BACKGROUND
among potential abiotic stresses, water deficit is considered to have the largest effect on agricultural productivity and is one of the main factors limiting the distribution of species worldwide  <cit> .
when plants are subjected to water deficit, numerous morphological and physiological responses are observed, and the amplitude of these responses depends on the plant genotype as well as the duration and severity of the stress  <cit> .
the plant response to water deficit involves several processes, beginning with the perception of stress, followed by modulation of the expression of specific genes, and finally, the appearance numerous transcriptomic, proteomic and metabolomic changes.
these changes result in the regulation of metabolism and the generation of regulatory networks that are involved in plant defense against the harmful effects of stress  <cit> .
transcriptomic studies have revealed that the expression of a wide range of genes is regulated in response to water deficit in citrus plants.
analysis of  <dig>  expressed sequence tags  in the roots of rangpur lime  subjected to osmotic stress resulted in the identification of genes involved in the water stress response, including those encoding aquaporins, dehydrins, sucrose synthase and enzymes related to the synthesis of proline  <cit> .
using a microarray containing  <dig>  genes, gimeno et al.
<cit>  investigated the response of the transcriptome of ‘clementine’ mandarin  grafted onto ‘cleopatra’ mandarin  to water deficit conditions.
as observed in other species, genes encoding proteins involved in lysine, proline and raffinose catabolism, hydrogen peroxide reduction, vacuolar malate transport, and defense  were induced.
analysis of the nac family of transcription factors resulted in the identification of one member, csnac <dig>  that was strongly induced by water deficit in the leaves of ‘cleopatra’ mandarin and rangpur lime and by salt stress, cold and abscisic acid  only in the leaves and roots of ‘cleopatra’ mandarin  <cit> .
in ‘cleopatra’ mandarin, xian et al.
<cit>  isolated a gene encoding crnced <dig>  which is an enzyme involved in aba synthesis, and produced transgenic plants that constitutively overexpressed this gene.
the transgenic lines displayed tolerance to dehydration, drought, salt, and oxidative damage compared with wild-type plants.
furthermore, low levels of reactive oxygen species  were detected in the transgenic plants under salt stress and dehydration.
in addition to studies addressing the effects of water deficit on the transcriptome, proteomic studies have revealed the role of proteins involved in the complex mechanisms underlying the stress responses of plants  <cit> .
indeed, many proteins related to stress defense, detoxification, carbohydrate metabolism and photosynthesis that participate in the process of adaptation and tolerance to stress have been identified  <cit> .
in a study that evaluated changes in the leaves of two contrasting populations of populus cathayana in response to water deficit,  <dig> drought-responsive proteins were identified: several of the proteins showing altered abundance were involved in transcriptional regulation, secondary metabolism, redox homeostasis and stress defense  <cit> .
an investigation of soybean  roots subjected to short-term water deficit revealed changes in the abundance of proteins involved in carbohydrate and nitrogen metabolism, cellular defense and programmed cell death  <cit> .
zadražnik et al.
<cit>  identified drought-responsive proteins in the leaves of two bean cultivars with differing responses to drought stress.
these proteins are primarily involved in energy metabolism, atp conversion, photosynthesis, protein synthesis and proteolysis and stress defence.
changes in protein levels in the leaves of ‘willow leaf’ and ‘cleopatra’ mandarin plants subjected to salt stress were analyzed by podda et al.
<cit> .
significant variations in the abundance of  <dig> protein spots were detected.
these salt-responsive proteins play roles in photosynthetic processes, ros scavenging, stress defense, and signaling.
however, there are few studies of the root proteome.
analysis of the root proteome of wild watermelon  has revealed that proteins involved in root morphogenesis, carbon/nitrogen metabolism, lignin synthesis and molecular chaperones are differentially regulated under drought stress  <cit> .
in the present study, we used proteomic approaches to analyses changes in the protein profiles of the roots of two citrus rootstock cultivars with contrasting responses to water deficit.
proteins showing significantly altered abundance were selected for identification via mass spectrometry and bioinformatics analysis.
in rangpur lime, the abundance of various proteins involved in protein metabolism, the stress response and proteolysis were modulated under water deficit conditions.
in contrast, repair-related proteins contributed more specifically to the response of ‘sunki maravilha’ mandarin to this stress.
this is the first report to examine the effects of water deficit on the abundance of proteins in citrus roots.
RESULTS
in the present study, root samples of rangpur lime and ‘sunki maravilha’ mandarin collected in a previous study by neves et al.
<cit>  were used.
considering the soil moisture data from the previous report  <cit> , two sampling points were selected for proteomic analysis as follows: 1) plants grown in soil with moisture ranging from  <dig> - <dig>  m3m− <dig> were defined as ‘control’ plants, whereas 2) the soil moisture for ‘drought-stressed’ plants ranged from  <dig> - <dig>  m3m− <dig>  according to a previous report by neves et al.
<cit> , stomatal resistance is more pronounced in both varieties at the selected drought stress sampling points.
in addition, they have reported that the leaf water potential decreases in water-stressed plants, reaching − <dig>  mpa and − <dig>  mpa in rangpur lime and ‘sunki maravilha’ mandarin, respectively.
interestingly, rangpur lime shows a higher growth rate when grown under water deficit compared with the rate observed for ‘sunki maravilha’.
when subjected to water deficit, the leaves and roots of ‘sunki maravilha’ display a progressive increase in the aba concentration.
the lower leaf growth rate that has been recorded for ‘sunki maravilha’ mandarin may be associated with its greater leaf aba concentration.
in contrast, in rangpur lime, alternations between high and low aba concentrations were observed  <cit> .
analysis of root protein profiles in response to water deficit
to elucidate the changes in protein abundance in response to water deficit, comparative analysis of the protein profiles of roots of rangpur lime and ‘sunki maravilha’ mandarin was performed via 2d gel electrophoresis.
the root protein profiles of both varieties that were grown under control conditions and subjected to water stress are shown in figure  <dig>  more than  <dig> spots were detected in both varieties via image analysis.
a total of  <dig> spots showed significant changes in abundance  in the rangpur lime roots.
these spots were subjected to mass spectrometry  analysis, and  <dig> proteins were identified.
among these proteins,  <dig> were increased and  <dig> were decreased in abundance, and seven proteins were unique to this genotype.
in ‘sunki maravilha’ mandarin,  <dig> spots showed significant changes in abundance.
among these spots,  <dig> proteins were identified,  <dig> of which increased and  <dig> of which decreased in abundance, and nine were unique to this genotype.figure  <dig>
2-de analysis of root proteins in rangpur lime under control conditions  and following water deficit  and in ‘sunki maravilha’ mandarin under control  and water deficit conditions .
the proteins indicated by the arrows were differentially expressed under the applied treatment.
the proteins in the squares are unique to rangpur lime, and those in the circles are exclusive to ‘sunki maravilha’ mandarin.
to understand the relationship between the two plant varieties as a function of water stress, multivariate analysis and principal component analysis  were performed .
pc <dig> represented 75% of the variance, suggesting that there were differences between rangpur lime and ‘sunki maravilha’ mandarin in response to water deficit.
pc <dig> accounted for 17% of the variance, indicating that in ‘sunki maravilha’ , there were differences between the well-watered plants and those under water stress.
interestingly, we observed only minor changes in the protein profiles of rangpur lime under control versus drought-stressed conditions, suggesting that protein abundance was less affected by water deficit in this variety.figure  <dig>
principal component analysis  and evaluation of variance under control conditions and drought in rangpur lime and ‘sunki maravilha’ mandarin.
hierarchical clustering of the experiments and  pca and eigenvalues table in control and water stress-treated samples from rangpur lime and ‘sunki maravilha’.
identification and analysis of differentially expressed proteins
spots showing differential intensities under water deficit were excised from the two-dimensional polyacrylamide gel electrophoresis  gels and identified via ms .
some proteins were identified more than once in different spots, reflecting different isoforms, post-translational modifications or alternative mrna splice forms  <cit> .
two spots were identified as epidermis-specific secreted glycoprotein ep1-like , five as germin-like , four as 2-phospho-d-glycerate hydrolase , three as mitochondrial processing peptidase alpha- <dig> subunit , two as putative mitochondrial processing peptidase , four as annexin  <dig> , two as annexin d <dig> , two as heat shock protein  <dig> , two as fructokinase  and two as lactoylglutathione lyase .
in addition, some of the proteins that were represented by different spots on the 2d gel showed opposite expression patterns .
the germin-like proteins, which were represented by five spots , and putative mitochondrial processing peptidase  exhibited opposite patterns of accumulation in ‘sunki maravilha’ mandarin .
in contrast, mitochondrial processing peptidase alpha  <dig> subunit  showed opposite accumulation patterns in rangpur lime.table  <dig>
identification of differentially expressed proteins in the roots of rangpur lime and ‘sunki maravilha’ mandarin subjected to water deficit
id spot
a
identified protein reference organism
b
accession number
c
mascot score/
p
-value
d
mr theor/exp
e
pi theor/exp
f
expression level
g
a b c d
fold change
h
rl sk
<dig>
1
<dig>   <dig>
<dig>   <dig>
<dig>
1
<dig>   <dig>
<dig>  ∞
<dig>   <dig>
<dig>  ∞
<dig>   <dig>
aspot id corresponding to the position in the 2d gel illustrated in figure  <dig>  bprotein accession number according to the ncbi database .
cbest matching protein identified by pblast analysis of the non-redundant  database.
dmascot score p value of the homology between citrus proteins and orthologous, homologous, or paralogous proteins, as annotated in ncbinr.
etheoretical and experimental masses  of identified proteins.
ftheoretical and experimental pis of identified proteins.
gexpression levels, presented as the % normalised volume, in the control and water deficit-stressed roots.
vertical bars indicate the mean ± se.
rangpur lime:  control; and  water deficit.
‘sunki maravilha’:  control; and  water deficit.
hfold change : bold = increased protein abundance; underlined = decreased protein abundance; italics = no significant difference; np = protein not found in gel; ∞ = present in one treatment in the genotype.
the functions of the identified proteins were inferred using the uniprot database .
the identified proteins were classified into the following seven major groups according to their possible biological functions: stress and defense response , metabolism , transport , energy , signal transduction , protein metabolism  and unknown  for rangpur lime and ‘sunki maravilha’ mandarin , respectively.
although the protein groups did not differ significantly between the two studied varieties, an additional class of proteins involved in dna repair was observed for ‘sunki maravilha’ mandarin .figure  <dig>
functional classifications of drought-responsive proteins.
functional categorization of proteins that showed significant changes in abundance in rangpur lime.
functional categorization of proteins with significantly altered levels in ‘sunki maravilha’ mandarin.
analysis of protein-protein interactions
analysis of interactomic data of a. thaliana orthologous proteins corresponding with the protein interaction profiles of rangpur lime and ‘sunki maravilha’ mandarin  allowed us to draw an interactome network.
the network developed for ‘sunki maravilha’ mandarin included  <dig> proteins and  <dig>  connectors, and that constructed for rangpur lime included  <dig> proteins and  <dig>  connectors .
based on analysis of the intersection between the networks of the two varieties,  <dig> proteins specific to the rangpur lime network,  <dig> proteins unique to the ‘sunki maravilha’ mandarin network, and  <dig> proteins shared between the two networks were observed .
the interactome networks obtained for each variety could be divided into several functional clusters.
evaluation of the highly connected regions and gene ontologies of each cluster revealed the presence of  <dig> clusters in the rangpur lime interactome network and  <dig> clusters in the ‘sunki maravilha’ mandarin interactome network .
to evaluate the proteins forming the most relevant network, centrality analysis was performed by sorting the proteins into hubs and/or bottlenecks.
among the  <dig> proteins showing altered abundance in rangpur lime and ‘sunki maravilha’ mandarin, mitochondrial malate dehydrogenase , mitochondrial processing peptidase , 2-phospho-d-glycerate hydrolase  and nucleoside diphosphate kinase  <dig>  were considered hubs/bottlenecks.
glyoxalase , annexin  <dig> , glutathione s-transferase  and putative l-galactose dehydrogenase  were only considered to be bottlenecks .
protein-protein interactions in rangpur lime
among the clusters of a. thaliana orthologous proteins corresponding with the differentially abundant proteins identified in the two studied citrus varieties, eight were exclusively related to rangpur lime .
fructokinase, which was a bottleneck protein in rangpur lime, was present in a sub-functional network involved in growth, development and the stress response  that contained the proteins cyp96a <dig>  cyp71a, cyp <dig>  and cyp <dig> and representatives of the cytochrome p <dig> superfamily.
this cluster also included wrky transcription factors .
the wrky <dig> transcription factors were associated with osmotin  <dig> , which interacted with chitinase , which is an enzyme involved in the response to various environmental stresses.
moreover, athchib was present in the clusters corresponding to metabolism and ethylene-dependent systemic resistance  and was related to beta-hexosaminidase , which was in turn associated with galactosidases , which are involved in carbohydrate metabolism.
the cluster related to the metabolism of amino acids and protein modification  contained cinnamyl alcohol dehydrogenases , which were linked to numerous peroxidases  associated with the oxidative stress response.
in the cluster related to the methylation and transposition of dna, methyl-cpg-binding proteins  and chromatin remodelling  <dig>  were found.figure  <dig>
interactome network of
a. thaliana
orthologous proteins related to water stress response of rangpur lime.
general network with inserts  that represent clusters .
a cluster  corresponding with proteins related to metabolism, development and the abiotic stress response.
a cluster  corresponding with proteins involved in carbohydrate metabolism and systemic responses that are dependent on ethylene.
a cluster  containing proteins related to protein modification.
a cluster  of proteins involved in the response to drought stress.
a cluster  corresponding with proteins related to dna methylation.
a cluster  related to proteins involved in amino acid metabolism.
a cluster  comprising amino acid precursor proteins.
a cluster  corresponding with proteins related to oxidative stress.
the circles indicate proteins involved in biological processes corresponding to the network, and the squares indicate proteins that were also differentially expressed during treatment.
the green nodes indicate proteins that were unique to the rangpur lime.
tyrosine aminotransferase  <dig>  was present in a cluster associated with hormone biosynthesis and responses to jasmonic acid and aba , and this protein interacted with coronatine-induced  <dig> , which is involved in methyl jasmonate signalling in guard cells .
cori <dig> was related to superroot  <dig>  and bisphosphate nucleotidase/inositol .
sal <dig> was also associated with a cluster of proteins involved in amino acid metabolism  and interacted with proteins involved in the biosynthesis of myoinositol, which is a signalling molecule involved in the stress response , and with phospholipase c , which is an important protein in the adaptation of plants to environmental stresses.
these proteins constituted the cluster related to the response to water deficit stress .
interactome network analysis for ‘sunki maravilha’ mandarin
gene ontology  analysis allowed us to identify the most representative biological processes in the protein interaction network of a. thaliana related to ‘sunki maravilha’ mandarin .
the interactome networks were divided into several sub-functional networks , and six of these clusters were found to be unique to ‘sunki maravilha’ mandarin orthologous proteins.
the main clusters were related to dna repair and amino acid and nucleic acid metabolism .
a large number of rna polymerases  were found in a cluster corresponding with dna repair and methylation .
these proteins were related to the ndpk <dig> protein, which was exclusive to ‘sunki maravilha’ and was considered to be a hub/bottleneck  of this genotype .
in turn, ndpk <dig> was associated with los <dig> and mppbeta , which were also considered to be hb proteins in the ‘sunki maravilha’ mandarin network .
this cluster was related to metabolism and biotic and abiotic stresses  and included several proteins that showed altered abundance in ‘sunki maravilha’ following exposure to water deficit that are known to be involved in the stress response.
these proteins included mitochondrial malate dehydrogenase , glyoxalase , 2-dehydro-3-deoxyphosphooctonate aldolase , fructokinase  and the dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex  <dig> .figure  <dig>
interactome network of
a. thaliana
orthologous proteins related to the water stress response of ‘sunki maravilha’ mandarin.
general network with inserts  that represent clusters .
a cluster  corresponding with proteins related to the metabolism of nucleotides and ubiquitination.
a cluster  comprising proteins involved in nucleotide metabolism.
a subgraph .
two clusters  corresponding with proteins involved in dna repair.
a cluster  containing proteins related to cell division.
the circles indicate proteins involved in biological processes corresponding to the network, and the network squares indicate proteins that were also differentially expressed during treatment.
the orange nodes indicate proteins that were unique to ‘sunki maravilha’ mandarin.
the cluster related to dna repair  contained rad <dig>  5′-flap endonuclease , chromatin remodelling  <dig> , and ultraviolet hypersensitive  proteins, which are involved in nucleotide excision from damaged dna, the regulation of replication and the response to uv light, respectively.
a large number of transcription factors were also found .
a cluster  related to cell division included the following proteins: cytidylyltransferase family protein , 3-deoxy-d-manno-octulosonic acid transferase , 3-deoxy-8-phosphooctulonate synthase , and maternal effect embryo arrest  <dig> .
a cluster related to energy, the metabolism of nucleic acids and ubiquitination  included an ubiquitin  protein that binds to atpc <dig>  which was in turn associated with the following proteins: glyceraldehyde 3-phosphate dehydrogenase subunit 2a , glycine dehydrogenase , serine transhydroxymethyltransferase  <dig> , a. thaliana glycine decarboxylase p-protein  <dig>  and sedoheptulose bisphosphatase , which is involved in the metabolism of osmoprotectants.
discussion
proteins showing altered abundance in response to water deficit in the roots of rangpur lime and ‘sunki maravilha’ mandarin were identified using a proteomic approach.
rangpur lime and ‘sunki maravilha’ were selected because in a previous study, these cultivars have been shown to differ in their use of available soil water, aba accumulation and expression of aba biosynthesis genes, suggesting that they use different systems to adapt to water restriction  <cit> .
protein changes associated with water deficit
water deficit caused alterations in protein abundance in rangpur lime and ‘sunki maravilha’ mandarin.
approximately 40% of the identified proteins were detected in multiple spots and had different isoelectric points  and molecular weights , suggesting the presence of isoforms and post-translational modifications or that these proteins were translated from different products of paralogous genes within a multigene family   <cit> .
the observed changes were related to phenotypic responses that determined the plant tolerance to water deficit  <cit> .
major changes in protein abundance caused by water stress were observed in ‘sunki maravilha’ mandarin compared with rangpur lime .
under control conditions, neves et al.
<cit>  have measured higher aba concentrations in the roots of unstressed ‘sunki maravilha’ mandarin compared with rangpur lime.
this finding can be considered to indicate increased physiological responsiveness to biotic and abiotic stresses, which enables better stomatal regulation and consequently reduces water use by ‘sunki maravilha’ mandarin plants subjected to water stress.
this physiological responsiveness leads to a series of changes at the protein level as an adaptive response to water stress.
the proteins identified in rangpur lime and ‘sunki maravilha’ mandarin were functionally categorized in terms of their roles in the response to water restriction.
comparative analysis of protein accumulation in rangpur lime and ‘sunki maravilha’ mandarin, together with the use of a systems biology approach, allowed us to establish a general profile of the biological processes involved in the response to water deficit in these plant varieties .
the main functional groups of proteins were examined in relation to water deficit.
proteins involved in metabolism and energy
the energy metabolism of proteins is often affected by water deficit.
in the present study, the abundance of some enzymes involved in the tricarboxylic acid  cycle and glycolysis was altered following water deficit in both evaluated varieties.
in rangpur lime and ‘sunki maravilha’ mandarin, the mitochondrial malate dehydrogenase levels  declined in response to water deficit.
this enzyme, which was considered to represent an hb in rangpur lime , is a key component of the tca cycle  <cit>  that is involved in central metabolism and redox homeostasis between organelle compartments  <cit> .
another protein in the energy metabolism class, atp synthase, showed decreased abundance in both varieties, which suggested that damage had occurred to the mitochondria and chloroplasts exposed to water deficit.
in addition, energy metabolism may have been weakened, which is a disadvantage due to the resulting decreases in the syntheses of atp and metabolites and feedback signaling.
thus, plants require additional energy to repair the damage caused by water stress.
mitochondrial processing protein accounted for approximately 11% of the proteome-level changes observed , suggesting that mitochondrial function and, hence, plant metabolism were altered and that the integrity of these processes must be protected from oxidative stress induced by drought  <cit> .
indeed, it is known that a key function of the mitochondria is defense against an excess of ros.
in fact, in plant cells, the mitochondria represent major sources of ros production and subsequent oxidative damage, as indicated in other proteomic studies  <cit> .
these findings suggest that plant tolerance to water deficit may be associated with efficient defense responses against oxidative stress at the cellular and subcellular levels.
los <dig> is an essential glycolytic enzyme that catalyses the interconversion of 2-phosphoglycerate and phosphoenolpyruvate and is induced by several types of abiotic stress, including water deficit and salinity  <cit> .
in the present study, los <dig> showed different patterns of expression and isoforms and was classified as an hb protein in both varieties.
in addition, it was identified in a unique ‘sunki maravilha’ mandarin cluster that was involved in the stress response and metabolism .
the opposite expression patterns observed for los <dig> indicate that this protein may play different roles during the water stress response in the two varieties.
systems biology analysis identified interactions between los <dig> and other proteins with important roles in the stress response and energy metabolism, such as ndpk <dig> , which was detected exclusively in ‘sunki maravilha’ mandarin and may be involved in the acclimation of this variety to water deficit due to its relationships with the general homeostasis of cellular nucleoside triphosphate  <cit> , oxidative stress responses  <cit>  and water deficit tolerance in bean  <cit> .
stress and defense proteins
approximately 37% of the proteins identified in this study were related to stress and defense .
plants have evolved antioxidant defense pathways to protect cells against the damage caused by high levels of ros under stress conditions  <cit> .
several enzymes involved in redox homeostasis were differentially regulated in the responses of rangpur lime and ‘sunki maravilha’ mandarin to water stress, including peroxidase , lactoylglutathione lyase  and glyoxalase .
in the two plant varieties, glyoxalase  showed opposite abundance patterns compared with those observed for lactoylglutathione lyase.
the excessive production of ros in stressed plants contributes to the accumulation of other toxic compounds, such as methylglyoxal, which is regulated by the glyoxalase system.
this system plays a role in tolerance to oxidative stress through the recycling of reduced glutathione  and specific changes in the absolute concentrations of ros  <cit> .
another protein that showed contrasting abundance between the two evaluated varieties was peroxidase, which, together with other enzymes, participates in the removal of h2o <dig> from cells.
in rangpur lime, we observed a decrease in peroxidase abundance under water stress conditions, whereas in ‘sunki maravilha’ mandarin, the abundance of this enzyme increased.
in addition, systems biology analysis identified a cluster related to the oxidative stress response that was unique to rangpur lime, in which peroxidase was directly associated with phenylalanine, aldehyde dehydrogenase and glutathione s-transferase, which are also involved in the detoxification of ros .
the changes in the abundance of several antioxidant enzymes observed in the rangpur lime and ‘sunki maravilha’ mandarin roots may also be necessary to balance the antioxidant system during water deficit.
choi and hwang  <cit>  have studied the expression of a peroxidase  in response to different biotic and abiotic stresses in pepper, demonstrating that its expression is strongly induced by drought, cold, salinity and osmotic stresses.
they also have shown that treatment with aba induces the expression of this enzyme.
the increased expression of peroxidase observed in ‘sunki maravilha’ mandarin may be associated with the accumulation of aba, which has been previously observed by neves et al.
<cit>  during water stress in this variety.
based on our results, it appears that the response of ros-scavenging enzymes to water deficit in citrus roots is genotype-dependent.
another protein involved in the stress response is annexin.
two isoforms of this protein with altered abundance following exposure to water stress were identified in our study .
the annat <dig> protein was up-regulated in ‘sunki maravilha’ mandarin but not in rangpur lime, whereas the abundance of the annat <dig> isoform was altered in both varieties .
annexins are directly involved in the regulation of signaling pathways that are activated by stress, and changes in the abundance of these proteins can alter plant tolerances to various types of abiotic stresses  <cit> .
in addition, the protein concentration of annexin in arabidopsis is increased by aba treatment, and this hormone may be a general regulator of annexin expression in several varieties of plant species  <cit> .
thus, based on the high levels of aba found in ‘sunki maravilha’ mandarin  <cit> , it can be speculated that this protein may function as a positive regulator of the accumulation of annexin in this variety.
moreover, annat <dig> was classified as a bottleneck protein in ‘sunki maravilha’ mandarin , and it is therefore an important candidate for future experiments designed to provide new insights regarding signaling pathways that are modulated by water stress.
repair and processing proteins
proteins related to the repair and processing of nucleotides, such as mrna-capping enzyme  and the histone ubiquitination protein group , demonstrated altered abundance in response to water deficit.
these proteins were identified exclusively in ‘sunki maravilha’ mandarin, and interestingly, they were present in the interactome sub-network exclusive to this variety.
systems biology analysis identified the interaction of mrna-capping protein with nucleotide excision repair .
the ner pathway is one of the most versatile repair pathways considering the diversity of dna lesions, including those induced by environmental factors, such as uv radiation  <cit> .
dna repair mechanisms are essential for maintaining genomic stability and integrity under stress conditions, allowing for greater genomic plasticity in response to environmental changes  <cit> .
protein modification represents a potential target in the engineering of plants with the goal of increasing tolerance to multiple stresses.
post-translational modifications of signaling proteins, such as phosphorylation and ubiquitination, are important for the regulation of gene expression in response to stress.
the ubiquitination of proteins can modulate stress response mechanisms involving the regulation and detection of hormone biosynthesis and the control of the abundance of proteins in signaling pathways, particularly transcription factors  <cit> .
studies have shown that aba is involved in controlling the processing of proteins and rna  <cit> .
analyses of the expression of genes in response to aba treatment in a. thaliana have revealed that this hormone alters the expression of ribosomal proteins and genes involved in proteolysis  <cit> .
in addition, liu and stone  <cit>  have found that the presence of aba promotes the self-ubiquitination of multi-domain ubiquitin e <dig> ligase  to increase the level of the transcription factor abscisic acid-insensitive <dig>  in a. thaliana.
the high levels of aba that have been found in ‘sunki maravilha’ mandarin by neves et al.
<cit>  may have induced the post-translational modifications of some proteins that are involved in the response to water restriction, contributing to the improved adaptation to drought of this variety.
putative mechanisms in rangpur lime and ‘sunki maravilha’ mandarin in response to water stress
the results of this study demonstrated that the dynamic stress response observed in rangpur lime included changes in the metabolism of carbohydrates and amino acids as well as alterations in the activation of ros detoxification and in the abundance of proteins involved in water stress defense .
for instance, l-galactose dehydrogenase  , which exhibited increased abundance in rangpur lime, held an important position in the network .
this enzyme was directly related to an ascorbate 5’-biphosphate nucleotide  that was associated with several proteins in cluster d  and is involved in the water deficit response, signal transduction and the response to aba .
sal <dig> acts as a negative regulator of drought tolerance in a. thaliana, and its inactivation increases the relative water content, improves water-use efficiency, reduces gas exchange and maintains viable tissues during prolonged water stress  <cit> .
in addition, plc <dig> is strongly induced under various environmental stresses, such as dehydration, salinity, and low temperature, and it plays a role in the inhibition of stomatal opening by aba  <cit> .
we hypothesize that the interactions of these proteins may be related to the faster response of rangpur to water stress, as demonstrated by its increased soil water collection efficiency.
the aba level increases in the roots of this plant during water deficit conditions in correlation with the activation of defense mechanisms  <cit> .
protein interaction network analysis suggested that the adaptation of ‘sunki maravilha’ plants to stress was aided by the activation of dna repair and processing proteins .
furthermore, our systems biology analysis revealed that the ndpk <dig> protein, which was considered to represent an hb in ‘sunki maravilha’ mandarin, interacted with proteins in the three clusters involved in the repair and processing of nucleotides .
ndpks are ubiquitous housekeeping enzymes involved in the response to heat stress  <cit> , uv-b light signaling, growth  <cit> , ros signaling  <cit>  and phytochrome signaling  <cit> .
changes in protein abundance and interactions with important proteins involved in dna repair pathways could be important factors determining the tolerance of ‘sunki maravilha’ mandarin to water deficit.
CONCLUSIONS
the present study revealed that the levels of a number of proteins involved in various cellular pathways are affected during water deficit in citrus roots.
the results showed that acclimatization to water deficit involved specific responses that differed between rangpur lime and ‘sunki maravilha’ mandarin.
proteins involved in metabolism, energy and signal transduction were down-regulated in ‘sunki maravilha’ mandarin.
in addition, proteins involved in the repair and processing of nucleotides were identified exclusively in this variety and showed higher levels in the drought-stressed roots compared with the control roots.
in contrast, the response to water deficit in rangpur lime included an increased abundance of proteins involved in transport, protein metabolism, the stress response and proteolysis and a decreased abundance of proteins related to metabolism and energy.
this study provides insights into the effects of drought on the abundance of proteins in the roots of two varieties of citrus rootstocks.
in addition, this work allows for a better understanding of the molecular basis of the response to water deficit in citrus.
further analyses are needed to elucidate the behaviors of the key target proteins that appear to be involved in this response.

 among potential abiotic stresses, water deficit is considered to have the largest effect on agricultural productivity and is one of the main factors limiting the distribution of species worldwide. when plants are subjected to water deficit, numerous morphological and physiological responses are observed, and the amplitude of these responses depends on the plant genotype as well as the duration and severity of the stress. the plant response to water deficit involves several processes, beginning with the perception of stress, followed by modulation of the expression of specific genes, and finally, the appearance numerous transcriptomic, proteomic and metabolomic changes. these changes result in the regulation of metabolism and the generation of regulatory networks that are involved in plant defense against the harmful effects of stress. transcriptomic studies have revealed that the expression of a wide range of genes is regulated in response to water deficit in citrus plants. analysis of expressed sequence tags in the roots of rangpur lime subjected to osmotic stress resulted in the identification of genes involved in the water stress response, including those encoding aquaporins, dehydrins, sucrose synthase and enzymes related to the synthesis of proline. using a microarray containing genes, gimeno et al. investigated the response of the transcriptome of ‘clementine’ mandarin grafted onto ‘cleopatra’ mandarin to water deficit conditions. as observed in other species, genes encoding proteins involved in lysine, proline and raffinose catabolism, hydrogen peroxide reduction, vacuolar malate transport, and defense were induced. analysis of the nac family of transcription factors resulted in the identification of one member, csnac that was strongly induced by water deficit in the leaves of ‘cleopatra’ mandarin and rangpur lime and by salt stress, cold and abscisic acid only in the leaves and roots of ‘cleopatra’ mandarin. in ‘cleopatra’ mandarin, xian et al. isolated a gene encoding crnced which is an enzyme involved in aba synthesis, and produced transgenic plants that constitutively overexpressed this gene. the transgenic lines displayed tolerance to dehydration, drought, salt, and oxidative damage compared with wild-type plants. furthermore, low levels of reactive oxygen species were detected in the transgenic plants under salt stress and dehydration. in addition to studies addressing the effects of water deficit on the transcriptome, proteomic studies have revealed the role of proteins involved in the complex mechanisms underlying the stress responses of plants. indeed, many proteins related to stress defense, detoxification, carbohydrate metabolism and photosynthesis that participate in the process of adaptation and tolerance to stress have been identified. in a study that evaluated changes in the leaves of two contrasting populations of populus cathayana in response to water deficit, drought-responsive proteins were identified: several of the proteins showing altered abundance were involved in transcriptional regulation, secondary metabolism, redox homeostasis and stress defense. an investigation of soybean roots subjected to short-term water deficit revealed changes in the abundance of proteins involved in carbohydrate and nitrogen metabolism, cellular defense and programmed cell death. zadražnik et al. identified drought-responsive proteins in the leaves of two bean cultivars with differing responses to drought stress. these proteins are primarily involved in energy metabolism, atp conversion, photosynthesis, protein synthesis and proteolysis and stress defence. changes in protein levels in the leaves of ‘willow leaf’ and ‘cleopatra’ mandarin plants subjected to salt stress were analyzed by podda et al.. significant variations in the abundance of protein spots were detected. these salt-responsive proteins play roles in photosynthetic processes, ros scavenging, stress defense, and signaling. however, there are few studies of the root proteome. analysis of the root proteome of wild watermelon has revealed that proteins involved in root morphogenesis, carbon/nitrogen metabolism, lignin synthesis and molecular chaperones are differentially regulated under drought stress. in the present study, we used proteomic approaches to analyses changes in the protein profiles of the roots of two citrus rootstock cultivars with contrasting responses to water deficit. proteins showing significantly altered abundance were selected for identification via mass spectrometry and bioinformatics analysis. in rangpur lime, the abundance of various proteins involved in protein metabolism, the stress response and proteolysis were modulated under water deficit conditions. in contrast, repair-related proteins contributed more specifically to the response of ‘sunki maravilha’ mandarin to this stress. this is the first report to examine the effects of water deficit on the abundance of proteins in citrus roots.  in the present study, root samples of rangpur lime and ‘sunki maravilha’ mandarin collected in a previous study by neves et al. were used. considering the soil moisture data from the previous report, two sampling points were selected for proteomic analysis as follows: 1) plants grown in soil with moisture ranging from - m3m− were defined as ‘control’ plants, whereas 2) the soil moisture for ‘drought-stressed’ plants ranged from - m3m− according to a previous report by neves et al., stomatal resistance is more pronounced in both varieties at the selected drought stress sampling points. in addition, they have reported that the leaf water potential decreases in water-stressed plants, reaching −  mpa and −  mpa in rangpur lime and ‘sunki maravilha’ mandarin, respectively. interestingly, rangpur lime shows a higher growth rate when grown under water deficit compared with the rate observed for ‘sunki maravilha’. when subjected to water deficit, the leaves and roots of ‘sunki maravilha’ display a progressive increase in the aba concentration. the lower leaf growth rate that has been recorded for ‘sunki maravilha’ mandarin may be associated with its greater leaf aba concentration. in contrast, in rangpur lime, alternations between high and low aba concentrations were observed. analysis of root protein profiles in response to water deficit to elucidate the changes in protein abundance in response to water deficit, comparative analysis of the protein profiles of roots of rangpur lime and ‘sunki maravilha’ mandarin was performed via 2d gel electrophoresis. the root protein profiles of both varieties that were grown under control conditions and subjected to water stress are shown in figure  more than spots were detected in both varieties via image analysis. a total of spots showed significant changes in abundance in the rangpur lime roots. these spots were subjected to mass spectrometry analysis, and proteins were identified. among these proteins, were increased and were decreased in abundance, and seven proteins were unique to this genotype. in ‘sunki maravilha’ mandarin, spots showed significant changes in abundance. among these spots, proteins were identified, of which increased and of which decreased in abundance, and nine were unique to this genotype.figure 2-de analysis of root proteins in rangpur lime under control conditions and following water deficit and in ‘sunki maravilha’ mandarin under control and water deficit conditions. the proteins indicated by the arrows were differentially expressed under the applied treatment. the proteins in the squares are unique to rangpur lime, and those in the circles are exclusive to ‘sunki maravilha’ mandarin. to understand the relationship between the two plant varieties as a function of water stress, multivariate analysis and principal component analysis were performed. pc represented 75% of the variance, suggesting that there were differences between rangpur lime and ‘sunki maravilha’ mandarin in response to water deficit. pc accounted for 17% of the variance, indicating that in ‘sunki maravilha’, there were differences between the well-watered plants and those under water stress. interestingly, we observed only minor changes in the protein profiles of rangpur lime under control versus drought-stressed conditions, suggesting that protein abundance was less affected by water deficit in this variety.figure principal component analysis and evaluation of variance under control conditions and drought in rangpur lime and ‘sunki maravilha’ mandarin. hierarchical clustering of the experiments and pca and eigenvalues table in control and water stress-treated samples from rangpur lime and ‘sunki maravilha’. identification and analysis of differentially expressed proteins spots showing differential intensities under water deficit were excised from the two-dimensional polyacrylamide gel electrophoresis gels and identified via ms. some proteins were identified more than once in different spots, reflecting different isoforms, post-translational modifications or alternative mrna splice forms. two spots were identified as epidermis-specific secreted glycoprotein ep1-like, five as germin-like, four as 2-phospho-d-glycerate hydrolase, three as mitochondrial processing peptidase alpha- subunit, two as putative mitochondrial processing peptidase, four as annexin, two as annexin d, two as heat shock protein, two as fructokinase and two as lactoylglutathione lyase. in addition, some of the proteins that were represented by different spots on the 2d gel showed opposite expression patterns. the germin-like proteins, which were represented by five spots, and putative mitochondrial processing peptidase exhibited opposite patterns of accumulation in ‘sunki maravilha’ mandarin. in contrast, mitochondrial processing peptidase alpha subunit showed opposite accumulation patterns in rangpur lime.table identification of differentially expressed proteins in the roots of rangpur lime and ‘sunki maravilha’ mandarin subjected to water deficit id spot a identified protein reference organism b accession number c mascot score/ p -value d mr theor/exp e pi theor/exp f expression level g a b c d fold change h rl sk 1 1 ∞ ∞ aspot id corresponding to the position in the 2d gel illustrated in figure  bprotein accession number according to the ncbi database. cbest matching protein identified by pblast analysis of the non-redundant database. dmascot score p value of the homology between citrus proteins and orthologous, homologous, or paralogous proteins, as annotated in ncbinr. etheoretical and experimental masses of identified proteins. ftheoretical and experimental pis of identified proteins. gexpression levels, presented as the % normalised volume, in the control and water deficit-stressed roots. vertical bars indicate the mean ± se. rangpur lime: control; and water deficit. ‘sunki maravilha’: control; and water deficit. hfold change: bold = increased protein abundance; underlined = decreased protein abundance; italics = no significant difference; np = protein not found in gel; ∞ = present in one treatment in the genotype. the functions of the identified proteins were inferred using the uniprot database. the identified proteins were classified into the following seven major groups according to their possible biological functions: stress and defense response, metabolism, transport, energy, signal transduction, protein metabolism and unknown for rangpur lime and ‘sunki maravilha’ mandarin, respectively. although the protein groups did not differ significantly between the two studied varieties, an additional class of proteins involved in dna repair was observed for ‘sunki maravilha’ mandarin.figure functional classifications of drought-responsive proteins. functional categorization of proteins that showed significant changes in abundance in rangpur lime. functional categorization of proteins with significantly altered levels in ‘sunki maravilha’ mandarin. analysis of protein-protein interactions analysis of interactomic data of a. thaliana orthologous proteins corresponding with the protein interaction profiles of rangpur lime and ‘sunki maravilha’ mandarin allowed us to draw an interactome network. the network developed for ‘sunki maravilha’ mandarin included proteins and connectors, and that constructed for rangpur lime included proteins and connectors. based on analysis of the intersection between the networks of the two varieties, proteins specific to the rangpur lime network, proteins unique to the ‘sunki maravilha’ mandarin network, and proteins shared between the two networks were observed. the interactome networks obtained for each variety could be divided into several functional clusters. evaluation of the highly connected regions and gene ontologies of each cluster revealed the presence of clusters in the rangpur lime interactome network and clusters in the ‘sunki maravilha’ mandarin interactome network. to evaluate the proteins forming the most relevant network, centrality analysis was performed by sorting the proteins into hubs and/or bottlenecks. among the proteins showing altered abundance in rangpur lime and ‘sunki maravilha’ mandarin, mitochondrial malate dehydrogenase, mitochondrial processing peptidase, 2-phospho-d-glycerate hydrolase and nucleoside diphosphate kinase were considered hubs/bottlenecks. glyoxalase, annexin, glutathione s-transferase and putative l-galactose dehydrogenase were only considered to be bottlenecks. protein-protein interactions in rangpur lime among the clusters of a. thaliana orthologous proteins corresponding with the differentially abundant proteins identified in the two studied citrus varieties, eight were exclusively related to rangpur lime. fructokinase, which was a bottleneck protein in rangpur lime, was present in a sub-functional network involved in growth, development and the stress response that contained the proteins cyp96a cyp71a, cyp and cyp and representatives of the cytochrome p superfamily. this cluster also included wrky transcription factors. the wrky transcription factors were associated with osmotin, which interacted with chitinase, which is an enzyme involved in the response to various environmental stresses. moreover, athchib was present in the clusters corresponding to metabolism and ethylene-dependent systemic resistance and was related to beta-hexosaminidase, which was in turn associated with galactosidases, which are involved in carbohydrate metabolism. the cluster related to the metabolism of amino acids and protein modification contained cinnamyl alcohol dehydrogenases, which were linked to numerous peroxidases associated with the oxidative stress response. in the cluster related to the methylation and transposition of dna, methyl-cpg-binding proteins and chromatin remodelling were found.figure interactome network of a. thaliana orthologous proteins related to water stress response of rangpur lime. general network with inserts that represent clusters. a cluster corresponding with proteins related to metabolism, development and the abiotic stress response. a cluster corresponding with proteins involved in carbohydrate metabolism and systemic responses that are dependent on ethylene. a cluster containing proteins related to protein modification. a cluster of proteins involved in the response to drought stress. a cluster corresponding with proteins related to dna methylation. a cluster related to proteins involved in amino acid metabolism. a cluster comprising amino acid precursor proteins. a cluster corresponding with proteins related to oxidative stress. the circles indicate proteins involved in biological processes corresponding to the network, and the squares indicate proteins that were also differentially expressed during treatment. the green nodes indicate proteins that were unique to the rangpur lime. tyrosine aminotransferase was present in a cluster associated with hormone biosynthesis and responses to jasmonic acid and aba, and this protein interacted with coronatine-induced, which is involved in methyl jasmonate signalling in guard cells. cori was related to superroot and bisphosphate nucleotidase/inositol. sal was also associated with a cluster of proteins involved in amino acid metabolism and interacted with proteins involved in the biosynthesis of myoinositol, which is a signalling molecule involved in the stress response, and with phospholipase c, which is an important protein in the adaptation of plants to environmental stresses. these proteins constituted the cluster related to the response to water deficit stress. interactome network analysis for ‘sunki maravilha’ mandarin gene ontology analysis allowed us to identify the most representative biological processes in the protein interaction network of a. thaliana related to ‘sunki maravilha’ mandarin. the interactome networks were divided into several sub-functional networks, and six of these clusters were found to be unique to ‘sunki maravilha’ mandarin orthologous proteins. the main clusters were related to dna repair and amino acid and nucleic acid metabolism. a large number of rna polymerases were found in a cluster corresponding with dna repair and methylation. these proteins were related to the ndpk protein, which was exclusive to ‘sunki maravilha’ and was considered to be a hub/bottleneck of this genotype. in turn, ndpk was associated with los and mppbeta, which were also considered to be hb proteins in the ‘sunki maravilha’ mandarin network. this cluster was related to metabolism and biotic and abiotic stresses and included several proteins that showed altered abundance in ‘sunki maravilha’ following exposure to water deficit that are known to be involved in the stress response. these proteins included mitochondrial malate dehydrogenase, glyoxalase, 2-dehydro-3-deoxyphosphooctonate aldolase, fructokinase and the dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex.figure interactome network of a. thaliana orthologous proteins related to the water stress response of ‘sunki maravilha’ mandarin. general network with inserts that represent clusters. a cluster corresponding with proteins related to the metabolism of nucleotides and ubiquitination. a cluster comprising proteins involved in nucleotide metabolism. a subgraph. two clusters corresponding with proteins involved in dna repair. a cluster containing proteins related to cell division. the circles indicate proteins involved in biological processes corresponding to the network, and the network squares indicate proteins that were also differentially expressed during treatment. the orange nodes indicate proteins that were unique to ‘sunki maravilha’ mandarin. the cluster related to dna repair contained rad 5′-flap endonuclease, chromatin remodelling, and ultraviolet hypersensitive proteins, which are involved in nucleotide excision from damaged dna, the regulation of replication and the response to uv light, respectively. a large number of transcription factors were also found. a cluster related to cell division included the following proteins: cytidylyltransferase family protein, 3-deoxy-d-manno-octulosonic acid transferase, 3-deoxy-8-phosphooctulonate synthase, and maternal effect embryo arrest. a cluster related to energy, the metabolism of nucleic acids and ubiquitination included an ubiquitin protein that binds to atpc which was in turn associated with the following proteins: glyceraldehyde 3-phosphate dehydrogenase subunit 2a, glycine dehydrogenase, serine transhydroxymethyltransferase, a. thaliana glycine decarboxylase p-protein and sedoheptulose bisphosphatase, which is involved in the metabolism of osmoprotectants. discussion proteins showing altered abundance in response to water deficit in the roots of rangpur lime and ‘sunki maravilha’ mandarin were identified using a proteomic approach. rangpur lime and ‘sunki maravilha’ were selected because in a previous study, these cultivars have been shown to differ in their use of available soil water, aba accumulation and expression of aba biosynthesis genes, suggesting that they use different systems to adapt to water restriction. protein changes associated with water deficit water deficit caused alterations in protein abundance in rangpur lime and ‘sunki maravilha’ mandarin. approximately 40% of the identified proteins were detected in multiple spots and had different isoelectric points and molecular weights, suggesting the presence of isoforms and post-translational modifications or that these proteins were translated from different products of paralogous genes within a multigene family. the observed changes were related to phenotypic responses that determined the plant tolerance to water deficit. major changes in protein abundance caused by water stress were observed in ‘sunki maravilha’ mandarin compared with rangpur lime. under control conditions, neves et al. have measured higher aba concentrations in the roots of unstressed ‘sunki maravilha’ mandarin compared with rangpur lime. this finding can be considered to indicate increased physiological responsiveness to biotic and abiotic stresses, which enables better stomatal regulation and consequently reduces water use by ‘sunki maravilha’ mandarin plants subjected to water stress. this physiological responsiveness leads to a series of changes at the protein level as an adaptive response to water stress. the proteins identified in rangpur lime and ‘sunki maravilha’ mandarin were functionally categorized in terms of their roles in the response to water restriction. comparative analysis of protein accumulation in rangpur lime and ‘sunki maravilha’ mandarin, together with the use of a systems biology approach, allowed us to establish a general profile of the biological processes involved in the response to water deficit in these plant varieties. the main functional groups of proteins were examined in relation to water deficit. proteins involved in metabolism and energy the energy metabolism of proteins is often affected by water deficit. in the present study, the abundance of some enzymes involved in the tricarboxylic acid cycle and glycolysis was altered following water deficit in both evaluated varieties. in rangpur lime and ‘sunki maravilha’ mandarin, the mitochondrial malate dehydrogenase levels declined in response to water deficit. this enzyme, which was considered to represent an hb in rangpur lime, is a key component of the tca cycle that is involved in central metabolism and redox homeostasis between organelle compartments. another protein in the energy metabolism class, atp synthase, showed decreased abundance in both varieties, which suggested that damage had occurred to the mitochondria and chloroplasts exposed to water deficit. in addition, energy metabolism may have been weakened, which is a disadvantage due to the resulting decreases in the syntheses of atp and metabolites and feedback signaling. thus, plants require additional energy to repair the damage caused by water stress. mitochondrial processing protein accounted for approximately 11% of the proteome-level changes observed, suggesting that mitochondrial function and, hence, plant metabolism were altered and that the integrity of these processes must be protected from oxidative stress induced by drought. indeed, it is known that a key function of the mitochondria is defense against an excess of ros. in fact, in plant cells, the mitochondria represent major sources of ros production and subsequent oxidative damage, as indicated in other proteomic studies. these findings suggest that plant tolerance to water deficit may be associated with efficient defense responses against oxidative stress at the cellular and subcellular levels. los is an essential glycolytic enzyme that catalyses the interconversion of 2-phosphoglycerate and phosphoenolpyruvate and is induced by several types of abiotic stress, including water deficit and salinity. in the present study, los showed different patterns of expression and isoforms and was classified as an hb protein in both varieties. in addition, it was identified in a unique ‘sunki maravilha’ mandarin cluster that was involved in the stress response and metabolism. the opposite expression patterns observed for los indicate that this protein may play different roles during the water stress response in the two varieties. systems biology analysis identified interactions between los and other proteins with important roles in the stress response and energy metabolism, such as ndpk, which was detected exclusively in ‘sunki maravilha’ mandarin and may be involved in the acclimation of this variety to water deficit due to its relationships with the general homeostasis of cellular nucleoside triphosphate, oxidative stress responses and water deficit tolerance in bean. stress and defense proteins approximately 37% of the proteins identified in this study were related to stress and defense. plants have evolved antioxidant defense pathways to protect cells against the damage caused by high levels of ros under stress conditions. several enzymes involved in redox homeostasis were differentially regulated in the responses of rangpur lime and ‘sunki maravilha’ mandarin to water stress, including peroxidase, lactoylglutathione lyase and glyoxalase. in the two plant varieties, glyoxalase showed opposite abundance patterns compared with those observed for lactoylglutathione lyase. the excessive production of ros in stressed plants contributes to the accumulation of other toxic compounds, such as methylglyoxal, which is regulated by the glyoxalase system. this system plays a role in tolerance to oxidative stress through the recycling of reduced glutathione and specific changes in the absolute concentrations of ros. another protein that showed contrasting abundance between the two evaluated varieties was peroxidase, which, together with other enzymes, participates in the removal of h2o from cells. in rangpur lime, we observed a decrease in peroxidase abundance under water stress conditions, whereas in ‘sunki maravilha’ mandarin, the abundance of this enzyme increased. in addition, systems biology analysis identified a cluster related to the oxidative stress response that was unique to rangpur lime, in which peroxidase was directly associated with phenylalanine, aldehyde dehydrogenase and glutathione s-transferase, which are also involved in the detoxification of ros. the changes in the abundance of several antioxidant enzymes observed in the rangpur lime and ‘sunki maravilha’ mandarin roots may also be necessary to balance the antioxidant system during water deficit. choi and hwang have studied the expression of a peroxidase in response to different biotic and abiotic stresses in pepper, demonstrating that its expression is strongly induced by drought, cold, salinity and osmotic stresses. they also have shown that treatment with aba induces the expression of this enzyme. the increased expression of peroxidase observed in ‘sunki maravilha’ mandarin may be associated with the accumulation of aba, which has been previously observed by neves et al. during water stress in this variety. based on our results, it appears that the response of ros-scavenging enzymes to water deficit in citrus roots is genotype-dependent. another protein involved in the stress response is annexin. two isoforms of this protein with altered abundance following exposure to water stress were identified in our study. the annat protein was up-regulated in ‘sunki maravilha’ mandarin but not in rangpur lime, whereas the abundance of the annat isoform was altered in both varieties. annexins are directly involved in the regulation of signaling pathways that are activated by stress, and changes in the abundance of these proteins can alter plant tolerances to various types of abiotic stresses. in addition, the protein concentration of annexin in arabidopsis is increased by aba treatment, and this hormone may be a general regulator of annexin expression in several varieties of plant species. thus, based on the high levels of aba found in ‘sunki maravilha’ mandarin, it can be speculated that this protein may function as a positive regulator of the accumulation of annexin in this variety. moreover, annat was classified as a bottleneck protein in ‘sunki maravilha’ mandarin, and it is therefore an important candidate for future experiments designed to provide new insights regarding signaling pathways that are modulated by water stress. repair and processing proteins proteins related to the repair and processing of nucleotides, such as mrna-capping enzyme and the histone ubiquitination protein group, demonstrated altered abundance in response to water deficit. these proteins were identified exclusively in ‘sunki maravilha’ mandarin, and interestingly, they were present in the interactome sub-network exclusive to this variety. systems biology analysis identified the interaction of mrna-capping protein with nucleotide excision repair. the ner pathway is one of the most versatile repair pathways considering the diversity of dna lesions, including those induced by environmental factors, such as uv radiation. dna repair mechanisms are essential for maintaining genomic stability and integrity under stress conditions, allowing for greater genomic plasticity in response to environmental changes. protein modification represents a potential target in the engineering of plants with the goal of increasing tolerance to multiple stresses. post-translational modifications of signaling proteins, such as phosphorylation and ubiquitination, are important for the regulation of gene expression in response to stress. the ubiquitination of proteins can modulate stress response mechanisms involving the regulation and detection of hormone biosynthesis and the control of the abundance of proteins in signaling pathways, particularly transcription factors. studies have shown that aba is involved in controlling the processing of proteins and rna. analyses of the expression of genes in response to aba treatment in a. thaliana have revealed that this hormone alters the expression of ribosomal proteins and genes involved in proteolysis. in addition, liu and stone have found that the presence of aba promotes the self-ubiquitination of multi-domain ubiquitin e ligase to increase the level of the transcription factor abscisic acid-insensitive in a. thaliana. the high levels of aba that have been found in ‘sunki maravilha’ mandarin by neves et al. may have induced the post-translational modifications of some proteins that are involved in the response to water restriction, contributing to the improved adaptation to drought of this variety. putative mechanisms in rangpur lime and ‘sunki maravilha’ mandarin in response to water stress the results of this study demonstrated that the dynamic stress response observed in rangpur lime included changes in the metabolism of carbohydrates and amino acids as well as alterations in the activation of ros detoxification and in the abundance of proteins involved in water stress defense. for instance, l-galactose dehydrogenase, which exhibited increased abundance in rangpur lime, held an important position in the network. this enzyme was directly related to an ascorbate 5’-biphosphate nucleotide that was associated with several proteins in cluster d and is involved in the water deficit response, signal transduction and the response to aba. sal acts as a negative regulator of drought tolerance in a. thaliana, and its inactivation increases the relative water content, improves water-use efficiency, reduces gas exchange and maintains viable tissues during prolonged water stress. in addition, plc is strongly induced under various environmental stresses, such as dehydration, salinity, and low temperature, and it plays a role in the inhibition of stomatal opening by aba. we hypothesize that the interactions of these proteins may be related to the faster response of rangpur to water stress, as demonstrated by its increased soil water collection efficiency. the aba level increases in the roots of this plant during water deficit conditions in correlation with the activation of defense mechanisms. protein interaction network analysis suggested that the adaptation of ‘sunki maravilha’ plants to stress was aided by the activation of dna repair and processing proteins. furthermore, our systems biology analysis revealed that the ndpk protein, which was considered to represent an hb in ‘sunki maravilha’ mandarin, interacted with proteins in the three clusters involved in the repair and processing of nucleotides. ndpks are ubiquitous housekeeping enzymes involved in the response to heat stress, uv-b light signaling, growth, ros signaling and phytochrome signaling. changes in protein abundance and interactions with important proteins involved in dna repair pathways could be important factors determining the tolerance of ‘sunki maravilha’ mandarin to water deficit.  the present study revealed that the levels of a number of proteins involved in various cellular pathways are affected during water deficit in citrus roots. the results showed that acclimatization to water deficit involved specific responses that differed between rangpur lime and ‘sunki maravilha’ mandarin. proteins involved in metabolism, energy and signal transduction were down-regulated in ‘sunki maravilha’ mandarin. in addition, proteins involved in the repair and processing of nucleotides were identified exclusively in this variety and showed higher levels in the drought-stressed roots compared with the control roots. in contrast, the response to water deficit in rangpur lime included an increased abundance of proteins involved in transport, protein metabolism, the stress response and proteolysis and a decreased abundance of proteins related to metabolism and energy. this study provides insights into the effects of drought on the abundance of proteins in the roots of two varieties of citrus rootstocks. in addition, this work allows for a better understanding of the molecular basis of the response to water deficit in citrus. further analyses are needed to elucidate the behaviors of the key target proteins that appear to be involved in this response. 
rootstocks play a major role in the tolerance of citrus plants to water deficit by controlling and adjusting the water supply to meet the transpiration demand of the shoots. alterations in protein abundance in citrus roots are crucial for plant adaptation to water deficit. we performed two-dimensional electrophoresis separation followed by lc/ms/ms to assess the proteome responses of the roots of two citrus rootstocks, rangpur lime and ‘sunki maravilha’ mandarin, which show contrasting tolerances to water deficits at the physiological and molecular levels. changes in the abundance of and proteins in rangpur lime and ‘sunki maravilha’ mandarin, respectively, were observed via lc/ms/ms in response to water deficit. multivariate principal component analysis of the data revealed major changes in the protein profile of ‘sunki maravilha’ in response to water deficit. additionally, proteomics and systems biology analyses allowed for the general elucidation of the major mechanisms associated with the differential responses to water deficit of both varieties. our study reveals that the levels of a number of proteins involved in various cellular pathways are affected during water deficit in the roots of citrus plants. the results show that acclimatization to water deficit involves specific responses in rangpur lime and ‘sunki maravilha’ mandarin. in addition, this work allows for a better understanding of the molecular basis of the response to water deficit in citrus. citrus rootstockwater deficitproteomicsprotein networkissue-copyright-statement© the author 2015 in contrast, the adaptation of ‘sunki maravilha’ to stress was aided by the activation of dna repair and processing proteins. this study provides insights into the effects of drought on the abundance of proteins in the roots of two varieties of citrus rootstocks. the defense mechanisms of rangpur lime included changes in the metabolism of carbohydrates and amino acids as well as in the activation of reactive oxygen species detoxification and in the levels of proteins involved in water stress defense. further analysis is needed to elucidate the behaviors of the key target proteins involved in this response.
 rootstocks play a major role in the tolerance of citrus plants to water deficit by controlling and adjusting the water supply to meet the transpiration demand of the shoots. alterations in protein abundance in citrus roots are crucial for plant adaptation to water deficit. we performed two-dimensional electrophoresis separation followed by lc/ms/ms to assess the proteome responses of the roots of two citrus rootstocks, rangpur lime and ‘sunki maravilha’ mandarin, which show contrasting tolerances to water deficits at the physiological and molecular levels.  changes in the abundance of and proteins in rangpur lime and ‘sunki maravilha’ mandarin, respectively, were observed via lc/ms/ms in response to water deficit. multivariate principal component analysis of the data revealed major changes in the protein profile of ‘sunki maravilha’ in response to water deficit. additionally, proteomics and systems biology analyses allowed for the general elucidation of the major mechanisms associated with the differential responses to water deficit of both varieties. the defense mechanisms of rangpur lime included changes in the metabolism of carbohydrates and amino acids as well as in the activation of reactive oxygen species detoxification and in the levels of proteins involved in water stress defense. in contrast, the adaptation of ‘sunki maravilha’ to stress was aided by the activation of dna repair and processing proteins.  our study reveals that the levels of a number of proteins involved in various cellular pathways are affected during water deficit in the roots of citrus plants. the results show that acclimatization to water deficit involves specific responses in rangpur lime and ‘sunki maravilha’ mandarin. this study provides insights into the effects of drought on the abundance of proteins in the roots of two varieties of citrus rootstocks. in addition, this work allows for a better understanding of the molecular basis of the response to water deficit in citrus. further analysis is needed to elucidate the behaviors of the key target proteins involved in this response. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords citrus rootstockwater deficitproteomicsprotein networkissue-copyright-statement© the author 2015 