BACKGROUND
the glyoxalase system is a two-enzyme driven pathway that detoxifies the highly cytotoxic compound, methylglyoxal  to d-lactate.
the detoxification is accomplished by the sequential action of two thiol-dependent enzymes; glyoxalase І  and glyoxalase ii .
in presence of reduced glutathione , mg is converted into hemithioacetal  spontaneously, and glyi catalyses the isomerization of this hta into s-d-lactoyl-glutathione .
glyii hydrolyses slg into d-lactate and recycles back one molecule of gsh to the system  <cit> .
both, the formation of mg and the glyoxalase enzymes have been ubiquitously found in all organisms from escherichia coli to homo sapiens  <cit> .
besides its proposed role in the detoxification of mg as metabolic enzyme, glyoxalase enzymes have been reported to be involved in various other functions.
glyoxalase system protects human from various vascular complications of diabetes, such as nephropathy, retinopathy, neuropathy and cardiovascular disease by resisting the increased accumulation of mg  <cit> .
moreover, glyoxalase pathway has also been shown to be involved in different important cellular functions of human, such as cell division and proliferation, microtubule assembly and protection against oxoaldehydes toxicity  <cit> .
for this, the pathway has been regarded as “marker for cell growth and division”.
similarly, stress tolerance potential of glyoxalase has been reported in plant by numerous studies  <cit> .
transgenic plants over-expressing glyi and/or glyii were found to provide significant tolerance against multiple abiotic stresses including salinity, drought and heavy metal toxicity  <cit> .
thus mg and glyoxalases are considered as potential biomarkers for plant stress tolerance  <cit> .
glyoxalase proteins have been extensively characterized from different genera such as escherichia coli, homo sapiens, saccharomyces cerevisiae, arabidopsis thaliana and oryza sativa  <cit> .
compared to other organisms, very little is known about plant glyoxalases.
the first plant glyoxalase activity was reported from douglas fir needles by smits and johnson  <cit> .
thereafter, presence of glyoxalase activity has been reported from various other plant species, such as rice, arabidopsis, tomato, wheat, sugarcane, brassica etc.
<cit> .
most of the genes of plant exist as family due to the expansion and gene duplication during the course of plant evolution  <cit> .
availability of the whole genome sequences has opened up the field to identify and characterize plant glyoxalase family substantially.
according to in silico genome wide analyses of rice and arabidopsis, there are eleven potential glyi and three glyii genes in rice; and eleven glyi and five glyii genes in arabidopsis  <cit> .
expression analysis of all these genes have been performed in different developmental tissues and stages, and in response to multiple abiotic stresses using publicly available mpss and microarray database.
it has been observed that atglyi- <dig>  osglyi- <dig>  atglyii- <dig>  atglyii- <dig>  osglyii- <dig> and osglyii- <dig> showed constitutive expression in all the tissues and stages, while atglyi- <dig>  osglyi- <dig>  and osglyi- <dig> expressed only in seed  <cit> .
on the other hand, atglyi- <dig>  osglyi- <dig>  atglyii- <dig> and osglyii- <dig> were the most stress inducible members  <cit> .
among these identified glyoxalase members, glyii genes have been extensively studied from both rice and arabidopsis but the research on glyi is still very limited.
to date, all five atglyii and three osglyii genes have been well characterized.
both osglyii- <dig> and osglyii- <dig> possessed typical glyii enzymatic activity and overexpression of these genes in tobacco provides enhanced tolerance against salinity stress  <cit> .
however, osglyii- <dig>  along with atglyii- <dig> showed functional divergence by possessing sulphur dioxygenase  activity instead of glyii  <cit> .
one of the rice glyi, osglyi- <dig>  have been studied extensively and found to possess ni2+-dependent glyi activity with stress modulation potential  <cit> .
soybean  is a legume plant of papilionoideae family  <cit> , major source of vegetable protein and edible oil.
it also has the capacity to fix atmospheric nitrogen through symbioses  <cit> .
however, production of soybean is under threat due to the unfavourable environmental stimuli such as drought, salinity and osmotic stresses  <cit> .
all these stresses severely affect the overall plant development in all the stages from germination to flowering and reduce the productivity and seed quality of soybean.
the yield has been reported to be reduced by about 40 % in response to drought  <cit> .
thus, there is an urgent need to identify novel stress responsive soybean genes using the available genome database  <cit> .
the soybean genome contains  <dig>  predicted protein-coding genes which are 70 % more than arabidopsis.
there have been two genome duplication events undergone in soybean at approximately  <dig> and  <dig> million years ago, that resulted a highly duplicated genome   <cit> .
a lot of gene families have been studied in soybean, such as erf, hd-zip, wrky, burp, mads-box, myb, nac, cyp .
genome wide analyses of glyoxalase gene family have been done in arabidopsis and rice  <cit> , but no such analysis has been performed in soybean in spite of having a handful genome sequences deposited in the publicly available database.
here, we present a detailed genome-wide identification of soybean glyi and glyii genes, their phylogenetic relationship, chromosomal distribution, structural and expressional analysis.
present results indicate that soybean genome contains  <dig> glyi and  <dig> glyii proteins, the largest family of glyoxalase known to date in any organism.
expression analysis of these genes based on publicly available microarray data indicates the differentially regulation of glyoxalase members in response to various developmental cues as well as stress treatments.
in particular gmglyi- <dig>  gmglyi- <dig> and gmglyii- <dig> are most up-regulated stress responsive members that might resist mg accumulation in stress by interacting with other members.
this study will facilitate the further investigation of soybean glyoxalase genes for the biological and molecular functions.
RESULTS
identification of glyi and glyii gene families in soybean
proteins having lactoylglutathione lyase domain  have been classified as glyi proteins and metallo-beta-lactamase domain  have been classified as glyii proteins  <cit> .
previously, glyoxalase proteins have been identified in two model plant genome, arabidopsis and rice  <cit> .
to identify all the putative members of the glyoxalase proteins in soybean, a blastp search of the soybean genome database g. max wm <dig> a <dig> v <dig>  was performed using the previously characterized protein sequence as a query.
glyi proteins have been primarily identified using a previously reported soybean glyi protein .
subsequently, each of the newly identified glyi protein sequences has been used as a query sequence individually in blastp search of soybean genome database.
subsequent searching process was repeated until there was no new member documented.
this search resulted in the identification of total  <dig> unique proteins.
all these identified proteins were analyzed using pfam to check the presence of unique lactoylglutathione lyase domain .
this analysis discarded two members due to the lack of lactoylglutathione lyase domain, and finally landed to a total of  <dig> soybean glyi proteins which is greater than the previously reported arabidopsis  and rice  glyi proteins.
these  <dig> glyi proteins have been coded by  <dig> unique genes located on  <dig> different chromosomes .
they were identified and named as gmglyi- <dig> to gmglyi- <dig> following the nomenclature proposed previously  <cit>  .fig.
<dig> chromosomal distribution of gmglyi  and gmglyii  genes on different soybean chromosomes.
only the chromosomes having glyoxalase genes are shown and their number is indicated above by roman numbers.
the scale is in mega base , and the centromeric regions are indicated by black ellipses.
red coloured boxes indicate the segmental duplicated genes connected by red lines, based on sequence similarities and divergence analysis .
black boxes indicate the non-duplicated genes
abbreviations: cds coding dna sequence, chro chromosome, pp polypeptide length, mw molecular weight, p
i isoelectric point, bp base pair, aa amino acid, kda kilodalton, ch chloroplast, cy cytosol, ec extracellular, mt mitochondria, nu nucleus, po peroxisome
alocalization prediction by cello v. <dig>
blocalization prediction by psort
cchloroplast localization signal confirmed by chlorop
similarly, soybean glyii proteins have been primarily identified using a previously characterized brassica juncea glyii protein  as query and secondarily by the newly identified members.
a total of  <dig> unique protein sequences have been identified and checked for the presence of unique metallo-beta-lactamase domain  using pfam.
three of them didn’t have this unique domain and were discarded from the list.
thus, a total of  <dig> soybean glyii proteins have been confirmed which is greater than the previously reported arabidopsis  and rice  glyii family members.
these  <dig> glyii proteins have been coded by  <dig> unique genes located on ten different chromosomes .
they were named as gmglyii- <dig> to gmglyii- <dig> like gmglyi genes .
in both gmglyi and gmglyii families, the number of proteins was greater than the number of genes ; indicating the existence of alternate splicing event in soybean glyoxalase genes.
most of the gmglyi genes  and gmglyii genes  showed only a single product.
however, rest seven gmglyi genes formed  <dig> alternative spliced products, whereas seven gmglyii genes lead to the generation of  <dig> proteins .table  <dig> list of identified glyii genes in soybean  along with their detailed information and localization
abbreviations: cds coding dna sequence, pp polypeptide length, mw molecular weight, p
i isoelectric point, bp base pair, aa amino acid, kda kilodalton, ch chloroplast, cy cytosol, ec extracellular, mt mitochondria, nu nucleus
alocalization prediction by cello v. <dig>
blocalization prediction by psort
cchloroplast localization signal confirmed by chlorop
detailed analysis of identified gmglyi and gmglyii members
all the newly identified gmglyi and gmglyii members were analyzed in detail.
the coding dna sequence  length of the gmglyi members vary from 333 bp  to 1101 bp  with an average of 740 bp.
consequently, gmglyi- <dig>  encodes for the largest protein of the family with a polypeptide length of  <dig> aa and molecular weight of  <dig>  kda; and the smallest protein  is  <dig> aa in length with  <dig>  kda in weight .
similar to the length and molecular weight variation, the proteins showed a wide range of deviation in their isoelectric point  value from  <dig>   to  <dig>  .
most of the gmglyi members showed acidic pi value , with only seven such as gmglyi- <dig> , gmglyi- <dig> , gmglyi- <dig> , gmglyi- <dig> , gmglyi- <dig> , gmglyi- <dig> , and gmglyi- <dig>  have showed basic pi value .
this ensures the presence of both positively and negatively charged gmglyi proteins at a certain physiological condition.
sub-cellular localization of all these predicted gmglyi proteins  were analyzed based on two different tools cello  <cit>  and wolf psort  <cit> , and the chloroplast localization was further confirmed by chlorop  <cit> .
different members were found to be localized at different sub-cellular compartments, such as chloroplast, cytosol, mitochondria, nucleus, extracellular, peroxisome.
most of the gmglyi proteins are found to be localized in cytosol, followed by chloroplast, mitochondria and nucleus .
similarly, the cds length of gmglyii transcripts varies from 432 bp  to 1584 bp  with an average of 850 bp .
the largest gmglyii- <dig>  protein is  <dig> aa in length with a molecular weight of  <dig>  kda; and the smallest protein  is  <dig> aa in length and  <dig>  kda in weight .
gmglyii proteins also show variation in their pi values ranging from  <dig>   to  <dig>  .
most of the gmglyii members  showed acidic pi value similar to gmglyi proteins, while only eight gmglyii members such as gmglyii- <dig> , gmglyii- <dig> , gmglyii- <dig> , gmglyii- <dig> , gmglyii- <dig> , gmglyii- <dig> , gmglyii- <dig> , and gmglyii- <dig>  have basic pi value .
similar to gmglyi, most of the gmglyii proteins are found to be localized in cytosol, followed by chloroplast , nucleus , and mitochondria .
chromosomal distribution and gene duplication
to determine the exact position and distribution of the identified gmglyi and gmglyii genes on different chromosomes, a detailed chromosome map was constructed.
soybean glyoxalase genes are found to be unevenly distributed throughout the chromosomes.
it has been found that  <dig> gmglyi genes are located on  <dig> different chromosomes .
the gene density per chromosome is highly uneven, where chromosome  <dig> and  <dig> contain the maximum occurrence of glyi genes .
however, chromosomes  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> have two glyi genes each, and only one glyi gene each is present on chromosomes  <dig>   <dig>   <dig>  and  <dig>  no glyi gene was found on chromosomes  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 20; thereafter not shown in the fig.
1a.
similarly,  <dig> gmglyii genes were found to be located on ten different chromosomes  and the gene density per chromosome is highly uneven.
chromosomes  <dig> and  <dig> contain the maximum glyii genes , whereas chromosomes  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> have only one glyii gene each.
no glyii gene was found on the rest of the chromosomes and as such not shown in the fig.
1b.
all the gmglyi and gmglyii genes were found to be located towards the chromosome ends , suggesting the possibility of inter-chromosomal genetic rearrangements between different soybean chromosomes during genome duplication.
due to two duplication events, soybean genome resulted in many paralogs within a gene family  <cit> .
out of the  <dig> gmglyi proteins ,  <dig> are clustered in pairs  and eight gmglyii proteins are clustered in pairs  out of a total of  <dig> gmglyii proteins in the phylogenetic tree .
the percentage of similarities between all these gmglyi  and gmglyii  proteins were combined separately.
it was observed that all the paired members of both glyi and glyii family  have very high level  of sequence similarities.
this high level of sequence similarities indicates the possibility of segmental duplication of the genes throughout evolution.
moreover, among the  <dig> gmglyi genes one gene pair  was present continuously  within a distance of less than 5 kb  on chromosome  <dig>  this indicates that these two genes might be duplicated by tandem duplication .
to identify the time course of gene duplication, all the identified duplicated gene pairs were analyzed using plant genome duplication database   <cit>  .
according to the ratio of nonsynonymous to synonymous substitutions , the evolutionary history of selection acting on different genes could be measured  <cit> .
this ratio could be used to interpret the direction and magnitude of natural selection enforcing on the various protein coding genes.
a pair of sequences having ka/ks <  <dig> implies purifying selection; ka/ks =  <dig> indicates both sequences are drifting neutrally; and lastly ka/ks >  <dig> implies positive or darwinian selection  <cit> .
the ka/ks of  <dig> glyoxalase duplicated gene pairs  was found to be less than  <dig> ; that indicates the influence of purifying selection in the evolution of these gene pairs.
considering the divergence rate of  <dig>  synonymous mutations per synonymous site per year for soybean  <cit> , the duplication time for each gene pairs was calculated.
it is observed that all the segmental duplicated pairs showed a time frame between  <dig>  and  <dig>  mya, except the tandem duplicated pair that occurred  <dig>  mya ago .table  <dig> divergence time between glyoxalase gene pairs in soybean
phylogenetic analysis of glyoxalase genes from various plant species
in the present study, a phylogenetic tree of all the identified gmglyi or gmglyii proteins along with other reported glyi or glyii proteins from other plant species were constructed using mega  <dig>  tool .
a neighbour joining phylogenetic tree was generated using a total of  <dig> full-length glyi protein sequences of soybean, rice and arabidopsis glyi family, and proteins from other plant species.
the tree was sub-divided into four subfamilies  as evident in fig.
2a.
all these subfamilies consist of representative member from both dicot arabidopsis and monocot rice, indicating that the evolution of plant glyi genes occurred before the split of dicot-monocot.
clade-iv has the largest glyi members from different plant species, while clade-ii has the lowest number of members only from arabidopsis and rice genome .
clade-i comprises of glyi members only from the complete genome database of three plants, arabidopsis, rice and soybean.
among them, osglyi- <dig> is functionally a diverge member of the rice glyi family and might possess some other activities than glyi .
in clade-iii, there are multiple members from arabidopsis, rice and soybean; and one member each from genlisea aurea and sorghum bicolor.
among them, three rice members osglyi- <dig>  osglyi- <dig> and osglyi-11; and two members of arabidopsis atglyi- <dig> and atglyi- <dig> have been already predicted to be ni2+-dependent glyi enzyme  <cit> .
thus rest of the members of this clade would be expected to have ni2+-dependent catalytic activity.
similarly, clade-iv has members from rice  and arabidopsis  which are zn2+-dependent glyi enzymes  <cit> .
thus rest of the glyi members from other species would require zn2+ for their optimum glyi activity.
this indicates that zn2+-dependent glyi enzymes are more diverse as they are present in many plant species .fig.
<dig> phylogenetic analyses of glyi  and glyii  proteins from various plant species.
glyoxalase protein sequences from various plant species were downloaded from various databases and provided as additional files  <dig> and  <dig>  an unrooted tree was generated using neighbor-joining method with  <dig> bootstrap by mega <dig>  software using the full-length amino acid sequences of eighty-three glyi  or forty-one glyii  proteins .
the numbers next to the branch shows the result of  <dig> bootstrap replicates expressed in percentage, and scores higher than 50 % are indicated on the nodes.
both trees were sub-divided into four classes  and indicated by different colours
to clarify the phylogenetic relationship among glyii proteins, we further constructed another tree for all full length sequences of gmglyii, osglyii, atglyii family and glyii sequences from other plant species .
this tree was subdivided into four classes  too like the previous one.
class-i consists of three proteins from soybean, and one each from rice  and arabidopsis .
among them, osglyii- <dig> has been reported to have sulphur dioxygenase  activity rather than glyii  <cit> .
so this sub class of proteins would be functionally diverse from glyii.
similarly, class-ii contains one protein each from rice , arabidopsis , and selaginella moellendorffii, and four proteins from soybean.
atglyii- <dig> has been reported to be the mitochondrial localized atglyii family member  <cit> .
division of class-iii and –iv is more interesting and evolutionarily more significant.
class-iii has glyii proteins from all monocot plants ; while class-iv has exclusively dicot members including arabidopsis, soybean, medicago, lotus etc.
.
apart from glyi, glyii proteins were found to be diversified after the split of monocot and dicot.
gene structures of gmglyi and gmglyii genes
detailed analysis of the exon-intron structure of gmglyi  and gmglyii  genes showed great variation among themselves.
all gmglyi and gmglyii genes contained at least one intron in their open reading frame , which means there is no intron less glyoxalase gene in soybean.
the number of introns varied from  <dig> to  <dig> in the orfs of different gmglyi genes .
the gmglyi- <dig> , gmglyi- <dig>  gmglyi- <dig>  and gmglyi- <dig> contained a single intron in their orf while the largest numbers of introns  were found in the gmglyi- <dig>  transcript.
in many cases, the borders of protein-coding sequence, 5′ and 3′ untranslated regions  also contain large numberof introns  <cit> .
out of  <dig> gmglyi transcripts, there was no intron in the 3′ utr of any of these genes and only eight of them contained a single intron in their 5′ utr region.
similarly, the number of introns varied from  <dig> to  <dig> in the orfs of different gmglyii genes .
the maximum number of introns  was observed in gmglyii- <dig> , followed by  <dig> each in gmglyii- <dig>  and gmglyii- <dig> .
gmglyii- <dig>  contained only a single intron in its orf while the rest have varied number of introns.
similar to gmglyi transcripts, there was no intron in the 3′ utr of gmglyii transcripts.
only six out of  <dig> transcripts  have a single intron in their 5′utr region.fig.
<dig> gene structures of gmglyi  and gmglyii  family members including the alternative spliced forms.
all the exons are shown in filled black boxes and the introns are indicated by black lines.
the 5’-utr regions are shown using empty boxes and the 3’-utr regions are shown in empty arrows which also indicate the direction of the gene.
left to right direction of transcript indicates “+” strand, while the right to left one indicates “-” strand, relative to the annotation of the genome sequence.
the size of the introns, exons, and utrs could be estimated from the scale at the bottom
longer introns are selectively advantageous that could counterbalance the mutational bias and improve the recombination frequency  <cit> .
a strong evidence for the presence of ancestral introns was reported by analyzing introns of animal, plant and fungus  <cit> .
moreover, the number of exons and introns were found to be similar in the paralogous genes  that clustered together in the phylogenetic analysis .
such as, gmglyi-1/- <dig>  gmglyi-4/- <dig>  gmglyi-10/- <dig>  and gmglyi-6/- <dig> have the same number of introns and exons.
analysis of gmglyi proteins for their domain architecture, catalytic conservance and metal ion dependency
all the predicted gmglyi  proteins were analyzed using pfam to reveal the presence of conserved glyoxalase domain  among them.
analyses of gmglyi proteins revealed that  <dig> out of forty-one contains two glyi domains, while the rest  <dig> have only single glyi domain .
presence of two glyi domain in a single protein have been previously reported from saccharomyces cerevisiae  <cit> , oryza sativa  <cit>  and plasmodium falciparum  <cit> .
presence of two domain forms two putative active sites on a single monomeric protein.
both the active sites are found to be functional, but allosterically regulated in plasmodium falciparum  <cit> , whereas one of the active site is found to be a pseudo-active site in oryza sativa  <cit> .
however, glyi proteins with single domain have also been reported from various species such as e. coli  <cit> , h. sapiens  <cit>  and function as homo-dimer.fig.
<dig> domain architectures of gmglyi proteins.
all forty-one soybean glyi proteins were analyzed for the presence of functional domain using pfam .
all the gmglyi proteins possess glyoxalase domain  that is represented by boxes.
the position of the domain is indicated by the amino acid number inside the box.
among the  <dig> gmglyi members,  <dig> of them have single glyoxalase domain, whereas rest  <dig> have two domains.
the length of full proteins is indicated by exact amino acid numbers and relative position of the domains could be interpreted by the scale given below
activity of glyi enzyme is highly dependent on divalent metal ions  <cit> .
on the basis of metal ion specificity glyi proteins could be divided into two classes; zn2+-dependent or zn2+-independent .
glyi from homo sapiens, saccharomyces cerevisiae and pseudomonas putida have been reported as zn2+-dependent , whereas glyi from e. coli and one of the rice glyi  showed ni2+-dependent activity  <cit> .
the metal dependency of the glyi enzymes could be easily predicted from the length of glyi domain, as ni2+-dependent glyi has a domain length of ~ <dig> aa and zn2+-dependent glyis are usually  <dig> aa in length  <cit> .
irrespective of the metal ion dependency, the active site of glyi proteins has a conserved motif of h/qeh/qe.
among them, the glutamate residues act as a base by accepting protons from the substrate and any mutation of this conserved residue resulted in the complete loss of activity  <cit> .
thus, to comment on the presence of enzymatic activity and metal ion dependency, glyi domain  of all the putative gmglyi proteins were aligned  along with known ni2+-dependent osglyi- <dig>  and zn2+-dependent osglyi- <dig>  <cit>  proteins.
all the metal binding sites were presented inside black boxes and the regions specific for zn2+-dependent glyi were presented by black arrows .fig.
<dig> multiple sequence alignment of glyi domain of all gmglyi proteins along with that of osglyi- <dig>  and osglyi- <dig>  glyi domain  of all gmglyi proteins were aligned with that of a ni2+-dependent osglyi- <dig>  and a zn2+-dependent osglyi- <dig> using clustalw program.
the alignment file was viewed using jalview multiple alignment editor program.
all four conserved metal binding sites were represented as black boxes and the specific region for zn2+-dependent glyi was marked with black arrow
based on the presence of all the four conserved metal binding site, the expected glyi enzyme activity of the putative gmglyi proteins was predicted .
out of a total  <dig> putative gmglyi proteins,  <dig> have all the four conserved residues and are expected to have functional glyi enzyme activity .
out of this  <dig> expected functional glyi enzymes,  <dig> are predicted to be ni2+-dependent as they have the domain length of around  <dig> aa and lack of the conserved regions specific for zn2+-dependent members.
the remaining four namely gmglyi- <dig> , gmglyi- <dig> , gmglyi- <dig>  and gmglyi- <dig>  are expected to be zn2+-dependent as their domain length is more than  <dig> aa and possessed the conserved regions .table  <dig> analysis of all putative gmglyi enzymes for their enzymatic activity and metal ion dependency
analysis of gmglyii proteins for their domain architecture and catalytic efficiency
genome wide analysis of soybean revealed the presence of  <dig> glyii proteins coded by  <dig> genes .
all these gmglyii proteins were analyzed using pfam to reveal the presence of conserved metallo-beta-lactamase domain  among them.
analysis of all gmglyii proteins revealed that  <dig> out of  <dig> have only metallo-beta-lactamase domain, while the rest eleven contain additional hydroxyacylglutathione hydrolase c-terminus  domain  along with metallo-beta-lactamase domain .
hagh-c domain is usually found to be present at the c-terminus of glyii enzymes that forms the substrate binding site along with the catalytic domain   <cit> .
however, glyii from various species such as e. coli, s. cerevisiae, s. typhimurium, l. infantum, a. thaliana, b. juncea, o. sativa and h. sapiens, contained the well conserved metal binding motif  and active site motif   <cit> .
both these motifs play an important role in the glyii enzyme activity of a protein.
therefore, to comment on the presence of enzymatic activity of the putative gmglyii proteins, their protein sequences were aligned by multiple sequence alignment .
both these motifs were indicated by black boxes ; their presence and absence were listed in table  <dig>  out of  <dig> putative gmglyii proteins only three of them do not possess the conserved metal binding residues, but all of them have the active site motif .
thus, it could be expected that all the predicted gmglyii proteins have the functional glyii enzyme activity except gmglyii- <dig> , gmglyii- <dig> , and gmglyii- <dig>  .fig.
<dig> domain architectures of gmglyii proteins.
all  <dig> soybean glyii proteins were analyzed for the presence of functional domain using pfam.
there were two types of domains observed in gmglyii proteins, such as β-lactamase domains  and hydroxyacylglutathione hydrolase c-terminus  domain .
the length of full protein and domain are indicated by exact amino acid numbers beside and inside of the shape, respectively.
the relative size could be identified by using the scale below
fig.
<dig> multiple sequence alignment of gmglyii proteins.
all gmglyii full length protein sequences were aligned using clustalw program and viewed using jalview multiple alignment editor program.
the black boxes indicate the most conserved metal binding motif  and active site motif
homology modelling of representative gmglyi and gmglyii members
to know the arrangement of active site residues and overall 3-d coordination, homology model of gmglyi- <dig>  gmglyi- <dig> and gmglyii- <dig> proteins was built  based on the closely related template structure of zea mays glyi   <cit> , mouse glyi , and atglyii- <dig>   <cit>  proteins, respectively.
gmglyi- <dig> is a ni2+-dependent monomeric glyi enzyme , while gmglyi- <dig> is a zn2+-dependent homodimeric enzyme .
gmglyi- <dig> has two putative active sites; one consists of h- <dig>  e- <dig>  q- <dig> and e- <dig>  and the other one consists of h- <dig>  e- <dig>  q- <dig> and a- <dig>  the second putative active site has lack of a highly conserved glu residues, thus might be inactive in nature like previously reported osglyi- <dig>   <cit> .
the zn2+-dependent gmglyi- <dig> consists of single glyi domain  and thus forms homo-dimer to create two putative active sites .
one putative active site has q <dig> and e <dig>  and h <dig> and e <dig> ; another one has opposite members from both chains.
here both the active sites have all four conserved residues and thus are predicted to be functionally active too.
on the other hand, gmglyii- <dig> is a monomeric protein consists of two structural orientations, an n-terminal domain  with two βββαβ topology and a c-terminal domain  with five α-helices .
the metal binding and active site residues are asn <dig>  his <dig>  asp <dig>  his <dig>  his <dig>  asp <dig> and his <dig>  are found to be conserved as compared to template atglyii- <dig> protein.fig.
<dig> three dimensional homology model structure of soybean glyoxalase proteins.
structures of gmglyi- <dig> , gmglyi- <dig>  and gmglyii- <dig>  were built using swiss-model server based on available close similar structure from protein data bank  zea mays glyi , mouse glyi , and atglyii- <dig>  proteins, respectively.
all the α-helices were marked with orange colour, while β-sheets were marked with cornflower blue.
the active sites residues were identified based on the alignment with template structure and shown by ball-stick model.
the structure and active residues were visualized and generated using chimera program
expression analysis of gmglyi and gmglyii genes at different soybean tissues
rna-seq atlas of glycine max provides high-resolution gene expression data in a diverse set of  <dig> soybean tissues such as young leaf, flower, one cm pod , pod-shell, seed , root and nodule.
all these tissues could be broadly divided into three classes; such as underground, aerial and seed.
rna-seq normalized expression data for all gmglyi and gmglyii genes were retrieved from soybase , except gmglyi- <dig> and gmglyi- <dig> due to lack of their appropriate probe .
data were analyzed and represented as heat maps generated using tigr mev software package .
expression analyses of all gmglyi genes revealed that the different members have different tissue specific expression.
among all the  <dig> analyzed genes, gmglyi- <dig> showed highest level of constitutive expression in all the tissues, followed by gmglyi- <dig>  gmglyi- <dig> and gmglyi- <dig>  this high level of constitutive expression indicates their significant role at all these plant tissues .
a cluster of genes showed medium to high level of expression in all the underground and aerial tissues only, followed by very low expression at the seed tissues.
they are gmglyi- <dig>  gmglyi- <dig>  gmglyi- <dig>  gmglyi- <dig>  gmglyi- <dig> and gmglyi- <dig>  previous studies on rice and arabidopsis showed the presence of highly seed specific glyi genes such as atglyi- <dig>  osglyi- <dig> and osglyi- <dig>  <cit> .
similarly, three of gmglyi genes such as gmglyi- <dig>  gmglyi- <dig> and gmglyi- <dig> showed medium level of expression in different seed tissues only , indicating the evolutionary conservance for the presence of seed specific glyi genes.fig.
<dig> expression profiling of soybean glyoxalase genes with hierarchical clustering in different developmental tissues and stages.
a, b rna-seq expression data of  <dig> developmental tissues, such as r , n , yl , f , p.1 cm , ps.10d , ps.14d , s.10d , s.14d , s.21d , s.25d , s.28d , s.35d , s.42d  was used in the analysis.
the normalized data was downloaded from soybase  and provided as additional file 2: table s <dig>  heatmap generation and hierarchical clustering was performed using mev software package.
the colour scale below the heat map indicates expression values; green indicates low transcript abundance while red indicates high level of transcript abundance.
c, d transcriptome data of all gmglyi and gmglyii genes at various developmental stages  were obtained from the national center for biotechnology information .
heatmaps generation and hierarchical clustering were performed using mev software package.
the colour scale given below the heat map indicates the expression values where blue indicates low transcript abundance and yellow indicates high transcript abundance
expression analyses of gmglyii genes indicate two clear clades .
out of  <dig> analyzed genes, five genes such as gmglyii- <dig>  gmglyii- <dig>  gmglyii- <dig>  gmglyii- <dig> and gmglyii- <dig> showed almost undetectable expression in all the tissues with few exceptions.
among others, gmglyii- <dig> showed highest level of constitutive expression in all the tissues, followed by gmglyii- <dig>  these two genes might play a major role in all tissues.
similar to gmglyi, a cluster of genes  showed medium level of expression in the underground and aerial tissues, except the seed .
no tissue specific expression pattern was observed in case of gmglyii genes.
from the expression data analysis of the identified paralogous pairs of gmglyi and gmglyii genes in  <dig> soybean tissues revealed a high level of expression divergence.
for example, gmglyi- <dig> showed high level constitutive expression while its paralogous gmglyi- <dig> showed detectable expression in a few tissues.
however, some of the paralogous gmglyi gene pairs namely gmglyi-1/- <dig>  gmglyi-2/- <dig>  gmglyi-4/- <dig>  gmglyi-10/- <dig>  and gmglyi-19/- <dig> showed similar pattern of expression.
the divergence is even more in case of gmglyii gene pairs.
for instance, gmglyii- <dig> is highly expressed in all the analyzed tissues while its paralogous counterpart gmglyii- <dig> remains mostly undetectable.
similar level of deviation was also observed in case of gmglyii-6/-10and gmglyii-7/- <dig> gene pairs.
expression analysis of gmglyi and gmglyii genes at different developmental stages
expression of gmglyi and gmglyii genes at different developmental stages was analyzed using publicly-available genome-wide transcript profiling data of soybean .
the dataset contains mainly two broad developmental sets, one at vegetative stages  and the other at reproductive stage .
as shown in fig.
9c, most of the gmglyi genes showed high level of expression, without any distinct pattern of expression.
out of the  <dig> gmglyi genes, only gmglyi- <dig> showed undetectable expression at all stages.
among others, gmglyi- <dig> showed maximum constitutive expression in all the developmental stages followed by gmglyi- <dig>  gmglyi- <dig>  gmglyi- <dig> and gmglyi- <dig> .
two of gmglyi members, gmglyi- <dig> and gmglyi- <dig> showed only reproductive stage specific expression.
this indicates the development specific modulation of gmglyi gene expression.
on the other hand, a distinct division is observed in the gmglyii gene expression through the developmental stages .
a cluster of genes such as gmglyii- <dig>  gmglyii- <dig>  gmglyii- <dig>  gmglyii- <dig>  gmglyii- <dig>  and gmglyii- <dig> showed either undetectable or very low level of expression in both vegetative and reproductive phases.
however, rest of the gmglyii members showed medium to high level of expression in all the developmental stages constitutively .
high level of expression of both gmglyi and gmglyii genes in all the developmental stages of soybean indicates the constitutive metabolic/cellular role of glyoxalase pathway throughout the life cycle of plant.
expression analysis of soybean glyoxalase genes under stress
to gain deep insights into the function of glyoxalase genes in the abiotic stress adaptation of soybean, the expression profiles of all gmglyi and gmglyii genes were analyzed in response to salinity and drought stresses using publicly available microarray data.
expression data sets were retrieved from gene expression omnibus database of gse <dig> and gse <dig> for salinity and drought stress, respectively.
data were not available for all these genes due to the limitation of respective probes.
among the total of  <dig> gmglyi and  <dig> gmglyii genes; data for  <dig> gmglyi and eight gmglyii genes were analyzed for salinity, while expression data of  <dig> gmglyi and  <dig> gmglyii genes were found for drought stress.
different glyoxalase members responded differentially in terms of their expression towards both these stresses .
in response to salinity stress, four gmglyi genes and two gmglyii genes showed up-regulation, while seven gmglyi genes and four gmglyii genes showed down-regulation and rest of them remained unaltered .
similarly, drought stress causes upregulation of seven gmglyi genes and five gmglyii genes, and down-regulation of eight gmglyi genes and six gmglyii genes .
among  <dig> gmglyi genes; gmglyi- <dig> and gmglyi- <dig> showed significant up-regulation, while other two of them  showed down-regulation in both the stresses .
in case of gmglyii; gmglyii- <dig> and gmglyii- <dig> showed up-regulation, while gmglyii- <dig> showed significant down-regulation in both salinity and drought stresses .
rest of the members of both gmglyi and gmglyii family showed variable expression pattern.
this indicates diverse role of different glyoxalase members in the stress modulation pathways of soybean plant.fig.
<dig> expression analyses of soybean glyoxalase genes in response to salinity, drought and hormonal treatment.
relative expression data of all available gmglyi  and gmglyii  genes under salinity and drought stresses were obtained from the national center for biotechnology information geo database .
expression data is presented as fold-change by comparing with the corresponding mock samples.
blue colour bar represents data for salinity stress, while red colour indicates drought stress.
c semiquantitative rt-pcr of four gmglyi genes , four gmglyii genes  and one house-keeping control gene, tubulin under different conditions such as control, salinity, drought and aba treatment.
d relative expression analysis of the representative eight glyoxalase genes were analyzed by measuring the pcr band intensity using image j software and represent as relative fold change in expression
to screen the role of soybean glyoxalase genes in response to salinity, drought and hormonal treatment , a semi quantitative rt-pcr was performed to validate four candidate gmglyi genes  and four candidate gmglyii genes  which were highly responsive in microarray data analysis .
for this purpose, 15 days old soybean seedlings were subjected to normal water , 200 mm nacl  or withdrawn of water  or 10 mm aba  for 8 h. expression of all the candidate genes were compared with that of tubulin  .
relative transcript abundance of all these transcripts was measured by scanning the gel image using image j software relative fold change in expression was calculated considering tubulin as internal control .
it could be clearly inferred from fig.
10d that gmglyi- <dig>  gmglyii- <dig> and gmglyii- <dig> showed strong up-regulation in response to both salinity and drought, while gmglyi- <dig>  gmglyii- <dig> and gmglyii- <dig> showed clear down-regulation .
the remaining two members, gmglyi- <dig> and gmglyi- <dig> showed slight up/down regulation as compared to control sample.
overall, the pattern of expression of these eight candidate genes  was found to be almost similar to that of microarray data .
identification of cis-elements in the promoter region of soybean glyoxalase genes
in order to comment on stress responsive expression of gmgly genes in response to salinity, drought and aba treatment, 1 kb upstream promoter region of each gmglyi and gmglyii genes were retrieved from soybase  and analyzed for the presence of cis-acting elements using plantcare  <cit> .
this analysis leads to the identification of several stress-responsive cis elements such as abscisic acid responsive element , auxin responsive element , fungal elicitor responsive element , ethylene responsive element , gibberellin-responsive element , heat shock element , jasmonate and elicitor responsive element , low temperature responsive element , myb-binding site , defence and stress responsive element , wounding and pathogen responsive elements , salicylic acid responsive element , methyl jasmonate-responsive element , element conferring high transcription level .
all these motifs are very crucial for plant stress modulation pathways and thus play important role to regulate the expression of various stress responsive genes  <cit> .
all these motifs were found to be distributed randomly in both the positive and negative strands of promoter sequences .
among gmglyi members, gmglyi- <dig> and gmglyi- <dig> have maximum cis-elements , while gmglyi- <dig> promoter has minimum two elements on it.
in case of gmglyii members, gmglyii- <dig> has maximum ten elements, while gmglyii- <dig> has only one element.
abre, hse, and tgacg motif are found to be present in almost every promoter of gmgly genes with few exceptions.
although correlation between the presence of cis-acting regulatory elements and the observed transcript abundance needs to be confirmed experimentally, these results indicated the stress-responsive nature of gmgly genes.fig.
<dig>
in silico promoter analysis of gmglyi and gmglyii genes.
one kb 5’ upstream sequence of all gmglyi and gmglyii genes was downloaded from soybase database and scanned through plantcare for the identification of number and position of various cis-acting regulatory elements.
different regulatory elements were indicated by different colour symbols and placed in their relative position on the promoter.
symbols presented above the line indicate forward strand of dna, while below one indicates the reverse strand
discussion
methylglyoxal  is a metabolic by-product generated naturally in all living cells  <cit> .
but the level of mg goes up in response to various abiotic stresses in plants  <cit> .
it has been established in literature that glyoxalase pathway plays a vital role in the detoxification of mg as well as provides tolerance against multiple abiotic stresses  <cit> .
genome wide analysis of glyoxalase pathway has been done preliminary in the monocot model plant rice and dicot model plant arabidopsis  <cit> .
however, this family has not been studied in any other species including legume.
in the present study, we have performed a genome wide analysis of soybean to identify glyoxalase gene families, including their chromosomal location, gene and protein structure, conserved active site and catalytic site motifs and expression profiles.
a total of  <dig> glyi and  <dig> glyii genes were identified in the soybean genome that codes for  <dig> glyi and  <dig> glyii proteins, respectively .
the number of gmglyi genes is  <dig>  times more than that of arabidopsis and rice ; and gmglyii shows  <dig>  times more abundance than that of arabidopsis , and  <dig> times more abundant than rice .
the possible reason behind this significant increase in gene number might be the two duplication events of soybean  <cit>  that has occurred after the monocot/dicot split, or most of the soybean genes expanded in a species-specific manner  <cit> .
to adopt with different adverse environmental conditions, plants tend to duplicate genes to generate novel members or increase number  <cit> .
there are three basic principal patterns of gene duplications, such as tandem duplication, segmental duplication and transposition.
in the present analysis, a total of ten duplicated pairs were observed in gmglyi family and five in gmglyii family .
all of them showed segmental duplication except one, that is the major pattern of gene duplication in plant.
the tandem duplication pair gmglyi-14/- <dig> was formed  <dig>  mya ago; while the segmental duplications of gmglyi genes have occurred between  <dig>  and  <dig>  mya and that of gmglyii occurred between  <dig>  and <dig>  mya.
this indicates that the tandem duplication event has occurred before the segmental duplication event.
similar pattern of duplication has been reported previously for hd-zip genes of soybean  <cit> .
soybean, including other plants has been found to possess greater number of glyi and glyii genes and proteins as compared to their animal counterpart to date.
one of the possible reasons behind this is gene duplication of plant during evolution that ultimately leads to functional divergence of genes  <cit> .
functional divergence might lead to either subfunctionalization or neofunctionalization, that in turn resulted in novel gene functions  <cit> .
in the present study, out of  <dig> predicted glyi proteins only twenty of them possess all four conserved metal binding sites and are expected to have functional glyi enzyme activity .
the other proteins might be functionally diverged and possess some other activities similar to glyi.
structurally glyi is the member of vicinal oxygen chelate  superfamily that includes extradiol dioxygenases, glyi and methylmalonyl‐coa epimerase  <cit> .
one of the earlier predicted gmglyi  was found to have glutathione s-transferase activity, too  <cit> .
apart from that, it has been well-characterized in literature that there are two metal activation classes of glyis; zn2+ and non-zn2+ .
both these classes possess same four conserved metal binding residues and octahedral metal co-ordination; regardless of the metal activation class  <cit> .
three dimensional structure of one of the predicted ni2+-dependent glyi, gmglyi- <dig>  and zn2+-dependent glyi, gmglyi- <dig>  confirms the presence of same active site residues in both.
however, ni2+-dependent gmglyi- <dig> found to be monomer consisting of two glyi domains that fold to create two putative active sites .
whereas, single glyi domain containing zn2+-dependent gmglyi- <dig> need to be homo- dimer to create two putative active sites .
interestingly, metal specificity of putative glyis could be easily predicted based on the protein’s amino acid length and sequence  <cit> .
zn2+- activated glyis are relatively larger in amino acid length than ni2+/co2+-activated ones and have unique region in their sequence .
based on these criteria,  <dig> out of  <dig> predicted functional gmglyi enzymes are expected be ni2+/co2+-activated.
same pattern of dominance by ni2+/co2+-activated forms was observed in case of rice glyis  and arabidopsis glyis   <cit> .
on contrary, glyii enzymes contain the β-lactamase fold structure that includes lactonase, rubredoxin:oxygen oxidoreductase, glyii, arylsulfatase, phosphodiesterase, carboxylesterase and trna maturase  <cit> .
previously, glyii family members from arabidopsis  and rice  have been reported to lack of glyii activity, instead they possess sulphur dioxygenase like ethylmalonic encephalopathy protein  <dig>  activity  <cit> .
similar to glyi, three out of  <dig> predicted gmglyii proteins did not possess the conserved metal binding motif that might have resulted in the absence of glyii activity and leads to the functional divergence.
expression of glyoxalase genes has been found to be highly specific towards certain tissue or developmental stages in arabidopsis and rice  <cit> .
thus, the expression pattern of gmglyi and gmglyi genes was analyzed at different developmental stages and tissues .
these data revealed the tissue specific expression pattern of glyoxalase genes in soybean too.
out of all, gmglyi- <dig>  gmglyi- <dig> and gmglyii- <dig> are found to be the constitutively expressed members of soybean glyoxalase system.
a cluster of gmglyi and gmglyii genes maintained high level of expression in all the underground and aerial tissues, followed by low level of expression in the different stages of seed development .
this indicates the presence of functional distribution among the multiple members in different tissue/developmental stimuli.
on contrary, some of the gmglyi genes such as gmglyi- <dig>  gmglyi- <dig>  and gmglyi- <dig> showed medium level of expression in the seed tissues only, with low or no expression in other parts .
this indicates development specific transition/regulation of gmglyi genes.
in case of gmglyii genes, two distinguishable clades were observed in their expression where one set showed high level expression in all the tissues and another have no expression at all.
these low expressive genes might have other cellular/metabolic regulation expect developmental/tissue regulation.
another interesting expression pattern was observed for soybean glyoxalase genes under abiotic stresses .
different members of gmglyi and gmglyii families responded differentially against salinity, drought and hormone  treatment .
gmglyii- <dig> and gmglyii- <dig> showed strong down-regulation in response to all three conditions, while gmglyii- <dig> showed sharp up-regulation .
presence of various cis-acting regulatory elements on the putative promoter sequence of gmglyi and gmglyii genes might be a probable reason behind this altered expression .
similar pattern of expression was observed previously in rice and arabidopsis glyoxalase genes  <cit> , where each member shows specific pattern of expression towards the particular type of stress treatment.
overall, the observed information in the present study will facilitate to find out the appropriate candidate gene for further functional characterization and raising stress-tolerant transgenic crop plants.
CONCLUSIONS
taken together, we have performed a comprehensive in silico analysis of soybean glyoxalase gene families , and provided detailed information about them.
specifically, our results show that soybean genome contains  <dig> gmglyi and  <dig> gmglyii genes that code for  <dig> gmglyi and twenty-three gmglyii proteins, respectively; the largest identified glyoxalase gene family to date in any species/organism.
present study indicates genome-wide duplication  of glyoxalase genes that lead to the expansion of family.
based on the presence of conserved motifs and sequence homology, we have provided insight into their putative function and metal dependency.
finally, expression data confirms the development, tissue and stress specific response of each and every gene in spite of having large multi-member family.

 the glyoxalase system is a two-enzyme driven pathway that detoxifies the highly cytotoxic compound, methylglyoxal to d-lactate. the detoxification is accomplished by the sequential action of two thiol-dependent enzymes; glyoxalase І and glyoxalase ii. in presence of reduced glutathione, mg is converted into hemithioacetal spontaneously, and glyi catalyses the isomerization of this hta into s-d-lactoyl-glutathione. glyii hydrolyses slg into d-lactate and recycles back one molecule of gsh to the system. both, the formation of mg and the glyoxalase enzymes have been ubiquitously found in all organisms from escherichia coli to homo sapiens. besides its proposed role in the detoxification of mg as metabolic enzyme, glyoxalase enzymes have been reported to be involved in various other functions. glyoxalase system protects human from various vascular complications of diabetes, such as nephropathy, retinopathy, neuropathy and cardiovascular disease by resisting the increased accumulation of mg. moreover, glyoxalase pathway has also been shown to be involved in different important cellular functions of human, such as cell division and proliferation, microtubule assembly and protection against oxoaldehydes toxicity. for this, the pathway has been regarded as “marker for cell growth and division”. similarly, stress tolerance potential of glyoxalase has been reported in plant by numerous studies. transgenic plants over-expressing glyi and/or glyii were found to provide significant tolerance against multiple abiotic stresses including salinity, drought and heavy metal toxicity. thus mg and glyoxalases are considered as potential biomarkers for plant stress tolerance. glyoxalase proteins have been extensively characterized from different genera such as escherichia coli, homo sapiens, saccharomyces cerevisiae, arabidopsis thaliana and oryza sativa. compared to other organisms, very little is known about plant glyoxalases. the first plant glyoxalase activity was reported from douglas fir needles by smits and johnson. thereafter, presence of glyoxalase activity has been reported from various other plant species, such as rice, arabidopsis, tomato, wheat, sugarcane, brassica etc.. most of the genes of plant exist as family due to the expansion and gene duplication during the course of plant evolution. availability of the whole genome sequences has opened up the field to identify and characterize plant glyoxalase family substantially. according to in silico genome wide analyses of rice and arabidopsis, there are eleven potential glyi and three glyii genes in rice; and eleven glyi and five glyii genes in arabidopsis. expression analysis of all these genes have been performed in different developmental tissues and stages, and in response to multiple abiotic stresses using publicly available mpss and microarray database. it has been observed that atglyi- osglyi- atglyii- atglyii- osglyii- and osglyii- showed constitutive expression in all the tissues and stages, while atglyi- osglyi- and osglyi- expressed only in seed. on the other hand, atglyi- osglyi- atglyii- and osglyii- were the most stress inducible members. among these identified glyoxalase members, glyii genes have been extensively studied from both rice and arabidopsis but the research on glyi is still very limited. to date, all five atglyii and three osglyii genes have been well characterized. both osglyii- and osglyii- possessed typical glyii enzymatic activity and overexpression of these genes in tobacco provides enhanced tolerance against salinity stress. however, osglyii- along with atglyii- showed functional divergence by possessing sulphur dioxygenase activity instead of glyii. one of the rice glyi, osglyi- have been studied extensively and found to possess ni2+-dependent glyi activity with stress modulation potential. soybean is a legume plant of papilionoideae family, major source of vegetable protein and edible oil. it also has the capacity to fix atmospheric nitrogen through symbioses. however, production of soybean is under threat due to the unfavourable environmental stimuli such as drought, salinity and osmotic stresses. all these stresses severely affect the overall plant development in all the stages from germination to flowering and reduce the productivity and seed quality of soybean. the yield has been reported to be reduced by about 40 % in response to drought. thus, there is an urgent need to identify novel stress responsive soybean genes using the available genome database. the soybean genome contains predicted protein-coding genes which are 70 % more than arabidopsis. there have been two genome duplication events undergone in soybean at approximately and million years ago, that resulted a highly duplicated genome. a lot of gene families have been studied in soybean, such as erf, hd-zip, wrky, burp, mads-box, myb, nac, cyp. genome wide analyses of glyoxalase gene family have been done in arabidopsis and rice, but no such analysis has been performed in soybean in spite of having a handful genome sequences deposited in the publicly available database. here, we present a detailed genome-wide identification of soybean glyi and glyii genes, their phylogenetic relationship, chromosomal distribution, structural and expressional analysis. present results indicate that soybean genome contains glyi and glyii proteins, the largest family of glyoxalase known to date in any organism. expression analysis of these genes based on publicly available microarray data indicates the differentially regulation of glyoxalase members in response to various developmental cues as well as stress treatments. in particular gmglyi- gmglyi- and gmglyii- are most up-regulated stress responsive members that might resist mg accumulation in stress by interacting with other members. this study will facilitate the further investigation of soybean glyoxalase genes for the biological and molecular functions.  identification of glyi and glyii gene families in soybean proteins having lactoylglutathione lyase domain have been classified as glyi proteins and metallo-beta-lactamase domain have been classified as glyii proteins. previously, glyoxalase proteins have been identified in two model plant genome, arabidopsis and rice. to identify all the putative members of the glyoxalase proteins in soybean, a blastp search of the soybean genome database g. max wm a v was performed using the previously characterized protein sequence as a query. glyi proteins have been primarily identified using a previously reported soybean glyi protein. subsequently, each of the newly identified glyi protein sequences has been used as a query sequence individually in blastp search of soybean genome database. subsequent searching process was repeated until there was no new member documented. this search resulted in the identification of total unique proteins. all these identified proteins were analyzed using pfam to check the presence of unique lactoylglutathione lyase domain. this analysis discarded two members due to the lack of lactoylglutathione lyase domain, and finally landed to a total of soybean glyi proteins which is greater than the previously reported arabidopsis and rice glyi proteins. these glyi proteins have been coded by unique genes located on different chromosomes. they were identified and named as gmglyi- to gmglyi- following the nomenclature proposed previously.fig. chromosomal distribution of gmglyi and gmglyii genes on different soybean chromosomes. only the chromosomes having glyoxalase genes are shown and their number is indicated above by roman numbers. the scale is in mega base, and the centromeric regions are indicated by black ellipses. red coloured boxes indicate the segmental duplicated genes connected by red lines, based on sequence similarities and divergence analysis. black boxes indicate the non-duplicated genes abbreviations: cds coding dna sequence, chro chromosome, pp polypeptide length, mw molecular weight, p i isoelectric point, bp base pair, aa amino acid, kda kilodalton, ch chloroplast, cy cytosol, ec extracellular, mt mitochondria, nu nucleus, po peroxisome alocalization prediction by cello v. blocalization prediction by psort cchloroplast localization signal confirmed by chlorop similarly, soybean glyii proteins have been primarily identified using a previously characterized brassica juncea glyii protein as query and secondarily by the newly identified members. a total of unique protein sequences have been identified and checked for the presence of unique metallo-beta-lactamase domain using pfam. three of them didn’t have this unique domain and were discarded from the list. thus, a total of soybean glyii proteins have been confirmed which is greater than the previously reported arabidopsis and rice glyii family members. these glyii proteins have been coded by unique genes located on ten different chromosomes. they were named as gmglyii- to gmglyii- like gmglyi genes. in both gmglyi and gmglyii families, the number of proteins was greater than the number of genes; indicating the existence of alternate splicing event in soybean glyoxalase genes. most of the gmglyi genes and gmglyii genes showed only a single product. however, rest seven gmglyi genes formed alternative spliced products, whereas seven gmglyii genes lead to the generation of proteins.table list of identified glyii genes in soybean along with their detailed information and localization abbreviations: cds coding dna sequence, pp polypeptide length, mw molecular weight, p i isoelectric point, bp base pair, aa amino acid, kda kilodalton, ch chloroplast, cy cytosol, ec extracellular, mt mitochondria, nu nucleus alocalization prediction by cello v. blocalization prediction by psort cchloroplast localization signal confirmed by chlorop detailed analysis of identified gmglyi and gmglyii members all the newly identified gmglyi and gmglyii members were analyzed in detail. the coding dna sequence length of the gmglyi members vary from 333 bp to 1101 bp with an average of 740 bp. consequently, gmglyi- encodes for the largest protein of the family with a polypeptide length of aa and molecular weight of  kda; and the smallest protein is aa in length with  kda in weight. similar to the length and molecular weight variation, the proteins showed a wide range of deviation in their isoelectric point value from to. most of the gmglyi members showed acidic pi value, with only seven such as gmglyi-, gmglyi-, gmglyi-, gmglyi-, gmglyi-, gmglyi-, and gmglyi- have showed basic pi value. this ensures the presence of both positively and negatively charged gmglyi proteins at a certain physiological condition. sub-cellular localization of all these predicted gmglyi proteins were analyzed based on two different tools cello and wolf psort, and the chloroplast localization was further confirmed by chlorop. different members were found to be localized at different sub-cellular compartments, such as chloroplast, cytosol, mitochondria, nucleus, extracellular, peroxisome. most of the gmglyi proteins are found to be localized in cytosol, followed by chloroplast, mitochondria and nucleus. similarly, the cds length of gmglyii transcripts varies from 432 bp to 1584 bp with an average of 850 bp. the largest gmglyii- protein is aa in length with a molecular weight of  kda; and the smallest protein is aa in length and  kda in weight. gmglyii proteins also show variation in their pi values ranging from to. most of the gmglyii members showed acidic pi value similar to gmglyi proteins, while only eight gmglyii members such as gmglyii-, gmglyii-, gmglyii-, gmglyii-, gmglyii-, gmglyii-, gmglyii-, and gmglyii- have basic pi value. similar to gmglyi, most of the gmglyii proteins are found to be localized in cytosol, followed by chloroplast, nucleus, and mitochondria. chromosomal distribution and gene duplication to determine the exact position and distribution of the identified gmglyi and gmglyii genes on different chromosomes, a detailed chromosome map was constructed. soybean glyoxalase genes are found to be unevenly distributed throughout the chromosomes. it has been found that gmglyi genes are located on different chromosomes. the gene density per chromosome is highly uneven, where chromosome and contain the maximum occurrence of glyi genes. however, chromosomes have two glyi genes each, and only one glyi gene each is present on chromosomes and no glyi gene was found on chromosomes and 20; thereafter not shown in the fig. 1a. similarly, gmglyii genes were found to be located on ten different chromosomes and the gene density per chromosome is highly uneven. chromosomes and contain the maximum glyii genes, whereas chromosomes and have only one glyii gene each. no glyii gene was found on the rest of the chromosomes and as such not shown in the fig. 1b. all the gmglyi and gmglyii genes were found to be located towards the chromosome ends, suggesting the possibility of inter-chromosomal genetic rearrangements between different soybean chromosomes during genome duplication. due to two duplication events, soybean genome resulted in many paralogs within a gene family. out of the gmglyi proteins, are clustered in pairs and eight gmglyii proteins are clustered in pairs out of a total of gmglyii proteins in the phylogenetic tree. the percentage of similarities between all these gmglyi and gmglyii proteins were combined separately. it was observed that all the paired members of both glyi and glyii family have very high level of sequence similarities. this high level of sequence similarities indicates the possibility of segmental duplication of the genes throughout evolution. moreover, among the gmglyi genes one gene pair was present continuously within a distance of less than 5 kb on chromosome this indicates that these two genes might be duplicated by tandem duplication. to identify the time course of gene duplication, all the identified duplicated gene pairs were analyzed using plant genome duplication database. according to the ratio of nonsynonymous to synonymous substitutions, the evolutionary history of selection acting on different genes could be measured. this ratio could be used to interpret the direction and magnitude of natural selection enforcing on the various protein coding genes. a pair of sequences having ka/ks <  implies purifying selection; ka/ks =  indicates both sequences are drifting neutrally; and lastly ka/ks >  implies positive or darwinian selection. the ka/ks of glyoxalase duplicated gene pairs was found to be less than; that indicates the influence of purifying selection in the evolution of these gene pairs. considering the divergence rate of synonymous mutations per synonymous site per year for soybean, the duplication time for each gene pairs was calculated. it is observed that all the segmental duplicated pairs showed a time frame between and mya, except the tandem duplicated pair that occurred mya ago.table divergence time between glyoxalase gene pairs in soybean phylogenetic analysis of glyoxalase genes from various plant species in the present study, a phylogenetic tree of all the identified gmglyi or gmglyii proteins along with other reported glyi or glyii proteins from other plant species were constructed using mega tool. a neighbour joining phylogenetic tree was generated using a total of full-length glyi protein sequences of soybean, rice and arabidopsis glyi family, and proteins from other plant species. the tree was sub-divided into four subfamilies as evident in fig. 2a. all these subfamilies consist of representative member from both dicot arabidopsis and monocot rice, indicating that the evolution of plant glyi genes occurred before the split of dicot-monocot. clade-iv has the largest glyi members from different plant species, while clade-ii has the lowest number of members only from arabidopsis and rice genome. clade-i comprises of glyi members only from the complete genome database of three plants, arabidopsis, rice and soybean. among them, osglyi- is functionally a diverge member of the rice glyi family and might possess some other activities than glyi. in clade-iii, there are multiple members from arabidopsis, rice and soybean; and one member each from genlisea aurea and sorghum bicolor. among them, three rice members osglyi- osglyi- and osglyi-11; and two members of arabidopsis atglyi- and atglyi- have been already predicted to be ni2+-dependent glyi enzyme. thus rest of the members of this clade would be expected to have ni2+-dependent catalytic activity. similarly, clade-iv has members from rice and arabidopsis which are zn2+-dependent glyi enzymes. thus rest of the glyi members from other species would require zn2+ for their optimum glyi activity. this indicates that zn2+-dependent glyi enzymes are more diverse as they are present in many plant species.fig. phylogenetic analyses of glyi and glyii proteins from various plant species. glyoxalase protein sequences from various plant species were downloaded from various databases and provided as additional files and an unrooted tree was generated using neighbor-joining method with bootstrap by mega software using the full-length amino acid sequences of eighty-three glyi or forty-one glyii proteins. the numbers next to the branch shows the result of bootstrap replicates expressed in percentage, and scores higher than 50 % are indicated on the nodes. both trees were sub-divided into four classes and indicated by different colours to clarify the phylogenetic relationship among glyii proteins, we further constructed another tree for all full length sequences of gmglyii, osglyii, atglyii family and glyii sequences from other plant species. this tree was subdivided into four classes too like the previous one. class-i consists of three proteins from soybean, and one each from rice and arabidopsis. among them, osglyii- has been reported to have sulphur dioxygenase activity rather than glyii. so this sub class of proteins would be functionally diverse from glyii. similarly, class-ii contains one protein each from rice, arabidopsis, and selaginella moellendorffii, and four proteins from soybean. atglyii- has been reported to be the mitochondrial localized atglyii family member. division of class-iii and –iv is more interesting and evolutionarily more significant. class-iii has glyii proteins from all monocot plants; while class-iv has exclusively dicot members including arabidopsis, soybean, medicago, lotus etc.. apart from glyi, glyii proteins were found to be diversified after the split of monocot and dicot. gene structures of gmglyi and gmglyii genes detailed analysis of the exon-intron structure of gmglyi and gmglyii genes showed great variation among themselves. all gmglyi and gmglyii genes contained at least one intron in their open reading frame, which means there is no intron less glyoxalase gene in soybean. the number of introns varied from to in the orfs of different gmglyi genes. the gmglyi-, gmglyi- gmglyi- and gmglyi- contained a single intron in their orf while the largest numbers of introns were found in the gmglyi- transcript. in many cases, the borders of protein-coding sequence, 5′ and 3′ untranslated regions also contain large numberof introns. out of gmglyi transcripts, there was no intron in the 3′ utr of any of these genes and only eight of them contained a single intron in their 5′ utr region. similarly, the number of introns varied from to in the orfs of different gmglyii genes. the maximum number of introns was observed in gmglyii-, followed by each in gmglyii- and gmglyii-. gmglyii- contained only a single intron in its orf while the rest have varied number of introns. similar to gmglyi transcripts, there was no intron in the 3′ utr of gmglyii transcripts. only six out of transcripts have a single intron in their 5′utr region.fig. gene structures of gmglyi and gmglyii family members including the alternative spliced forms. all the exons are shown in filled black boxes and the introns are indicated by black lines. the 5’-utr regions are shown using empty boxes and the 3’-utr regions are shown in empty arrows which also indicate the direction of the gene. left to right direction of transcript indicates “+” strand, while the right to left one indicates “-” strand, relative to the annotation of the genome sequence. the size of the introns, exons, and utrs could be estimated from the scale at the bottom longer introns are selectively advantageous that could counterbalance the mutational bias and improve the recombination frequency. a strong evidence for the presence of ancestral introns was reported by analyzing introns of animal, plant and fungus. moreover, the number of exons and introns were found to be similar in the paralogous genes that clustered together in the phylogenetic analysis. such as, gmglyi-1/- gmglyi-4/- gmglyi-10/- and gmglyi-6/- have the same number of introns and exons. analysis of gmglyi proteins for their domain architecture, catalytic conservance and metal ion dependency all the predicted gmglyi proteins were analyzed using pfam to reveal the presence of conserved glyoxalase domain among them. analyses of gmglyi proteins revealed that out of forty-one contains two glyi domains, while the rest have only single glyi domain. presence of two glyi domain in a single protein have been previously reported from saccharomyces cerevisiae, oryza sativa and plasmodium falciparum. presence of two domain forms two putative active sites on a single monomeric protein. both the active sites are found to be functional, but allosterically regulated in plasmodium falciparum, whereas one of the active site is found to be a pseudo-active site in oryza sativa. however, glyi proteins with single domain have also been reported from various species such as e. coli, h. sapiens and function as homo-dimer.fig. domain architectures of gmglyi proteins. all forty-one soybean glyi proteins were analyzed for the presence of functional domain using pfam. all the gmglyi proteins possess glyoxalase domain that is represented by boxes. the position of the domain is indicated by the amino acid number inside the box. among the gmglyi members, of them have single glyoxalase domain, whereas rest have two domains. the length of full proteins is indicated by exact amino acid numbers and relative position of the domains could be interpreted by the scale given below activity of glyi enzyme is highly dependent on divalent metal ions. on the basis of metal ion specificity glyi proteins could be divided into two classes; zn2+-dependent or zn2+-independent. glyi from homo sapiens, saccharomyces cerevisiae and pseudomonas putida have been reported as zn2+-dependent, whereas glyi from e. coli and one of the rice glyi showed ni2+-dependent activity. the metal dependency of the glyi enzymes could be easily predicted from the length of glyi domain, as ni2+-dependent glyi has a domain length of ~ aa and zn2+-dependent glyis are usually aa in length. irrespective of the metal ion dependency, the active site of glyi proteins has a conserved motif of h/qeh/qe. among them, the glutamate residues act as a base by accepting protons from the substrate and any mutation of this conserved residue resulted in the complete loss of activity. thus, to comment on the presence of enzymatic activity and metal ion dependency, glyi domain of all the putative gmglyi proteins were aligned along with known ni2+-dependent osglyi- and zn2+-dependent osglyi- proteins. all the metal binding sites were presented inside black boxes and the regions specific for zn2+-dependent glyi were presented by black arrows.fig. multiple sequence alignment of glyi domain of all gmglyi proteins along with that of osglyi- and osglyi- glyi domain of all gmglyi proteins were aligned with that of a ni2+-dependent osglyi- and a zn2+-dependent osglyi- using clustalw program. the alignment file was viewed using jalview multiple alignment editor program. all four conserved metal binding sites were represented as black boxes and the specific region for zn2+-dependent glyi was marked with black arrow based on the presence of all the four conserved metal binding site, the expected glyi enzyme activity of the putative gmglyi proteins was predicted. out of a total putative gmglyi proteins, have all the four conserved residues and are expected to have functional glyi enzyme activity. out of this expected functional glyi enzymes, are predicted to be ni2+-dependent as they have the domain length of around aa and lack of the conserved regions specific for zn2+-dependent members. the remaining four namely gmglyi-, gmglyi-, gmglyi- and gmglyi- are expected to be zn2+-dependent as their domain length is more than aa and possessed the conserved regions.table analysis of all putative gmglyi enzymes for their enzymatic activity and metal ion dependency analysis of gmglyii proteins for their domain architecture and catalytic efficiency genome wide analysis of soybean revealed the presence of glyii proteins coded by genes. all these gmglyii proteins were analyzed using pfam to reveal the presence of conserved metallo-beta-lactamase domain among them. analysis of all gmglyii proteins revealed that out of have only metallo-beta-lactamase domain, while the rest eleven contain additional hydroxyacylglutathione hydrolase c-terminus domain along with metallo-beta-lactamase domain. hagh-c domain is usually found to be present at the c-terminus of glyii enzymes that forms the substrate binding site along with the catalytic domain. however, glyii from various species such as e. coli, s. cerevisiae, s. typhimurium, l. infantum, a. thaliana, b. juncea, o. sativa and h. sapiens, contained the well conserved metal binding motif and active site motif. both these motifs play an important role in the glyii enzyme activity of a protein. therefore, to comment on the presence of enzymatic activity of the putative gmglyii proteins, their protein sequences were aligned by multiple sequence alignment. both these motifs were indicated by black boxes; their presence and absence were listed in table  out of putative gmglyii proteins only three of them do not possess the conserved metal binding residues, but all of them have the active site motif. thus, it could be expected that all the predicted gmglyii proteins have the functional glyii enzyme activity except gmglyii-, gmglyii-, and gmglyii-.fig. domain architectures of gmglyii proteins. all soybean glyii proteins were analyzed for the presence of functional domain using pfam. there were two types of domains observed in gmglyii proteins, such as β-lactamase domains and hydroxyacylglutathione hydrolase c-terminus domain. the length of full protein and domain are indicated by exact amino acid numbers beside and inside of the shape, respectively. the relative size could be identified by using the scale below fig. multiple sequence alignment of gmglyii proteins. all gmglyii full length protein sequences were aligned using clustalw program and viewed using jalview multiple alignment editor program. the black boxes indicate the most conserved metal binding motif and active site motif homology modelling of representative gmglyi and gmglyii members to know the arrangement of active site residues and overall 3-d coordination, homology model of gmglyi- gmglyi- and gmglyii- proteins was built based on the closely related template structure of zea mays glyi, mouse glyi, and atglyii- proteins, respectively. gmglyi- is a ni2+-dependent monomeric glyi enzyme, while gmglyi- is a zn2+-dependent homodimeric enzyme. gmglyi- has two putative active sites; one consists of h- e- q- and e- and the other one consists of h- e- q- and a- the second putative active site has lack of a highly conserved glu residues, thus might be inactive in nature like previously reported osglyi-. the zn2+-dependent gmglyi- consists of single glyi domain and thus forms homo-dimer to create two putative active sites. one putative active site has q and e and h and e; another one has opposite members from both chains. here both the active sites have all four conserved residues and thus are predicted to be functionally active too. on the other hand, gmglyii- is a monomeric protein consists of two structural orientations, an n-terminal domain with two βββαβ topology and a c-terminal domain with five α-helices. the metal binding and active site residues are asn his asp his his asp and his are found to be conserved as compared to template atglyii- protein.fig. three dimensional homology model structure of soybean glyoxalase proteins. structures of gmglyi-, gmglyi- and gmglyii- were built using swiss-model server based on available close similar structure from protein data bank zea mays glyi, mouse glyi, and atglyii- proteins, respectively. all the α-helices were marked with orange colour, while β-sheets were marked with cornflower blue. the active sites residues were identified based on the alignment with template structure and shown by ball-stick model. the structure and active residues were visualized and generated using chimera program expression analysis of gmglyi and gmglyii genes at different soybean tissues rna-seq atlas of glycine max provides high-resolution gene expression data in a diverse set of soybean tissues such as young leaf, flower, one cm pod, pod-shell, seed, root and nodule. all these tissues could be broadly divided into three classes; such as underground, aerial and seed. rna-seq normalized expression data for all gmglyi and gmglyii genes were retrieved from soybase, except gmglyi- and gmglyi- due to lack of their appropriate probe. data were analyzed and represented as heat maps generated using tigr mev software package. expression analyses of all gmglyi genes revealed that the different members have different tissue specific expression. among all the analyzed genes, gmglyi- showed highest level of constitutive expression in all the tissues, followed by gmglyi- gmglyi- and gmglyi- this high level of constitutive expression indicates their significant role at all these plant tissues. a cluster of genes showed medium to high level of expression in all the underground and aerial tissues only, followed by very low expression at the seed tissues. they are gmglyi- gmglyi- gmglyi- gmglyi- gmglyi- and gmglyi- previous studies on rice and arabidopsis showed the presence of highly seed specific glyi genes such as atglyi- osglyi- and osglyi-. similarly, three of gmglyi genes such as gmglyi- gmglyi- and gmglyi- showed medium level of expression in different seed tissues only, indicating the evolutionary conservance for the presence of seed specific glyi genes.fig. expression profiling of soybean glyoxalase genes with hierarchical clustering in different developmental tissues and stages. a, b rna-seq expression data of developmental tissues, such as r, n, yl, f, p.1 cm, ps.10d, ps.14d, s.10d, s.14d, s.21d, s.25d, s.28d, s.35d, s.42d was used in the analysis. the normalized data was downloaded from soybase and provided as additional file 2: table s heatmap generation and hierarchical clustering was performed using mev software package. the colour scale below the heat map indicates expression values; green indicates low transcript abundance while red indicates high level of transcript abundance. c, d transcriptome data of all gmglyi and gmglyii genes at various developmental stages were obtained from the national center for biotechnology information. heatmaps generation and hierarchical clustering were performed using mev software package. the colour scale given below the heat map indicates the expression values where blue indicates low transcript abundance and yellow indicates high transcript abundance expression analyses of gmglyii genes indicate two clear clades. out of analyzed genes, five genes such as gmglyii- gmglyii- gmglyii- gmglyii- and gmglyii- showed almost undetectable expression in all the tissues with few exceptions. among others, gmglyii- showed highest level of constitutive expression in all the tissues, followed by gmglyii- these two genes might play a major role in all tissues. similar to gmglyi, a cluster of genes showed medium level of expression in the underground and aerial tissues, except the seed. no tissue specific expression pattern was observed in case of gmglyii genes. from the expression data analysis of the identified paralogous pairs of gmglyi and gmglyii genes in soybean tissues revealed a high level of expression divergence. for example, gmglyi- showed high level constitutive expression while its paralogous gmglyi- showed detectable expression in a few tissues. however, some of the paralogous gmglyi gene pairs namely gmglyi-1/- gmglyi-2/- gmglyi-4/- gmglyi-10/- and gmglyi-19/- showed similar pattern of expression. the divergence is even more in case of gmglyii gene pairs. for instance, gmglyii- is highly expressed in all the analyzed tissues while its paralogous counterpart gmglyii- remains mostly undetectable. similar level of deviation was also observed in case of gmglyii-6/-10and gmglyii-7/- gene pairs. expression analysis of gmglyi and gmglyii genes at different developmental stages expression of gmglyi and gmglyii genes at different developmental stages was analyzed using publicly-available genome-wide transcript profiling data of soybean. the dataset contains mainly two broad developmental sets, one at vegetative stages and the other at reproductive stage. as shown in fig. 9c, most of the gmglyi genes showed high level of expression, without any distinct pattern of expression. out of the gmglyi genes, only gmglyi- showed undetectable expression at all stages. among others, gmglyi- showed maximum constitutive expression in all the developmental stages followed by gmglyi- gmglyi- gmglyi- and gmglyi-. two of gmglyi members, gmglyi- and gmglyi- showed only reproductive stage specific expression. this indicates the development specific modulation of gmglyi gene expression. on the other hand, a distinct division is observed in the gmglyii gene expression through the developmental stages. a cluster of genes such as gmglyii- gmglyii- gmglyii- gmglyii- gmglyii- and gmglyii- showed either undetectable or very low level of expression in both vegetative and reproductive phases. however, rest of the gmglyii members showed medium to high level of expression in all the developmental stages constitutively. high level of expression of both gmglyi and gmglyii genes in all the developmental stages of soybean indicates the constitutive metabolic/cellular role of glyoxalase pathway throughout the life cycle of plant. expression analysis of soybean glyoxalase genes under stress to gain deep insights into the function of glyoxalase genes in the abiotic stress adaptation of soybean, the expression profiles of all gmglyi and gmglyii genes were analyzed in response to salinity and drought stresses using publicly available microarray data. expression data sets were retrieved from gene expression omnibus database of gse and gse for salinity and drought stress, respectively. data were not available for all these genes due to the limitation of respective probes. among the total of gmglyi and gmglyii genes; data for gmglyi and eight gmglyii genes were analyzed for salinity, while expression data of gmglyi and gmglyii genes were found for drought stress. different glyoxalase members responded differentially in terms of their expression towards both these stresses. in response to salinity stress, four gmglyi genes and two gmglyii genes showed up-regulation, while seven gmglyi genes and four gmglyii genes showed down-regulation and rest of them remained unaltered. similarly, drought stress causes upregulation of seven gmglyi genes and five gmglyii genes, and down-regulation of eight gmglyi genes and six gmglyii genes. among gmglyi genes; gmglyi- and gmglyi- showed significant up-regulation, while other two of them showed down-regulation in both the stresses. in case of gmglyii; gmglyii- and gmglyii- showed up-regulation, while gmglyii- showed significant down-regulation in both salinity and drought stresses. rest of the members of both gmglyi and gmglyii family showed variable expression pattern. this indicates diverse role of different glyoxalase members in the stress modulation pathways of soybean plant.fig. expression analyses of soybean glyoxalase genes in response to salinity, drought and hormonal treatment. relative expression data of all available gmglyi and gmglyii genes under salinity and drought stresses were obtained from the national center for biotechnology information geo database. expression data is presented as fold-change by comparing with the corresponding mock samples. blue colour bar represents data for salinity stress, while red colour indicates drought stress. c semiquantitative rt-pcr of four gmglyi genes, four gmglyii genes and one house-keeping control gene, tubulin under different conditions such as control, salinity, drought and aba treatment. d relative expression analysis of the representative eight glyoxalase genes were analyzed by measuring the pcr band intensity using image j software and represent as relative fold change in expression to screen the role of soybean glyoxalase genes in response to salinity, drought and hormonal treatment, a semi quantitative rt-pcr was performed to validate four candidate gmglyi genes and four candidate gmglyii genes which were highly responsive in microarray data analysis. for this purpose, 15 days old soybean seedlings were subjected to normal water, 200 mm nacl or withdrawn of water or 10 mm aba for 8 h. expression of all the candidate genes were compared with that of tubulin. relative transcript abundance of all these transcripts was measured by scanning the gel image using image j software relative fold change in expression was calculated considering tubulin as internal control. it could be clearly inferred from fig. 10d that gmglyi- gmglyii- and gmglyii- showed strong up-regulation in response to both salinity and drought, while gmglyi- gmglyii- and gmglyii- showed clear down-regulation. the remaining two members, gmglyi- and gmglyi- showed slight up/down regulation as compared to control sample. overall, the pattern of expression of these eight candidate genes was found to be almost similar to that of microarray data. identification of cis-elements in the promoter region of soybean glyoxalase genes in order to comment on stress responsive expression of gmgly genes in response to salinity, drought and aba treatment, 1 kb upstream promoter region of each gmglyi and gmglyii genes were retrieved from soybase and analyzed for the presence of cis-acting elements using plantcare. this analysis leads to the identification of several stress-responsive cis elements such as abscisic acid responsive element, auxin responsive element, fungal elicitor responsive element, ethylene responsive element, gibberellin-responsive element, heat shock element, jasmonate and elicitor responsive element, low temperature responsive element, myb-binding site, defence and stress responsive element, wounding and pathogen responsive elements, salicylic acid responsive element, methyl jasmonate-responsive element, element conferring high transcription level. all these motifs are very crucial for plant stress modulation pathways and thus play important role to regulate the expression of various stress responsive genes. all these motifs were found to be distributed randomly in both the positive and negative strands of promoter sequences. among gmglyi members, gmglyi- and gmglyi- have maximum cis-elements, while gmglyi- promoter has minimum two elements on it. in case of gmglyii members, gmglyii- has maximum ten elements, while gmglyii- has only one element. abre, hse, and tgacg motif are found to be present in almost every promoter of gmgly genes with few exceptions. although correlation between the presence of cis-acting regulatory elements and the observed transcript abundance needs to be confirmed experimentally, these results indicated the stress-responsive nature of gmgly genes.fig. in silico promoter analysis of gmglyi and gmglyii genes. one kb 5’ upstream sequence of all gmglyi and gmglyii genes was downloaded from soybase database and scanned through plantcare for the identification of number and position of various cis-acting regulatory elements. different regulatory elements were indicated by different colour symbols and placed in their relative position on the promoter. symbols presented above the line indicate forward strand of dna, while below one indicates the reverse strand discussion methylglyoxal is a metabolic by-product generated naturally in all living cells. but the level of mg goes up in response to various abiotic stresses in plants. it has been established in literature that glyoxalase pathway plays a vital role in the detoxification of mg as well as provides tolerance against multiple abiotic stresses. genome wide analysis of glyoxalase pathway has been done preliminary in the monocot model plant rice and dicot model plant arabidopsis. however, this family has not been studied in any other species including legume. in the present study, we have performed a genome wide analysis of soybean to identify glyoxalase gene families, including their chromosomal location, gene and protein structure, conserved active site and catalytic site motifs and expression profiles. a total of glyi and glyii genes were identified in the soybean genome that codes for glyi and glyii proteins, respectively. the number of gmglyi genes is times more than that of arabidopsis and rice; and gmglyii shows times more abundance than that of arabidopsis, and times more abundant than rice. the possible reason behind this significant increase in gene number might be the two duplication events of soybean that has occurred after the monocot/dicot split, or most of the soybean genes expanded in a species-specific manner. to adopt with different adverse environmental conditions, plants tend to duplicate genes to generate novel members or increase number. there are three basic principal patterns of gene duplications, such as tandem duplication, segmental duplication and transposition. in the present analysis, a total of ten duplicated pairs were observed in gmglyi family and five in gmglyii family. all of them showed segmental duplication except one, that is the major pattern of gene duplication in plant. the tandem duplication pair gmglyi-14/- was formed mya ago; while the segmental duplications of gmglyi genes have occurred between and mya and that of gmglyii occurred between and mya. this indicates that the tandem duplication event has occurred before the segmental duplication event. similar pattern of duplication has been reported previously for hd-zip genes of soybean. soybean, including other plants has been found to possess greater number of glyi and glyii genes and proteins as compared to their animal counterpart to date. one of the possible reasons behind this is gene duplication of plant during evolution that ultimately leads to functional divergence of genes. functional divergence might lead to either subfunctionalization or neofunctionalization, that in turn resulted in novel gene functions. in the present study, out of predicted glyi proteins only twenty of them possess all four conserved metal binding sites and are expected to have functional glyi enzyme activity. the other proteins might be functionally diverged and possess some other activities similar to glyi. structurally glyi is the member of vicinal oxygen chelate superfamily that includes extradiol dioxygenases, glyi and methylmalonyl‐coa epimerase. one of the earlier predicted gmglyi was found to have glutathione s-transferase activity, too. apart from that, it has been well-characterized in literature that there are two metal activation classes of glyis; zn2+ and non-zn2+. both these classes possess same four conserved metal binding residues and octahedral metal co-ordination; regardless of the metal activation class. three dimensional structure of one of the predicted ni2+-dependent glyi, gmglyi- and zn2+-dependent glyi, gmglyi- confirms the presence of same active site residues in both. however, ni2+-dependent gmglyi- found to be monomer consisting of two glyi domains that fold to create two putative active sites. whereas, single glyi domain containing zn2+-dependent gmglyi- need to be homo- dimer to create two putative active sites. interestingly, metal specificity of putative glyis could be easily predicted based on the protein’s amino acid length and sequence. zn2+- activated glyis are relatively larger in amino acid length than ni2+/co2+-activated ones and have unique region in their sequence. based on these criteria, out of predicted functional gmglyi enzymes are expected be ni2+/co2+-activated. same pattern of dominance by ni2+/co2+-activated forms was observed in case of rice glyis and arabidopsis glyis. on contrary, glyii enzymes contain the β-lactamase fold structure that includes lactonase, rubredoxin:oxygen oxidoreductase, glyii, arylsulfatase, phosphodiesterase, carboxylesterase and trna maturase. previously, glyii family members from arabidopsis and rice have been reported to lack of glyii activity, instead they possess sulphur dioxygenase like ethylmalonic encephalopathy protein activity. similar to glyi, three out of predicted gmglyii proteins did not possess the conserved metal binding motif that might have resulted in the absence of glyii activity and leads to the functional divergence. expression of glyoxalase genes has been found to be highly specific towards certain tissue or developmental stages in arabidopsis and rice. thus, the expression pattern of gmglyi and gmglyi genes was analyzed at different developmental stages and tissues. these data revealed the tissue specific expression pattern of glyoxalase genes in soybean too. out of all, gmglyi- gmglyi- and gmglyii- are found to be the constitutively expressed members of soybean glyoxalase system. a cluster of gmglyi and gmglyii genes maintained high level of expression in all the underground and aerial tissues, followed by low level of expression in the different stages of seed development. this indicates the presence of functional distribution among the multiple members in different tissue/developmental stimuli. on contrary, some of the gmglyi genes such as gmglyi- gmglyi- and gmglyi- showed medium level of expression in the seed tissues only, with low or no expression in other parts. this indicates development specific transition/regulation of gmglyi genes. in case of gmglyii genes, two distinguishable clades were observed in their expression where one set showed high level expression in all the tissues and another have no expression at all. these low expressive genes might have other cellular/metabolic regulation expect developmental/tissue regulation. another interesting expression pattern was observed for soybean glyoxalase genes under abiotic stresses. different members of gmglyi and gmglyii families responded differentially against salinity, drought and hormone treatment. gmglyii- and gmglyii- showed strong down-regulation in response to all three conditions, while gmglyii- showed sharp up-regulation. presence of various cis-acting regulatory elements on the putative promoter sequence of gmglyi and gmglyii genes might be a probable reason behind this altered expression. similar pattern of expression was observed previously in rice and arabidopsis glyoxalase genes, where each member shows specific pattern of expression towards the particular type of stress treatment. overall, the observed information in the present study will facilitate to find out the appropriate candidate gene for further functional characterization and raising stress-tolerant transgenic crop plants.  taken together, we have performed a comprehensive in silico analysis of soybean glyoxalase gene families, and provided detailed information about them. specifically, our results show that soybean genome contains gmglyi and gmglyii genes that code for gmglyi and twenty-three gmglyii proteins, respectively; the largest identified glyoxalase gene family to date in any species/organism. present study indicates genome-wide duplication of glyoxalase genes that lead to the expansion of family. based on the presence of conserved motifs and sequence homology, we have provided insight into their putative function and metal dependency. finally, expression data confirms the development, tissue and stress specific response of each and every gene in spite of having large multi-member family. 
the present study identifies the largest family of glyoxalase proteins to date with gmglyi and gmglyii members in soybean. moreover, this study provides a strong basis about the biological role and function of gmglyi and gmglyii members in soybean growth, development and stress physiology. detailed analysis of gmglyi and gmglyii genes strongly indicates the genome-wide segmental and tandem duplication of the glyoxalase members. previously genome-wide analysis of glyoxalase genes has been conducted in model plants arabidopsis and rice, but no such study was performed in any legume species. role of glyoxalase enzymes has been extensively investigated in various plant species which showed their crucial role in salinity, drought and heavy metal stress tolerance. expression profiling of these genes has been performed in different tissues and developmental stages as well as under salinity and drought stresses using publicly available rnaseq and microarray data. detailed analysis of these identified members was conducted including their nomenclature and classification, chromosomal distribution and duplication, exon-intron organization, and protein domain and motifs identification. glyoxalase pathway consists of two enzymes, glyoxalase i and glyoxalase ii which detoxifies a highly cytotoxic metabolite methylglyoxal to its non-toxic form. in the present study, a comprehensive genome database analysis of soybean was performed and identified a total of putative glyi and glyii proteins encoded by and genes, respectively. the study revealed that gmglyi- and gmglyii- have been expressed intensively in all the developmental stages and tissues; while gmglyi- gmglyi- gmglyi- gmglyii- and gmglyii- were highly abiotic stress responsive members.
 glyoxalase pathway consists of two enzymes, glyoxalase i and glyoxalase ii which detoxifies a highly cytotoxic metabolite methylglyoxal to its non-toxic form. mg may form advanced glycation end products with various cellular macro-molecules such as proteins, dna and rna; that ultimately lead to their inactivation. role of glyoxalase enzymes has been extensively investigated in various plant species which showed their crucial role in salinity, drought and heavy metal stress tolerance. previously genome-wide analysis of glyoxalase genes has been conducted in model plants arabidopsis and rice, but no such study was performed in any legume species.  in the present study, a comprehensive genome database analysis of soybean was performed and identified a total of putative glyi and glyii proteins encoded by and genes, respectively. detailed analysis of these identified members was conducted including their nomenclature and classification, chromosomal distribution and duplication, exon-intron organization, and protein domain and motifs identification. expression profiling of these genes has been performed in different tissues and developmental stages as well as under salinity and drought stresses using publicly available rnaseq and microarray data. the study revealed that gmglyi- and gmglyii- have been expressed intensively in all the developmental stages and tissues; while gmglyi- gmglyi- gmglyi- gmglyii- and gmglyii- were highly abiotic stress responsive members.  the present study identifies the largest family of glyoxalase proteins to date with gmglyi and gmglyii members in soybean. detailed analysis of gmglyi and gmglyii genes strongly indicates the genome-wide segmental and tandem duplication of the glyoxalase members. moreover, this study provides a strong basis about the biological role and function of gmglyi and gmglyii members in soybean growth, development and stress physiology. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords glyoxalaseglycine maxabiotic stressfunctional divergencegene duplicationmicroarraymetal dependencyrna seq-atlassemiquantitative rt-pcrissue-copyright-statement© the author 2016 