BACKGROUND
environmental stresses, such as drought and high salinity, can cause severe damage to plants, leading to considerable reduction in their productivity.
to survive under such conditions, plants have developed a series of defense responsive pathways.
among them, ca2+-dependent signaling leads to the activation of late embryogenesis abundant -type genes, which may function in protection and damage repair of plants  <cit> .
lea proteins were first identified in cotton seeds  <cit> .
the proteins accumulated to high levels in the late stages of seed development  <cit> .
subsequently, they were found to be expressed in vegetative tissues  <cit>  and could be induced by abscisic acid  and various abiotic stresses, such as drought and cold  <cit> .
with the development of deep sequencing technology, an increasing number of lea proteins have been identified.
on the basis of their amino acid sequence similarities and conserved motifs, lea proteins are categorized into different groups  <cit> .
in this work, we adopt the classification introduced by battaglia’s group, in which lea proteins are categorized into seven distinct families  <cit> .
groups  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  which share specific motifs within each group, are considered to be hydrophilic or “typical” lea proteins.
conversely, group  <dig> corresponds to atypical lea proteins.
this group includes all lea proteins with higher content of hydrophobic residues than typical lea proteins.
on the basis of their sequence similarity, group  <dig> lea proteins are divided into the subgroups 5a, 5b, and 5c, corresponding to the first described proteins d- <dig>  d- <dig>  and d- <dig>  <cit>  in this group, respectively.
physicochemical properties show that group  <dig> lea proteins are not soluble after boiling, suggesting that they may adopt a globular conformation and are not heat stable  <cit> .
subsequent reports show that subgroup 5c lea proteins are natively folded and have more β-sheets than α-helixes  <cit> , which is different from subgroups 5a and 5b lea proteins that are intrinsically unstructured  <cit> .
for example, arabidopsis lea <dig> has an αβ-fold consisting of one α-helix and seven β-strands that form two antiparallel β-sheets as determined by nuclear magnetic resonance spectroscopy  <cit> .
moreover, subgroup 5c lea proteins have other outstanding characteristics, such as lower instability index, narrower range of gravy values, and lower proportion of polar  and small residues, but higher proportion of non-polar residues than subgroup 5a and 5b members  <cit> .
all of these differences indicate that subgroup 5c lea proteins may function differently from other lea proteins in plants.
at present, only a small number of subgroup 5c lea genes have been characterized.
their transcripts can be upregulated in response to diverse stresses, as reported for cotton lea14-a  <cit> , craterostigma plantagineum pcc27- <dig>  <cit> , soybean d95- <dig>  <cit> , tomato er <dig>  <cit> , hot pepper calea <dig>  <cit> , arabidopsis lea <dig>  <cit>  and at2g <dig>  <cit> , and sweetpotato iblea <dig>  <cit> .
overexpression of calea <dig> in tobacco improves tolerance to dehydration and nacl but not to low temperature  <cit> .
transgenic sweetpotato non-embryogenic calli that overexpress iblea <dig> show increased tolerance to drought and salt stress by enhancing lignification  <cit> .
recently, rice oslea <dig> has been reported to enhance resistance against diverse abiotic stresses in recombinant escherichia coli cells.
in vitro analysis showed that oslea <dig> was able to protect lactate dehydrogenase from aggregation under different abiotic stresses  <cit> .
all these results suggest that subgroup 5c lea proteins are closely associated with resistance to multiple abiotic stresses.
foxtail millet  beauv.
), a member of the poaceae family, has a long history in cultivation of about 7000 years.
it has been widely planted in northern china and other asian countries.
recently, a draft genome sequence for foxtail millet has been completed  <cit> , which enables foxtail millet to be a tractable experimental grass model  <cit> .
as a diploid c <dig> panicoid crop species, foxtail millet is well known for its remarkable drought resistance.
however, the molecular mechanisms underlying this tolerance are not well defined.
in this study, we isolated and functionally characterized a novel member of the atypical subgroup 5c lea gene, silea <dig>  from foxtail millet.
the expression of silea <dig> was induced by aba, polyethylene glycol  and nacl.
overexpression of silea <dig> resulted in enhanced resistance to abiotic stresses in e. coli, arabidopsis and foxtail millet.
the silea <dig> promoter mediated remarkable induction of β-glucuronidase  expression in transgenic arabidopsis under various stresses.
cis-acting regulatory elements in the silea <dig> promoter were also predicted.
these data reveal the potential application of silea <dig> in the genetic engineering of other crops.
RESULTS
silea <dig> is an atypical lea protein
the full-length sequence of silea <dig> was determined by 5′ and 3′ rapid amplification of cdna ends  .
the sequence is 821 bp in length, with a 100 bp 5′ untranslated region  and a 208 bp 3′ utr  .
silea <dig> harbors two exons separated by an intron , and encodes an open reading frame of  <dig> aa with a predicted molecular mass of  <dig>  kd and pi of  <dig> .
it is rich in ser , lys , and ile , but contains low quantities of trp , asn , cys , and gln .
three hydrophobic regions  were identified in the silea <dig> protein .
a motif search of the silea <dig> protein in interproscan revealed that it contains a “lea_2” motif , which was classified into subgroup 5c  according to battaglia’s classification of lea proteins  <cit> .
further analysis showed that silea <dig> contains a lower percentage of polar amino acids  and higher percentage of non-polar amino acids  than other group lea proteins   <cit> .
the gravy value and the instability index of silea <dig> is − <dig>  and  <dig> , respectively .
all these characteristics were in accordance with those of other members of subgroup 5c  <cit> .
two additional silea <dig> homologs  existed in foxtail millet.
they showed  <dig> % and  <dig> % identity to silea <dig>  respectively.
homologous sequences of silea <dig> were also present in other plant species, such as rice oslea <dig> , maize lea14-a  and arabidopsis atlea <dig> .
the homologs shared different levels of sequence similarities, indicating different levels of evolutionary relationship among these proteins .
a phylogenetic tree for silea <dig> and its homologs was constructed from protein sequences .
the proteins were mainly separated into two clades comprising dicot and monocot species, respectively.
the silea <dig> protein shared the closest relationship with the pearl millet lea-like protein, followed by oslea <dig> .
sequence logos were produced to examine the conservation level at each residue position.
the sequences lacked significant signature motifs or consensus sequences, except for several conserved residues at specific positions, such as proline at positions  <dig> and  <dig> .
this is a common feature of subgroup 5c lea proteins  <cit> .figure  <dig>
multiple sequence alignment and phylogenetic relationship for the silea <dig> protein and its homologs.
multiple sequence alignment of silea <dig> with its homologs from various plant species.
the conserved “lea_2” motif  is boxed.
phylogenetic relationships of silea <dig> and its homologs.
the divergence of the clades between the monocots and dicots is highlighted.
silea <dig> and its two homologs in foxtail millet are highlighted by solid dark circle and triangle, respectively.
the genbank accession numbers are as follows: silea <dig> , atlea <dig> , lea14-a , d95- <dig> , iblea <dig> , er <dig> , lemmi <dig> , calea <dig> , oslea <dig> , pcc27- <dig> , pclea <dig> , at1g <dig> , lea14-a , lea <dig> homolog , at2g <dig> , os01g <dig> , lea14-a-like , loc <dig> , sorbidraft 09 g <dig> , lea-like protein , osi <dig> , os05g <dig> , os05g <dig> , at2g <dig> , loc <dig>  and umc <dig> .
the sequences used for alignment and phylogenetic tree construction were shown in additional file  <dig>
subcellular localization of silea14
to investigate the potential role of silea <dig>  we examined the subcellular localization of silea <dig> fused to gfp and gfp alone  in onion epidermal cells and foxtail millet root protoplasts.
the constructs used were shown in figure 2a.
when observed by confocal microscopy, the green fluorescent protein  fluorescence of silea14-gfp was distributed throughout the cell, consistent with the gfp control, both in the onion cells  and the foxtail millet root protoplasts .
these results indicated that silea <dig> was localized in the cytosol.figure  <dig>
subcellular localization of silea <dig> protein.
diagram of the constructs of prok219-gfp and prok219-silea14-gfp used for subcellular localization.
35s-pro, cauliflower mosaic virus 35s promoter; silea <dig>  the silea <dig> coding region without the stop codon; gfp, green fluorescent protein; nos, nopaline synthase terminator.
fluorescent microscopic images of gfp and silea14-gfp fusion protein in the onion epidermal cells   and in the foxtail millet root protoplasts  .
silea <dig> expression profiles under normal and stress conditions
quantitative reverse-transcription pcr  was carried out to reveal the temporal and spatial expression of silea <dig> in foxtail millet .
silea <dig> was expressed at the highest level in roots, followed by stems and leaves, and at the lowest level in inflorescences.
after pollination, silea <dig> transcription was gradually upregulated with seed maturation, which indicated a potential role for silea <dig> in the maturation and desiccation phases of foxtail millet seed development.figure  <dig>
silea14
expression patterns in foxtail millet determined by qrt-pcr.
tissue-specific expression of silea <dig>  total rna was extracted from roots , stems , leaves , inflorescences  and seeds at  <dig> ,  <dig>  and  <dig>  days after pollination, respectively.
foxtail millet actin <dig> was used as an internal control.
silea <dig> transcripts accumulation in response to various treatments.
two-week-old foxtail millet seedlings were treated with 100 μm aba , 20%  peg  <dig>  and 250 mm nacl  for the indicated times, respectively.
total rna was extracted from the above-ground parts of ten plants at the indicated times after the treatments.
data represent means and standard errors for three biological replicates.
the expression level of silea <dig> was also examined by qrt-pcr under various stresses.
under aba treatment, silea <dig> transcription abruptly reached its highest level  after 1 h treatment, and then decreased gradually and reverted almost to the control level at 24 h .
under peg stress, silea <dig> transcription was rapidly induced within  <dig>  h and increased to a peak level  at 12 h .
salt treatment resulted in increased accumulation of silea <dig> to a maximum level  only after 3 h .
these results indicated that silea <dig> might play important roles in the responses to salt and drought stresses.
silea <dig> enhances salt tolerance in transformed e. coli
in both prokaryotes and eukaryotes lea proteins might have similar protective mechanisms  <cit> .
therefore, the up-regulation of silea <dig> in response to nacl prompted us to evaluate the salt stress tolerance of recombinant e. coli overexpressing silea <dig> .
there are no significant differences in colony number between transformed e. coli harboring silea <dig> and the control under normal conditions, indicating that overexpression of silea <dig> did not affect the growth of e. coli recombinants in non-stress conditions.
however, when grown on luria–bertani  plates supplemented with 600 mm nacl, the number of transformed cells was much higher than that of the control.
a similar result was obtained on lb medium supplemented with 600 mm kcl.
these results demonstrated that overexpression of silea <dig> in e. coli significantly enhanced tolerance to salt stress.figure  <dig>
assay for salt stress tolerance of silea <dig> transformed
e. coli
.
transformant e. coli  with pet30a-silea <dig> were grown on lb plates with or without 600 mm nacl and kcl for  <dig> and 24 h, respectively.
transformant with empty vector pet30a was used as a control.
the number of colonies on the plate was calculated.
the 200×, 500×, 1000×, 2000× and 4000× represent the dilution fold.
bar = 1 cm.
data represent means and standard errors for three biological replicates.
statistical significance was determined by student’s t-test.
**p <  <dig> .
silea <dig> improves the abiotic stress resistance of transgenic arabidopsis
to assess the function of silea <dig> in plants, it was overexpressed in arabidopsis under the control of super promoter .
at least  <dig> transgenic plants were obtained, and two independent homozygous t <dig> transgenic lines  with high expression levels of silea <dig>  were chosen for further investigation.figure  <dig>
assay for salinity and mannitol stress tolerance of
silea14
overexpression arabidopsis.
diagram of the t-dna region of the binary vector ps1300-silea <dig>  lb, left border; rb, right border; 35s-pro, cauliflower mosaic virus 35s promoter; hpt, hygromycin b phosphotransferase gene; super-pro, super promoter; silea <dig>  silea <dig> gene; nos, nopaline synthase terminator.
rt-pcr analysis of silea <dig> in transgenic arabidopsis lines .
wt was used as a control.
actin <dig> was a loading control.
the phenotype of transgenic and wt arabidopsis germinated on ms, ms + 125 mm nacl and ms + 250 mm mannitol, respectively, for 7 days.
this experiment had three replicates, and each experiment comprised 30– <dig> plants.
bar = 1 cm.
fresh/dry weights of seedlings germinated on ms, ms + 125 mm nacl and ms + 250 mm mannitol, respectively.
data represent means and standard errors for three biological replications.
statistical significance was determined by student’s t-test.
*p <  <dig> ; **p <  <dig> .
under non-stress conditions, no significant differences in the fresh and dry weights were observed between the wt and transgenic plants .
however, when grown on murashige and skoog  medium supplemented with 125 mm nacl, the transgenic seedlings showed significantly larger cotyledons and longer roots than those of the wt, even though growth of these organs of the transgenic and wt plants was inhibited compared with normal conditions .
consistently, the fresh and dry weights of transgenic seedlings were significantly higher than that of the wt .
similar results were obtained when seedlings were treated with 250 mm mannitol .
these results suggest that silea <dig> may enhance the osmotic stress resistance of transgenic arabidopsis.
overexpression of silea <dig> increases the salt tolerance of transgenic foxtail millet
to further analyze the function of silea <dig>  it was transformed into foxtail millet under the control of ubiquitin promoter .
the silea <dig> integration was confirmed by genomic pcr using two pairs of primer sets specific to hpt and 35s promoter, respectively .
qrt-pcr analysis showed higher expression level of silea <dig> in transgenic lines l <dig>  l <dig>  and l <dig>  which showed a 3: <dig> segregation ratio at t <dig> generation, than that of the wt .
the t <dig> generation of these three lines were used for further analysis.figure  <dig>
assay for nacl, peg and aba stress on
silea14
overexpression foxtail millet germination.
diagram of the t-dna region of the binary vector pcou-silea14-flag.
lb, left border; rb, right border; 35s-pro, cauliflower mosaic virus 35s promoter; hpt, hygromycin b phosphotransferase gene; ubi-pro, maize ubiquitin promoter; silea14-flag, silea <dig> gene fused with a flag tag; nos, nopaline synthase terminator.
qrt-pcr analysis of silea <dig> in transgenic foxtail millet lines .
actin <dig> was used as an internal control.
the phenotype of transgenic and wt foxtail millet under various abiotic stress treatment during the germination stage.
the t <dig> seeds soaked in water  or in water containing 150 mm nacl, 250 mm nacl, 10% peg, 20% peg and 10 μm aba for 1 day at 30°c and then placed on the filter paper in a petri dish wet with the same solutions mentioned above for  <dig> and 9 days, respectively.
this experiment had three replicates, and each experiment comprised 30– <dig> plants.
bar = 1 cm.
the shoot length of transgenic and wt foxtail millet germinated under above conditions for  <dig> and 9 days, respectively.
the root length of transgenic and wt foxtail millet germinated under above conditions for  <dig> and 9 days, respectively.
data in b, e-h represent means and standard errors for three biological replicates.
statistical significance was determined by student’s t-test.
*p <  <dig> ; **p <  <dig> .
first, we examined the salt tolerance of transgenic foxtail millet during germination, and the results were shown in figure  <dig>  compared with the wt plants, transgenic lines showed better growth performance  with enhanced shoot and root growth  when germinated in water for 4 days.
under salt stress for 4 days, the transgenic lines showed significantly longer shoot and root than the wt , even though the growth of both wt and transgenic lines were seriously suppressed .
similar results were obtained for 9 days salt stress treatment .
further, we examined the salt tolerance of transgenic foxtail millet seedlings in soil.
no striking differences in the height were observed between the transgenic and the wt plants under non-stress conditions, but wider leaves were observed in the transgenic foxtail millet compared with those of the wt .
this finding is in accordance with the result that transgenic lines showed longer shoot and root lengths when germinated in water compared with the wt .
under 150 mm nacl stress for 6 days, the first and second leaves of wt plants became curled, whereas there was no obvious effect on the transgenic lines.
the wt plants were shorter than those of the transgenic lines.
under 250 mm nacl stress for 6 days, the growth of wt plants was severely inhibited and leaves were curled, bleached, and senescent.
however, the transgenic lines showed healthy growth except that only a few old leaves became bleached .figure  <dig>
salt tolerance of
silea14
overexpressing foxtail millet seedling.
the phenotype of transgenic and wt foxtail millet under normal and salt stress condition.
two-week-old seedlings in soil were irrigated with water,  <dig> and 250 mm nacl solution every 3 days.
after 6 days, the phenotypes of the transgenic lines and wt were investigated.
this experiment had three replicates; and six to eight plants grown in one plot were used in each experiment.
bar = 10 cm.
relative electrolyte leakage in wt and transgenic lines after salt stress.
proline content in wt and transgenic foxtail millet after salt stress.
soluble sugar content in wt and transgenic foxtail millet after salt stress.
data in b-d represent means and standard errors for three biological replicates.
statistical significance was determined by student’s t-test.
*p <  <dig> ; **p <  <dig> .
electrolyte leakage always occurs following membrane damage under salinity stress  <cit> .
furthermore, plants accumulate several metabolites, such as amino acids , sugars, and sugar alcohols , to prevent detrimental changes caused by severe osmotic stress  <cit> .
therefore, we measured the electrolyte leakage and change in free proline and soluble sugar contents in control and silea <dig> transgenic lines with or without salinity stress, respectively.
under optimal or salt-stress conditions, the amount of electrolyte leakage in the transgenic lines was significantly lower than that of the wt plants .
this finding is in agreement with the result that the silea <dig> transgenic lines showed enhanced germination and growth compared with the wt under both normal and salt-stress conditions .
no significant differences in free proline and soluble sugar contents were observed between the wt and the transgenic lines under normal conditions.
under  <dig> and 250 mm nacl stress, the free proline and soluble sugar contents increased in all plants.
however, this increase was significantly more pronounced in the transgenic lines than in the wt plants .
all of these increased osmotic protectants are beneficial for protection of the plants against salt stress.
moreover, we noted that the transgenic line l <dig> demonstrated better salt tolerance than the other two transgenic lines , which correlated with the higher accumulation of silea <dig> transcripts in l <dig>  all of these results indicated that silea <dig> overexpressing foxtail millet is more tolerant to salt stress compared with the wt.
overexpression of silea <dig> increases drought resistance of transgenic foxtail millet
we also examined the drought resistance of transgenic foxtail millet during germination.
when germinated in water for 4 days, the transgenic lines showed better growth performance than wt as described above .
compared with in water, no obvious suppression under 10% peg stress but serious suppression under 20% peg stress for 4 days were observed .
the shoot and root length of transgenic lines was longer than that of the wt no matter germinated in water, 10% or 20% peg .
when germinated in water or peg for 9 days, similar results were obtained .
the observation that silea <dig> improved osmotic stress resistance during germination prompted us to investigate the drought resistance of transgenic foxtail millet seedlings in soil.
as it is shown in figure 8a, no significant differences in the height were observed between the transgenic lines and the wt when watered well, except that the leaves of the transgenic lines were wider.
however, under drought stress for 7 days, more than 90% of wt leaves, but less than 20% of transgenic plant lines leaves, became curled and wilted.
after rewatering, 74–100% plants of trangenic lines survived in contrast to only 14% of wt .figure  <dig>
drought tolerance of
silea14
overexpression foxtail millet in soil.
the phenotypes of wt and transgenic foxtail millet under normal condition, drought stress for 7 days and after rewatering for 3 days.
two-week-old seedlings were used.
six to ten plants grown in one plot were used in each experiment.
this experiment had three replicates.
bar = 10 cm.
the survival rate of wt and transgenic foxtail millet plants in  under drought stress.
free proline content in wt and transgenic plants after drought stress.
soluble sugar content in wt and transgenic plants after drought stress.
data in b-d represent means and standard errors for three biological replicates.
statistical significance was determined by student’s t-test.
*p <  <dig> ; **p <  <dig> .
the change in free proline and soluble sugar contents in control and silea <dig> transgenic lines with or without drought stress was examined.
no significant differences in free proline and soluble sugar contents were observed between the wt and the transgenic lines under non-stress conditions.
under drought stress for 3 days, the free proline and soluble sugar contents increased in all wt and transgenic plants.
however, a significantly higher increase in free proline content was observed in the transgenic lines l <dig> and l <dig> when compared to the wt.
in addition, compared with the wt, the soluble sugar content increased significantly in the transgenic lines l <dig> and l <dig> .
taken together these results indicate that silea <dig> overexpressing foxtail millet showed improved drought resistance.
gus activity is induced by various stresses in prosilea14::gus transgenic arabidopsis
the putative silea <dig> promoter region was isolated from the foxtail millet genome using pcr.
this fragment consisted in nt − <dig> to + <dig>  upstream of silea <dig> coding sequence.
the silea <dig> promoter was fused to the gus gene and transformed into arabidopsis.
then, qrt-pcr was carried out to examine the gus expression level under various stresses.
as it is shown in figure  <dig>  gus transcription was up-regulated to its highest level  after 6 h treatment with aba and nacl, respectively.
for peg stress, gus expression gradually increased and peaked  after 18 h treatment.
to further confirm these results, a histochemical gus-staining assay was performed.
no visible gus activity was noted for the control.
however, induction of gus activity was observed with aba, peg or nacl application for  <dig> to 18 h, although the gus signals were not particularly strong .
nevertheless, collectively these results suggest that the selected silea <dig> promoter sequence is enough to regulate aba, peg and nacl induction of the gene.figure  <dig>
expression of gus gene in pro
silea14
::gus transgenic arabidopsis upon various stresses determined by qrt-pcr.
one-week-old seedlings were treated with 100 μm aba, 250 mm nacl and 20%  peg  <dig> solution for  <dig>   <dig>   <dig> and 18 h, respectively.
total rna was extracted from at least  <dig> whole seedlings per treatment.
the expression of gus in prosilea14::gus transgenic arabidopsis was determined by qrt-pcr using gus-specific primers .
actin <dig> was used to normalize the gene expression.
data represent means and standard errors for three biological replicates.
silea <dig> promoter contains stress-associated cis-elements
to characterize the mechanism of silea <dig> function, a 1273 bp silea <dig> promoter fragment  was subjected to a putative cis-acting regulatory element search using the plant cis-acting regulatory dna elements  database  <cit> .
cis-acting elements reported to be involved in aba- and dehydration-mediated gene expression were identified .
abscisic acid-responsive elements  are common motifs for aba- and dehydration-mediated regulation of transcription  <cit> .
in the silea <dig> promoter, six abre-like sequences and four aba- and dehydration-responsive acgt-box motifs  <cit>  were identified.
a dehydration-responsive element -like site, as a cis-acting element involved in aba induction, drought, salinity, or cold stresses  <cit> , was also identified at position − <dig>  myc and myb binding sites have been identified as regulatory elements associated with aba, drought, or cold responsiveness in plants  <cit> .
in the silea <dig> promoter, as many as  <dig> and three myc- and myb-like sequences, respectively, were observed.
these results suggest that silea <dig> may be under the regulation of dreb-, dbf-, cbf-, myc- or myb-like transcription factors as in arabidopsis and rice.
in addition, other regulatory cis-acting elements were identified in the silea <dig> promoter, such as low temperature-responsive, pathogen-, defense- and wounding-associated elements.
however, whether silea <dig> participates in these abiotic or biotic stresses awaits further investigation.table  <dig>
putative
cis-
acting regulatory elements associated with aba and various stresses in
silea14
promoter region
cis
-element name
sequence
position
function
ref.
m = c/a; y = t/c; b = t/c/g; n = a/t/g/c; r = a/g; w = a/t; v = a/c/g; k = g/t.
discussion
typical lea proteins can retain water molecules and protect other proteins from aggregation or desiccation because of their highly hydrophilic properties  <cit> .
conversely, atypical lea proteins have higher content of hydrophobic residues than typical leas.
the latter have been speculated to be involved in diverse stress tolerances, although few studies have been carried out to characterize their functions  <cit> .
here, we reported the identification and characterization of silea <dig>  a novel atypical lea member, in foxtail millet.
as a key phytohormone, aba plays an important role in plant stress responses.
osmotic stress-regulated genes can be activated through both aba-dependent and aba-independent pathways  <cit> .
however, it is considered that stress-signalling pathways for the activation of lea-like genes completely independent of aba may not exist  <cit> .
in our study, silea <dig> accumulation was remarkably induced by aba and peaked rapidly after 1 h treatment .
in addition, silea <dig> promoter-driven gus activity was distinctly stimulated by aba .
compared with the wt, transgenic foxtail millet seeds showed better germination in aba solution .
all of these results indicate that activation of silea <dig> under salt and drought stresses may be dependent on aba.
yeast and e. coli heterologous systems have been widely used to investigate lea gene functions  <cit> .
in the present study, overexpression of silea <dig> protected e. coli cells from damage caused by salt stress .
overexpression of silea <dig> in arabidopsis imparted increased tolerance to salt and mannitol stresses .
this result suggests that silea <dig> from foxtail millet, a monocot, can function properly in the dicot arabidopsis.
interestingly, transgenic foxtail millet overexpressing silea <dig> exhibited superior germination and subsequent growth in soil compared with the wt even under normal conditions.
under salt and drought stresses, these differences were more remarkable, which were indicative of the key roles of silea <dig> in foxtail millet .
consistent with these findings, both silea <dig> transcription and silea <dig> promoter-driven gus activity were remarkably induced by nacl and peg .
however, it should be noted that some discrepancies were observed between the expression pattern of the endogenous silea <dig> upon abiotic stress and aba treatment  and the silea <dig> promoter-driven gus transcript accumulation responsive to abiotic stress and aba driven by silea <dig> promoter region in transgenic arabidopsis .
for example, the silea <dig> transcripts accumulated to the highest level after 250 mm nacl treatment for 3 h , whereas the gus transcripts reached the peak after 6 h treatment .
additionally, the histochemical analysis showed strong gus signals in the petioles of transgenic arabidopsis , whereas the endogenous silea <dig> was highly expressed in roots .
this is probably due to that silea <dig> is from the monocot foxtail millet and the gus expression driven by silea <dig> promoter is performed in the dicot arabidopsis.
so far, there is only one report on the functional mechanism of the atypical lea protein in plants  <cit> .
overexpression of iblea <dig>  a homologous gene of silea <dig>  enhanced tolerance to drought and salt stress in the sweetpotato calli.
the contents of lignin in the iblea14-overexpressing calli were increased under normal conditions.
the authors inferred that iblea <dig> may be involved in these functions as a consequence of regulating increased lignin production.
in our study, we found that overexpression of silea <dig> in arabidopsis and foxtail millet obviously improved the osmotic stress resistance of transgenic plants.
meanwhile, the proline and sugar accumulated at a higher level in transgenic lines, especially in l <dig> which accumulated higher silea <dig> transcripts, than wt after osmotic and nacl stress.
these results implied that overexpression of silea <dig> might up-regulate these metabolites.
however, further study is needed.
the identification of cis-acting elements in the silea <dig> promoter may help to provide insight into the molecular mechanism of silea <dig> function.
as a major cis-acting element, abre has been identified in the promoters of many aba-inducible genes of plants such as the cotton lea gene d- <dig>  <cit> .
the bzip transcription factors areb/abf can bind to abre and activate aba-inducible gene expression  <cit> .
in the present study, ten cis-elements containing the abre acgt-core were identified in the − <dig> to −77 bp region of the silea <dig> promoter.
the dre element was first identified in the promoter of rd29a, a gene responsive to dehydration stress in arabidopsis  <cit> .
in plants, the ap <dig> transcription factors dreb/cbf specifically bind to the dre element to regulate the expression of the downstream stress-responsive genes  <cit> .
there are interactions between abre and dre in arabidopsis rd29a gene expression in response to dehydration and high-salinity stresses  <cit> .
one dre-like element was predicted in the silea <dig> promoter.
in arabidopsis, an myc transcription factor, atmyc <dig> and an myb transcription factor, atmyb <dig>  have been shown to bind the cis-elements canntg and c/taacna/g, respectively, to regulate expression of the dehydration-responsive gene rd <dig>  <cit> .
ten myc and three myb-like sequences were identified in the silea <dig> promoter.
proteins that bind to these elements remain to be isolated.
it is necessary to further elucidate the detailed functions of these putative regulatory cis-elements.
in addition, pathogen-related elements were also identified.
however, whether silea <dig> participates in biotic stress responses needs to be investigated.
CONCLUSIONS
in conclusion, this study characterizes a novel atypical lea gene silea <dig> from foxtail millet.
silea <dig> is responsive to aba, peg and nacl and the silea <dig> is localized in the cytosol.
silea <dig> improves the salt tolerance of e. coli transformant and transgenic arabidopsis.
furthermore, overexpression of silea <dig> significantly enhances the salt and drought tolerances of transgenic foxtail millet.
silea <dig> plays important roles in plant abiotic stress resistance and could be used in crops genetic engineering with the aim of improving stress tolerance.

 environmental stresses, such as drought and high salinity, can cause severe damage to plants, leading to considerable reduction in their productivity. to survive under such conditions, plants have developed a series of defense responsive pathways. among them, ca2+-dependent signaling leads to the activation of late embryogenesis abundant -type genes, which may function in protection and damage repair of plants. lea proteins were first identified in cotton seeds. the proteins accumulated to high levels in the late stages of seed development. subsequently, they were found to be expressed in vegetative tissues and could be induced by abscisic acid and various abiotic stresses, such as drought and cold. with the development of deep sequencing technology, an increasing number of lea proteins have been identified. on the basis of their amino acid sequence similarities and conserved motifs, lea proteins are categorized into different groups. in this work, we adopt the classification introduced by battaglia’s group, in which lea proteins are categorized into seven distinct families. groups and which share specific motifs within each group, are considered to be hydrophilic or “typical” lea proteins. conversely, group corresponds to atypical lea proteins. this group includes all lea proteins with higher content of hydrophobic residues than typical lea proteins. on the basis of their sequence similarity, group lea proteins are divided into the subgroups 5a, 5b, and 5c, corresponding to the first described proteins d- d- and d- in this group, respectively. physicochemical properties show that group lea proteins are not soluble after boiling, suggesting that they may adopt a globular conformation and are not heat stable. subsequent reports show that subgroup 5c lea proteins are natively folded and have more β-sheets than α-helixes, which is different from subgroups 5a and 5b lea proteins that are intrinsically unstructured. for example, arabidopsis lea has an αβ-fold consisting of one α-helix and seven β-strands that form two antiparallel β-sheets as determined by nuclear magnetic resonance spectroscopy. moreover, subgroup 5c lea proteins have other outstanding characteristics, such as lower instability index, narrower range of gravy values, and lower proportion of polar and small residues, but higher proportion of non-polar residues than subgroup 5a and 5b members. all of these differences indicate that subgroup 5c lea proteins may function differently from other lea proteins in plants. at present, only a small number of subgroup 5c lea genes have been characterized. their transcripts can be upregulated in response to diverse stresses, as reported for cotton lea14-a, craterostigma plantagineum pcc27-, soybean d95-, tomato er, hot pepper calea, arabidopsis lea and at2g, and sweetpotato iblea. overexpression of calea in tobacco improves tolerance to dehydration and nacl but not to low temperature. transgenic sweetpotato non-embryogenic calli that overexpress iblea show increased tolerance to drought and salt stress by enhancing lignification. recently, rice oslea has been reported to enhance resistance against diverse abiotic stresses in recombinant escherichia coli cells. in vitro analysis showed that oslea was able to protect lactate dehydrogenase from aggregation under different abiotic stresses. all these results suggest that subgroup 5c lea proteins are closely associated with resistance to multiple abiotic stresses. foxtail millet beauv.), a member of the poaceae family, has a long history in cultivation of about 7000 years. it has been widely planted in northern china and other asian countries. recently, a draft genome sequence for foxtail millet has been completed, which enables foxtail millet to be a tractable experimental grass model. as a diploid c panicoid crop species, foxtail millet is well known for its remarkable drought resistance. however, the molecular mechanisms underlying this tolerance are not well defined. in this study, we isolated and functionally characterized a novel member of the atypical subgroup 5c lea gene, silea from foxtail millet. the expression of silea was induced by aba, polyethylene glycol and nacl. overexpression of silea resulted in enhanced resistance to abiotic stresses in e. coli, arabidopsis and foxtail millet. the silea promoter mediated remarkable induction of β-glucuronidase expression in transgenic arabidopsis under various stresses. cis-acting regulatory elements in the silea promoter were also predicted. these data reveal the potential application of silea in the genetic engineering of other crops.  silea is an atypical lea protein the full-length sequence of silea was determined by 5′ and 3′ rapid amplification of cdna ends. the sequence is 821 bp in length, with a 100 bp 5′ untranslated region and a 208 bp 3′ utr. silea harbors two exons separated by an intron, and encodes an open reading frame of aa with a predicted molecular mass of kd and pi of. it is rich in ser, lys, and ile, but contains low quantities of trp, asn, cys, and gln. three hydrophobic regions were identified in the silea protein. a motif search of the silea protein in interproscan revealed that it contains a “lea_2” motif, which was classified into subgroup 5c according to battaglia’s classification of lea proteins. further analysis showed that silea contains a lower percentage of polar amino acids and higher percentage of non-polar amino acids than other group lea proteins. the gravy value and the instability index of silea is − and, respectively. all these characteristics were in accordance with those of other members of subgroup 5c. two additional silea homologs existed in foxtail millet. they showed % and % identity to silea respectively. homologous sequences of silea were also present in other plant species, such as rice oslea, maize lea14-a and arabidopsis atlea. the homologs shared different levels of sequence similarities, indicating different levels of evolutionary relationship among these proteins. a phylogenetic tree for silea and its homologs was constructed from protein sequences. the proteins were mainly separated into two clades comprising dicot and monocot species, respectively. the silea protein shared the closest relationship with the pearl millet lea-like protein, followed by oslea. sequence logos were produced to examine the conservation level at each residue position. the sequences lacked significant signature motifs or consensus sequences, except for several conserved residues at specific positions, such as proline at positions and. this is a common feature of subgroup 5c lea proteins.figure multiple sequence alignment and phylogenetic relationship for the silea protein and its homologs. multiple sequence alignment of silea with its homologs from various plant species. the conserved “lea_2” motif is boxed. phylogenetic relationships of silea and its homologs. the divergence of the clades between the monocots and dicots is highlighted. silea and its two homologs in foxtail millet are highlighted by solid dark circle and triangle, respectively. the genbank accession numbers are as follows: silea, atlea, lea14-a, d95-, iblea, er, lemmi, calea, oslea, pcc27-, pclea, at1g, lea14-a, lea homolog, at2g, os01g, lea14-a-like, loc, sorbidraft 09 g, lea-like protein, osi, os05g, os05g, at2g, loc and umc. the sequences used for alignment and phylogenetic tree construction were shown in additional file subcellular localization of silea14 to investigate the potential role of silea we examined the subcellular localization of silea fused to gfp and gfp alone in onion epidermal cells and foxtail millet root protoplasts. the constructs used were shown in figure 2a. when observed by confocal microscopy, the green fluorescent protein fluorescence of silea14-gfp was distributed throughout the cell, consistent with the gfp control, both in the onion cells and the foxtail millet root protoplasts. these results indicated that silea was localized in the cytosol.figure subcellular localization of silea protein. diagram of the constructs of prok219-gfp and prok219-silea14-gfp used for subcellular localization. 35s-pro, cauliflower mosaic virus 35s promoter; silea the silea coding region without the stop codon; gfp, green fluorescent protein; nos, nopaline synthase terminator. fluorescent microscopic images of gfp and silea14-gfp fusion protein in the onion epidermal cells and in the foxtail millet root protoplasts. silea expression profiles under normal and stress conditions quantitative reverse-transcription pcr was carried out to reveal the temporal and spatial expression of silea in foxtail millet. silea was expressed at the highest level in roots, followed by stems and leaves, and at the lowest level in inflorescences. after pollination, silea transcription was gradually upregulated with seed maturation, which indicated a potential role for silea in the maturation and desiccation phases of foxtail millet seed development.figure silea14 expression patterns in foxtail millet determined by qrt-pcr. tissue-specific expression of silea total rna was extracted from roots, stems, leaves, inflorescences and seeds at, and days after pollination, respectively. foxtail millet actin was used as an internal control. silea transcripts accumulation in response to various treatments. two-week-old foxtail millet seedlings were treated with 100 μm aba, 20% peg and 250 mm nacl for the indicated times, respectively. total rna was extracted from the above-ground parts of ten plants at the indicated times after the treatments. data represent means and standard errors for three biological replicates. the expression level of silea was also examined by qrt-pcr under various stresses. under aba treatment, silea transcription abruptly reached its highest level after 1 h treatment, and then decreased gradually and reverted almost to the control level at 24 h. under peg stress, silea transcription was rapidly induced within  h and increased to a peak level at 12 h. salt treatment resulted in increased accumulation of silea to a maximum level only after 3 h. these results indicated that silea might play important roles in the responses to salt and drought stresses. silea enhances salt tolerance in transformed e. coli in both prokaryotes and eukaryotes lea proteins might have similar protective mechanisms. therefore, the up-regulation of silea in response to nacl prompted us to evaluate the salt stress tolerance of recombinant e. coli overexpressing silea. there are no significant differences in colony number between transformed e. coli harboring silea and the control under normal conditions, indicating that overexpression of silea did not affect the growth of e. coli recombinants in non-stress conditions. however, when grown on luria–bertani plates supplemented with 600 mm nacl, the number of transformed cells was much higher than that of the control. a similar result was obtained on lb medium supplemented with 600 mm kcl. these results demonstrated that overexpression of silea in e. coli significantly enhanced tolerance to salt stress.figure assay for salt stress tolerance of silea transformed e. coli. transformant e. coli with pet30a-silea were grown on lb plates with or without 600 mm nacl and kcl for and 24 h, respectively. transformant with empty vector pet30a was used as a control. the number of colonies on the plate was calculated. the 200×, 500×, 1000×, 2000× and 4000× represent the dilution fold. bar = 1 cm. data represent means and standard errors for three biological replicates. statistical significance was determined by student’s t-test. **p < . silea improves the abiotic stress resistance of transgenic arabidopsis to assess the function of silea in plants, it was overexpressed in arabidopsis under the control of super promoter. at least transgenic plants were obtained, and two independent homozygous t transgenic lines with high expression levels of silea were chosen for further investigation.figure assay for salinity and mannitol stress tolerance of silea14 overexpression arabidopsis. diagram of the t-dna region of the binary vector ps1300-silea lb, left border; rb, right border; 35s-pro, cauliflower mosaic virus 35s promoter; hpt, hygromycin b phosphotransferase gene; super-pro, super promoter; silea silea gene; nos, nopaline synthase terminator. rt-pcr analysis of silea in transgenic arabidopsis lines. wt was used as a control. actin was a loading control. the phenotype of transgenic and wt arabidopsis germinated on ms, ms + 125 mm nacl and ms + 250 mm mannitol, respectively, for 7 days. this experiment had three replicates, and each experiment comprised 30– plants. bar = 1 cm. fresh/dry weights of seedlings germinated on ms, ms + 125 mm nacl and ms + 250 mm mannitol, respectively. data represent means and standard errors for three biological replications. statistical significance was determined by student’s t-test. *p < ; **p < . under non-stress conditions, no significant differences in the fresh and dry weights were observed between the wt and transgenic plants. however, when grown on murashige and skoog medium supplemented with 125 mm nacl, the transgenic seedlings showed significantly larger cotyledons and longer roots than those of the wt, even though growth of these organs of the transgenic and wt plants was inhibited compared with normal conditions. consistently, the fresh and dry weights of transgenic seedlings were significantly higher than that of the wt. similar results were obtained when seedlings were treated with 250 mm mannitol. these results suggest that silea may enhance the osmotic stress resistance of transgenic arabidopsis. overexpression of silea increases the salt tolerance of transgenic foxtail millet to further analyze the function of silea it was transformed into foxtail millet under the control of ubiquitin promoter. the silea integration was confirmed by genomic pcr using two pairs of primer sets specific to hpt and 35s promoter, respectively. qrt-pcr analysis showed higher expression level of silea in transgenic lines l l and l which showed a 3: segregation ratio at t generation, than that of the wt. the t generation of these three lines were used for further analysis.figure assay for nacl, peg and aba stress on silea14 overexpression foxtail millet germination. diagram of the t-dna region of the binary vector pcou-silea14-flag. lb, left border; rb, right border; 35s-pro, cauliflower mosaic virus 35s promoter; hpt, hygromycin b phosphotransferase gene; ubi-pro, maize ubiquitin promoter; silea14-flag, silea gene fused with a flag tag; nos, nopaline synthase terminator. qrt-pcr analysis of silea in transgenic foxtail millet lines. actin was used as an internal control. the phenotype of transgenic and wt foxtail millet under various abiotic stress treatment during the germination stage. the t seeds soaked in water or in water containing 150 mm nacl, 250 mm nacl, 10% peg, 20% peg and 10 μm aba for 1 day at 30°c and then placed on the filter paper in a petri dish wet with the same solutions mentioned above for and 9 days, respectively. this experiment had three replicates, and each experiment comprised 30– plants. bar = 1 cm. the shoot length of transgenic and wt foxtail millet germinated under above conditions for and 9 days, respectively. the root length of transgenic and wt foxtail millet germinated under above conditions for and 9 days, respectively. data in b, e-h represent means and standard errors for three biological replicates. statistical significance was determined by student’s t-test. *p < ; **p < . first, we examined the salt tolerance of transgenic foxtail millet during germination, and the results were shown in figure  compared with the wt plants, transgenic lines showed better growth performance with enhanced shoot and root growth when germinated in water for 4 days. under salt stress for 4 days, the transgenic lines showed significantly longer shoot and root than the wt, even though the growth of both wt and transgenic lines were seriously suppressed. similar results were obtained for 9 days salt stress treatment. further, we examined the salt tolerance of transgenic foxtail millet seedlings in soil. no striking differences in the height were observed between the transgenic and the wt plants under non-stress conditions, but wider leaves were observed in the transgenic foxtail millet compared with those of the wt. this finding is in accordance with the result that transgenic lines showed longer shoot and root lengths when germinated in water compared with the wt. under 150 mm nacl stress for 6 days, the first and second leaves of wt plants became curled, whereas there was no obvious effect on the transgenic lines. the wt plants were shorter than those of the transgenic lines. under 250 mm nacl stress for 6 days, the growth of wt plants was severely inhibited and leaves were curled, bleached, and senescent. however, the transgenic lines showed healthy growth except that only a few old leaves became bleached.figure salt tolerance of silea14 overexpressing foxtail millet seedling. the phenotype of transgenic and wt foxtail millet under normal and salt stress condition. two-week-old seedlings in soil were irrigated with water, and 250 mm nacl solution every 3 days. after 6 days, the phenotypes of the transgenic lines and wt were investigated. this experiment had three replicates; and six to eight plants grown in one plot were used in each experiment. bar = 10 cm. relative electrolyte leakage in wt and transgenic lines after salt stress. proline content in wt and transgenic foxtail millet after salt stress. soluble sugar content in wt and transgenic foxtail millet after salt stress. data in b-d represent means and standard errors for three biological replicates. statistical significance was determined by student’s t-test. *p < ; **p < . electrolyte leakage always occurs following membrane damage under salinity stress. furthermore, plants accumulate several metabolites, such as amino acids, sugars, and sugar alcohols, to prevent detrimental changes caused by severe osmotic stress. therefore, we measured the electrolyte leakage and change in free proline and soluble sugar contents in control and silea transgenic lines with or without salinity stress, respectively. under optimal or salt-stress conditions, the amount of electrolyte leakage in the transgenic lines was significantly lower than that of the wt plants. this finding is in agreement with the result that the silea transgenic lines showed enhanced germination and growth compared with the wt under both normal and salt-stress conditions. no significant differences in free proline and soluble sugar contents were observed between the wt and the transgenic lines under normal conditions. under and 250 mm nacl stress, the free proline and soluble sugar contents increased in all plants. however, this increase was significantly more pronounced in the transgenic lines than in the wt plants. all of these increased osmotic protectants are beneficial for protection of the plants against salt stress. moreover, we noted that the transgenic line l demonstrated better salt tolerance than the other two transgenic lines, which correlated with the higher accumulation of silea transcripts in l all of these results indicated that silea overexpressing foxtail millet is more tolerant to salt stress compared with the wt. overexpression of silea increases drought resistance of transgenic foxtail millet we also examined the drought resistance of transgenic foxtail millet during germination. when germinated in water for 4 days, the transgenic lines showed better growth performance than wt as described above. compared with in water, no obvious suppression under 10% peg stress but serious suppression under 20% peg stress for 4 days were observed. the shoot and root length of transgenic lines was longer than that of the wt no matter germinated in water, 10% or 20% peg. when germinated in water or peg for 9 days, similar results were obtained. the observation that silea improved osmotic stress resistance during germination prompted us to investigate the drought resistance of transgenic foxtail millet seedlings in soil. as it is shown in figure 8a, no significant differences in the height were observed between the transgenic lines and the wt when watered well, except that the leaves of the transgenic lines were wider. however, under drought stress for 7 days, more than 90% of wt leaves, but less than 20% of transgenic plant lines leaves, became curled and wilted. after rewatering, 74–100% plants of trangenic lines survived in contrast to only 14% of wt.figure drought tolerance of silea14 overexpression foxtail millet in soil. the phenotypes of wt and transgenic foxtail millet under normal condition, drought stress for 7 days and after rewatering for 3 days. two-week-old seedlings were used. six to ten plants grown in one plot were used in each experiment. this experiment had three replicates. bar = 10 cm. the survival rate of wt and transgenic foxtail millet plants in under drought stress. free proline content in wt and transgenic plants after drought stress. soluble sugar content in wt and transgenic plants after drought stress. data in b-d represent means and standard errors for three biological replicates. statistical significance was determined by student’s t-test. *p < ; **p < . the change in free proline and soluble sugar contents in control and silea transgenic lines with or without drought stress was examined. no significant differences in free proline and soluble sugar contents were observed between the wt and the transgenic lines under non-stress conditions. under drought stress for 3 days, the free proline and soluble sugar contents increased in all wt and transgenic plants. however, a significantly higher increase in free proline content was observed in the transgenic lines l and l when compared to the wt. in addition, compared with the wt, the soluble sugar content increased significantly in the transgenic lines l and l. taken together these results indicate that silea overexpressing foxtail millet showed improved drought resistance. gus activity is induced by various stresses in prosilea14::gus transgenic arabidopsis the putative silea promoter region was isolated from the foxtail millet genome using pcr. this fragment consisted in nt − to + upstream of silea coding sequence. the silea promoter was fused to the gus gene and transformed into arabidopsis. then, qrt-pcr was carried out to examine the gus expression level under various stresses. as it is shown in figure  gus transcription was up-regulated to its highest level after 6 h treatment with aba and nacl, respectively. for peg stress, gus expression gradually increased and peaked after 18 h treatment. to further confirm these results, a histochemical gus-staining assay was performed. no visible gus activity was noted for the control. however, induction of gus activity was observed with aba, peg or nacl application for to 18 h, although the gus signals were not particularly strong. nevertheless, collectively these results suggest that the selected silea promoter sequence is enough to regulate aba, peg and nacl induction of the gene.figure expression of gus gene in pro silea14::gus transgenic arabidopsis upon various stresses determined by qrt-pcr. one-week-old seedlings were treated with 100 μm aba, 250 mm nacl and 20% peg solution for and 18 h, respectively. total rna was extracted from at least whole seedlings per treatment. the expression of gus in prosilea14::gus transgenic arabidopsis was determined by qrt-pcr using gus-specific primers. actin was used to normalize the gene expression. data represent means and standard errors for three biological replicates. silea promoter contains stress-associated cis-elements to characterize the mechanism of silea function, a 1273 bp silea promoter fragment was subjected to a putative cis-acting regulatory element search using the plant cis-acting regulatory dna elements database. cis-acting elements reported to be involved in aba- and dehydration-mediated gene expression were identified. abscisic acid-responsive elements are common motifs for aba- and dehydration-mediated regulation of transcription. in the silea promoter, six abre-like sequences and four aba- and dehydration-responsive acgt-box motifs were identified. a dehydration-responsive element -like site, as a cis-acting element involved in aba induction, drought, salinity, or cold stresses, was also identified at position − myc and myb binding sites have been identified as regulatory elements associated with aba, drought, or cold responsiveness in plants. in the silea promoter, as many as and three myc- and myb-like sequences, respectively, were observed. these results suggest that silea may be under the regulation of dreb-, dbf-, cbf-, myc- or myb-like transcription factors as in arabidopsis and rice. in addition, other regulatory cis-acting elements were identified in the silea promoter, such as low temperature-responsive, pathogen-, defense- and wounding-associated elements. however, whether silea participates in these abiotic or biotic stresses awaits further investigation.table putative cis- acting regulatory elements associated with aba and various stresses in silea14 promoter region cis -element name sequence position function ref. m = c/a; y = t/c; b = t/c/g; n = a/t/g/c; r = a/g; w = a/t; v = a/c/g; k = g/t. discussion typical lea proteins can retain water molecules and protect other proteins from aggregation or desiccation because of their highly hydrophilic properties. conversely, atypical lea proteins have higher content of hydrophobic residues than typical leas. the latter have been speculated to be involved in diverse stress tolerances, although few studies have been carried out to characterize their functions. here, we reported the identification and characterization of silea a novel atypical lea member, in foxtail millet. as a key phytohormone, aba plays an important role in plant stress responses. osmotic stress-regulated genes can be activated through both aba-dependent and aba-independent pathways. however, it is considered that stress-signalling pathways for the activation of lea-like genes completely independent of aba may not exist. in our study, silea accumulation was remarkably induced by aba and peaked rapidly after 1 h treatment. in addition, silea promoter-driven gus activity was distinctly stimulated by aba. compared with the wt, transgenic foxtail millet seeds showed better germination in aba solution. all of these results indicate that activation of silea under salt and drought stresses may be dependent on aba. yeast and e. coli heterologous systems have been widely used to investigate lea gene functions. in the present study, overexpression of silea protected e. coli cells from damage caused by salt stress. overexpression of silea in arabidopsis imparted increased tolerance to salt and mannitol stresses. this result suggests that silea from foxtail millet, a monocot, can function properly in the dicot arabidopsis. interestingly, transgenic foxtail millet overexpressing silea exhibited superior germination and subsequent growth in soil compared with the wt even under normal conditions. under salt and drought stresses, these differences were more remarkable, which were indicative of the key roles of silea in foxtail millet. consistent with these findings, both silea transcription and silea promoter-driven gus activity were remarkably induced by nacl and peg. however, it should be noted that some discrepancies were observed between the expression pattern of the endogenous silea upon abiotic stress and aba treatment and the silea promoter-driven gus transcript accumulation responsive to abiotic stress and aba driven by silea promoter region in transgenic arabidopsis. for example, the silea transcripts accumulated to the highest level after 250 mm nacl treatment for 3 h, whereas the gus transcripts reached the peak after 6 h treatment. additionally, the histochemical analysis showed strong gus signals in the petioles of transgenic arabidopsis, whereas the endogenous silea was highly expressed in roots. this is probably due to that silea is from the monocot foxtail millet and the gus expression driven by silea promoter is performed in the dicot arabidopsis. so far, there is only one report on the functional mechanism of the atypical lea protein in plants. overexpression of iblea a homologous gene of silea enhanced tolerance to drought and salt stress in the sweetpotato calli. the contents of lignin in the iblea14-overexpressing calli were increased under normal conditions. the authors inferred that iblea may be involved in these functions as a consequence of regulating increased lignin production. in our study, we found that overexpression of silea in arabidopsis and foxtail millet obviously improved the osmotic stress resistance of transgenic plants. meanwhile, the proline and sugar accumulated at a higher level in transgenic lines, especially in l which accumulated higher silea transcripts, than wt after osmotic and nacl stress. these results implied that overexpression of silea might up-regulate these metabolites. however, further study is needed. the identification of cis-acting elements in the silea promoter may help to provide insight into the molecular mechanism of silea function. as a major cis-acting element, abre has been identified in the promoters of many aba-inducible genes of plants such as the cotton lea gene d-. the bzip transcription factors areb/abf can bind to abre and activate aba-inducible gene expression. in the present study, ten cis-elements containing the abre acgt-core were identified in the − to −77 bp region of the silea promoter. the dre element was first identified in the promoter of rd29a, a gene responsive to dehydration stress in arabidopsis. in plants, the ap transcription factors dreb/cbf specifically bind to the dre element to regulate the expression of the downstream stress-responsive genes. there are interactions between abre and dre in arabidopsis rd29a gene expression in response to dehydration and high-salinity stresses. one dre-like element was predicted in the silea promoter. in arabidopsis, an myc transcription factor, atmyc and an myb transcription factor, atmyb have been shown to bind the cis-elements canntg and c/taacna/g, respectively, to regulate expression of the dehydration-responsive gene rd. ten myc and three myb-like sequences were identified in the silea promoter. proteins that bind to these elements remain to be isolated. it is necessary to further elucidate the detailed functions of these putative regulatory cis-elements. in addition, pathogen-related elements were also identified. however, whether silea participates in biotic stress responses needs to be investigated.  in conclusion, this study characterizes a novel atypical lea gene silea from foxtail millet. silea is responsive to aba, peg and nacl and the silea is localized in the cytosol. silea improves the salt tolerance of e. coli transformant and transgenic arabidopsis. furthermore, overexpression of silea significantly enhances the salt and drought tolerances of transgenic foxtail millet. silea plays important roles in plant abiotic stress resistance and could be used in crops genetic engineering with the aim of improving stress tolerance. 
silea was expressed in roots, stems, leaves, inflorescences and seeds at different levels under normal growth conditions. late embryogenesis abundant proteins are involved in protecting higher plants from damage caused by environmental stresses. similarly, the transgenic foxtail millet showed improved growth under salt and drought stresses compared with the wt. taken together, our results indicated that silea is a novel atypical lea protein and plays important roles in resistance to abiotic stresses in plants. overexpression of silea improved escherichia coli growth performance compared with the control under salt stress. however, the molecular mechanisms underlying tolerance to these conditions are not well defined. foxtail millet is an important cereal crop for food and feed in semi-arid areas. here, we characterized a novel atypical lea gene named silea from foxtail millet. to further assess the function of silea in plants, transgenic arabidopsis and foxtail millet plants that overexpressed silea were obtained. we characterized a novel atypical lea gene silea from foxtail millet, which plays important roles in plant abiotic stress resistance. the transgenic arabidopsis seedlings showed higher tolerance to salt and osmotic stress than the wild type. modification of silea expression may improve abiotic stress resistance in agricultural crops.
 late embryogenesis abundant proteins are involved in protecting higher plants from damage caused by environmental stresses. foxtail millet is an important cereal crop for food and feed in semi-arid areas. however, the molecular mechanisms underlying tolerance to these conditions are not well defined.  here, we characterized a novel atypical lea gene named silea from foxtail millet. it contains two exons separated by one intron. silea was expressed in roots, stems, leaves, inflorescences and seeds at different levels under normal growth conditions. in addition, silea was dramatically induced by osmotic stress, nacl and exogenous abscisic acid. the silea protein was localized in the nucleus and the cytoplasm. overexpression of silea improved escherichia coli growth performance compared with the control under salt stress. to further assess the function of silea in plants, transgenic arabidopsis and foxtail millet plants that overexpressed silea were obtained. the transgenic arabidopsis seedlings showed higher tolerance to salt and osmotic stress than the wild type. similarly, the transgenic foxtail millet showed improved growth under salt and drought stresses compared with the wt. taken together, our results indicated that silea is a novel atypical lea protein and plays important roles in resistance to abiotic stresses in plants.  we characterized a novel atypical lea gene silea from foxtail millet, which plays important roles in plant abiotic stress resistance. modification of silea expression may improve abiotic stress resistance in agricultural crops. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2014 