BACKGROUND
histone modifications and histone variants play critical roles in regulating gene expression, modulating the cell cycle, and are responsible for maintaining genome stability  <cit> .
the fundamental structural unit of chromatin in eukaryotic cells is the nucleosome, that consists of  <dig> base pairs  of dna wrapped around a histone octamer, each of which is formed by two copies of h2a, h2b, h <dig> and h <dig>  <cit> .
an additional histone, h <dig> links these nucleosomes together along the chromatin chain.
in general, the n terminus of histone h <dig> and h <dig>  and n and c terminus of h2a and h2b are prone to being covalently modified by many enzymes, such as hmt  and hat .
these modifications include methylation, acetylation, phosphorylation, ubiquitination, glycosylation, adp ribosylation, carbonylation, sumoylation and biotinylation.
most of these modifications are dynamic and can be reversed by other enzymes, such as histone demethylase and hdac .
using techniques such as western blotting and mass spectrometry, increasing number of histone modification sites have been identified in mouse, yeast, drosophila melanogaster, tetrahymena thermophila and a. thaliana  <cit> .
mass spectrometry  allows us not only to deduce the amino acid sequence of a peptide, but also to identify the exact sites and type of modifications in the peptide via the modified peptide mass shifts.
epigenetic studies of chromatin in model organisms have provided insights into the modifications of histones, ranging from the identification of several enzymes and related effectors associated with histone modifications to their biological functions in cell development  <cit> .
it is currently proposed that histone modifications play vital roles in many fundamental biological processes by rearranging the structure and composition of chromatin.
in eukaryotes, such chromatin re-structuring events can help partition the genome into distinct domains such as euchromatin and heterochromatin and result in dna transcription, dna repair and dna replication  <cit> .
nonetheless, some histone modifications may also participate in chromosome condensation, indicating their importantce in the cell cycle and cell mitosis  <cit> .
interestingly, corresponding to their different functions, different histone modifications have different distribution patterns along the chromatin.
for example, acetylated histones and methylated histone h <dig> lysine  <dig> mainly locate at the actively transcribing genes  <cit> , while histone h <dig> lysine  <dig> methylation is a marker of heterochromatin  <cit> .
although histone modifications and their functions are well studied in yeast and mammals  <cit> , similar studies in plants are just at its infancy stage.
recently, the variants of histone h2a, h2b, h <dig> and h <dig> and their modifications in a. thaliana have been identified using mass spectrometry  <cit> .
these studies reveal modifications at sites that are unique to plant  <cit> .
the genomic distribution patterns of several histone posttranslational modifications  in a. thaliana have been determined by microarray combined with chromatin immunoprecipitation , and those distribution patterns are consistent with their functions  <cit> .
posttranslational modifications  can regulate the plant's responses to internal and external signals, such as cell differentiation, development, light, temperature, and other abiotic and biotic stresses  <cit> .
for example, methylation and acetylation of histone h <dig> regulate the expression of frigida , flowering locus c  and other vernalization related genes to ensure flowering at proper time in a. thaliana  <cit> .
studies have shown that histone modifications may also be involved in plant responses to abiotic stresses, such as salinity stress and drought stress  <cit> .
in addition, phosphorylation of histone h <dig> is involved in chromosome condensation and sister chromatid cohesion  <cit> .
histone acetylation can also affect cellular pattern in arabidopsis root epidermis by regulating the expression of cellular patterning genes  <cit> .
however, the ptms of histone in other plant species are still elusive, including several important crops, like soybean, rice and wheat.
investigation on histone epigenetics in other higher plant systems will contribute to deciphering of the "histone code" hypothesis  <cit> .
soybean is an important economic crop with a diploidized tetraploid genome  which is much larger than that of a. thaliana   <cit> .
here, we report the first identification of the variants of soybean histone h <dig> and h <dig> and their ptms using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry , in combination with nano-liquid chromatography .
our investigations reveal some important features of histone modifications in soybean, including acetylation at histone h <dig> lysine  <dig>  lysine  <dig>  lysine 23; and histone h <dig> lysine  <dig> and lysine 12; methylations at histone h <dig> lysine  <dig>  lysine  <dig> and lysine  <dig>  surprisingly, histone h <dig> lysine  <dig> is also methylated in soybean, which is not reported in a. thaliana  <cit> .
in addition, variants of histone h <dig>  and histone h <dig>  are also identified and different modifications of the two variants of histone h <dig> are also studied.
RESULTS
isolation and identification of core histones of soybean
using reversed phase high-performance liquid chromatography , core histones of soybean were separated and eluted in the order of h2b, h <dig>  h2a and h <dig> between 38–55% of buffer b, and collected according to the uv signal  .
maldi-tof ms  was employed to monitor the isolated histones in the collected fractions and the calculated mass of histone h <dig>  h <dig>  h2a and h2b were approximately  <dig> ,  <dig> ,  <dig>  and  <dig>  kda, respectively.
according to the results of the rp-hplc analysis , several variants of h2b and h2a were detected.
triton-urea-acetic acid  gel indicated that at least  <dig> variants of histone h2b and  <dig> variants of histone h2a were present in soybean .
by extending the slope of gradient of buffer b from 35% to 65% acn in  <dig> min, two variants of histone h <dig>  h <dig>  and h <dig> , were also separated .
core histones were also isolated by fast protein liquid chromatography and the individual histone protein was then separated via sds-page .
protein bands containing the corresponding core histones were excised and followed by endoproteinase in-gel digestion.
each histone protein band was divided into two portions and subjected to trypsin or lys-c digestion respectively before ms analysis.
ms analysis covered most of the amino acid sequence of histone h <dig>  which consists of  <dig> amino acid residues.
most of the  <dig> amino acid residues in soybean histone h <dig> were also identified using ms analysis.
histone modifications of soybean histone h <dig> and its variants
two variants of histone h <dig> were determined in soybean.
although the amino acid sequences of the two variants of d. melanogaster histone h <dig> were very similar and with only four amino acid differences, they could be separated by extending the slope of gradient of buffer b during rp-hplc separation  <cit> .
similar methods were adopted to isolate soybean histone h <dig> variants .
two consecutive peaks were eluted between  <dig> % –  <dig> % of buffer b. these two peaks were collected, digested by trypsin and analyzed by nano-lc/ms/ms separately.
in the mass spectrum of the first peak, the histone peptide with the mass of  <dig>  containing 27ksapa31tggvk <dig> was detected .
in the mass spectrum of the second peak, another histone peptide with the mass of  <dig> , corresponding to 27ksapt31tggvk <dig> was identified .
these two histone peptides were different in the amino acid residue  <dig>  so the first and second peaks were designated histone h <dig>  and h <dig> , respectively.
we further analyzed the variants of histone h <dig> using the information from soybean genome database .
data from soybean genome showed that these two histone h <dig> variants in soybean differed in four amino acids at the position of amino acid  <dig>   <dig>   <dig> and  <dig>  they were a31f41s87s <dig> and t31y41h87l <dig> in histone h <dig>  and h <dig> , respectively.
three more peptides from our ms analysis further confirmed this conclusion: peptide precursor ion at m/z  <dig>  containing 84fqss87avs90alqeaaeaylv <dig> and peptide precursor ion at m/z  <dig>  containing 41frpgtvalr <dig> in the mass spectrum of histone h <dig> , peptide precursor ion at m/z  <dig>  corresponding to 41yrpgtvalr <dig> in the mass spectrum of histone h <dig>  .
in the soybean genome, we also found another histone h <dig> variant, centromere specific histone h <dig>  which differed greatly in amino acid sequence from the other two variants .
next, the modifications of histone h <dig> were investigated.
modifications of histone h <dig> were complicated due to its high abundance of both lysine and arginine in its primary amino acid sequence .
from the ms analysis, mono-, di- and tri-methylation of lysine  <dig> were detected in both histone h <dig> variants; with mono-methylation as the predominant modification .
in the trypsin digestion, peptide precursor ions with the mass of m/z  <dig> ,  <dig>  and  <dig>  represented the mono-, di-, and tri-methylated peptides 27ksapttggvk <dig> of histone variant h <dig>  respectively .
although such peptide contained two potential methylation sites , de novo sequencing clearly indicated that methylation were mainly located at lysine  <dig> .
methylated lysine  <dig> was determined by other peptides whose mass were m/z  <dig> ,  <dig>  and  <dig>  containing 28sapttggvkkphr <dig> of histone variant h <dig> .
de novo sequencing showed that it could also be mono-, di- and tri-methylated .
more interestingly, most of histone h <dig> lysine  <dig> methylation did not appear in those peptides which contained histone h <dig> lysine  <dig> methylation, since only two very small peaks whose mass were m/z  <dig>  and  <dig>  were detected in the ms spectrum , which may be corresponding to the peptides containing methylation at both lysine  <dig> and lysine  <dig>  in addition, no peptide that contained both tri-methylated lysine  <dig> and lysine  <dig> was identified because of the absence of peptide precursor ion at m/z  <dig> in figure 3a.
similar results were also obtained in histone variant h <dig>  .
other ptms were also observed in the peptides of histone h <dig>  peptide 3tkqtar <dig> containing mono-, di- and tri-methylated histone h <dig> lysine  <dig>  of which mass were m/z  <dig> ,  <dig>  and  <dig>  respectively, were detected .
of these three modifications, histone h <dig> lysine  <dig> mono-methylation was the dominant one, and this result was similar to that in a. thaliana  <cit> .
lysine acetylation in soybean histone h <dig> was also identified.
peptides 10stggk14acapr <dig> at the m/z  <dig>  and 18kacqlatk <dig> at the m/z  <dig>  containing acetylated lysine  <dig> and lysine  <dig> respectively were shown in figure 7b and 7c.
another peptide at the m/z  <dig>  containing acetylated lysine  <dig> was also detected, which was 19qlatk23acaark <dig> .
since the mass shift of acetylation and tri-methylation were very similar , western blotting with specific antibodies to these acetylation and tri-methylation sites was performed and further confirmed our ms results .
nd, not detected; +, modification present.
methylation of histone h <dig> lysine  <dig> was observed in our studies.
such methylation was frequently found in mammals  <cit> .
compared with the mass of the peptide at m/z  <dig> , the mass of the peptides at m/z  <dig>  and  <dig>  shifted about  <dig> da and  <dig> da .
this indicated that these peptides might be methylated.
fragmentation of the methylated peptide at m/z  <dig>  resulted in a ms/ms spectrum containing both complete b-ion series and y-ion series.
according to this spectrum , the amino acid sequence of 73eiaqdfk79monotdlr <dig> could be assigned to this peptide, which revealed that there was mono-methylation at lysine  <dig> in soybean histone h <dig>  western blotting was performed to confirm this result .
consequently, the peptide with the mass  <dig>  should contain di-methylated histone h <dig> lysine  <dig>  due to their low abundance, de novo sequence was not successful; however, western blotting supported this prediction .
the differences of the modification patterns found in these histone h <dig> variants were obvious.
although most of their acetylation patterns were similar, their methylation patterns exhibited several differences.
almost all of lysine  <dig> in histone variant h <dig>  were methylated, whereas some histone variant h <dig>  were not methylated at lysine  <dig>  a peptide precursor ion at m/z  <dig>  which contained the unmethylated lysine  <dig> was detected in histone h <dig>   while the peptide containing unmethylated lysine  <dig> of histone h <dig>   were absent in figure 3a.
on the other hand, the peptide containing unmethylated lysine  <dig> was not detected in both histone h <dig> variants.
while lysine  <dig> methylation can be easily detected in histone h <dig>  , such methylation was not detected in histone h <dig> .
another difference between these two variants was that mono-, di- and tri- methylated lysine  <dig> were also only present in histone h <dig>  .
although the modifications of the soybean centromere specific histone h <dig> were not identified in this study, the amino acid residues at all the acetylated sites and two methylated sites  of histone h <dig>  and h <dig>  were different in the centromere specific histone h <dig> , indicating that the centromere specific histone h <dig> might have distinct histone modification patterns from that of h <dig>  and h <dig> .
histone modifications of soybean histone h <dig> and its variants
purified histone h <dig> was digested separately with either trypsin or lys-c and the corresponding digested fractions were separated and analyzed by nano-lc combined with ms/ms.
most of the potential ptm sites were examined and compared to other species.
acetylation of histone h <dig> was observed.
as shown in table  <dig>  lysine  <dig> of histone h <dig> was acetylated in the peptide 6ggk8acglgk <dig> with the mass of  <dig>  .
lysine  <dig> was acetylated in the histone h <dig> peptide 9glgk12acggak <dig> with mass at m/z  <dig>  .
none of the two unacetylated or di-acetylated peptide precursor ions was detected.
we also detected a peptide precursor ion with mass at m/z  <dig> , which corresponded to the peptide 1sgrgkggkglgk12acggak <dig>  and further proved that lysine  <dig> could be acetylated.
similarly, these acetylation sites were further verified by western blotting with specific antibodies to histone h <dig> lysine  <dig> acetylation and lysine  <dig> acetylation .
however, acetylation of lysine  <dig> and  <dig> were not detected.
our data thus indicated that lysine  <dig> and  <dig> were the main acetylation sites in the n terminus of soybean histone h <dig> and their acetylation might not happen simultaneously; a result that is differed from those found in histone h <dig> of a. thaliana and mammals  <cit> .
in our ms analysis, we cannot detect histone h <dig> lysine  <dig> modification, whereas the western blotting results showed that histone h <dig> lysine  <dig> methylation did present in soybean .
nd, not detected; +, modification present.
two variants of histone h <dig> were identified , which varied at the amino acid residue i <dig> of histone h <dig>  and v <dig> of histone h <dig>  .
the trypsin digested peptides of histone h <dig> were directly applied to maldi-tof/tof analysis and after peptide mass fingerprinting search, the peptide precursor ion at m/z  <dig>  was readily detected.
further de novo sequencing showed that it contained the amino acid sequence of 60iflenvir <dig>  however, in the nano-lc fractionated histone h <dig> peptides, another peptide with the amino acid sequence of 60vflenvir <dig> with the mass of  <dig>  was detected.
although only one peak representing histone h <dig> was observed in the rp-hplc spectrum , it may be due to the high similarity in the hydrophobicity of the two variants so that they can not be separated using such method.
discussion
in general, the amino acid sequences of histones in eukaryote are highly conserved and the posttranslational modification  patterns on specific amino acid residues are also quite similar.
characterization of histone modifications of histones h <dig> and h <dig> in soybean showed similarities to that of a. thaliana and other organisms.
high density acetylations in the n-terminal tails of histone h <dig> and h <dig> were detected in both soybean and other organisms  <cit> .
it is suggested that these acetylations play important roles in the transcriptional regulation of many physiological processes in plants, including cold tolerance, floral development and light responsiveness  <cit> .
however, histone modification patterns in different eukaryotes may also have some distinct properties.
for example, previous studies indicated that histone h <dig> lysine  <dig> modifications were quite distinct between animal and plant.
histone h <dig> lysine  <dig> methylation is evolutionarily conserved from yeast to mammals and is very critical in dna repair and genome integrity  <cit> .
however, histone h <dig> lysine  <dig> was acetylated in a. thaliana  <cit> .
our results also showed some differences that exist between soybean and the model dicot a. thaliana: mono- and di- methylation of lysine  <dig> were detected in soybean but such ptms were not found in a. thaliana  <cit> .
western blotting results also showed that methylated histone h <dig> lysine  <dig> might not be widely distributed throughout the whole soybean genome, since when equal amount of histone was applied, the signals of histone h <dig> lysine  <dig> methylation were much weaker than that of other modifications of histone h <dig> .
studies in yeast and mammals show that histone h <dig> lysine  <dig> is hypermethylated at silenced loci and is important in dna repair and genome stability  <cit> .
whether this modification is also crucial in maintaining soybean genome integrity requires further investigations.
the patterns of histone h <dig> lysine  <dig> and lysine  <dig> methylation were also different between soybean and a. thaliana.
previous studies indicate that methylation of lysine  <dig> and lysine  <dig> carry independent functions: histone h <dig> lysine  <dig> methylation is mainly involved in gene silencing and heterochromatin formation while methylated histone h <dig> lysine  <dig> is found to be associated with the phosphorylated ctd of pol ii, suggesting a role in gene expression and elongation  <cit> .
in a. thaliana, the mads-box transcription repressor flowering locus c  is a crucial regulator in controlling flowering time.
histone h <dig> lysine  <dig> methylation usually represses flc expression while histone h <dig> lysine  <dig> methylation has an opposite effect, suggesting that the modifications at these two sites must be carefully regulated in order to flower properly  <cit> .
in a. thaliana, it was reported about 15% of the peptides from histone variant h <dig>  were modified with both histone h <dig> lysine  <dig> di-methylation and lysine  <dig> mono-methylation  <cit> .
so it seems that methylated lysine  <dig> and lysine  <dig> can coexist on the same histone h <dig> n-terminus in a. thaliana.
however, our present ms data revealed that most of the methylated lysine  <dig> and methylated lysine  <dig> were unlikely to coexist on the same histone h <dig> molecule in soybean.
therefore, we speculate that soybean and arabidopsis may regulate the occurrence of histone h <dig> lysine  <dig> and lysine  <dig> methylation by different ways, although so far little about the relationship between histone h <dig> lysine  <dig> and lysine  <dig> has been known.
analysis of the public database of soybean genome revealed that at least  <dig> variants of h2a and  <dig> variants of h2b were present in soybean.
it may be due to the gene duplications and reshuffling events happened during soybean diploidized tetraploid genome formation, which occurred at about 8– <dig> million years ago and 40– <dig> million years ago respectively .
however, we have not identified any ptms of soybean histone h2b and h2a in our studies so far.
genomic analysis also found  <dig> variants of histone h <dig> in soybean: h <dig> , h <dig>  and centromere specific histone h <dig>  but we could not isolate the centromere specific histone h <dig>  other studies indicate that the expression of this variant peaks in late s/g <dig> and it is mainly deposited at functional centromeres  <cit> .
it may account for the absence of centromere specific histone h <dig> in soybean leaves which do not undergo active cell division.
the modification patterns of the other two histone h <dig> variants in soybean were different from those in a. thaliana.
only tri-methylation at histone h <dig> lysine  <dig> was found in histone h <dig>  of a. thaliana  <cit>  while methylated histone h <dig> lysine  <dig> including tri-methylation was absent in soybean histone h <dig>  and mono-, di- and tri-methylation of histone h <dig> lysine  <dig> were found in soybean histone h <dig> .
besides, histone h <dig> lysine  <dig> methylation was only detected in histone variant h <dig> .
histone h <dig> lysine  <dig> methylation is suggested to be associated with euchromatin region and viewed as a marker of transcriptionally active genes  <cit> .
in addition, methylated lysine  <dig> is also associated with gene transcription  <cit> .
previous studies suggested that different variants of histone h <dig> might carry different functions  <cit> .
in d. melanogaster and a. thaliana, the replication-independent histone h <dig> variants which are usually associated with actively transcribing regions are rich in active modifications, including histone h <dig> lysine  <dig> methylation and acetylations  <cit> .
the presence of modifications  in soybean histone h <dig>  suggested that the soybean histone h <dig>  might also be related to actively transcribing genes.
two soybean histone h <dig> variants were identified in our study, although histone h <dig> was the most conserved core histone, and no variant of histone h <dig> was found previously  <cit> .
the significance of these two novel histone h <dig> variants of soybean awaits further investigations.
our study expands the map of histone ptms in higher plant.
however, some ptms identified in other organisms, such as the histone h <dig> lysine  <dig> and histone h <dig> lysine  <dig> modifications were not detected in our ms analysis.
our western blotting results indicated that the above ptms did present in soybean .
the sensitivity of our existing ms machine may limit the coverage of our study.
therefore, more sensitive and higher resolution ms machinery is definitely preferred for future consideration.
besides, histone phosphorylation was also not detected because the phospho-histones could decompose when they were extracted by acid  <cit> .
in addition, since individual histone ptms may vary in different tissues and developmental stages, our mass spectrometry analysis here may not be capable of identifying all modification sites along the amino acid sequence of every histone in soybean.
CONCLUSIONS
we present the first report of histone h <dig> and h <dig> variants and their ptms in the legume plant soybean using nano-lc combined with mass spectrometry, mainly focusing on the acetylation and methylation of histone h <dig> and h <dig> and their variants.
significant differences are found in histone modifications between soybean and a. thaliana, which show that although the amino acid sequences of histones are conserved in evolution, their modification patterns can be quite different.
the modifications in the variants of soybean histone h <dig> are also different, further proving that histone variants have distinct biological functions which are consistent with their specific modification patterns.
our results present comprehensive information for future studies on understanding the biological functions of histone modifications in soybean, such as regulating the dna transcription and dna repair.
further investigations in soybean histone modifications may shed light to better understanding the mechanism of epigenetics in plant, a task that cannot be accomplished by solely investigating a. thaliana.

 histone modifications and histone variants play critical roles in regulating gene expression, modulating the cell cycle, and are responsible for maintaining genome stability. the fundamental structural unit of chromatin in eukaryotic cells is the nucleosome, that consists of base pairs of dna wrapped around a histone octamer, each of which is formed by two copies of h2a, h2b, h and h. an additional histone, h links these nucleosomes together along the chromatin chain. in general, the n terminus of histone h and h and n and c terminus of h2a and h2b are prone to being covalently modified by many enzymes, such as hmt and hat. these modifications include methylation, acetylation, phosphorylation, ubiquitination, glycosylation, adp ribosylation, carbonylation, sumoylation and biotinylation. most of these modifications are dynamic and can be reversed by other enzymes, such as histone demethylase and hdac. using techniques such as western blotting and mass spectrometry, increasing number of histone modification sites have been identified in mouse, yeast, drosophila melanogaster, tetrahymena thermophila and a. thaliana. mass spectrometry allows us not only to deduce the amino acid sequence of a peptide, but also to identify the exact sites and type of modifications in the peptide via the modified peptide mass shifts. epigenetic studies of chromatin in model organisms have provided insights into the modifications of histones, ranging from the identification of several enzymes and related effectors associated with histone modifications to their biological functions in cell development. it is currently proposed that histone modifications play vital roles in many fundamental biological processes by rearranging the structure and composition of chromatin. in eukaryotes, such chromatin re-structuring events can help partition the genome into distinct domains such as euchromatin and heterochromatin and result in dna transcription, dna repair and dna replication. nonetheless, some histone modifications may also participate in chromosome condensation, indicating their importantce in the cell cycle and cell mitosis. interestingly, corresponding to their different functions, different histone modifications have different distribution patterns along the chromatin. for example, acetylated histones and methylated histone h lysine mainly locate at the actively transcribing genes, while histone h lysine methylation is a marker of heterochromatin. although histone modifications and their functions are well studied in yeast and mammals, similar studies in plants are just at its infancy stage. recently, the variants of histone h2a, h2b, h and h and their modifications in a. thaliana have been identified using mass spectrometry. these studies reveal modifications at sites that are unique to plant. the genomic distribution patterns of several histone posttranslational modifications in a. thaliana have been determined by microarray combined with chromatin immunoprecipitation, and those distribution patterns are consistent with their functions. posttranslational modifications can regulate the plant's responses to internal and external signals, such as cell differentiation, development, light, temperature, and other abiotic and biotic stresses. for example, methylation and acetylation of histone h regulate the expression of frigida, flowering locus c and other vernalization related genes to ensure flowering at proper time in a. thaliana. studies have shown that histone modifications may also be involved in plant responses to abiotic stresses, such as salinity stress and drought stress. in addition, phosphorylation of histone h is involved in chromosome condensation and sister chromatid cohesion. histone acetylation can also affect cellular pattern in arabidopsis root epidermis by regulating the expression of cellular patterning genes. however, the ptms of histone in other plant species are still elusive, including several important crops, like soybean, rice and wheat. investigation on histone epigenetics in other higher plant systems will contribute to deciphering of the "histone code" hypothesis. soybean is an important economic crop with a diploidized tetraploid genome which is much larger than that of a. thaliana. here, we report the first identification of the variants of soybean histone h and h and their ptms using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, in combination with nano-liquid chromatography. our investigations reveal some important features of histone modifications in soybean, including acetylation at histone h lysine lysine lysine 23; and histone h lysine and lysine 12; methylations at histone h lysine lysine and lysine surprisingly, histone h lysine is also methylated in soybean, which is not reported in a. thaliana. in addition, variants of histone h and histone h are also identified and different modifications of the two variants of histone h are also studied.  isolation and identification of core histones of soybean using reversed phase high-performance liquid chromatography, core histones of soybean were separated and eluted in the order of h2b, h h2a and h between 38–55% of buffer b, and collected according to the uv signal. maldi-tof ms was employed to monitor the isolated histones in the collected fractions and the calculated mass of histone h h h2a and h2b were approximately,, and kda, respectively. according to the results of the rp-hplc analysis, several variants of h2b and h2a were detected. triton-urea-acetic acid gel indicated that at least variants of histone h2b and variants of histone h2a were present in soybean. by extending the slope of gradient of buffer b from 35% to 65% acn in min, two variants of histone h h and h, were also separated. core histones were also isolated by fast protein liquid chromatography and the individual histone protein was then separated via sds-page. protein bands containing the corresponding core histones were excised and followed by endoproteinase in-gel digestion. each histone protein band was divided into two portions and subjected to trypsin or lys-c digestion respectively before ms analysis. ms analysis covered most of the amino acid sequence of histone h which consists of amino acid residues. most of the amino acid residues in soybean histone h were also identified using ms analysis. histone modifications of soybean histone h and its variants two variants of histone h were determined in soybean. although the amino acid sequences of the two variants of d. melanogaster histone h were very similar and with only four amino acid differences, they could be separated by extending the slope of gradient of buffer b during rp-hplc separation. similar methods were adopted to isolate soybean histone h variants. two consecutive peaks were eluted between % – % of buffer b. these two peaks were collected, digested by trypsin and analyzed by nano-lc/ms/ms separately. in the mass spectrum of the first peak, the histone peptide with the mass of containing 27ksapa31tggvk was detected. in the mass spectrum of the second peak, another histone peptide with the mass of, corresponding to 27ksapt31tggvk was identified. these two histone peptides were different in the amino acid residue so the first and second peaks were designated histone h and h, respectively. we further analyzed the variants of histone h using the information from soybean genome database. data from soybean genome showed that these two histone h variants in soybean differed in four amino acids at the position of amino acid and they were a31f41s87s and t31y41h87l in histone h and h, respectively. three more peptides from our ms analysis further confirmed this conclusion: peptide precursor ion at m/z containing 84fqss87avs90alqeaaeaylv and peptide precursor ion at m/z containing 41frpgtvalr in the mass spectrum of histone h, peptide precursor ion at m/z corresponding to 41yrpgtvalr in the mass spectrum of histone h. in the soybean genome, we also found another histone h variant, centromere specific histone h which differed greatly in amino acid sequence from the other two variants. next, the modifications of histone h were investigated. modifications of histone h were complicated due to its high abundance of both lysine and arginine in its primary amino acid sequence. from the ms analysis, mono-, di- and tri-methylation of lysine were detected in both histone h variants; with mono-methylation as the predominant modification. in the trypsin digestion, peptide precursor ions with the mass of m/z, and represented the mono-, di-, and tri-methylated peptides 27ksapttggvk of histone variant h respectively. although such peptide contained two potential methylation sites, de novo sequencing clearly indicated that methylation were mainly located at lysine. methylated lysine was determined by other peptides whose mass were m/z, and containing 28sapttggvkkphr of histone variant h. de novo sequencing showed that it could also be mono-, di- and tri-methylated. more interestingly, most of histone h lysine methylation did not appear in those peptides which contained histone h lysine methylation, since only two very small peaks whose mass were m/z and were detected in the ms spectrum, which may be corresponding to the peptides containing methylation at both lysine and lysine in addition, no peptide that contained both tri-methylated lysine and lysine was identified because of the absence of peptide precursor ion at m/z in figure 3a. similar results were also obtained in histone variant h. other ptms were also observed in the peptides of histone h peptide 3tkqtar containing mono-, di- and tri-methylated histone h lysine of which mass were m/z, and respectively, were detected. of these three modifications, histone h lysine mono-methylation was the dominant one, and this result was similar to that in a. thaliana. lysine acetylation in soybean histone h was also identified. peptides 10stggk14acapr at the m/z and 18kacqlatk at the m/z containing acetylated lysine and lysine respectively were shown in figure 7b and 7c. another peptide at the m/z containing acetylated lysine was also detected, which was 19qlatk23acaark. since the mass shift of acetylation and tri-methylation were very similar, western blotting with specific antibodies to these acetylation and tri-methylation sites was performed and further confirmed our ms results. nd, not detected; +, modification present. methylation of histone h lysine was observed in our studies. such methylation was frequently found in mammals. compared with the mass of the peptide at m/z, the mass of the peptides at m/z and shifted about da and da. this indicated that these peptides might be methylated. fragmentation of the methylated peptide at m/z resulted in a ms/ms spectrum containing both complete b-ion series and y-ion series. according to this spectrum, the amino acid sequence of 73eiaqdfk79monotdlr could be assigned to this peptide, which revealed that there was mono-methylation at lysine in soybean histone h western blotting was performed to confirm this result. consequently, the peptide with the mass should contain di-methylated histone h lysine due to their low abundance, de novo sequence was not successful; however, western blotting supported this prediction. the differences of the modification patterns found in these histone h variants were obvious. although most of their acetylation patterns were similar, their methylation patterns exhibited several differences. almost all of lysine in histone variant h were methylated, whereas some histone variant h were not methylated at lysine a peptide precursor ion at m/z which contained the unmethylated lysine was detected in histone h while the peptide containing unmethylated lysine of histone h were absent in figure 3a. on the other hand, the peptide containing unmethylated lysine was not detected in both histone h variants. while lysine methylation can be easily detected in histone h, such methylation was not detected in histone h. another difference between these two variants was that mono-, di- and tri- methylated lysine were also only present in histone h. although the modifications of the soybean centromere specific histone h were not identified in this study, the amino acid residues at all the acetylated sites and two methylated sites of histone h and h were different in the centromere specific histone h, indicating that the centromere specific histone h might have distinct histone modification patterns from that of h and h. histone modifications of soybean histone h and its variants purified histone h was digested separately with either trypsin or lys-c and the corresponding digested fractions were separated and analyzed by nano-lc combined with ms/ms. most of the potential ptm sites were examined and compared to other species. acetylation of histone h was observed. as shown in table lysine of histone h was acetylated in the peptide 6ggk8acglgk with the mass of. lysine was acetylated in the histone h peptide 9glgk12acggak with mass at m/z. none of the two unacetylated or di-acetylated peptide precursor ions was detected. we also detected a peptide precursor ion with mass at m/z, which corresponded to the peptide 1sgrgkggkglgk12acggak and further proved that lysine could be acetylated. similarly, these acetylation sites were further verified by western blotting with specific antibodies to histone h lysine acetylation and lysine acetylation. however, acetylation of lysine and were not detected. our data thus indicated that lysine and were the main acetylation sites in the n terminus of soybean histone h and their acetylation might not happen simultaneously; a result that is differed from those found in histone h of a. thaliana and mammals. in our ms analysis, we cannot detect histone h lysine modification, whereas the western blotting results showed that histone h lysine methylation did present in soybean. nd, not detected; +, modification present. two variants of histone h were identified, which varied at the amino acid residue i of histone h and v of histone h. the trypsin digested peptides of histone h were directly applied to maldi-tof/tof analysis and after peptide mass fingerprinting search, the peptide precursor ion at m/z was readily detected. further de novo sequencing showed that it contained the amino acid sequence of 60iflenvir however, in the nano-lc fractionated histone h peptides, another peptide with the amino acid sequence of 60vflenvir with the mass of was detected. although only one peak representing histone h was observed in the rp-hplc spectrum, it may be due to the high similarity in the hydrophobicity of the two variants so that they can not be separated using such method. discussion in general, the amino acid sequences of histones in eukaryote are highly conserved and the posttranslational modification patterns on specific amino acid residues are also quite similar. characterization of histone modifications of histones h and h in soybean showed similarities to that of a. thaliana and other organisms. high density acetylations in the n-terminal tails of histone h and h were detected in both soybean and other organisms. it is suggested that these acetylations play important roles in the transcriptional regulation of many physiological processes in plants, including cold tolerance, floral development and light responsiveness. however, histone modification patterns in different eukaryotes may also have some distinct properties. for example, previous studies indicated that histone h lysine modifications were quite distinct between animal and plant. histone h lysine methylation is evolutionarily conserved from yeast to mammals and is very critical in dna repair and genome integrity. however, histone h lysine was acetylated in a. thaliana. our results also showed some differences that exist between soybean and the model dicot a. thaliana: mono- and di- methylation of lysine were detected in soybean but such ptms were not found in a. thaliana. western blotting results also showed that methylated histone h lysine might not be widely distributed throughout the whole soybean genome, since when equal amount of histone was applied, the signals of histone h lysine methylation were much weaker than that of other modifications of histone h. studies in yeast and mammals show that histone h lysine is hypermethylated at silenced loci and is important in dna repair and genome stability. whether this modification is also crucial in maintaining soybean genome integrity requires further investigations. the patterns of histone h lysine and lysine methylation were also different between soybean and a. thaliana. previous studies indicate that methylation of lysine and lysine carry independent functions: histone h lysine methylation is mainly involved in gene silencing and heterochromatin formation while methylated histone h lysine is found to be associated with the phosphorylated ctd of pol ii, suggesting a role in gene expression and elongation. in a. thaliana, the mads-box transcription repressor flowering locus c is a crucial regulator in controlling flowering time. histone h lysine methylation usually represses flc expression while histone h lysine methylation has an opposite effect, suggesting that the modifications at these two sites must be carefully regulated in order to flower properly. in a. thaliana, it was reported about 15% of the peptides from histone variant h were modified with both histone h lysine di-methylation and lysine mono-methylation. so it seems that methylated lysine and lysine can coexist on the same histone h n-terminus in a. thaliana. however, our present ms data revealed that most of the methylated lysine and methylated lysine were unlikely to coexist on the same histone h molecule in soybean. therefore, we speculate that soybean and arabidopsis may regulate the occurrence of histone h lysine and lysine methylation by different ways, although so far little about the relationship between histone h lysine and lysine has been known. analysis of the public database of soybean genome revealed that at least variants of h2a and variants of h2b were present in soybean. it may be due to the gene duplications and reshuffling events happened during soybean diploidized tetraploid genome formation, which occurred at about 8– million years ago and 40– million years ago respectively. however, we have not identified any ptms of soybean histone h2b and h2a in our studies so far. genomic analysis also found variants of histone h in soybean: h, h and centromere specific histone h but we could not isolate the centromere specific histone h other studies indicate that the expression of this variant peaks in late s/g and it is mainly deposited at functional centromeres. it may account for the absence of centromere specific histone h in soybean leaves which do not undergo active cell division. the modification patterns of the other two histone h variants in soybean were different from those in a. thaliana. only tri-methylation at histone h lysine was found in histone h of a. thaliana while methylated histone h lysine including tri-methylation was absent in soybean histone h and mono-, di- and tri-methylation of histone h lysine were found in soybean histone h. besides, histone h lysine methylation was only detected in histone variant h. histone h lysine methylation is suggested to be associated with euchromatin region and viewed as a marker of transcriptionally active genes. in addition, methylated lysine is also associated with gene transcription. previous studies suggested that different variants of histone h might carry different functions. in d. melanogaster and a. thaliana, the replication-independent histone h variants which are usually associated with actively transcribing regions are rich in active modifications, including histone h lysine methylation and acetylations. the presence of modifications in soybean histone h suggested that the soybean histone h might also be related to actively transcribing genes. two soybean histone h variants were identified in our study, although histone h was the most conserved core histone, and no variant of histone h was found previously. the significance of these two novel histone h variants of soybean awaits further investigations. our study expands the map of histone ptms in higher plant. however, some ptms identified in other organisms, such as the histone h lysine and histone h lysine modifications were not detected in our ms analysis. our western blotting results indicated that the above ptms did present in soybean. the sensitivity of our existing ms machine may limit the coverage of our study. therefore, more sensitive and higher resolution ms machinery is definitely preferred for future consideration. besides, histone phosphorylation was also not detected because the phospho-histones could decompose when they were extracted by acid. in addition, since individual histone ptms may vary in different tissues and developmental stages, our mass spectrometry analysis here may not be capable of identifying all modification sites along the amino acid sequence of every histone in soybean.  we present the first report of histone h and h variants and their ptms in the legume plant soybean using nano-lc combined with mass spectrometry, mainly focusing on the acetylation and methylation of histone h and h and their variants. significant differences are found in histone modifications between soybean and a. thaliana, which show that although the amino acid sequences of histones are conserved in evolution, their modification patterns can be quite different. the modifications in the variants of soybean histone h are also different, further proving that histone variants have distinct biological functions which are consistent with their specific modification patterns. our results present comprehensive information for future studies on understanding the biological functions of histone modifications in soybean, such as regulating the dna transcription and dna repair. further investigations in soybean histone modifications may shed light to better understanding the mechanism of epigenetics in plant, a task that cannot be accomplished by solely investigating a. thaliana. 
in soybean leaves, mono-, di- and tri-methylation at lysine lysine and lysine and acetylation at lysine and were detected in histone h lysine was prone to being mono-methylated, while tri-methylation was predominant at lysine we also observed that lysine methylation and lysine methylation usually excluded each other in histone h although methylation at histone h lysine was not reported in a. thaliana, mono- and di-methylated histone h lysine were detected in soybean. histone modifications and histone variants are of importance in many biological processes. to understand the biological functions of the global dynamics of histone modifications and histone variants in higher plants, we elucidated the variants and post-translational modifications of histones in soybean, a legume plant with a much bigger genome than that of arabidopsis thaliana. this work revealed several distinct variants of soybean histone and their modifications that were different from a. thaliana, thus providing important biological information toward further understanding of the histone modifications and their functional significance in higher plants. the methylation patterns in these two histone h variants also exhibited differences. using a combination of mass spectrometry and nano-liquid chromatography, two variants of histone h were detected and their modifications were determined.
 histone modifications and histone variants are of importance in many biological processes. to understand the biological functions of the global dynamics of histone modifications and histone variants in higher plants, we elucidated the variants and post-translational modifications of histones in soybean, a legume plant with a much bigger genome than that of arabidopsis thaliana.  in soybean leaves, mono-, di- and tri-methylation at lysine lysine and lysine and acetylation at lysine and were detected in histone h lysine was prone to being mono-methylated, while tri-methylation was predominant at lysine we also observed that lysine methylation and lysine methylation usually excluded each other in histone h although methylation at histone h lysine was not reported in a. thaliana, mono- and di-methylated histone h lysine were detected in soybean. besides, acetylation at lysine and of histone h in soybean were identified. using a combination of mass spectrometry and nano-liquid chromatography, two variants of histone h were detected and their modifications were determined. they were different at positions of a31f41s87s and t31y41h87l, respectively. the methylation patterns in these two histone h variants also exhibited differences. lysine and lysine methylation were only detected in histone h, suggesting that histone variant h might be associated with actively transcribing genes. in addition, two variants of histone h were also detected, which were missing in other organisms. in the histone variant h and h, the amino acid was isoleucine and valine, respectively.  this work revealed several distinct variants of soybean histone and their modifications that were different from a. thaliana, thus providing important biological information toward further understanding of the histone modifications and their functional significance in higher plants. 