BACKGROUND
plants are constantly challenged by a wide range of environmental stresses such as drought, high salinity, and temperature fluctuations due to their sessile nature.
response to abiotic stresses is very complex, as various stages of plant growth and development can be affected by a particular stress and often several stresses simultaneously
<cit> .
molecular responses to abiotic stresses include stress perception, signal transduction to cellular components, gene expression, and metabolic changes, which help the plants to adapt to stress environments
<cit> .
post-translational modifications of proteins play an important role in most cellular processes by rapidly altering the functions of preexisting proteins and protein complexes.
sumoylation or sumo  conjugation is one of the essential post translational regulatory process essentially found in all eukaryotes.
it is a three step enzymatic cascade mediated by sumo activating enzyme , sumo conjugating enzyme , and sumo ligase  for the attachment of sumo to its target protein
<cit> .
sumo plays an important role in regulation of protein–protein interactions and subcellular locations in yeast and animals
<cit> .
increased level of sumo conjugates in response to heat shock, oxidative stress, and dna damaging agents has been reported in mammalian cell cultures
<cit> .
similarly, increased accumulation of sumo conjugates upon exposure of arabidopsis seedlings to heat shock, h2o <dig>  ethanol, and amino acid analog canavanine suggests its important role in stress response and protection in plants
<cit> .
several studies in arabidopsis highlighted the importance of sumoylation in post translational regulation in response to stresses such as salt, cold, drought, heat, copper toxicity, and nutrient deprivation
<cit> .
importance of sumoylation for abiotic stress response is strengthened from the reports on drastic reduction in tolerance to stresses due to mutation of genes involved in sumo conjugation
<cit> .
further, embryo lethality caused by insertional mutation in atsae <dig> or atsce <dig> or double mutations in atsumo <dig> and atsumo <dig> genes of arabidopsis inferred its role in plant growth and development
<cit> .the reversible conjugation of the sumo peptide to protein substrates is emerging as a major post-translational regulatory process in plants
<cit> .
arabidopsis sumo and related enzymes have been implicated in abscisic acid  responses, flowering time regulation as well as stress responses
<cit> .
in rice , transcripts for the sce is regulated by cold, salt, aba, and heat
<cit> .
however, detailed understanding of sce for abiotic stress tolerance in plant is still in its infancy.
spartina alterniflora, a halophytic grass commonly known as smooth cordgrass, possesses all the known mechanisms of salt tolerance
<cit> .
in this investigation, we have functionally characterized a sce gene, sasce <dig>  previously obtained from the salinity stressed cdna library of spartina alterniflora <cit> .
sasce <dig> is a functional homolog of sumo conjugating enzyme, ubc <dig> of saccharomyces cerevisiae.
salt, drought, cold, and aba induced the expression of sasce <dig> in leaves and roots of spartina alterniflora.
furthermore, sasce <dig> overexpression in arabidopsis improved abiotic stress tolerance by regulating the stress responsive genes involved in ion homeostasis, proline accumulation, and detoxification of reactive oxygen radicals.
this study demonstrates the potential and superiority of orthologous stress responsive genes from a halophyte grass spartina alterniflora in improving salt and drought stress tolerance in plants.
RESULTS
sasce <dig> of spartina alterniflora is an ortholog of sumo conjugating enzyme
a full length cdna clone of  <dig> bp open reading frame, encoding a sumo conjugating enzyme, named as sasce <dig>  was obtained from the salt stressed cdna library of spartina alterniflora constructed in our laboratory
<cit> .
sasce <dig> gene codes for a polypeptide of  <dig> amino acids with a molecular weight of ~ <dig> kd and isoelectric point  <dig> .
amino acid sequence comparisons of sasce <dig> with its orthologs from other species showed 90-93% similarity with sce of monocots such as triticum durum, oryza sativa, brachypodium distachyon, 85-89% to dicots, nicotiana benthamiana, glycine max, vitis vinifera, medicago truncatula, arabidopsis thaliana, 81% to bryophyte selaginella moellendorffii, 53% to entamoeba histolytica, and 57% with ubc <dig> of saccharomyces cerevisiae .
clustering of sasce <dig> with monocots in phylogenetic analysis and sharing of high degree of homology with counterpart proteins from dicots, yeast, and amoeba, suggested evolutionary conservation of sce proteins and possible evolution from a common ancestor .
we also evaluated the conservation of tertiary structure of sasce <dig> by modeling of predicted tertiary structure based on crystallographic data deposited on the swissprot
<cit> .
comparison of predicted tertiary structure of sasce <dig> with that based on crystallographic analysis of the human counterpart hsubc9
<cit> , revealed the presence of four alpha helices and four beta sheets in sasce <dig> with overall 65% identity with hsubc <dig> .
sasce <dig> functionally complemented ubc <dig> mutant of saccharomyces cerevisiae
we analyzed the catalytically conserved role of sasce <dig> using ubc <dig> mutant strain of s. cerevisiae.
ubc <dig> mutant is temperature sensitive, which normally grows at 25°c but unable to grow at restrictive temperature of 37°c
<cit> .
sasce <dig> orf was cloned into the yeast expression vector pvtl <dig> under the regulation of an endogenous alcohol dehydrogenase  promoter to produce pvtl260-sasce <dig> , transformed into yeast ubc <dig> mutant, and grown on synthetic dropout medium lacking leucine, supplemented with 2% glucose at 25°c as well as at 37°c.
we found that only pvtl260-sasce <dig> transformed ubc <dig> mutant was able to grow at 37°c, but not the un-transformed or only vector  transformed ubc <dig>  at 25°c, growth of all the strains  was normal .
this experiment indicated the functional complementation of ubc <dig> mutant by sasce <dig> .
regulation of expression of sasce <dig> by multiple stresses in spartina alterniflora
quantitative real-time pcr was used to analyze the expression patterns of sasce <dig> in leaves and roots of spartina alterniflora.
as shown in figure
<dig>  sasce <dig> was constitutively expressed in both leaves and roots of spartina alterniflora but differentially expressed by abiotic stresses such as salt, drought, cold, and aba.
under salt stress, sasce <dig> expression was gradually upregulated in leaves and roots starting from  <dig> min up to  <dig> h of stress.
in root, salinity led to highest expression of sasce <dig> within  <dig> h, whereas in leaves it was observed at  <dig> h of stress.
under drought stress in root, transcript induction peaked within  <dig> h, but in leaves it was seen at  <dig> h of drought stress.
under cold stress, expression in leaves began at  <dig> h, peaked at  <dig> h, declines gradually after that, and reached to basal level at  <dig> h. however, in root, transcript accumulation slightly increased after  <dig> min, maintained up to  <dig> h before doubling at  <dig> h, and reached a maximum peak at  <dig> h. aba stress in leaves led to little induction of transcripts up to  <dig> h, then continued to increase up to  <dig> h, whereas root showed early and abrupt increase  of transcript accumulation just after  <dig> min of aba treatment, then increased up to  <dig> h and maintained up to  <dig> h. increased expression of sasce <dig> transcripts by salinity, drought, cold and aba revealed the stress responsive nature and possible aba mediated regulation of sasce <dig> in spartina alterniflora.
overexpression of sasce <dig> improved salinity tolerance
to investigate the possible role of sasce <dig> in imparting salinity tolerance in plant, sasce <dig> orf was cloned into binary vector pcambia <dig> under 35s promoter to produce 35s-sasce <dig> , and was transformed into arabidopsis ecotype columbia by floral dip method.
t <dig> homozygous sasce <dig> transgenic plants were analyzed for stress tolerance.
two transgenic lines  were chosen for further analysis based on their high level expression of sasce <dig> transcripts .
four-week old soil grown wild type  and 35s:sasce <dig> plants were irrigated with  <dig> mm nacl until the salt stress induced injury symptoms such as yellowing of rosette leaves and reduced plant heights were visible.
as shown in figure
4b, 35s-sasce <dig> arabidopsis plants had less visual salt induced stress injury in comparison to wt plants even after three weeks of stress, while wt plants did not survive.
to further evaluate the role of salt stress on ionic balance in 35s-sasce <dig> arabidopsis, leaves from wt and transgenic lines were collected after three weeks of salt stress and na+, k+ concentration was estimated on dry weight basis.
the sasce <dig> transgenic lines, t16- <dig> and t17- <dig>  showed higher k+/na+ ratio under non-stress, and also maintained significantly higher k+/na+ ratio than the wt under salinity stress, which indicated the possible role of sasce <dig> in regulating ion homeostasis .
the sasce <dig> overexpressing transgenic plants accumulated more chlorophyll and proline than the wt under salinity stress .
overexpression of sasce <dig> improved drought tolerance
to further characterize the function of sasce <dig> for drought tolerance, 4-week-old soil grown wt and sasce <dig> transgenic arabidopsis plants were subjected to water stress for  <dig> d. at the 14th day of water withholding, most of the wt plants appeared dehydrated and weak, but the sasce <dig> overexpressing plants grew normally and were healthier than the wt .
to study the membrane stability due to overexpression of sasce <dig>  uniform sized rosette leaves were detached from non-stressed wt and sasce <dig> arabidopsis plants and after seven days of continuous drought stress, electrolyte leakage was measured.
electrolyte leakage was relatively less in t16- <dig>  t17- <dig> plants than wt, under non stress and drought stress .
also, chlorophyll levels in sasce <dig> transgenic plants after withholding water for  <dig> d were significantly higher compared to wt .
similarly, the amount of proline after seven days of drought stress was more in t16- <dig> and t17- <dig> plants compared to the wt .these results showed that overexpression of sasce <dig> improved drought tolerance.
sasce <dig> reduced reactive oxygen species  under drought stress
we assayed the wt and sasce <dig> transgenic arabidopsis plants for detection of ros levels using nitroblue tetrazolium  staining.
leaves from four week old plants after seven days of drought stress and non-stress were incubated into nbt solution.
without drought treatment, leaves of wt and 35s-sasce <dig> transgenic lines, t16- <dig> and t17- <dig>  showed minimal nbt staining indicating low superoxide levels whereas drought stressed wt plants showed higher level of staining than sasce <dig> overexpressing plants .
expression pattern of stress-responsive genes in sasce <dig> transgenic arabidopsis
we further elucidated the possible molecular mechanism of sasce <dig> in stress response by analyzing the expression levels of a set of selected ion transporter genes , stress responsive genes  and antioxidants genes   in sasce <dig> transgenic arabidopsis plants.
transcript expression levels for genes belonging to all the three categories were upregulated in sasce <dig> transgenic plants compared to the wt plants .
discussion
sumoylation pathway is involved in post translational modification of proteins through employment of three key enzymes, sumo activating enzyme , sumo conjugating enzyme , and sumo ligase  by regulating gene expression, nuclear localization, and signal transduction
<cit> .
sumoylation pathway begins with the activation of the sumo c-terminus through exposing glycine by a sumo-specific e <dig> activating enzyme.
activated sumo is subsequently transferred to the cysteine residue of a sumo-conjugating enzyme e <dig>  and then with the help of an e <dig> ligase, sumo is finally conjugated to the target lysine residue of a substrate protein
<cit> .
in arabidopsis, sumoylation of proteins has been reported in environmental response, flowering time regulation, and phosphorus starvation
<cit> .
members of sumoylation pathway such as atsce <dig> , atsiz <dig> , atsumo1and atsumo <dig> have been identified and found to be involved in aba signaling and drought response in arabidopsis <cit> .
in rice, regulation of sumo conjugating enzyme genes by temperature stress has also been reported
<cit> .
but whether sumo conjugating enzyme imparts stress tolerance to plant is still not clear.
in this study, we characterized a sumo conjugating enzyme gene “sasce9” from a halophytic grass, spartina alterniflora, and investigated its role in abiotic stress tolerance mechanisms in plant.
sasce <dig>  an ortholog of sce, induced by multiple stresses in halophytic grass spartina alterniflora
we have identified a sumo conjugating enzyme gene, sasce <dig> from spartina alterniflora, which has same sequence characteristics as the reported yeast sumo conjugating enzyme, ubc <dig>  the deduced protein sequence analysis of this gene revealed the presence of high structural homology with sce orthologs from amoeba, yeast, and plants, suggesting evolutionary conserved function of sce across the eukaryotes.
predicted tertiary structure of sasce <dig> using swiss-model software suggested the presence of four alpha helices and four beta sheets similar to the known x-ray crystal structure of sce, hssce <dig> of human
<cit> .
to further validate the functionally conserved role of sasce <dig>  a temperature sensitive mutant strain of saccharomyces cerevisiae, ubc9-ts was used
<cit> .
sasce <dig> was able to functionally complement the ubc <dig> mutant of yeast, indicating the potential evolutionary conserved function of sasce <dig> between spartina and yeast.
there are several reports for functional complementation of ubc <dig> mutant phenotype by expression of dmubc <dig> from d. melanogaster <cit> , nbubc <dig> from n. benthamiana <cit> , hsubc <dig> from h. sapiens <cit> , and ossce <dig> from o. sativa <cit> .
sumoylation status of the proteins involved in stress response is reported to be altered as they travel between the nucleus and the cytosol
<cit> .
as the sasce <dig> gene was obtained from salinity stressed library of spartina alterniflora, we further wanted to check its organ specific inducibility in leaves and roots of spartina alterniflora under salt, drought, cold, and exogenously supplied aba stress.
interestingly, the increased expression of sasce <dig> transcript was observed under all the stresses, as shown by qrt-pcr, with highest early induction  found in leaf by salinity and drought and in root by drought and aba, suggesting the stress responsive transcript accumulation of sasce <dig> in spartina alterniflora.
the inducibility of sasce <dig> gene by aba indicated that the expression of sasce <dig> may be regulated through aba responsive pathway in spartina alterniflora.
aba mediates the responses of plant to stress conditions such as salinity, drought, and cold
<cit> .
moreover, salt, cold, and aba stress induced accumulation of sumo conjugates and high expression of atsce1a has been observed in different tissues of arabidopsis <cit> .
ossce <dig> was found to be induced by temperature stress in the seedlings of rice
<cit> .
chaikam and karlson
<cit>  reported developmental and stress responsive regulation of sumo cascade components with highest level of expression in reproductive tissues suggesting possible role of sumoylation during flowering in rice.
sasce <dig> enhances salinity and drought tolerance in arabidopsis
considering the stress inducible response of sasce <dig> in spartina alterniflora, sasce <dig> gene was further tested for its role in-planta towards abiotic stress.
under normal condition, growth and morphology of wt, sasce <dig> transgenic lines were similar.
under  <dig> mm nacl stress, both transgenic lines could grow and set seed successfully, while most of the wt plants died.
higher k+/na+ ratio in sasce <dig> transgenic lines under non-stress and salinity stress suggested improved basal uptake of k+ due to overexpression of sasce <dig>  several studies have demonstrated the importance of increased k+/na+ ratio for tolerance of plants against salinity stress
<cit> .
enhanced expression of two ion transporters, atnhx <dig> and atsos <dig>  in sasce <dig> transgenic plants compared to wt thus confirmed our hypothesis for the preferential uptake of k+ by sasce <dig> transgenic plants.
under continuous drought stress, sasce <dig> transgenic plants were healthy, turgid, and green, while wt plants lost its vigor and growth.
sasce <dig> expression improved drought tolerance of sasce <dig> arabidopsis by maintaining membrane stability as its electrolyte leakage was lower than the wt.
retention of chlorophyll in plants under stress is an important aspect for increased stress tolerance.
this study demonstrated that the increased level of chlorophyll under salinity and drought stresses might be an important mechanism for the improvement of stress tolerance due to expression of sasce <dig> in arabidopsis.
sasce <dig> enhances abiotic stress tolerance through scavenging oxygen radicals and proline production
proline plays a vital role in maintaining osmotic balance and stabilizing cellular structures in plants
<cit> .
increased free proline content in transgenic arabidopsis under salinity and drought stress conditions indicated the role of sasce <dig> in proline accumulation, thereby protecting the plants against the stress.
enhanced expression of p5cs <dig>  in sasce <dig> plants compared to the wt plants further supported our hypothesis for the role of sasce <dig> in osmotic stress tolerance.
in an earlier report, transgenic plants overexpressing the p5cs gene from vigna aconitifolia accumulated more proline and were more tolerant to osmotic stress
<cit> .
furthermore, histochemical staining of leaves using nbt showed that even without stress o2- could be stained, but the transgenic lines had lower levels of ros  relative to wt.
drought stress resulted in increased ros levels in wt but transgenic lines accumulated remarkably less o2- as evidenced by the accumulation of less blue products.
ros were produced in both non-stress and stress conditions but the balance between the production and removal of ros determines its effect on cellular system
<cit> .
plants evolve a complex antioxidant system in order to detoxify stress-induced ros in which ros scavenging enzymes such as superoxide dismutase and peroxidase play essential role
<cit> .
increased expression of atsod <dig> and atcat in sasce <dig> transgenic plants might be responsible for scavenging ros produced under drought stress in this study.
these analyses further validated the role of sasce <dig> for improving stress tolerance in plant by affecting different stress related pathways.
our observation clearly showed the indirect role of sasce <dig> in scavenging of oxygen radicals resulting in protection of plant against osmotic stress.
CONCLUSIONS
this study demonstrated for the first time that manipulation of a sasce <dig> gene, a member of the sumoylation pathway, through transgenic approach, can lead to improved abiotic stress tolerance in plants.
although, lois et al., <cit>  developed atsce1a expressing transgenic arabidopsis plants, the focus of their study was to correlate the level of atsce1a expression with the level of sumoylation which in turn attenuates aba-mediated growth inhibition and induces aba- and stress-responsive genes.
thus, we hypothesize that overexpression of sasce <dig> might have increased the sumoylation status in sasce <dig> transgenic plants and improved the stress tolerance by regulating stress responsive genes.
further investigations on identifying interacting partner of sumoylation pathway members and its target protein by comparing sce mutant would reveal the exact molecular mechanisms for possible role in imparting stress tolerance through manipulation of post translational modifications in plant.

 plants are constantly challenged by a wide range of environmental stresses such as drought, high salinity, and temperature fluctuations due to their sessile nature. response to abiotic stresses is very complex, as various stages of plant growth and development can be affected by a particular stress and often several stresses simultaneously. molecular responses to abiotic stresses include stress perception, signal transduction to cellular components, gene expression, and metabolic changes, which help the plants to adapt to stress environments. post-translational modifications of proteins play an important role in most cellular processes by rapidly altering the functions of preexisting proteins and protein complexes. sumoylation or sumo conjugation is one of the essential post translational regulatory process essentially found in all eukaryotes. it is a three step enzymatic cascade mediated by sumo activating enzyme, sumo conjugating enzyme, and sumo ligase for the attachment of sumo to its target protein. sumo plays an important role in regulation of protein–protein interactions and subcellular locations in yeast and animals. increased level of sumo conjugates in response to heat shock, oxidative stress, and dna damaging agents has been reported in mammalian cell cultures. similarly, increased accumulation of sumo conjugates upon exposure of arabidopsis seedlings to heat shock, h2o ethanol, and amino acid analog canavanine suggests its important role in stress response and protection in plants. several studies in arabidopsis highlighted the importance of sumoylation in post translational regulation in response to stresses such as salt, cold, drought, heat, copper toxicity, and nutrient deprivation. importance of sumoylation for abiotic stress response is strengthened from the reports on drastic reduction in tolerance to stresses due to mutation of genes involved in sumo conjugation. further, embryo lethality caused by insertional mutation in atsae or atsce or double mutations in atsumo and atsumo genes of arabidopsis inferred its role in plant growth and development.the reversible conjugation of the sumo peptide to protein substrates is emerging as a major post-translational regulatory process in plants. arabidopsis sumo and related enzymes have been implicated in abscisic acid responses, flowering time regulation as well as stress responses. in rice, transcripts for the sce is regulated by cold, salt, aba, and heat. however, detailed understanding of sce for abiotic stress tolerance in plant is still in its infancy. spartina alterniflora, a halophytic grass commonly known as smooth cordgrass, possesses all the known mechanisms of salt tolerance. in this investigation, we have functionally characterized a sce gene, sasce previously obtained from the salinity stressed cdna library of spartina alterniflora. sasce is a functional homolog of sumo conjugating enzyme, ubc of saccharomyces cerevisiae. salt, drought, cold, and aba induced the expression of sasce in leaves and roots of spartina alterniflora. furthermore, sasce overexpression in arabidopsis improved abiotic stress tolerance by regulating the stress responsive genes involved in ion homeostasis, proline accumulation, and detoxification of reactive oxygen radicals. this study demonstrates the potential and superiority of orthologous stress responsive genes from a halophyte grass spartina alterniflora in improving salt and drought stress tolerance in plants.  sasce of spartina alterniflora is an ortholog of sumo conjugating enzyme a full length cdna clone of bp open reading frame, encoding a sumo conjugating enzyme, named as sasce was obtained from the salt stressed cdna library of spartina alterniflora constructed in our laboratory. sasce gene codes for a polypeptide of amino acids with a molecular weight of ~ kd and isoelectric point. amino acid sequence comparisons of sasce with its orthologs from other species showed 90-93% similarity with sce of monocots such as triticum durum, oryza sativa, brachypodium distachyon, 85-89% to dicots, nicotiana benthamiana, glycine max, vitis vinifera, medicago truncatula, arabidopsis thaliana, 81% to bryophyte selaginella moellendorffii, 53% to entamoeba histolytica, and 57% with ubc of saccharomyces cerevisiae. clustering of sasce with monocots in phylogenetic analysis and sharing of high degree of homology with counterpart proteins from dicots, yeast, and amoeba, suggested evolutionary conservation of sce proteins and possible evolution from a common ancestor. we also evaluated the conservation of tertiary structure of sasce by modeling of predicted tertiary structure based on crystallographic data deposited on the swissprot. comparison of predicted tertiary structure of sasce with that based on crystallographic analysis of the human counterpart hsubc9, revealed the presence of four alpha helices and four beta sheets in sasce with overall 65% identity with hsubc. sasce functionally complemented ubc mutant of saccharomyces cerevisiae we analyzed the catalytically conserved role of sasce using ubc mutant strain of s. cerevisiae. ubc mutant is temperature sensitive, which normally grows at 25°c but unable to grow at restrictive temperature of 37°c. sasce orf was cloned into the yeast expression vector pvtl under the regulation of an endogenous alcohol dehydrogenase promoter to produce pvtl260-sasce, transformed into yeast ubc mutant, and grown on synthetic dropout medium lacking leucine, supplemented with 2% glucose at 25°c as well as at 37°c. we found that only pvtl260-sasce transformed ubc mutant was able to grow at 37°c, but not the un-transformed or only vector transformed ubc at 25°c, growth of all the strains was normal. this experiment indicated the functional complementation of ubc mutant by sasce. regulation of expression of sasce by multiple stresses in spartina alterniflora quantitative real-time pcr was used to analyze the expression patterns of sasce in leaves and roots of spartina alterniflora. as shown in figure  sasce was constitutively expressed in both leaves and roots of spartina alterniflora but differentially expressed by abiotic stresses such as salt, drought, cold, and aba. under salt stress, sasce expression was gradually upregulated in leaves and roots starting from min up to h of stress. in root, salinity led to highest expression of sasce within h, whereas in leaves it was observed at h of stress. under drought stress in root, transcript induction peaked within h, but in leaves it was seen at h of drought stress. under cold stress, expression in leaves began at h, peaked at h, declines gradually after that, and reached to basal level at h. however, in root, transcript accumulation slightly increased after min, maintained up to h before doubling at h, and reached a maximum peak at h. aba stress in leaves led to little induction of transcripts up to h, then continued to increase up to h, whereas root showed early and abrupt increase of transcript accumulation just after min of aba treatment, then increased up to h and maintained up to h. increased expression of sasce transcripts by salinity, drought, cold and aba revealed the stress responsive nature and possible aba mediated regulation of sasce in spartina alterniflora. overexpression of sasce improved salinity tolerance to investigate the possible role of sasce in imparting salinity tolerance in plant, sasce orf was cloned into binary vector pcambia under 35s promoter to produce 35s-sasce, and was transformed into arabidopsis ecotype columbia by floral dip method. t homozygous sasce transgenic plants were analyzed for stress tolerance. two transgenic lines were chosen for further analysis based on their high level expression of sasce transcripts. four-week old soil grown wild type and 35s:sasce plants were irrigated with mm nacl until the salt stress induced injury symptoms such as yellowing of rosette leaves and reduced plant heights were visible. as shown in figure  4b, 35s-sasce arabidopsis plants had less visual salt induced stress injury in comparison to wt plants even after three weeks of stress, while wt plants did not survive. to further evaluate the role of salt stress on ionic balance in 35s-sasce arabidopsis, leaves from wt and transgenic lines were collected after three weeks of salt stress and na+, k+ concentration was estimated on dry weight basis. the sasce transgenic lines, t16- and t17- showed higher k+/na+ ratio under non-stress, and also maintained significantly higher k+/na+ ratio than the wt under salinity stress, which indicated the possible role of sasce in regulating ion homeostasis. the sasce overexpressing transgenic plants accumulated more chlorophyll and proline than the wt under salinity stress. overexpression of sasce improved drought tolerance to further characterize the function of sasce for drought tolerance, 4-week-old soil grown wt and sasce transgenic arabidopsis plants were subjected to water stress for d. at the 14th day of water withholding, most of the wt plants appeared dehydrated and weak, but the sasce overexpressing plants grew normally and were healthier than the wt. to study the membrane stability due to overexpression of sasce uniform sized rosette leaves were detached from non-stressed wt and sasce arabidopsis plants and after seven days of continuous drought stress, electrolyte leakage was measured. electrolyte leakage was relatively less in t16- t17- plants than wt, under non stress and drought stress. also, chlorophyll levels in sasce transgenic plants after withholding water for d were significantly higher compared to wt. similarly, the amount of proline after seven days of drought stress was more in t16- and t17- plants compared to the wt.these results showed that overexpression of sasce improved drought tolerance. sasce reduced reactive oxygen species under drought stress we assayed the wt and sasce transgenic arabidopsis plants for detection of ros levels using nitroblue tetrazolium staining. leaves from four week old plants after seven days of drought stress and non-stress were incubated into nbt solution. without drought treatment, leaves of wt and 35s-sasce transgenic lines, t16- and t17- showed minimal nbt staining indicating low superoxide levels whereas drought stressed wt plants showed higher level of staining than sasce overexpressing plants. expression pattern of stress-responsive genes in sasce transgenic arabidopsis we further elucidated the possible molecular mechanism of sasce in stress response by analyzing the expression levels of a set of selected ion transporter genes, stress responsive genes and antioxidants genes in sasce transgenic arabidopsis plants. transcript expression levels for genes belonging to all the three categories were upregulated in sasce transgenic plants compared to the wt plants. discussion sumoylation pathway is involved in post translational modification of proteins through employment of three key enzymes, sumo activating enzyme, sumo conjugating enzyme, and sumo ligase by regulating gene expression, nuclear localization, and signal transduction. sumoylation pathway begins with the activation of the sumo c-terminus through exposing glycine by a sumo-specific e activating enzyme. activated sumo is subsequently transferred to the cysteine residue of a sumo-conjugating enzyme e and then with the help of an e ligase, sumo is finally conjugated to the target lysine residue of a substrate protein. in arabidopsis, sumoylation of proteins has been reported in environmental response, flowering time regulation, and phosphorus starvation. members of sumoylation pathway such as atsce, atsiz, atsumo1and atsumo have been identified and found to be involved in aba signaling and drought response in arabidopsis. in rice, regulation of sumo conjugating enzyme genes by temperature stress has also been reported. but whether sumo conjugating enzyme imparts stress tolerance to plant is still not clear. in this study, we characterized a sumo conjugating enzyme gene “sasce9” from a halophytic grass, spartina alterniflora, and investigated its role in abiotic stress tolerance mechanisms in plant. sasce an ortholog of sce, induced by multiple stresses in halophytic grass spartina alterniflora we have identified a sumo conjugating enzyme gene, sasce from spartina alterniflora, which has same sequence characteristics as the reported yeast sumo conjugating enzyme, ubc the deduced protein sequence analysis of this gene revealed the presence of high structural homology with sce orthologs from amoeba, yeast, and plants, suggesting evolutionary conserved function of sce across the eukaryotes. predicted tertiary structure of sasce using swiss-model software suggested the presence of four alpha helices and four beta sheets similar to the known x-ray crystal structure of sce, hssce of human. to further validate the functionally conserved role of sasce a temperature sensitive mutant strain of saccharomyces cerevisiae, ubc9-ts was used. sasce was able to functionally complement the ubc mutant of yeast, indicating the potential evolutionary conserved function of sasce between spartina and yeast. there are several reports for functional complementation of ubc mutant phenotype by expression of dmubc from d. melanogaster, nbubc from n. benthamiana, hsubc from h. sapiens, and ossce from o. sativa. sumoylation status of the proteins involved in stress response is reported to be altered as they travel between the nucleus and the cytosol. as the sasce gene was obtained from salinity stressed library of spartina alterniflora, we further wanted to check its organ specific inducibility in leaves and roots of spartina alterniflora under salt, drought, cold, and exogenously supplied aba stress. interestingly, the increased expression of sasce transcript was observed under all the stresses, as shown by qrt-pcr, with highest early induction found in leaf by salinity and drought and in root by drought and aba, suggesting the stress responsive transcript accumulation of sasce in spartina alterniflora. the inducibility of sasce gene by aba indicated that the expression of sasce may be regulated through aba responsive pathway in spartina alterniflora. aba mediates the responses of plant to stress conditions such as salinity, drought, and cold. moreover, salt, cold, and aba stress induced accumulation of sumo conjugates and high expression of atsce1a has been observed in different tissues of arabidopsis. ossce was found to be induced by temperature stress in the seedlings of rice. chaikam and karlson reported developmental and stress responsive regulation of sumo cascade components with highest level of expression in reproductive tissues suggesting possible role of sumoylation during flowering in rice. sasce enhances salinity and drought tolerance in arabidopsis considering the stress inducible response of sasce in spartina alterniflora, sasce gene was further tested for its role in-planta towards abiotic stress. under normal condition, growth and morphology of wt, sasce transgenic lines were similar. under mm nacl stress, both transgenic lines could grow and set seed successfully, while most of the wt plants died. higher k+/na+ ratio in sasce transgenic lines under non-stress and salinity stress suggested improved basal uptake of k+ due to overexpression of sasce several studies have demonstrated the importance of increased k+/na+ ratio for tolerance of plants against salinity stress. enhanced expression of two ion transporters, atnhx and atsos in sasce transgenic plants compared to wt thus confirmed our hypothesis for the preferential uptake of k+ by sasce transgenic plants. under continuous drought stress, sasce transgenic plants were healthy, turgid, and green, while wt plants lost its vigor and growth. sasce expression improved drought tolerance of sasce arabidopsis by maintaining membrane stability as its electrolyte leakage was lower than the wt. retention of chlorophyll in plants under stress is an important aspect for increased stress tolerance. this study demonstrated that the increased level of chlorophyll under salinity and drought stresses might be an important mechanism for the improvement of stress tolerance due to expression of sasce in arabidopsis. sasce enhances abiotic stress tolerance through scavenging oxygen radicals and proline production proline plays a vital role in maintaining osmotic balance and stabilizing cellular structures in plants. increased free proline content in transgenic arabidopsis under salinity and drought stress conditions indicated the role of sasce in proline accumulation, thereby protecting the plants against the stress. enhanced expression of p5cs in sasce plants compared to the wt plants further supported our hypothesis for the role of sasce in osmotic stress tolerance. in an earlier report, transgenic plants overexpressing the p5cs gene from vigna aconitifolia accumulated more proline and were more tolerant to osmotic stress. furthermore, histochemical staining of leaves using nbt showed that even without stress o2- could be stained, but the transgenic lines had lower levels of ros relative to wt. drought stress resulted in increased ros levels in wt but transgenic lines accumulated remarkably less o2- as evidenced by the accumulation of less blue products. ros were produced in both non-stress and stress conditions but the balance between the production and removal of ros determines its effect on cellular system. plants evolve a complex antioxidant system in order to detoxify stress-induced ros in which ros scavenging enzymes such as superoxide dismutase and peroxidase play essential role. increased expression of atsod and atcat in sasce transgenic plants might be responsible for scavenging ros produced under drought stress in this study. these analyses further validated the role of sasce for improving stress tolerance in plant by affecting different stress related pathways. our observation clearly showed the indirect role of sasce in scavenging of oxygen radicals resulting in protection of plant against osmotic stress.  this study demonstrated for the first time that manipulation of a sasce gene, a member of the sumoylation pathway, through transgenic approach, can lead to improved abiotic stress tolerance in plants. although, lois et al., developed atsce1a expressing transgenic arabidopsis plants, the focus of their study was to correlate the level of atsce1a expression with the level of sumoylation which in turn attenuates aba-mediated growth inhibition and induces aba- and stress-responsive genes. thus, we hypothesize that overexpression of sasce might have increased the sumoylation status in sasce transgenic plants and improved the stress tolerance by regulating stress responsive genes. further investigations on identifying interacting partner of sumoylation pathway members and its target protein by comparing sce mutant would reveal the exact molecular mechanisms for possible role in imparting stress tolerance through manipulation of post translational modifications in plant. 
in addition, this study demonstrates the potential of the halophyte grass s. alterniflora as a reservoir of abiotic stress related genes for crop improvement. amino acid sequence comparisons of sasce with its orthologs from other plant species showed high degree of structural conservation among each other. these results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. expression analysis of stress responsive genes in sasce arabidopsis plants revealed the increased expression of antioxidant genes, atsod and atcat, ion antiporter genes, atnhx and atsos a gene involved in proline biosynthesis, atp5cs, and a gene involved in aba dependent signaling pathway, atrd the study provides evidence that the overexpression of sasce in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ros, accumulation of an osmolyte, proline, and expression of stress responsive genes. sasce overexpressing arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. although the mechanism for regulation of sumo conjugation pathway genes under abiotic stress has been studied to certain extent, the role of sumo conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. here, we have characterized a sumo conjugating enzyme gene ‘sasce9’ from a halophytic grass spartina alterniflora and investigated its role in imparting abiotic stress tolerance. abiotic stress tolerancehalophytestress induciblesumo conjugating genespartina alterniflora constitutive overexpression of sasce in arabidopsis through agrobacterium mediated transformation improved salinity and drought tolerance of arabidopsis. sumo conjugation is a post translational regulatory process found in all eukaryotes, mediated by sumo activating enzyme, sumo conjugating enzyme, and sumo ligase for the attachment of sumo to its target protein. sasce transgenic plants accumulated lower levels of reactive oxygen under salinity stress. sasce gene encodes for a polypeptide of amino acids with a molecular weight of ~ kd and isoelectric point.
 sumo conjugation is a post translational regulatory process found in all eukaryotes, mediated by sumo activating enzyme, sumo conjugating enzyme, and sumo ligase for the attachment of sumo to its target protein. although the mechanism for regulation of sumo conjugation pathway genes under abiotic stress has been studied to certain extent, the role of sumo conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. here, we have characterized a sumo conjugating enzyme gene ‘sasce9’ from a halophytic grass spartina alterniflora and investigated its role in imparting abiotic stress tolerance.  sasce gene encodes for a polypeptide of amino acids with a molecular weight of ~ kd and isoelectric point. amino acid sequence comparisons of sasce with its orthologs from other plant species showed high degree of structural conservation among each other. complementation analysis using yeast sce mutant, ubc revealed functional conservation of sasce between yeast and s. alterniflora. sasce transcript was inducible by salinity, drought, cold, and exogenously supplied aba both in leaves and roots of s. alterniflora. constitutive overexpression of sasce in arabidopsis through agrobacterium mediated transformation improved salinity and drought tolerance of arabidopsis. sasce overexpressing arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. sasce transgenic plants accumulated lower levels of reactive oxygen under salinity stress. expression analysis of stress responsive genes in sasce arabidopsis plants revealed the increased expression of antioxidant genes, atsod and atcat, ion antiporter genes, atnhx and atsos a gene involved in proline biosynthesis, atp5cs, and a gene involved in aba dependent signaling pathway, atrd  these results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. the study provides evidence that the overexpression of sasce in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ros, accumulation of an osmolyte, proline, and expression of stress responsive genes. in addition, this study demonstrates the potential of the halophyte grass s. alterniflora as a reservoir of abiotic stress related genes for crop improvement. abiotic stress tolerancehalophytestress induciblesumo conjugating genespartina alterniflora 