. mn-toxicity decreased p and mg concentrations in c. grandis leaves, but did not significantly affect them in c. sinensis leaves. leaf total soluble protein, mda and chl concentrations and gas exchange total soluble protein concentration was decreased by mn-toxicity in c. grandis leaves, but was not significantly affected in c. sinensis ones. as shown in figure 4b, mn-toxicity did not significantly affect leaf concentration of mda. mn-toxicity decreased co assimilation, transpiration and stomatal conductance in leaves of c. grandis and c. sinensis, especially in the former. mn-toxicity increased intercellular co concentration in c. grandis leaves, but did not significantly affect it in c. sinensis leaves. mn-toxicity did not significantly affect leaf concentrations of chl a+b, chl a and chl b, and the ratio of chl a to chl b in the two citrus species. identification of differentially expressed genes by cdna-aflp a total of selective primer combinations were used for the cdna-aflp analysis in order to identify the genes responsive to mn-toxicity in the leaves of two citrus species differing in mn-tolerance. for c. grandis leaves, a total of 16– clear and unambiguous transcript-derived fragments were detected with each premier combination, which resulted in approximately tdfs, ranging from 100– bp. a total of differentially expressed and reproducible tdfs were recovered from the silver-stained cdna-aflp gels based on their presence, absence or difference in the levels of expression. all these tdfs were re-amplified, ligated and sequenced, and cdna fragments produced useable sequence data. tdf sequences were compared with those present in the genbank database. of the tdf sequences, tdfs showed significant homology to genes encoding known or putative proteins, and tdfs were homologous to genes encoding uncharacterized proteins, hypothetical proteins or unknown proteins. the remaining tdfs did not show homology to any nucleotide or amino sequence in the public databases. of these matched tdfs, tdfs increased and decreased in response to mn-toxicity. according to the biological functional properties, these tdfs were classified into the following functional categories: biological regulation and signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, lipid metabolism, cell wall metabolism, stress responses, cell transport, other and unknown biological processes. for c. sinensis leaves, a total of 16– clear and unambiguous tdfs were obtained with each premier pair, which yield approximately tdfs, ranging from 100– bp. a total of differentially expressed and reproducible tdfs were isolated from the silver-stained cdna-aflp gels. all these tdfs were sequenced, and produced readable sequences. of the tdfs, tdfs were homologous to genes encoding known or putative proteins, and three tdfs belonged to genes encoding hypothetical proteins. the remaining five tdfs had no database matches. among the matched tdfs, seven tdfs were up-regulated and eight was down-regulated by mn-toxicity. these tdfs were involved in biological regulation and signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, cell transport, other and unknown biological processes. qrt-pcr analysis of some mn-toxicity-responsive genes to validate the cdna-aflp expression patterns, tdfs from c. grandis leaves and one tdf from c. sinensis ones were selected for qrt-pcr analysis. the expression levels of all these tdfs except for two tdfs matched well with the expression profiles observed with cdna-aflp. the discrepancy in expression patterns for two tdfs between qrt-pcr and cdna-aflp analysis might be due to gene family complexity. nevertheless, the cdna-aflp technique allowed us to isolate the differentially expressed genes under mn-toxicity. discussion c. sinensis is more tolerant to mn-toxicity than c. grandis as shown in figures  and 2a-c, μm mn treatment greatly inhibited c. grandis plant growth, especially the shoots and increased the concentration of mn in roots, stems and leaves, and foliar mn concentration for μm mn treatment was far more than the sufficiency range of 25– mg kg- dw for sweet orange leaves. based on these results, plants that received μm mn are considered mn-excess. mn-toxicity-induced decrease in plant dw and increase in root dw/shoot dw ratio agree with the previous results obtained on c. grandis and with the view that mn-toxicity affects plant tops more than root systems. mn-toxicity, however, had no influence on the ratio of root dw to shoot dw in lucerne plants, despite mn-toxicity depressed growth of shoots and roots. our results showed that mn-toxicity c. grandis plants had decreased root, shoot and root + shoot dw, and increased ratio of root dw to shoot dw, while mn-toxicity did not significantly affect c. sinensis growth except for increased root dw/shoot dw ratio, meaning that c. sinensis is more tolerant to mn-toxicity than c. grandis. this is also supported by our data that the gas exchange in c. grandis leaves was affected by mn-toxicity far more than in c. sinensis ones, and that mn-toxicity decreased total soluble protein, p and mg concentrations only in c. grandis leaves. like that of previous workers, the observed lower co assimilation in mn-toxicity leaves of c. grandis and c. sinensis was primarily caused by non-stomatal factors because the decreases in both co assimilation and stomatal conductance was accompanied by unchanged or increased intercellular co assimilation. it is noteworthy that the reduction in co assimilation in leaves of mn-toxicity plants could not attributed to photo-oxidative damage and decreased chl, because there were no significant differences in leaf concentrations of mda, chl a+b, chl a and chl b between control and mn-toxicity leaves. similar results have been obtained on c. grandis. under mn-toxicity, the majority of mn in c. sinensis and c. grandis plants was retained in the roots, as previously found for c. grandis, lucerne and douglas fir. however, in rice exposed to mn-toxicity, mn was predominantly accumulated in leaves compared with roots. the tolerance of plants to mn is associated not only with low mn uptake, but also with relatively little mn translocation from roots to shoots. our results showed that under mn-toxicity, c. sinensis plants accumulated more mn in roots and less mn in shoots than c. grandis ones, and that the concentration of mn was lower in mn-toxicity c. sinensis leaves than in c. grandis ones. this might contribute to the mn-tolerance of c. sinensis. we isolated differentially expressed tdfs from mn-toxicity c. grandis leaves, which belong to different functional categories, meaning that mn-toxicity affected different physiological and biochemical pathways. in contrast, we only identified differentially expressed tdfs from mn-toxicity c. sinensis leaves. obviously, the transcript profile was less affected by mn-toxicity in c. sinensis leaves compared to c. grandis ones, which may be associated with the less mn accumulation in mn-toxicity c. sinensis leaves. these data also support above inference that c. sinensis is more tolerant to mn-toxicity than c. grandis. genes involved in biological regulation and signal transduction protein phosphorylation, a versatile post-translational modification, is involved in almost all plant signal pathways and plays important roles in regulation of abiotic stress responses. phosphorylation and dephosphorylation of a protein often serve as an “on-and-off” switch in the regulation of cellular activities. for optimal regulation, kinases and phosphatases must strike a balance in any given cell. as shown in additional file mn-toxicity decreased the expression levels of genes involved in phosphorylation and dephosphorylation except for increased expression of a mitogen-activated protein kinase gene in c. grandis leaves. this means that the balance between phosphorylation and dephosphorylation was upset and phosphorylation of some proteins might be impaired in mn-toxicity c. grandis ones. mapk cascades are important signal modules that convert signals generated at the receptors/sensors to appropriate cellular responses. increasing evidence demonstrates the role of mapk signal in different heavy metal stresses for different plant species. the observed higher expression level of mapk in mn-toxicity leaves agrees with the previous reports that excess copper, cadmium and mercury led to the activation of a novel mapk gene osmsrmk from japonica-type rice, and that mapk pathways were activated by excess cu and cd in m. sativa seedlings. thus, mapk cascade might play a role in the responses of plants to mn-toxicity. in contrast to c. grandis, vh1-interacting kinase gene in c. sinensis leaves was induced by mn-toxicity. mapks are able to phosphorylate different substrates in different cellular compartments, including transcription factors in the nucleus. thus, genes related to tfs might be affected in mn-toxicity c. grandis leaves due to altered expression level of mapk gene. as expected, mn-toxicity increased the expression levels of genes encoding tf jumonji domain-containing protein and myb family transcription factor, and decreased the expression levels of genes encoding tf ilr, c3h type zinc finger protein, putative tf and dna-binding storekeeper protein-related transcriptional regulator in c. grandis leaves. jumonji c domain-containing proteins have been shown to function as demethylases and to involve in chromatin structure and gene expression. recently, govind et al. showed that two jumonji tfs and other genes were induced in drought-stressed peanut plants, which agrees with our results that mn-toxicity c. grandis leaves had higher mrna levels of gene encoding tf jumonji domain-containing protein. they also found that silencing of jumonji made the transgenic tobacco plants more tolerant to drought, while down-regulation of hsp resulted in susceptibility. rampey et al. reported that ilr3- arabidopsis seedlings were less sensitive than wild type to mn-toxicity. ilr is a basic helix–loop–helix type tf, which seems to regulate metal homeostasis in part through the action of putative fe/mn ccc1-like transporters. therefore, the down-regulation of ilr in mn-toxicity c. grandis leaves might be an adaptive response. like c. grandis, the expression of dna-binding storekeeper protein-related transcriptional regulator was inhibited in c. sinensis leaves. ilr, however, was up-regulated in mn-toxicity c. sinensis leaves. calcium is a secondary messenger and has been suggested to participate in heavy metal signal. indeed, ca concentration in runner bean cells greatly increased in response to cd stress. ca may interact with calmodulin to propagate the signal and ultimately to regulate downstream genes involved in heavy metal transport, metabolism, and tolerance. in this study, mn-toxicity decreased the expression levels of ca-dependent protein kinase 1-like, calmodulin-binding transcription activator and ca2+-binding protein genes in c. grandis leaves, and increased the expression levels of ca-binding ef-hand domain-containing protein gene in c. grandis leaves and calmodulin-binding transcription activator in c. sinensis ones. thus, genes related to ca2+ signal might be involved in response to mn-toxicity. busov et al. characterized a 5ng gene from juvenile loblolly pine shoots, which was highly and specifically induced by auxin prior to adventitious root formation. toxicity of mn, on the other hand, led to auxin deficiency caused by activation of indole-acetic acid oxidase under excess mn. this supports our data that auxin-induced protein 5ng gene was down-regulated in c. sinensis leaves. similarly, the transcript abundance of auxin-response factor in c. grandis leaves also decreased in response to mn toxicity. however, mn-toxicity increased 5ng mrna level in c. grandis ones. transducin/wd- repeat-containing protein is a g protein involved in a wide range of functions that include signal transduction, transcription and stress-tolerant function. it was up-regulated by excess cu in germinating rice seeds, but was down-regulated by mn-toxicity in c. grandis leaves. vq motif-containing proteins play important roles in plant growth, development and defense responses. loss-of function mutants and/or overexpression lines for the vq genes are altered in seed size, tolerance to abiotic stress, or resistance to pathogen infection. liu et al. reported that transgenic arabidopsis plants overexpressing the atarvq a gene encoding a plant-specific vq motif-containing protein, were more resistant to arsenate stress. recently, cheng et al. showed that plant vq motif-containing proteins played critical roles in the network of wrky-mediated gene expression. we found that under mn-toxicity, the expression level of vq genes was decreased in c. grandis, which is in agreement with the previous report that atarvq expression was strongly down-regulated by arsenate stress. zhang et al. observed that a. thaliana fes1a could prevent cytosolic hsp degradation. thus, the observed lower mrna level of hsp nucleotide exchange factor fes in mn-toxicity c. grandis leaves agrees with the results that the expression levels of hsp and stromal kda heat shock-related protein, chloroplastic-like gene in c. grandis ones decreased in response to mn-toxicity. similar result has been obtained on b-deficient c. sinensis roots. phosphatidylinositol 4-kinase catalyzes the phosphorylation of ptdins in the d- position of the inositol ring. this is the committed step in the synthetic pathway leading to ptdins -bisphosphate. apart from this function in signal transduction, both ptdins 4-p and ptdins -p appear to have other cellular functions in c. grandis leaves decreased in response to mn-toxicity, which disagrees with mn-stimulated ptdins 4-kinase from dunaliella parva. based on these results above mentioned, we conclude that the responses of citrus plants to mn-toxicity might be regulated in multiple signal pathways. genes involved in carbohydrate and energy metabolism thirteen genes in c. grandis leaves and two genes in c. sinensis ones related to carbohydrate and energy metabolism were altered under mn-toxicity. atp synthase is an important enzyme that provides energy for the cell to use through the synthesis of atp. hamilton et al. showed that exposure of wheat to al increased mitochondrial f1f0-atpase activity only in the al-tolerant wheat variety, and concluded that the al-induced increase in atp synthase activity was an adaptive response involved in al tolerance. similar to al-toxicity, mn-toxicity increased the expression levels of synthase subunit alpha in c. grandis and c. sinensis leaves and atp synthase subunit alpha in c. grandis leaves. aconitate hydrolyase involved in glycolysis catalyses the interconversion of citrate and isocitrate, via a cis-aconitate. our finding that the expression of ah was induced in mn-toxicity c. grandis leaves means that its activity was enhanced by mn-toxicity, which is in agreement with the previous report that ah was up-regulated by cd stress. in addition, mn-toxicity also increased the expression levels of nadph-ferrihemoprotein reductase and cytochrome c biogenesis orf genes related to respiration in c. grandis leaves. overall, the metabolic pathways related to atp synthesis and reducing power production might be activated in mn-stressed leaves to yield more energy needed to meet the high-energy demand of stressed cell. by contrast, the mrna level of gene encoding 2-phospho-d-glycerate hydrolase, an essential and ubiquitous glycolytic enzyme responsible for the catalysis of the conversion of 2-phosphoglycerate to phosphoenolpyruvate, was down-regulated in mn-toxicity c. grandis leaves. this agrees with the previous report that the abundance of enolase in agrostis stolonifera leaves was decreased after d of heat stress. ferreira et al. in populus euphratica leaves observed that the level of enolase was transiently increased after h of heat stress, followed by a decrease after h of heat stress. however, other studies showed that leaf enolase was induced in various plants by different abiotic stresses, such as water stress in maize, cold stress in tobacco, salt stress in ice plant and cd-stress in phytolacca americana. like to enolase, the expression level of gene encoding cyt p, an enzyme typically catalyzing the reaction: rh + o + nadph + h+ → roh + h2o + nadp+, was reduced in mn-toxicity c. grandis leaves, which might contribute to maintaining nadph homeostasis by decreasing its utilization. however, the expression of cyt p family subfamily a, polypeptide gene in c. sinsnsis leaves was induced by mn-toxicity, which is in agreement with the report that at least of the cyt p450s were induced in arabidopsis by abiotic and biotic stress 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 cyt p tolerated better oxidative stress after herbicide treatment. therefore, the observed higher mrna level of cyt p family subfamily a, polypeptide might be advantegous to mn-toxicity tolerance of c. sinensis plants. thioredoxins are small proteins, which are involved in the cell redox regulation. trx m, a chloroplastic protein, preferentially activates nadp-malate dehydrogenase, a key enzyme involved in carbon fixation and sugar biosynthesis during photosynthesis. historically the m-type trxs were so named because they were able to activate nadp-mdh. the activity of nadp-mdh might be down-regulated in mn-toxicity c. grandis leaves due to lower mrna levels of trx m and trx m. evidence shows that trx m m and m could act as antioxidants, i.e. possibly by serving as hydrogen donors for a trx-dependent peroxidase. trehalose-6-phosphate synthase is a key enzyme for synthesizing trehalose, which plays an important role in abiotic stress tolerance of plants. the observed lower level of tps in mn-xicity c. grandis leaves indicates that mn-toxicity might decrease leaf concentration of trehalose, thus lowering the stress tolerance. atp sulfurylase activity were enhanced by sulphur deprivation and reduced after resupply of so42–. heiss et al. reported that cd stress strongly increased the expression levels of atps and 5’-adenylysulfate reductase in roots and leaves of 6-week-old brassica juncea, accompanied by enhanced cysteine concentration in roots and leaves, and concluded that roots and leaves responded to cd stress with a coordinative up-regulation of several s assimilation enzymes to meet an increased demand for cysteine during phytochelatin synthesis. however, the expression of atps was down-regulated in mn-toxicity c. grandis leaves. udp-glycosyltransferases, which glycosylate a broad array of aglycones, including plant hormones, all major classes of plant secondary metabolites, and xenobiotics such as herbicides, play an important role for the stabilization, enhancement of water solubility and detoxification of natural products. cytokinins can be glucosylated to form o-glucosides and n-glucosides. the glycoconjugates are inactive and are considered to play a role in hormonal homeostasis. hou et al. showed that ugt76c and ugt76c recognized all cytokinins and glucosylated them at the n and n positions. veselov et al. reported that cd stress sharply lowered the concentration of cytokinin in wheat roots and shoots caused by elevated activity of cytokinin oxidase. obviously, the down-regulation of udp-glycosyltransferase 76f1-like gene in mn-toxicity c. grandis leaves is advantageous to cytokinin homeostasis under mn-toxicity if leaf concentration of cytokinin decreased in response to mn-toxicity. genes involved in nucleic acid metabolism evidence shows that heavy metals inhibit plant nucleic acid metabolism. as expected, the expression levels of genes encoding tho complex, subunit, dna polymerase phi subunit, histone h, dna - methyltransferase drm2-like and luc7-like protein 3-like in c. grandis leaves and genes encoding tho complex, subunit and histone h in c. sinensis ones decreased in response to mn-toxicity, indicating that leaf nucleic acid metabolism might be impaired by mn-toxicity. genes involved in protein metabolism mn-toxicity has been demonstrated to affect protein metabolism in plants. we found that the expression levels of three ribosomal genes encoding ribosomal protein s, ribosomal protein s and 60s ribosomal protein l mitochondrial-like, which are involved in mature ribosome assembly and translation processes, were down-regulated in mn-toxicity c. grandis leaves, while only one ribosomal gene encoding 60s ribosomal protein l mitochondrial-like in c. sinensis leaves was down-regulated by mn-toxicity. this indicates that the biosynthesis of protein in mn-toxicity c. grandis leaves might be impaired more severe than in mn-toxicity c. sinensis ones, which is in agreement with our data that mn-toxicity decreased the concentration of total soluble protein in c. grandis leaves, but did not affect its concentration in c. sinensis ones. however, the expression of 30s ribosomal protein s gene was induced in mn-toxicity c. grandis leaves. translational control of protein synthesis depends on numerous eukaryotic initiation factors. our finding that the expression of gene encoding similar to translation initiation factor if was down-regulated in mn-toxicity c. grandis leaves agrees with our previous report that the abundances of eif4b eif subunit and eif3g in c. sinensis roots decreased in response to b-deficiency. however, salt and heavy metal induced the accumulation of rice eif5a- and eif5a- mrnas in rice cells. elongation factors whose expressions have been demonstrated to correlate to the high rate of protein synthesis in developing plant tissues facilitate translational elongation, from the formation of the first peptide bond to the formation of the last one in the ribosome. guo et al. isolated a translation elongation factor 2-like protein gene from a cold defective arabidopsis mutant, los 1– the los1- mutant plants were impaired in protein synthesis under cold stress. we found that the expression level of one gene encoding translation elongation factor- alpha, partial in c. grandis leaves decreased in response to mn-toxicity, which agrees with the previous results obtained on drought-stressed soybean nodules, anoxia maize roots, and b-deficient roots of c. sinensis, brassica napus and lupinus albus. however, the abundances of chloroplast translational elongation factor tu and elongation factor p in rice leaves increased in response to cd stress. thus, it appears that the influence of abiotic stress on translation elongation factors depends on plant species and kinds of stresses. in addition, the expression level of gene encoding tetratricopeptide repeat-containing protein involved in protein synthesis was down-regulated in mn-toxicity c. grandis leaves. these results further demonstrate that protein biosynthesis is impaired in mn-toxicity c. grandis leaves. mn-toxicity increased the expression levels of genes encoding atp-dependent clp protease, cysteine proteinase and xylem cysteine proteinase in c. grandis leaves. this indicates that the hydrolysis of some proteins might be up-regulated in mn-toxicity leaves, thus decreasing leaf concentration of total soluble protein. this is also supported by our data that mn-toxicity leaves had lower concentration of total soluble protein. however, the expression levels of genes encoding carboxyl-terminal peptidase, papain family cysteine protease, cathepsin b-like cysteine proteinase like protein and α/β-hydrolase-like protein decreased in response to mn-toxicity. like other ptms, ubiquitination, which serves as a versatile ptm, plays a key role in regulating plant response to abiotic stresses. the up-regulation of genes encoding mnd1-interacting protein and ubiquitin-conjugating enzyme e in mn-toxicity c. grandis leaves might be an adaptive response to mn-toxicity. however, the expression levels of ubiquitin-correlative genes such as ubiquitin-protein ligase and btb and math domain-containing protein in c. grandis leaves decreased in response to mn-toxicity, meaning that the ubiquitination of some proteins might be impaired in mn-toxicity c. grandis leaves. three genes involved in amino acid metabolism was down-regulated in mn-toxicity c. grandis leaves. this means that the biosynthesis of amino acids might be impaired in mn-toxicity c. grandis leaves. chorismate synthase catalyzes the last of the seven steps in the shikimate pathway which is used in prokaryotes, fungi and plants for the biosynthesis of aromatic amino acids. cbs catalyzes the first step of the transsulfuration pathway from homocysteine to cystathionine. jung et al. observed that cbsx directly modulated trx in chloroplasts, which affected the level of h2o and, consequently, the expression of the genes involved in secondary cell-wall thickening, concluding that cbsx protein played a critical role in thickening of the secondary cell walls of the endothecium during anther dehiscence in arabidopsis. transgenic tobacco plants overexpressing oscbsx isolated from rice displayed enhanced tolerance to salinity, heavy metal, and oxidative stress. this observed lower expression level of gene encoding cbs domain-containing protein in c. grandis leaves disagrees with our previous data that the abundance of cbs family protein in c. sinensis roots increased in response to b-deficiency. genes involved in lipid metabolism lecithin-cholesterol acyltransferase catalyzes the transacylation of acyl groups from phospholipids to sterols in mammals and yeast. sterol acylation is an essential process of sterol homeostasis in eukaryotic cells. in arabidopsis, phospholipid sterol acyltransferase, which displays homology with the mammalian lcat, catalyzes a phospholipid-dependent formation of sterol esters. recent work with arabidopsis showed that sterol ester concentration decreased in leaves of psat1- or psat1- mutants accompanied by an early leaf senescence phenotype, suggesting a major contribution of the psat in maintaining both free sterol homeostasis in plant cell membranes and leaf viability during developmental aging. the up-regulation of lecithin-cholesterol acyltransferase-like in mn-toxicity c. grandis leaves might enhance the leaf concentration of sterol ester, thus preventing leaf senescence. genes involved in cell wall metabolism our results showed that the expression levels of genes involved in cell wall biosynthesis were down-regulated in mn-toxicity c. grandis leaves. in addition, the mrna levels of gene encoding glycoside hydrolase family protein, which catalyze the hydrolytic cleavage of the pectin and gene encoding cell wall-associated hydrolase were up-regulated in mn-toxicity c. grandis leaves. therefore, the formation of cell wall might be impaired in mn-toxicity leaves. however, the expression of gene encoding protein trichome birefringence-like was induced in mn-toxicity c. grandis leaves. genes involved in stress responses mn-toxicity has been demonstrated to stimulate ros production in plants. since mn-toxicity did not affect the concentration of mda in citrus leaves, some protective antioxidant enzymes should be up-regulated to meet the increased requirement for scavenging ros. our results showed that the expression of gene encoding catalase, an enzyme involved in scavenging h2o was induced in mn-toxicity c. grandis leaves, which agrees with the previous report that mn-toxicity c. grandis leaves had increased specific activity of cat and similar leaf area-based activity of cat. besides ros scavenger enzymes, this detoxification mechanism also involves “house-keeping” enzymes. the family of nudix hydrolases, which catalyze the hydrolytic breakdown of nucleoside diphosphates linked to some other moieties such as a phosphate, sugar or nucleoside, are one of these “house-keeping” enzyme families. ogawa et al. showed that atnudx a chloroplastic atnudx, played an important role in modulation of the nadh and/or nadph pools through the hydrolysis of nadh to reduced nicotinamide mononucleotide in arabidopsis chloroplasts. transgenic arabidopsis plants overexpressing atnudx and atnudx displayed enhanced tolerance to oxidative stress, resulting from the maintenance of nad+ and atp levels by nucleotide recycling of free adp-ribose molecules. the higher expression level of nudx in c. grandis leaves agrees with the previous reports that the abundance of nudx homolog in c. sinensis roots increased in response to b-deficiency and chrysanthemum lavandulifolium clnudx clnudx clnudx and clnudx were induced by salt, drought, heat, and cold stresses. however, the expression levels of genes encoding monodehydroascorbate reductase, peroxidase, glutathione s-transferase tau, nadp-dependent alkenal double bond reductase p, trx m, trx m chloroplastic-like, and cbs domain-containing protein related to oxidative defense in c. grandis leaves decreased in response to mn-toxicity. it is noteworthy that mn-toxicity c. grandis leaves had higher specific activity of mdar and leaf area-based activity of mdar compared with controls. the discrepancy between the expression level of mdar and the activity of the corresponding enzyme indicates that ptms might affect mdar activity. senescence is a genetically programmed decline in various cellular processes and involves in the hydrolysis of macromolecules such as proteins and lipids. it is governed by the developmental age and is induced or enhanced by biotic and abiotic stresses. generally in plants the term senescence and programmed cell death denote the processes that initiate the programmed death of individual cell. the senescence can be considered as one of the examples for pcd. as expected, the expression level of one gene encoding for putative senescence-associated protein was up-regulated by mn-toxicity in the less mn-tolerant c. grandis leaves. this agrees with the previous report that the gene was highly differentially expressed only in the heat-sensitive fescue genotype at 44°c. in addition, the expression levels of apoptosis linked genes were enhanced in mn-toxicity c. grandis leaves. thus, it is reasonable to assume that the mn-toxicity sensitive c. grandis plants were under great stress and leaf senescence was accelerated, which might contribute to plant survival by using more metabolites through glycolysis, and protein and lipid degradation. as shown in figure  the mn-toxicity-induced senescence in c. grandis leaves was a very complicated process. hsps/chaperones have been demonstrated to play a key role in protecting plants against heavy metal stress. the observed lower expression levels of genes encoding heat shock protein-related, partial, heat shock protein 60-3a, hsp and stromal kda heat shock-related protein, chloroplastic-likes in mn-toxicity c. grandis leaves indicate decreased protein synthesis in these leaves, as indicated by decreased leaf concentration of total soluble proteins. this is similar to the previous report that the abundance of hsp was enhanced in leaves of high cd-accumulating soybean cultivar, but was lowered in low cd-accumulating one. one crucial adaptive mechanism of plants to p-deficiency is the immediate cleavage of phosphate from phosphorylated substrates. phosphoethanolamine/phosphocholine phosphatase may release pi from organic to maintain pi homeostasis of plant cells. the down-regulation of ppspase, putative gene in c. grandis leaves might decrease the release of pi from phosphorylated substrates, thus lowering leaf p concentration. this agrees with the previous report that mn-toxicity decreased p concentration in tea leaves. genes involved in cell transport eight genes in c. grandis leaves and one gene in c. sinensis ones were regulated by mn-toxicity. we found that the expression of citrus sucrose transporter was induced in mn-toxicity c. grandis leaves, which is in agreement with the previous report that the gene strongly expressed in source, sugar exporting organs, because mn-toxicity increased or did not affect the concentrations of non-structural carbohydrates in c. grandis leaves. the up-regulation of citrus sucrose transporter might be helpful to decrease sugar accumulation in mn-toxicity leaves, which might in turn lead to feedback suppression of co assimilation. one of strategies of plants to deal with heavy metals is to transport them out of the cells, thereby removing them from the cytosol. kim et al. observed that transgenic a. thaliana plants overexpressing the abc transporter atpdr were more resistant to cd or lead and displayed lower cd concentration in roots and shoots than wild-type plants, while atpdr rnai transgenic plants and t-dna insertion lines were more sensitive to cd or pb and had higher cd concentration, concluding that the abc transporter atpdr is a cd extrusion pump conferring heavy metal resistance. the down-regulation of genes encoding abc-transporter-like protein and abc transporter family protein in c. grandis leaves indicates that the extrusion of mn from leaves might be lessened in response to mn-toxicity, which might be one of the causes that mn-toxicity c. grandis leaves accumulated more mn than c. sinensis ones. evidence has shown that cora-like proteins may represent the major transport systems in eukaryotes such as yeast, animals, and plants. the down-regulation of mg transporter cora-like protein gene in mn-toxicity c. grandis and c. sinensis leaves might reduce mg transport, hence decreasing leaf mg concentration. this agrees with the previous report that mn-toxicity decreased leaf concentration of mg. interestingly, mg concentration was decreased by mn-toxicity only in c. grandis leaves, but was not significantly affected in c. sinensis leaves. this means that leaf mg concentration is also regulated by other factors. plant cyclic nucleotide gated channels, which comprise a large gene family in arabidopsis, have been proposed to be involved in multiple plant physiological processes including plant growth and heavy metal toxicity tolerance. evidence suggests that cngcs paly a role in heavy metal homeostasis, for example, in tobacco, overexpression of tobacco cngc led to hypersensitivity to pb. however, sunkar et al. showed that transgenic tobacco plants overexpressing a truncated ntcbp exhibited improved tolerance to pb2+ and decreased uptake of this metal ion. we found that the expression of cyclic nucleotide gated channel gene was down-regulated in mn-toxicity c. grandis leaves, suggesting that the gene might be involved in the response to mn-toxicity. our results showed that the expression of gene encoding protease inhibitor/seed storage/lipid transfer protein family protein was down-regulated in mn-toxicity c. grandis leaves, indicating that the exchange of lipids between membranes might be decreased.  our results clearly demonstrated that c. sinensis was more tolerant to mn-toxicity than c. grandis. under mn-toxicity, c. sinensis plants accumulated more mn in roots and less mn in shoots than c. grandis ones, and that the leaf concentration of mn was lower in the former. this might contribute to the mn-tolerance of c. sinensis. in this study, we first used the cdna-aflp technique to compare the mrna levels of genes from control and mn-toxicity leaves of two citrus species differing in mn-tolerance. in mn-toxicity c. grandis leaves, up-regulated and down-regulated genes were isolated, while only seven up-regulated and eight down-regulated genes were identified in mn-toxicity c. sinensis ones. obviously, mn-toxicity affected gene expression far less in c. sinensis leaves than in c. grandis ones, which might be associated with less leaf mn concentration in mn-toxicity c. grandis leaves. cdna-aflp analysis suggests that the responses of c. grandis leaves to mn-toxicity might include following several aspects: accelerating leaf senescence; activating the metabolic pathway related to atpase synthesis and reducing power production; decreasing cell transport; inhibiting protein and nucleic acid metabolisms; impairing the formation of cell wall; and triggering multiple signal transduction pathways. we also identified many new mn-toxicity-responsive genes involved in biological and signal transduction, carbohydrate metabolism, protein metabolism, stress responses and cell transport. further studies will elucidate the roles of these genes in response to mn-toxicity, which will help me to design mn-tolerant transgenic crops. 
using cdna-aflp, we isolated up-regulated and down-regulated genes in mn-toxicity c. grandis leaves. they were grouped into the following functional categories: biological regulation and signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, lipid metabolism, cell wall metabolism, stress responses and cell transport. the responses of c. grandis leaves to mn-toxicity might include following several aspects: accelerating leaf senescence; activating the metabolic pathway related to atpase synthesis and reducing power production; decreasing cell transport; inhibiting protein and nucleic acid metabolisms; impairing the formation of cell wall; and triggering multiple signal transduction pathways. our results demonstrated that c. sinensis was more tolerant to mn-toxicity than c. grandis, and that mn-toxicity affected gene expression far less in c. sinensis leaves. under mn-toxicity, the majority of mn in seedlings was retained in the roots; c. sinensis seedlings accumulated more mn in roots and less mn in shoots than c. grandis ones and mn concentration was lower in mn-toxicity c. sinensis leaves compared to mn-toxicity c. grandis ones. this might be associated with more mn accumulation in roots and less mn accumulation in leaves of mn-toxicity c. sinensis seedlings than those of c. grandis seedlings. we also identified many new mn-toxicity-responsive genes involved in biological and signal transduction, carbohydrate and protein metabolisms, stress responses and cell transport. seedlings of ‘xuegan’ and ‘sour pummelo’ were irrigated for weeks with nutrient solution containing μm or μm mnso the objectives of this study were to understand the mechanisms of citrus mn-tolerance and to identify differentially expressed genes, which might be involved in mn-tolerance. very little is known about manganese -toxicity-responsive genes in citrus plants. mn-toxicity affected c. grandis seedling growth, leaf co assimilation, total soluble concentration, phosphorus and magenisum more than c. sinensis. however, only up-regulated and down-regulated genes were identified in mn-toxicity c. sinensis ones.
 very little is known about manganese -toxicity-responsive genes in citrus plants. seedlings of ‘xuegan’ and ‘sour pummelo’ were irrigated for weeks with nutrient solution containing μm or μm mnso the objectives of this study were to understand the mechanisms of citrus mn-tolerance and to identify differentially expressed genes, which might be involved in mn-tolerance.  under mn-toxicity, the majority of mn in seedlings was retained in the roots; c. sinensis seedlings accumulated more mn in roots and less mn in shoots than c. grandis ones and mn concentration was lower in mn-toxicity c. sinensis leaves compared to mn-toxicity c. grandis ones. mn-toxicity affected c. grandis seedling growth, leaf co assimilation, total soluble concentration, phosphorus and magenisum more than c. sinensis. using cdna-aflp, we isolated up-regulated and down-regulated genes in mn-toxicity c. grandis leaves. they were grouped into the following functional categories: biological regulation and signal transduction, carbohydrate and energy metabolism, nucleic acid metabolism, protein metabolism, lipid metabolism, cell wall metabolism, stress responses and cell transport. however, only up-regulated and down-regulated genes were identified in mn-toxicity c. sinensis ones. the responses of c. grandis leaves to mn-toxicity might include following several aspects: accelerating leaf senescence; activating the metabolic pathway related to atpase synthesis and reducing power production; decreasing cell transport; inhibiting protein and nucleic acid metabolisms; impairing the formation of cell wall; and triggering multiple signal transduction pathways. we also identified many new mn-toxicity-responsive genes involved in biological and signal transduction, carbohydrate and protein metabolisms, stress responses and cell transport.  our results demonstrated that c. sinensis was more tolerant to mn-toxicity than c. grandis, and that mn-toxicity affected gene expression far less in c. sinensis leaves. this might be associated with more mn accumulation in roots and less mn accumulation in leaves of mn-toxicity c. sinensis seedlings than those of c. grandis seedlings. our findings increase our understanding of the molecular mechanisms involved in the responses of plants to mn-toxicity. cdna-aflpcitrus grandiscitrus sinensisleavesmanganese 