the date palm ( phoenix dactylifera l. ) is of economic importance to the kingdom of saudi arabia which is the second largest producer of dates worldwide .
although date palms can grow under a variety of environmental conditions , production is impeded by various biotic and abiotic stress factors .
most importantly , water shortage and salinity of the ground water provide abiotic stresses which decrease date production .
this is a worldwide problem with some 20% of the world 's cultivated land and approximately 50% of all irrigated land being affected . in consequence ,
adaptation of crop plants to water deficit and salt stress is of high priority in worldwide programmes for breeding modern varieties ( for a review see ) .
for date palm thousands of different cultivars are known which have been selected by the producers mainly for improved crop yield and quality .
thus , a high degree of variability is presumably present in date palm germplasms with respect to drought and salinity ( desiccation and salt tolerance ) .
the high salinity exhibits negative effects on the critical biochemical processes of the plant : salt stress affects the whole plant as well as tissues and cells .
it can lead to water deficit stress , metabolic toxicity , and nutritional deficiencies and finally drastically reduce production . as studied extensively in arabidopsis and rice , three aspects of adaptive responses in plants can be considered under conditions of salt and drought : ( a ) ion and osmotic homeostasis , ( b ) growth control / inhibition , and ( c ) control and repair of stress damage ( detoxification ) . the findings on mechanisms of adaptation to abiotic stresses in model plants such as arabidopsis are relevant to crop plants .
transduction of extracellular , abiotic stress signals via the cell wall and membrane into the cytoplasm and subcellular compartments follows various pathways and triggers various responses .
two of the principal elements in these pathways of plant cells are ( i ) intracellular ca ions and ( ii ) protein kinases . consequently , the sensing of abiotic stress such as drought or salinity results in changes of the phosphorylation status of cellular proteins . as revealed by mutant analysis in arabidopsis ,
abscisic acid ( aba ) is another key regulator of signal cascades that are triggered by salt and water deficit .
molecular studies in date palm for understanding some basic molecular mechanisms in response to drought and salinity have been rather limited [ 917 ] .
recent date proteome analyses , however , open the way to the identification of important biomarkers .
identified an abc superfamily atp - binding cassette transporter as a putative biomarker for male date palms .
palms affected by the leaf brittle disease express a manganese - stabilizing 33  kda protein not detectable in healthy plants .
the recent sequencing and annotation of date palm genomes   opens the possibility for the application of further high throughput technologies to the study of stress - related gene functions in date palm .
proteome analysis , for example , opens the possibility to identify date palm proteins involved in transduction network regulation via posttranslational protein modification by phosphorylation / dephosphorylation under abiotic stress conditions   as studied for example , in the desiccation - tolerant plant craterostigma plantagineum [ 22 , 23 ] .
the effort described here relates to proteome analysis of salinity and water stress - related sensitive protein resulting from the salinity and drought gene expression in leaves of young date palm seedlings .
seeds of the cultivar  sagie  were scarified with sulfuric acid ( 96% ) for 5  min and washed 5 times with sterile distilled water , followed by sterilization with 1% ( v / v ) mercuric chloride for 3  min , washed 5 times with sterile distilled water , and imbibed for 48  h in distilled water .
the seeds were sterilized a second time with calcium hypochlorite ( 5% , v / v ) for 4  min and washed 4 times with sterile distilled water .
seeds were germinated between wet layers of tissue papers until the radical reached 1  cm and then transferred to pots containing organic soil and irrigated with tap water and grown in growth chambers under controlled light conditions ( 12  h light/12  h dark ) at 30c until the age of three months .
twelve 3-month - old date palm seedlings were selected and divided as follows : 4 seedlings were daily irrigated with distilled water for one month as control , 4 seedlings were subjected to drought ( 27.5  g / l peg 6000 ) for one month , and the other 4 seedlings were subjected to salt stress with 16  g / l nacl , according to a modified method of san et al . .
at the end of the stress period , samples were washed with distilled water , frozen in liquid nitrogen , and stored at 80c until use .
four replicates of the frozen shoot materials of stressed and control plantlets were ground into a fine powder under liquid nitrogen , and the proteins were precipitated by the addition of 1.8  ml of ice - cold acetone containing 0.07% ( v / v ) mercaptoethanol .
one hundred mg of each of the lyophilized raw extract was dissolved in 400600  l of ief buffer ( 7  m urea , 2  m thiourea , 2% ( w / v ) chaps , and 30  mm tris ph 8.0 ) .
the mixture was then centrifuged for 10 minutes at 4c at 16,100  g and total soluble protein in the supernatants was quantified using the 2d quant kit ( ge healthcare , munich , germany ) .
equal amounts of all single samples were pooled to get dige internal standard ( is ) .
amount of 50  g of is was used for each analytical gel and 300  g of is for each preparative gel ( necessary for protein identification by ms ) .
50  g of each protein sample as well as needed amount of internal standard was labeled with fluorescent dyes using the refraction-2dtm labeling kit ( nh dyeagnostics gmbh , halle , germany ) according to the manufacturer 's protocol .
the internal standard was labeled with g100 , whereas the single analyzed samples were labeled with g200 or g300 before mixing .
2d gel electrophoresis was briefly performed as follows : samples mixture was separated in the first dimension according to their isoelectric point ( pi ) using immobilized ph gradient strips ( immobiline drystrip , 24  cm , ph 47 , ge healthcare ) focused by ipgphor 3 ( ge healthcare ) and in the second dimension according to their molecular weight by sds - page using the ettan dalttwelve gel system ( ge healthcare ) . for preparative gels , glass plates were silanized on one side prior to gel casting and the gels were run in parallel to the analytical gels .
the fluorescent scans of the analytical gels were generated using ettan dige imager ( ge healthcare ) immediately after electrophoresis .
preparative gels were stained with 1  mm ruthenium(ii)-tris(bathophenanthroline disulfonate ) fluorescence stain and reference markers were attached to the glass plates prior to scanning , thus enabling blind picking of the protein spots after the difference gel electrophoresis ( dige ) analysis .
the preparative gels were scanned directly after destaining and stored wet at 4c before spot cutting .
the internal standard included all proteins in the analysis and as it was run on every gel along with all analyzed samples , it was used for spot matching across all the gels . the biological variation analysis ( bva ) module allowed quantitative comparisons of protein expression across multiple gels .
the extended data analysis ( eda ) was used for multivariate analysis of protein expression data derived from the bva module in order to perform a principal component analysis ( pca ) and to identify protein spots of interest with differential expression analysis .
all automatically identified spots were checked manually to confirm that they are real spots and marked for picking . for protein identification in a single spot ,
proteins fixed in the polyacrylamide gel plug were reduced , alkylated , and digested with trypsin ( promega , mannheim , germany ) .
the resulting peptides were analyzed by nano - hplc ( ultimate 3000  hplc system , lc packings , dionex , idstein , germany ) coupled to an amazon etd ms ion trap spectrometer ( bruker daltonics , bremen , germany ) using nano - esi spray .
the nano - hplc system and the ion trap spectrometer were controlled using the bruker compass hystar v3.2-sr2 software .
the liquid chromatography system was supplied with reversed - phase precolumn ( lc packings , dionex ) for sample desalting and a 15  cm pepmap 100 reversed - phase c18 column , 75  m inner diameter ( lc packings , dionex ) , for peptide fractionation .
the peptides were separated using a 45  min linear gradient from 96% ( v / v ) solution a ( 2% ( v / v ) acetonitrile , 0.1% ( v / v ) formic acid in high purity water ) and 4% ( v / v ) solution b ( 98% ( v / v ) acetonitrile , 0.1% ( v / v ) formic acid in high purity water ) to 50% ( v / v ) solution a and 50% ( v / v ) solution b at a flow rate of 300  nl / min .
the electrospray was operated in positive ion mode with 4000  v spray voltage and 10  psi gas pressure .
the end plate offset of the mass spectrometer was set to 500  v and for the acquisition the standard method proteomics auto msms alternating spectra cid - etd bruker trap control v7.0 was used .
raw data files were evaluated using compass data analysis v4.0-sr5 software with embedded search engine mascot search 2.3.01 ( matrix science ltd . , london , uk ) .
swiss prot ( all species ) and ncbinr ( green plants ) databases were involved in the protein search using the following parameters : enzyme trypsin , up to one missed cleavage permitted , no fixed modifications and variable modifications carbamidomethyl ( c ) , oxidation ( m ) and propionamide ( c ) allowed , and mass tolerance for both precursor ion and fragment ion 0.3  da . only the protein hit with highest protein score was used for further analysis .
when the protein was identified with one peptide only or several proteins with similar protein score were identified in a spot , the spots were excluded from the analysis .
the objective of this experiment is comparison of protein spots of salt - stressed and drought - stressed date palm shoots by searching for new protein spots or proteins spots differing in their intensity due to stress . in this experiment ,
4 salt stress samples ( 6164 ) , 4 drought stress samples ( 7376 ) , and 4 control samples ( 14 ) of lyophilized raw protein extracts of date palm were analyzed .
samples 6164 represent the date palm seedlings that were exposed to a high concentration of nacl ( 16 
g / l ) for one month , whereas samples 7376 represent seedlings that were exposed to a high peg concentration ( 27.5 
all the samples were solubilized in ief1 buffer ( 7  m urea , 2  m thiourea , 2% chaps , and 30  mm tris ph 8.0 ) according to the data shown in table 1 .
after overnight solubilization and subsequent centrifugation , the soluble proteins were quantified using the 2d quant kit .
estimated total protein concentrations are listed in table 1 . for successful labelling of the samples with g200 or g300 ,
all the samples were diluted with ief 1 buffer to reach the concentration of the sample with lowest concentration ( 1.1  g/l in this case , table 1 ) .
an internal standard was prepared by mixing all 12 samples in the same weight ratio and the standard was labelled with g100 .
the presence of an internal standard in every gel provided an intrinsic link between samples . each protein spot in a sample was compared to its representative spot within the internal standard on the same gel to generate a ratio of relative protein levels .
quantitative comparison of samples between gels was based on the relative change of a sample to its in - gel internal standard .
the labelling and mixing scheme for the performed dige experiments is shown in table 2 .
part of the internal standard was saved before labelling with g100 and this unlabeled part was applied to 2 preparative gels that were in the end used for the protein identification by mass spectrometry .
proteins from the extracts were separated in the first dimension according to their protein intensity using ief on immobiline drystrip , 24  cm , ph 47 , and in the second dimension according to their molecular weight using sds - page .
all of the 6 analytical gels and the 2 preparative gels were run at the same time .
the preparative gels were fixed for 1 hour in fixation solution ( 40% ethanol and 10% acetic acid in water ) and then stained with rubps ( 1  mm in fixation solution ) for 20 minutes and destained overnight in fixation solution .
the preparative gels were scanned directly after destaining and stored wet at 4c before spot cutting .
for dige analysis , spots were detected on all scans and intergel matching was performed using the decyder software .
the matching was manually checked and corrected in mismatched regions . in the next step ,
pca helps to identify some underlying sources of variation and gives first impression on how well experimental groups can be separated .
spots that could be localized on 80% of spot maps ( gel scans ) were included in the analysis ( figure 2 ) . 

scores of the spot maps for salt stress ( red circles in figure 2(a ) ) were localized closely to each other in the graph and thus showed a good reproducibility for the salt stress biological replicates .
in addition , scores of the spot maps for controls ( green circles in figure 2(a ) ) had an outlier in spot map for sample 2 .
other three spot maps were well separated from the salt stress spot maps in a compact group .
furthermore , scores of the spot maps for drought stress ( blue circles in figure 2(a ) ) were located between controls and salt stress with an outlier for sample 76 .
position of other three replicates to each other indicated a lower reproducibility of the drought stress compared to the salt stress .
position closer to the control suggested smaller differences in drought stress as compared to salt stress .
for this analysis , the standardized abundance of each spot on the gel was calculated for two samples in the gel using the spot intensities of the internal standard .
significant differences between the samples were visualized showing blue or red spot contours for higher or lower standardized abundance ( threshold 3 ) of the spots , respectively .
figure 3 shows fluorescence scan of the control versus salt stress , figure 4 shows fluorescence scan of control versus drought stress , and figure 5 shows fluorescence scan of the salt stress versus drought stress .
several regions with significant spot changes were localized and may indicate potential effects on the proteome due to stress .
t - test ( p value calculated using student 's t - test ) , one - way anova ( p value calculated using one - way anova statistical test ) , and average ratio ( average ratio between the groups selected in the protein statistics ) values were calculated for all matched spots . the spots were filtered for one - way anova value lower than 0.2 , t - test value lower than 0.1 , and average ratio < 2 and > 2 .
eda is proteomic software for multivariate analysis of protein expression data derived from bva module .
it was used for pca analysis presented in section 1 and to find the protein spots of interest with differential expression analysis .
all automatically chosen spots were checked manually if they are real spots and marked for picking ( figure 6 ) .
using low level statistical parameters , 47 spots were chosen for protein identification , most of them with lower spot volume under stress conditions compared to the standard .
detailed results of dige analysis are found in the supplementary excel tables ( in supplementary material available online at http://dx.doi.org/10.1155/2015/407165 ) .
proteins in the gel plugs were reduced with 10  mm dithiothreitol and alkylated using 55  mm iodoacetamide in 0.1  m to open s - s bridges for action of trypsin .
digestion with trypsin ( 12.5  ng/l of trypsin in 50  mm nh4hco3 ) was performed overnight at 37c .
the resulting peptides were extracted from the gel plugs in two extraction steps : first one with 25  mm nh4hco3 and second one with 5% formic acid .
collected extracts were dried down and resolubilized in 2% acetonitrile with 0.1% formic acid in water ( ms grade ) for ms analysis .
the resulting peptides were separated according to their hydrophobicity by nano - hplc ( c18 column , ultimate 3000  hplc system , dionex ) and sprayed directly into an ion trap spectrometer ( amazon etd , bruker daltonics ) using nano - esi sprayer .
processed ms / ms spectra were used for the protein identification with in - house mascot search server ( matrix science software ) .
swiss prot ( all species ) and ncbinr ( green plants ) databases were involved in the protein search ( figure 7 and table 3 ) . 
 ms results .
biological replicates for salt stress showed good reproducibility and were well separated from control samples in the pca score plot .
several regions with significant spots changing probably due to the applied stress were localized in the gel scans using differential in - gel analysis ( dia , decyder ) .
most of the spots from these regions were decreased in the relative abundance under stress conditions .
only low quality statistics allowed the finding of some more intensive spots sensitive to stress .
proteins in 47 spots were analyzed by mass spectrometry ; for 8 spots no protein could be identified .
levels of atp synthase alpha and beta subunits , unknown protein 18 and some of rubisco fragments were significantly changed under both stress conditions .
changes in abundance of superoxide dismutase , chlorophyll a - b binding protein , light - harvesting complex i protein lhca1 , rubisco activase , phosphoglycerate kinase , chloroplast light - harvesting chlorophyll a / b - binding protein , phosphoribulokinase , transketolase , rubisco , and some of rubisco fragments were significant only for salt stress .
proteome analysis provides one of the best options for the functional analysis of translated regions of the genome .
the levels of atp synthase alpha ( accession number gi|158325128 ) and beta subunits ( accession number gi|292559515 ) , unknown protein ( accession number gi|205830697 ) , and some of rubisco fragments ( accession numbers gi|28195663 , gi|292559516 , gi|3152721 , gi|209417491 , gi|209417489 , gi|55785631 , and gi|16565309 ) were significantly changed under both stress conditions compared to the control which indicates that these protein subunits are associated with drought and salinity stress [ 2527 ] . in the current study ,
changes in abundance of superoxide dismutase , chlorophyll a - b binding protein , light - harvesting complex i protein lhca1 , rubisco activase , phosphoglycerate kinase , chloroplast light - harvesting chlorophyll a / b - binding protein , phosphoribulokinase , transketolase , rubisco , and some of rubisco fragments were significant only in salt stress condition .
this result is consistent with many salinity stress tolerance studies in other organisms [ 2830 ] .
the superfamily of light - harvesting chlorophyll a / b - binding ( lhc ) proteins in higher plants and green algae is composed of more than 20 different members associated with photosystem i ( psi ) or photosystem ii ( psii ) . in this study , chlorophyll a - b binding protein ( cab ) and light - harvesting complex i protein lhca1 were upregulated .
accumulation of cab and lhca1 in psi exposed to salt and drought stress might represent one of the strategies to prevent or lower light stress - induced damage [ 25 , 26 , 28 ] .
the accumulation of chl has been proposed as one of the potential biochemical indicators of salt tolerance in different crops .
it was also proposed that these proteins might have a protective function within psii under stress conditions either by binding free chlorophyll molecules and preventing the formation of free radicals and/or by acting as sinks for excitation energy , because under stress conditions , a mobile pool of lhcb1 and lhcb2 moves from psii to psi due to the reversible phosphorylation of lhc proteins by a thylakoid - bound kinase [ 26 , 32 ] .
decrease in the initial activity and activation state of rubisco as a result of drought stress was encountered in mediterranean species . in the present study , the expression of superoxide dismutases ( sod )
the superoxide dismutases are metalloenzymes that catalyze the dismutation of ion superoxide into oxygen and hydrogen peroxide ( sods ) and constitute the first line of defence against reactive oxygen species .
the superoxide radical is a reactive oxygen species ( ros ) whose production increases under abiotic and biotic stresses , including drought [ 28 , 35 ]
. meanwhile , alscher et al .   stated that sods play a critical role in protecting plant tissues from ros .
roveda - hoyos and fonseca - moreno   reported that eight proteins were overregulated in wheat leaves . in response to salt stress , among them a protein complex of psii , a protein oee2 ( oxygen - evolving enhancer protein 2 ) , and superoxide dismutase .
the change in regulation of phosphoribulokinase in the current study is also correlated with salt stress .
the expression of prk ( phosphoribulokinase ) in ice plant was affected under salt stress conditions .
the amount of mrna was declined by a factor of approximately three within days , followed by an increase to approximately prestress levels . in conclusion ,
proteome of salinity and drought - stressed palm seedlings was compared with that of nonstressed plants of the same age .
the ms analysis of 47 sensitive protein spots showed that 12 proteins could be identified , 3 of them were significantly changed in both stresses , and 9 proteins were significantly changed only in salt - stressed plants .
proteins could not be identified in 8 spots , whereas in 26 spots rubisco and its fragments were identified .
 this study was carried out to study the proteome of date palm under salinity and drought stress conditions to possibly identify proteins involved in stress tolerance . for this purpose , three - month - old seedlings of date palm cultivar  
 sagie  were subjected to drought ( 27.5  g / l polyethylene glycol 6000 ) and salinity stress conditions ( 16  g / l nacl ) for one month . 
 dige analysis of protein extracts identified 47 differentially expressed proteins in leaves of salt- and drought - treated palm seedlings . 
 mass spectrometric analysis identified 12 proteins ; three out of them were significantly changed under both salt and drought stress , while the other nine were significantly changed only in salt - stressed plants . 
 the levels of atp synthase alpha and beta subunits , an unknown protein and some of rubisco fragments were significantly changed under both salt and drought stress conditions . 
 changes in abundance of superoxide dismutase , chlorophyll a - b binding protein , light - harvesting complex1 protein lhca1 , rubisco activase , phosphoglycerate kinase , chloroplast light - harvesting chlorophyll a / b - binding protein , phosphoribulokinase , transketolase , rubisco , and some of rubisco fragments were significant only for salt stress . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion