BACKGROUND
in natural environments, plant growth and crop productivity are reduced by abiotic stresses such as high salinity, drought, heavy metals, and oxidative stress.
although these stresses might elicit different plant responses, most induce the accumulation of reactive oxygen species   <cit> , including hydroxyl radicals , superoxide anions , and hydrogen peroxide .
the production of large amounts of ros in plant cells leads to oxidative damage .
in our previous study, we prepared a cdna library from seedlings of a salt-tolerant plant, puccinellia tenuiflora that had been treated with 150 mm nahco <dig>  <cit> .
one of the sequenced genes, putoxr, was found to confer enhanced tolerance to multiple abiotic stresses in yeast.
arabidopsis thaliana has a homologous gene, which in our preliminary studies was found to be associated with multiple abiotic and oxidative stress.
we thus named it atoxr , furthermore, the study of atoxr gene have not been reported.
h2o2-scavenging in plants is achieved through several mechanisms, for example, the water–water cycle, catalase  enzymatic reactions, the glutathione peroxidase  cycle, and the ascorbate-glutathione  cycle  <cit> .
these pathways involve a number of enzymes, such as superoxide dismutase , ascorbate peroxidase , catalase , and glutathione peroxidase  , and anti-oxidants such as glutathione  <cit>  and ascorbic acid   <cit> .
in plants, h2o <dig> levels can be changed via the asa/glutathione  cycle, apx plays a central role in the cycle and is emerging as a key enzyme in cellular h2o <dig> metabolism.
asa is not only an important component of human nutrition but also an antioxidant and h2o2-scavenger that defends plants against abiotic stresses .
for example, enhanced asa accumulation confers tolerance to oxidative, salt and drought stresses in potato and maintenance of a high asa level is required for oxidative stress tolerance in arabidopsis  <cit> .
asa is synthesized through multiple biosynthetic pathways in plants , while the major pathway is smirnoff–wheeler pathway  <cit> .
previous studies have shown that overexpression of asa biosynthetic pathway related genes resulted in increased vitamin c content .
in addition, co-expression of nced and alo improves vitamin c content and tolerance to abiotic stresses in transgenic tobacco and stylo plants  <cit> .
although vitamin c is essential, humans are one of the few mammalian species unable to synthesis the vitamin and must obtain it through dietary sources.
in recent years, the physiological function that asa plays in abiotic stress tolerance and nutritional quality has garnered increasing attention.
in the present study, the expression pattern of atoxr in seedlings treated by abiotic stresses was studied using quantitative real-time pcr .
the subcellular localization of the atoxr protein and the phenotypes of atoxr gene transformants in yeast and arabidopsis were also analyzed.
additionally, the h2o <dig> and vitamin c content of atoxr transgenic arabidopsis and wt plants were determined in plants grown under abiotic stresses.
methods
plasmid constructs and plant materials
the open reading frame  of atoxr was amplified from arabidopsis cdna using the primers atoxr-fw and atoxr-rv .
the amplified product atoxr was digested with kpni and bamhi and cloned into the yeast expression vector pyes <dig>  to form pyes2-atoxr plasmid, which was transferred into saccharomyces cerevisiae strain invsc <dig>  for the construction of gfp fusion proteins, atoxr without its stop codon was amplified with the primers atoxr-fw-bamhi and atoxr-rv-kpni  by using atoxr cdna as a template, the amplified product was digested with bamhi and kpni, and cloned into the pegfp vector .
the construct plasmid pegfp-atoxr-gfp was digested with bamhi and ecori, and cloned into the pyes <dig> vector to obtain the plasmid pyes2-atoxr-gfp, which was transferred into invsc <dig> using a lithium acetate-based method  <cit> .
atoxr-gfp was amplified from pegfp-atoxr-gfp with primers atoxr-fw-bamhi and gfp-rv-saci .
the product was then digested with bamhi and saci, and cloned into the pbi <dig> vector to obtain the plasmid pbi121-atoxr-gfp.
the constructs pbi121-atoxr, pbi121-atoxr-gfp and pbi121-gfp as control were transformed into agrobacterium tumefaciens strain eha <dig> to obtain the transgenic arabidopsis by the floral dip method  <cit> .table  <dig> sequence of the primers used for pcr
atoxr-fw
atoxr-rv
pbi121-atoxr-fw
ggatcctatgtatttcgccg
pbi121-atoxr-rv
gagctcctcctctaatagtc
atoxr-fw-bamhi
ggtaccatgtatttcgccgccatagc
atoxr-rv-kpni
ggatccgcctctctccgttttctcc
gfp-rv-saci
gagctcctagagtcgcggccgc
pyes2-atoxr-fw
ggtacctatgtatttcgccg
pyes2-atoxr-rv
ggatccctcctctaatagtc
atoxr-rt-fw
atoxr-rt-rv
actin-fw
actin-rv
phylogenetic analyses, yeast transformations, and growth conditions
full-length amino acids sequences were aligned using clustalx, and then imported into the molecular evolutionary genetics analysis  package version  <dig>   <cit> .
phylogenetic analyses were conducted using the neighbor joining method in mega.
the following accession numbers were used: atoxr , oryza sativa , glycine max , zea mays , setaria italica , medicago truncatula , eutrema salsugineum , capsella rubella , theobroma cacao , and ricinus communis .
yeast transformations were performed using a lithium acetate-based method  <cit> .
the empty vector plasmids pyes2-atoxr and pyes <dig> were used as controls, and were introduced into the yeast strain invsci.
the transformed yeast strains were grown in synthetic defined  medium lacking the appropriate amino acids for the selective growth for the expression plasmids.
for the response assays, yeast transformants were cultured in liquid yeast extract peptone dextrose  medium  until they reached an optical density at 600 nm of ≈  <dig> , and were then diluted 10−1-, 10−2-, 10−3-,10−4-, and 10−5-fold using double distilled h2o.
then, aliquots of each dilution were spotted onto solid yeast ypd and ypg medium  supplemented with different concentrations of nacl , mannitol , h2o <dig> , mncl <dig> , mgcl <dig> , cdcl <dig> , bacl <dig> , cucl <dig> , fecl <dig> , alcl <dig> .
a yeast transformant of the pyes <dig> empty vector was used as a control, and growth was monitored for 3–7 days at 30 °c.
analysis of gene expression using quantitative real-time pcr
arabidopsis seeds were surface sterilized and plated on solid half ms medium.
after 2 days stratification at 4 °c, the plates were stored in a 22 °c incubator for propagation.
the seedlings were transferred from the plates to a 1: <dig> mixture of soil and vermiculite and grown to maturity at 22 °c.
the plants were cultured under a 16-h-light/8-h-dark cycle in a growth chamber.
roots, stems, leaves, panicle, and siliques of 2-month-old plants were sampled for qrt-pcr.
a second batch of seedlings were pre-cultured for 2 weeks on 1/ <dig> solid medium, and then treated with different concentrations of various stresses , the shoots and roots were sampled after 0 h, 6 h, 12 h, and 24 h treatment and used for qrt-pcr analyses.
total rna was isolated using the rneasy plant mini kit , and treated with rnase-free dnasei .
first-strand cdna was synthesized using superscript iii reverse transcriptase .
gene-specific primers pairs atoxr-rt-fw and atoxr-rt-rv were used for atoxr, while actin-fw and actin-rv were used for actin .
relative quantification using qrt-pcr reactions were performed with sybr green i using the lightcycler® <dig> system ii .
localization analysis of atoxr protein in yeast and plant cells
yeast transformants carrying the plasmids pyes2-atoxr-gfp and pyes2-gfp were pre-cultured in liquid ypd medium overnight at 30 °c, washed three times with distilled water to remove the glucose and cultured in liquid sd uracil medium with galactose at 30 °c to induce the expression of gfp and atoxr-gfp under the control of gal promoter.
live cells were incubated with er tracker, and the localization of atoxr-gfp was observed with a confocal microscope .
five-day-old transgenic roots carrying pbi121-gfp and pbi121-atoxr-gfp were incubated with 1 μm endoplasmic reticulum  tracker for 15 min at 37 °c, and the localization of atoxr-gfp was observed with a confocal microscope .
gfp fluorescence was detected between  <dig> and 550 nm with excitation at 488 nm, er tracker dyes signals were detected using a 615 nm emission filter with excitation at 587 nm.
analysis of the transgenic arabidopsis plants
t <dig> homozygous transgenic plants overexpressing-atoxr in a col- <dig> backgrounds were selected with kanamycin.
total rnas of the individual lines were obtained using trizol.
denaturing gel electrophoresis was performed to examine the transformation of the atoxr gene, which was identified using a probe labeled with digoxigenin  followed by rna gel blotting according to the methods described by  <cit> .
signals were detected using a luminescent image analyzer .
the single lines were named # <dig>  # <dig>  # <dig>  respectively.
the seeds of transgenic plants were treated 10 days with different concentrations of nacl ,mannitol ,cucl <dig>  and h2o <dig> .
t <dig> seeds from the arabidopsis col- <dig> lines were surface-sterilized, grown on 1/ <dig> ms plates, and supplemented with different concentrations of nacl  and h2o <dig>  for 2 weeks.
the seedling were grown under 16 h/8 h light/dark cycles at 22 °c, the root lengths were measured.
statistical analyses were performed using student’s t-tests.
h2o <dig> content measurement
the h2o <dig> content was measured in 10-day-old wt and t <dig> generation transgenic plants that overexpress atoxr and that had been treated with 150 mm nacl, 300 mm mannitol, 50 μm cucl <dig>  and 3 mm h2o <dig> for 12 h, 24 h, or 48 h. approximately  <dig>  g fresh weight of each sample was harvested and immediately ground in liquid nitrogen with a mortar and pestle.
the h2o <dig> content was measured colorimetrically at 415 nm by the titanium tetrachloride reaction method  <cit> .
measurement of total asa content
to measure the asa levels, seedlings of wt and t <dig> generation transgenic a.thaliana were grown on ms medium for  <dig> d, and that had been treated with 150 mm nacl, 300 mm mannitol, 50 μm cucl <dig>  and 3 mm h2o <dig> for 12 h, 24 h, or 48 h. approximately  <dig>  g fresh weight of each sample was harvested and immediately ground in liquid nitrogen with a mortar and pestle.
the asa content was determined using an asa content test kit .
briefly, samples were ground under liquid nitrogen and homogenized in 1 ml of cold extraction buffer .
the homogenate was centrifuged at 10000 rpm for 20 min at 4 °c, then the supernatant or standard solution  were incubated with 800 μl solution ii and 100 μl solution iii, respectively, and pipetting immediately at room temperature.
then, the absorption values of 30 s and 150 s at 265 nm with uv spectrophotometer were used for calculated the total asa levels of plants.
three biological replications were used for statistical analyses.
statistical significance was determined using student’s t-tests.
RESULTS
atoxr, a single copy gene encoding an er protein in arabidopsis
a blast search of the ncbi database for matches to putoxr identified one candidate , which we named atoxr.
the sequence is from a gene of unknown function with 61 % identity to putoxr at the amino acid sequence level.
atoxr is a single copy gene with a 564-bp open reading frame  encoding  <dig> amino acids with a predicted molecular mass of  <dig>  kda.
homologous proteins are found in several other plants .
in a phylogenetic tree based on the amino acid sequences of the conserved region, atoxr is most closely related to eutrema salsugineum and capsella rubella of the family brassicaceae .
atoxr is predicted to have two transmembrane domains by the tmhmm algorithm .fig.
<dig> sequence and bioinformatic analyses of atoxr.
alignment of the amino acid sequence of atoxr from arabidopsis thaliana  with those of oryza sativa , glycine max , zea mays , setaria italica , medicago truncatula , eutrema salsugineum , capsella rubella , theobroma cacao , and ricinus communis .
b phylogenetic trees based on the amino acid sequence of atoxr and homologous sequences from the genbank database.
the accession numbers are listed in the experimental procedures section.
transmembrane domains in atoxr were predicted by the tmhmm algorithm , and are underlined in black in
the er was visualized using er tracker, an er-specific dye for living cells.
because eukaryotic yeast shares similar cellular structures with plant cells, the subcellular localization of atoxr in yeast and plant cells were both analysed with an er tracker in this study.
we observed that the green fluorescence of gfp alone was almost evenly distributed throughout the yeast  and arabidopsis root cells , whereas the green fluorescence of atoxr-gfp fusion protein  and the red fluorescence of the er tracker  overlapped  in both yeast cells and arabidopsis root cells .
these results suggested that the atoxr-gfp was localized in the er in yeast and arabidopsis cells , atoxr is a single copy gene ecoding an er protein in arabidopsis genome.fig.
<dig> subcellular localization of atoxr-gfp.
a expression of gfp protein  and atoxr-gfp fusion constructs  in yeast cells.
scale bars = 5 μm.
b expression of gfp protein  and atoxr-gfp fusion constructs  in a. thaliana  root cells.
the gfp column shows the signal detected in the green channel; the er tracker column shows the signal detected in the red channel; the gfp+ er tracker column corresponds to the merging of the green and red channels, in which yellow represents the superposition of green and red.
dic: differential interference contrast.
scale bars = 20 μm
gene expression of atoxr is induced by abiotic stresses in arabidopsis
to examine the biological functions of atoxr, we first investigated its expression pattern in various organs by qrt-pcr.
this analysis showed that atoxr was expressed in all arabidopsis plant organs , with the highest levels of expression in leaves under normal conditions .
when plants were grown in the presence of 150 mm nacl, atoxr mrna expression, as shown by qrt-pcr, was induced within 6 h of treatment, declined at 12 h, and then increased again after 24 h in the leaves.
in roots, atoxr mrna was slightly declined at 6 h after treatment, and then increased gradually and peaked at 24 h. in the presence of 300 mm mannitol, atoxr mrna expression peaked at 24 h in leaves, whereas expression was induced at 6 h after inititation of treatment in roots, and then began to decline gradually.
after stressing with 50 μm cucl <dig>  atoxr mrna expression peaked at 24 h in both the leaves and roots.
in the presence of 3 mm h2o <dig>  atoxr mrna expression peaked at 24 h in both the leaves; in contrast, expression peaked at  <dig>  and then declined in roots .
overall, these results suggest that the atoxr gene confers a response to multiple abiotic stresses in both leves and roots of arabidopsis.fig.
<dig> expression analysis of atoxr gene from a. thaliana.
quantitative rt-pcr analysis of atoxr in different organs of a. thaliana.
quantitative rt-pcr analysis of the expression of atoxr in response to various abiotic stresses .
atoxr expression was normalized against actin mrna levels; the reported data are the means of three replicate experiments ± s.e
overexpression of atoxr gene enhances tolerance to abiotic stresses in yeast and arabidopsis
growth of transgenic yeast cells carrying atoxr was better on solid yeast ypg medium than that of control cells on media containing nacl, mannitol, or h2o <dig> .
the transformants also grew much better than did the empty vector transformants on media containing al3+, mg2+, cu2+,mn2+, ba2+, or fe3+ , suggesting that atoxr is associated with the oxidative stress caused by high levels of cations.fig.
<dig> growth of atoxr yeast transformants in response to a variety of abiotic stresses.
yeast transformants were grown on  ypd medium containing nacl , mannitol , and h2o <dig>  in the presence of 2 %  galactose and  ypd medium containing different concentrations of the following metal ions: mgcl <dig> , mncl <dig> , bacl <dig> , cdcl <dig> , cucl <dig> , fecl <dig> , alcl <dig>  in the presence of 2 %  galactose.
growth was monitored for 3–6 days at 30 °c
three arabidopsis transgenic plants that overexpressed atoxr under the control of the camv35s promoter  were identified by northern blotting .
control samples showed weak atoxr signals, whereas the transgenic plants had higher expression confirming that the plants had been successfully transformed with atoxr.
in seedlings exposed to nacl , mannitol , cucl <dig> , or h2o <dig> , both the primary roots and leaves grew better in the atoxr transgenic lines compared to wt plants , whereas the growth of wt was similar to that of the transgenic lines on control medium .
measurements confirmed that the root lengths  of atoxr transgenic lines were higher than those of wt plants under stress conditions.
these results suggest that overexpression of atoxr improved the tolerance to multiple abiotic stresses in arabidopsis.fig.
<dig> relative abiotic stress tolerance of wild-type  and atoxr-transgenic plants.
rna gel blot analysis of t <dig> transgenic plants expressing atoxr.
wt: arabidopsis thaliana ecotype columbia-0; # <dig>  # <dig>  and #3: t <dig> seedlings with atoxr on a columbia- <dig> background.
growth of wt plants and transgenic atoxr plants  on medium containing 1/ <dig> ms ,  <dig> and 125 mm nacl ,  <dig> and 200 mm mannitol ,  <dig> and 50 μm cucl <dig> , and  <dig> and 2 mm h2o <dig>  for 14 days.
root lengths of wt and atoxr transgenic plants were measured after treatment.
each value represents the means ± se of  <dig> plants.
statistical significance was determined using student’s t-tests.
*represents p <  <dig>  and **represents p <  <dig>
determination of h2o <dig> and asa content in atoxr transgenic arabidopsis plants under abiotic stresses
on the control medium, the h2o <dig> contents were slightly lower in three transgenic plants than those of wt plants, whereas h2o <dig> levels began to significant decrease in the three transgenic plants within 12 h of exposure to 150 mm nacl, 300 mm mannitol, 50 μm cucl <dig>  or 3 mm h2o <dig>  comparable to that of wt plants .
these results suggest that atoxr is associated with h2o <dig> scavenging to lower oxidative stress.fig.
<dig> effect of abiotic stresses on h2o <dig> content in wt and transgenic plants.
the h2o <dig> content of 10-day-old wt and t <dig> generation transgenic plants overexpressing atoxr were assessed after  <dig>   <dig>  or 48 h of treatment:  150 mm nacl;  300 mm mannitol;  50 μm cucl2;  3 mm h2o <dig>  the means ± standard deviations  of three replicates are shown.
statistical significance was determined using student’s t-tests.
*represents p <  <dig>  and **represents p <  <dig>
in addition, we also test the asa content in atoxr transgenic plants under abiotic stresses conditions.
anylsis the phenotype of asa levels in the normal condition , the asa content was higher in the transgenic plants than in the wt, while in the presence of stresses , it was significantly higher after 12 h, 24 h, and 48 h treatment .
to further confirm the content of asa in wt and atoxr transgenic plants under abiotic stresses conditions, hplc method was used to determine the total asa content in the tissues under 150 mm nacl and 3 mm h2o <dig> conditions for  <dig> and 48 h treatment.
the hplc analysis showed similar trend to the results of test kit analysis .
overall, these results suggest that overexpression of atoxr improves the accumulation of asa content in transgenic plants compared with wt plants under abiotic stress conditions.fig.
<dig> effect of abiotic stresses on asa content in wt and transgenic plants.
asa content of 10-day-old wt and t <dig> generation transgenic plants overexpressing atoxr were assessed after 12 h, 24 h, or 48 h treatment:  150 mm nacl;  300 mm mannitol;  50 μm cucl2;  3 mm h2o <dig>  the means ± sds of three replicates are shown.
statistical significance was determined using student’s t-tests.
* represents p <  <dig>  and ** represents p <  <dig>
discussion
asa is known to play a role in response to oxidative stress, although the regulatory molecular mechanism of asa synthesis has not been yet well understood.
here, we showed that atoxr, an arabidopsis gene of unknown function, is related to the levels of asa and h2o <dig>  atoxr is predicted to have two transmembrane domains.
atoxr-gfp was localized to the er in yeast and arabidopsis cells , the localization pattern of atoxr-gfp is agreement with previous reports  <cit> .
treatment with nacl, mannitol, metal ions, or h2o <dig> induced atoxr expression in both the leaves and roots .
in plants, salt, osmotic stress, and metal ions can be indirect produce reactive oxygen species , which leads to oxidative stress .
together, these findings suggest that the expression of atoxr might be associated with oxidative stress.
in addition, overexpression of atoxr in yeast and arabidopsis enhanced their tolerances to multiple abiotic stresses .
high levels of cations , salt, osmotic stress, etc.
can be indirect produce ros, which lead to oxidative stress  <cit> .
overexpressing atoxr also improved the tolerance to metal ions in yeast and arabidopsis .
thus, these results suggest that the atoxr gene has a role in the response to multiple abiotic stress, and associated with oxidative stress in yeast and plants.
although moderate levels of ros have a role in regulating various biological processes such as hormone signaling, and biotic and abiotic stress responses  <cit> , excessive ros can cause irreversible damage in plants .
our finding that atoxr-overexpressing transgenic plants accumulated less h2o <dig> than did wt after challenge with nacl, mannitol, cucl <dig>  and h2o <dig>  is similarly to previous reports that the overexpression of stress resistance-related genes resulted in less h2o <dig> accumulation in response to abiotic stresses .
plants have four major h2o2-scavenging pathways.
two of these pathways, the water-water cycle and the ascorbate-glutathione cycle  <cit> , are related to asa, which is known to have roles in plant stress responses  <cit> .
therefore, the effect of atoxr on conferring tolerance to multiple stresses might be caused by the increased asa content in atoxr transgenic plants.
in addition, asa also has a role in ros detoxification, as an antioxidant and h2o2-scavenger in plant cell to avoid accumulation of ros under stress conditions  <cit> .
in our study, the increased asa content in atoxr-expressing arabidopsis in response to abiotic stresses  suggests that atoxr improves tolerance to abiotic stresses by increasing the asa, which in turn promotes the scavenging of excess h2o <dig>  similar result was obtained in the transgenic plants expressing dhar, galdh and co-expression of nced and alo increased tolerance to abiotic stresses with elevated levels of asa  <cit> .
therefore, our result suggest that overexpression of atoxr is an effective way for use in crops improvement for increased tolerance to abiotic stresses and nutrition quality.
CONCLUSIONS
in this study, our results suggest that expression of atoxr gene is response to abiotic stresses in roots and leaves of arabidopsis.
the h2o <dig> and asa content of atoxr-overexpressing transgenic plants were significantly lower and significantly higher, respectively, than those of wt plants under abiotic stress conditions, furthermore, overexpression of atoxr gene improves abiotic stresses tolerance in arabidopsis.
in addition to this, because asa is an important component of human nutrition, modified expression of atoxr offers potential for the development of crop varieties with elevated asa.
hence, increasing the asa content in crops is important for further agriculture production through moleculer breeding.
additional file
additional file 1: total asa content in wt and transgenic plants were performed by hplc analysis.
asa content of 10-day-old wt and t <dig> generation transgenic plants overexpressing atoxr were assessed after 24 h, or 48 h treatment:  150 mm nacl;  3 mm h2o <dig>  the clear extracts  were injected directly into the hplc instrument , and chromatographic separation was achieved on an sepax gp-c <dig>  column and detected at 254 nm with a uv detector.
the means ± sds of three replicates are shown.
statistical significance was determined using student’s t-tests.
* represents p <  <dig>  and ** represents p <  <dig> .
abbreviations
apxascorbate peroxidase
asal-ascorbic acid
catcatalase
digdigoxigenin
erendoplasmic reticulum.
gpxglutathione peroxidase
gshglutathione
h2o2hydrogen peroxide
msmurashige and skoogmedium
o2−superoxide anions
oh−hydroxyl radicals
qrt-pcrquantitative real-time pcr
rosreactive oxygen species
sdsynthetic defined medium
sodsuperoxide dismutase
ypdyeast extract peptone dextrose medium
ypgyeast extract peptone galactose medium
we thank dr. raymond for english editing.
funding
this work was supported by china postdoctoral science foundation , heilongjiang province government postdoctoral science foundation  and heilongjiang province foundation for returnees  awarded to yuanyuan bu.
further supported by the program for changjiang scholars and innovative research team in university  awarded to shenkui liu.
the funding bodies were not involved in design of the studies; collection, analysis, nor interpretation of the data; nor writing of the manuscript.
availability of data and materials
amino acids sequences of atoxr , oryza sativa , glycine max , zea mays , setaria italica , medicago truncatula , eutrema salsugineum , capsella rubella , theobroma cacao , and ricinus communis  are available in the ncbi-genbank database.
the datasets supporting the conclusions of this article are included within the article and its additional file  <dig>
authors’ contributions
yb, bs and sl designed the study.
yb and bs performed the experiments and drofted the manuscript.
yb and bs analyzed the data.
az and xz provide the materials.
sl and tt supervised the study and critically revised the manuscript.
all authors read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.
consent for publication
not applicable.
ethics approval and consent to participate
not applicable.

 in natural environments, plant growth and crop productivity are reduced by abiotic stresses such as high salinity, drought, heavy metals, and oxidative stress. although these stresses might elicit different plant responses, most induce the accumulation of reactive oxygen species, including hydroxyl radicals, superoxide anions, and hydrogen peroxide. the production of large amounts of ros in plant cells leads to oxidative damage. in our previous study, we prepared a cdna library from seedlings of a salt-tolerant plant, puccinellia tenuiflora that had been treated with 150 mm nahco. one of the sequenced genes, putoxr, was found to confer enhanced tolerance to multiple abiotic stresses in yeast. arabidopsis thaliana has a homologous gene, which in our preliminary studies was found to be associated with multiple abiotic and oxidative stress. we thus named it atoxr, furthermore, the study of atoxr gene have not been reported. h2o2-scavenging in plants is achieved through several mechanisms, for example, the water–water cycle, catalase enzymatic reactions, the glutathione peroxidase cycle, and the ascorbate-glutathione cycle. these pathways involve a number of enzymes, such as superoxide dismutase, ascorbate peroxidase, catalase, and glutathione peroxidase, and anti-oxidants such as glutathione and ascorbic acid. in plants, h2o levels can be changed via the asa/glutathione cycle, apx plays a central role in the cycle and is emerging as a key enzyme in cellular h2o metabolism. asa is not only an important component of human nutrition but also an antioxidant and h2o2-scavenger that defends plants against abiotic stresses. for example, enhanced asa accumulation confers tolerance to oxidative, salt and drought stresses in potato and maintenance of a high asa level is required for oxidative stress tolerance in arabidopsis. asa is synthesized through multiple biosynthetic pathways in plants, while the major pathway is smirnoff–wheeler pathway. previous studies have shown that overexpression of asa biosynthetic pathway related genes resulted in increased vitamin c content. in addition, co-expression of nced and alo improves vitamin c content and tolerance to abiotic stresses in transgenic tobacco and stylo plants. although vitamin c is essential, humans are one of the few mammalian species unable to synthesis the vitamin and must obtain it through dietary sources. in recent years, the physiological function that asa plays in abiotic stress tolerance and nutritional quality has garnered increasing attention. in the present study, the expression pattern of atoxr in seedlings treated by abiotic stresses was studied using quantitative real-time pcr. the subcellular localization of the atoxr protein and the phenotypes of atoxr gene transformants in yeast and arabidopsis were also analyzed. additionally, the h2o and vitamin c content of atoxr transgenic arabidopsis and wt plants were determined in plants grown under abiotic stresses. methods plasmid constructs and plant materials the open reading frame of atoxr was amplified from arabidopsis cdna using the primers atoxr-fw and atoxr-rv. the amplified product atoxr was digested with kpni and bamhi and cloned into the yeast expression vector pyes to form pyes2-atoxr plasmid, which was transferred into saccharomyces cerevisiae strain invsc for the construction of gfp fusion proteins, atoxr without its stop codon was amplified with the primers atoxr-fw-bamhi and atoxr-rv-kpni by using atoxr cdna as a template, the amplified product was digested with bamhi and kpni, and cloned into the pegfp vector. the construct plasmid pegfp-atoxr-gfp was digested with bamhi and ecori, and cloned into the pyes vector to obtain the plasmid pyes2-atoxr-gfp, which was transferred into invsc using a lithium acetate-based method. atoxr-gfp was amplified from pegfp-atoxr-gfp with primers atoxr-fw-bamhi and gfp-rv-saci. the product was then digested with bamhi and saci, and cloned into the pbi vector to obtain the plasmid pbi121-atoxr-gfp. the constructs pbi121-atoxr, pbi121-atoxr-gfp and pbi121-gfp as control were transformed into agrobacterium tumefaciens strain eha to obtain the transgenic arabidopsis by the floral dip method.table sequence of the primers used for pcr atoxr-fw atoxr-rv pbi121-atoxr-fw ggatcctatgtatttcgccg pbi121-atoxr-rv gagctcctcctctaatagtc atoxr-fw-bamhi ggtaccatgtatttcgccgccatagc atoxr-rv-kpni ggatccgcctctctccgttttctcc gfp-rv-saci gagctcctagagtcgcggccgc pyes2-atoxr-fw ggtacctatgtatttcgccg pyes2-atoxr-rv ggatccctcctctaatagtc atoxr-rt-fw atoxr-rt-rv actin-fw actin-rv phylogenetic analyses, yeast transformations, and growth conditions full-length amino acids sequences were aligned using clustalx, and then imported into the molecular evolutionary genetics analysis package version. phylogenetic analyses were conducted using the neighbor joining method in mega. the following accession numbers were used: atoxr, oryza sativa, glycine max, zea mays, setaria italica, medicago truncatula, eutrema salsugineum, capsella rubella, theobroma cacao, and ricinus communis. yeast transformations were performed using a lithium acetate-based method. the empty vector plasmids pyes2-atoxr and pyes were used as controls, and were introduced into the yeast strain invsci. the transformed yeast strains were grown in synthetic defined medium lacking the appropriate amino acids for the selective growth for the expression plasmids. for the response assays, yeast transformants were cultured in liquid yeast extract peptone dextrose medium until they reached an optical density at 600 nm of ≈ , and were then diluted 10−1-, 10−2-, 10−3-,10−4-, and 10−5-fold using double distilled h2o. then, aliquots of each dilution were spotted onto solid yeast ypd and ypg medium supplemented with different concentrations of nacl, mannitol, h2o, mncl, mgcl, cdcl, bacl, cucl, fecl, alcl. a yeast transformant of the pyes empty vector was used as a control, and growth was monitored for 3–7 days at 30 °c. analysis of gene expression using quantitative real-time pcr arabidopsis seeds were surface sterilized and plated on solid half ms medium. after 2 days stratification at 4 °c, the plates were stored in a 22 °c incubator for propagation. the seedlings were transferred from the plates to a 1: mixture of soil and vermiculite and grown to maturity at 22 °c. the plants were cultured under a 16-h-light/8-h-dark cycle in a growth chamber. roots, stems, leaves, panicle, and siliques of 2-month-old plants were sampled for qrt-pcr. a second batch of seedlings were pre-cultured for 2 weeks on 1/ solid medium, and then treated with different concentrations of various stresses, the shoots and roots were sampled after 0 h, 6 h, 12 h, and 24 h treatment and used for qrt-pcr analyses. total rna was isolated using the rneasy plant mini kit, and treated with rnase-free dnasei. first-strand cdna was synthesized using superscript iii reverse transcriptase. gene-specific primers pairs atoxr-rt-fw and atoxr-rt-rv were used for atoxr, while actin-fw and actin-rv were used for actin. relative quantification using qrt-pcr reactions were performed with sybr green i using the lightcycler® system ii. localization analysis of atoxr protein in yeast and plant cells yeast transformants carrying the plasmids pyes2-atoxr-gfp and pyes2-gfp were pre-cultured in liquid ypd medium overnight at 30 °c, washed three times with distilled water to remove the glucose and cultured in liquid sd uracil medium with galactose at 30 °c to induce the expression of gfp and atoxr-gfp under the control of gal promoter. live cells were incubated with er tracker, and the localization of atoxr-gfp was observed with a confocal microscope. five-day-old transgenic roots carrying pbi121-gfp and pbi121-atoxr-gfp were incubated with 1 μm endoplasmic reticulum tracker for 15 min at 37 °c, and the localization of atoxr-gfp was observed with a confocal microscope. gfp fluorescence was detected between and 550 nm with excitation at 488 nm, er tracker dyes signals were detected using a 615 nm emission filter with excitation at 587 nm. analysis of the transgenic arabidopsis plants t homozygous transgenic plants overexpressing-atoxr in a col- backgrounds were selected with kanamycin. total rnas of the individual lines were obtained using trizol. denaturing gel electrophoresis was performed to examine the transformation of the atoxr gene, which was identified using a probe labeled with digoxigenin followed by rna gel blotting according to the methods described by. signals were detected using a luminescent image analyzer. the single lines were named # # # respectively. the seeds of transgenic plants were treated 10 days with different concentrations of nacl,mannitol,cucl and h2o. t seeds from the arabidopsis col- lines were surface-sterilized, grown on 1/ ms plates, and supplemented with different concentrations of nacl and h2o for 2 weeks. the seedling were grown under 16 h/8 h light/dark cycles at 22 °c, the root lengths were measured. statistical analyses were performed using student’s t-tests. h2o content measurement the h2o content was measured in 10-day-old wt and t generation transgenic plants that overexpress atoxr and that had been treated with 150 mm nacl, 300 mm mannitol, 50 μm cucl and 3 mm h2o for 12 h, 24 h, or 48 h. approximately  g fresh weight of each sample was harvested and immediately ground in liquid nitrogen with a mortar and pestle. the h2o content was measured colorimetrically at 415 nm by the titanium tetrachloride reaction method. measurement of total asa content to measure the asa levels, seedlings of wt and t generation transgenic a.thaliana were grown on ms medium for d, and that had been treated with 150 mm nacl, 300 mm mannitol, 50 μm cucl and 3 mm h2o for 12 h, 24 h, or 48 h. approximately  g fresh weight of each sample was harvested and immediately ground in liquid nitrogen with a mortar and pestle. the asa content was determined using an asa content test kit. briefly, samples were ground under liquid nitrogen and homogenized in 1 ml of cold extraction buffer. the homogenate was centrifuged at 10000 rpm for 20 min at 4 °c, then the supernatant or standard solution were incubated with 800 μl solution ii and 100 μl solution iii, respectively, and pipetting immediately at room temperature. then, the absorption values of 30 s and 150 s at 265 nm with uv spectrophotometer were used for calculated the total asa levels of plants. three biological replications were used for statistical analyses. statistical significance was determined using student’s t-tests.  atoxr, a single copy gene encoding an er protein in arabidopsis a blast search of the ncbi database for matches to putoxr identified one candidate, which we named atoxr. the sequence is from a gene of unknown function with 61 % identity to putoxr at the amino acid sequence level. atoxr is a single copy gene with a 564-bp open reading frame encoding amino acids with a predicted molecular mass of  kda. homologous proteins are found in several other plants. in a phylogenetic tree based on the amino acid sequences of the conserved region, atoxr is most closely related to eutrema salsugineum and capsella rubella of the family brassicaceae. atoxr is predicted to have two transmembrane domains by the tmhmm algorithm.fig. sequence and bioinformatic analyses of atoxr. alignment of the amino acid sequence of atoxr from arabidopsis thaliana with those of oryza sativa, glycine max, zea mays, setaria italica, medicago truncatula, eutrema salsugineum, capsella rubella, theobroma cacao, and ricinus communis. b phylogenetic trees based on the amino acid sequence of atoxr and homologous sequences from the genbank database. the accession numbers are listed in the experimental procedures section. transmembrane domains in atoxr were predicted by the tmhmm algorithm, and are underlined in black in the er was visualized using er tracker, an er-specific dye for living cells. because eukaryotic yeast shares similar cellular structures with plant cells, the subcellular localization of atoxr in yeast and plant cells were both analysed with an er tracker in this study. we observed that the green fluorescence of gfp alone was almost evenly distributed throughout the yeast and arabidopsis root cells, whereas the green fluorescence of atoxr-gfp fusion protein and the red fluorescence of the er tracker overlapped in both yeast cells and arabidopsis root cells. these results suggested that the atoxr-gfp was localized in the er in yeast and arabidopsis cells, atoxr is a single copy gene ecoding an er protein in arabidopsis genome.fig. subcellular localization of atoxr-gfp. a expression of gfp protein and atoxr-gfp fusion constructs in yeast cells. scale bars = 5 μm. b expression of gfp protein and atoxr-gfp fusion constructs in a. thaliana root cells. the gfp column shows the signal detected in the green channel; the er tracker column shows the signal detected in the red channel; the gfp+ er tracker column corresponds to the merging of the green and red channels, in which yellow represents the superposition of green and red. dic: differential interference contrast. scale bars = 20 μm gene expression of atoxr is induced by abiotic stresses in arabidopsis to examine the biological functions of atoxr, we first investigated its expression pattern in various organs by qrt-pcr. this analysis showed that atoxr was expressed in all arabidopsis plant organs, with the highest levels of expression in leaves under normal conditions. when plants were grown in the presence of 150 mm nacl, atoxr mrna expression, as shown by qrt-pcr, was induced within 6 h of treatment, declined at 12 h, and then increased again after 24 h in the leaves. in roots, atoxr mrna was slightly declined at 6 h after treatment, and then increased gradually and peaked at 24 h. in the presence of 300 mm mannitol, atoxr mrna expression peaked at 24 h in leaves, whereas expression was induced at 6 h after inititation of treatment in roots, and then began to decline gradually. after stressing with 50 μm cucl atoxr mrna expression peaked at 24 h in both the leaves and roots. in the presence of 3 mm h2o atoxr mrna expression peaked at 24 h in both the leaves; in contrast, expression peaked at and then declined in roots. overall, these results suggest that the atoxr gene confers a response to multiple abiotic stresses in both leves and roots of arabidopsis.fig. expression analysis of atoxr gene from a. thaliana. quantitative rt-pcr analysis of atoxr in different organs of a. thaliana. quantitative rt-pcr analysis of the expression of atoxr in response to various abiotic stresses. atoxr expression was normalized against actin mrna levels; the reported data are the means of three replicate experiments ± s.e overexpression of atoxr gene enhances tolerance to abiotic stresses in yeast and arabidopsis growth of transgenic yeast cells carrying atoxr was better on solid yeast ypg medium than that of control cells on media containing nacl, mannitol, or h2o. the transformants also grew much better than did the empty vector transformants on media containing al3+, mg2+, cu2+,mn2+, ba2+, or fe3+, suggesting that atoxr is associated with the oxidative stress caused by high levels of cations.fig. growth of atoxr yeast transformants in response to a variety of abiotic stresses. yeast transformants were grown on ypd medium containing nacl, mannitol, and h2o in the presence of 2 % galactose and ypd medium containing different concentrations of the following metal ions: mgcl, mncl, bacl, cdcl, cucl, fecl, alcl in the presence of 2 % galactose. growth was monitored for 3–6 days at 30 °c three arabidopsis transgenic plants that overexpressed atoxr under the control of the camv35s promoter were identified by northern blotting. control samples showed weak atoxr signals, whereas the transgenic plants had higher expression confirming that the plants had been successfully transformed with atoxr. in seedlings exposed to nacl, mannitol, cucl, or h2o, both the primary roots and leaves grew better in the atoxr transgenic lines compared to wt plants, whereas the growth of wt was similar to that of the transgenic lines on control medium. measurements confirmed that the root lengths of atoxr transgenic lines were higher than those of wt plants under stress conditions. these results suggest that overexpression of atoxr improved the tolerance to multiple abiotic stresses in arabidopsis.fig. relative abiotic stress tolerance of wild-type and atoxr-transgenic plants. rna gel blot analysis of t transgenic plants expressing atoxr. wt: arabidopsis thaliana ecotype columbia-0; # # and #3: t seedlings with atoxr on a columbia- background. growth of wt plants and transgenic atoxr plants on medium containing 1/ ms, and 125 mm nacl, and 200 mm mannitol, and 50 μm cucl, and and 2 mm h2o for 14 days. root lengths of wt and atoxr transgenic plants were measured after treatment. each value represents the means ± se of plants. statistical significance was determined using student’s t-tests. *represents p <  and **represents p <  determination of h2o and asa content in atoxr transgenic arabidopsis plants under abiotic stresses on the control medium, the h2o contents were slightly lower in three transgenic plants than those of wt plants, whereas h2o levels began to significant decrease in the three transgenic plants within 12 h of exposure to 150 mm nacl, 300 mm mannitol, 50 μm cucl or 3 mm h2o comparable to that of wt plants. these results suggest that atoxr is associated with h2o scavenging to lower oxidative stress.fig. effect of abiotic stresses on h2o content in wt and transgenic plants. the h2o content of 10-day-old wt and t generation transgenic plants overexpressing atoxr were assessed after or 48 h of treatment: 150 mm nacl; 300 mm mannitol; 50 μm cucl2; 3 mm h2o the means ± standard deviations of three replicates are shown. statistical significance was determined using student’s t-tests. *represents p <  and **represents p <  in addition, we also test the asa content in atoxr transgenic plants under abiotic stresses conditions. anylsis the phenotype of asa levels in the normal condition, the asa content was higher in the transgenic plants than in the wt, while in the presence of stresses, it was significantly higher after 12 h, 24 h, and 48 h treatment. to further confirm the content of asa in wt and atoxr transgenic plants under abiotic stresses conditions, hplc method was used to determine the total asa content in the tissues under 150 mm nacl and 3 mm h2o conditions for and 48 h treatment. the hplc analysis showed similar trend to the results of test kit analysis. overall, these results suggest that overexpression of atoxr improves the accumulation of asa content in transgenic plants compared with wt plants under abiotic stress conditions.fig. effect of abiotic stresses on asa content in wt and transgenic plants. asa content of 10-day-old wt and t generation transgenic plants overexpressing atoxr were assessed after 12 h, 24 h, or 48 h treatment: 150 mm nacl; 300 mm mannitol; 50 μm cucl2; 3 mm h2o the means ± sds of three replicates are shown. statistical significance was determined using student’s t-tests. * represents p <  and ** represents p <  discussion asa is known to play a role in response to oxidative stress, although the regulatory molecular mechanism of asa synthesis has not been yet well understood. here, we showed that atoxr, an arabidopsis gene of unknown function, is related to the levels of asa and h2o atoxr is predicted to have two transmembrane domains. atoxr-gfp was localized to the er in yeast and arabidopsis cells, the localization pattern of atoxr-gfp is agreement with previous reports. treatment with nacl, mannitol, metal ions, or h2o induced atoxr expression in both the leaves and roots. in plants, salt, osmotic stress, and metal ions can be indirect produce reactive oxygen species, which leads to oxidative stress. together, these findings suggest that the expression of atoxr might be associated with oxidative stress. in addition, overexpression of atoxr in yeast and arabidopsis enhanced their tolerances to multiple abiotic stresses. high levels of cations, salt, osmotic stress, etc. can be indirect produce ros, which lead to oxidative stress. overexpressing atoxr also improved the tolerance to metal ions in yeast and arabidopsis. thus, these results suggest that the atoxr gene has a role in the response to multiple abiotic stress, and associated with oxidative stress in yeast and plants. although moderate levels of ros have a role in regulating various biological processes such as hormone signaling, and biotic and abiotic stress responses, excessive ros can cause irreversible damage in plants. our finding that atoxr-overexpressing transgenic plants accumulated less h2o than did wt after challenge with nacl, mannitol, cucl and h2o is similarly to previous reports that the overexpression of stress resistance-related genes resulted in less h2o accumulation in response to abiotic stresses. plants have four major h2o2-scavenging pathways. two of these pathways, the water-water cycle and the ascorbate-glutathione cycle, are related to asa, which is known to have roles in plant stress responses. therefore, the effect of atoxr on conferring tolerance to multiple stresses might be caused by the increased asa content in atoxr transgenic plants. in addition, asa also has a role in ros detoxification, as an antioxidant and h2o2-scavenger in plant cell to avoid accumulation of ros under stress conditions. in our study, the increased asa content in atoxr-expressing arabidopsis in response to abiotic stresses suggests that atoxr improves tolerance to abiotic stresses by increasing the asa, which in turn promotes the scavenging of excess h2o similar result was obtained in the transgenic plants expressing dhar, galdh and co-expression of nced and alo increased tolerance to abiotic stresses with elevated levels of asa. therefore, our result suggest that overexpression of atoxr is an effective way for use in crops improvement for increased tolerance to abiotic stresses and nutrition quality.  in this study, our results suggest that expression of atoxr gene is response to abiotic stresses in roots and leaves of arabidopsis. the h2o and asa content of atoxr-overexpressing transgenic plants were significantly lower and significantly higher, respectively, than those of wt plants under abiotic stress conditions, furthermore, overexpression of atoxr gene improves abiotic stresses tolerance in arabidopsis. in addition to this, because asa is an important component of human nutrition, modified expression of atoxr offers potential for the development of crop varieties with elevated asa. hence, increasing the asa content in crops is important for further agriculture production through moleculer breeding. additional file additional file 1: total asa content in wt and transgenic plants were performed by hplc analysis. asa content of 10-day-old wt and t generation transgenic plants overexpressing atoxr were assessed after 24 h, or 48 h treatment: 150 mm nacl; 3 mm h2o the clear extracts were injected directly into the hplc instrument, and chromatographic separation was achieved on an sepax gp-c column and detected at 254 nm with a uv detector. the means ± sds of three replicates are shown. statistical significance was determined using student’s t-tests. * represents p <  and ** represents p < . abbreviations apxascorbate peroxidase asal-ascorbic acid catcatalase digdigoxigenin erendoplasmic reticulum. gpxglutathione peroxidase gshglutathione h2o2hydrogen peroxide msmurashige and skoogmedium o2−superoxide anions oh−hydroxyl radicals qrt-pcrquantitative real-time pcr rosreactive oxygen species sdsynthetic defined medium sodsuperoxide dismutase ypdyeast extract peptone dextrose medium ypgyeast extract peptone galactose medium we thank dr. raymond for english editing. funding this work was supported by china postdoctoral science foundation, heilongjiang province government postdoctoral science foundation and heilongjiang province foundation for returnees awarded to yuanyuan bu. further supported by the program for changjiang scholars and innovative research team in university awarded to shenkui liu. the funding bodies were not involved in design of the studies; collection, analysis, nor interpretation of the data; nor writing of the manuscript. availability of data and materials amino acids sequences of atoxr, oryza sativa, glycine max, zea mays, setaria italica, medicago truncatula, eutrema salsugineum, capsella rubella, theobroma cacao, and ricinus communis are available in the ncbi-genbank database. the datasets supporting the conclusions of this article are included within the article and its additional file authors’ contributions yb, bs and sl designed the study. yb and bs performed the experiments and drofted the manuscript. yb and bs analyzed the data. az and xz provide the materials. sl and tt supervised the study and critically revised the manuscript. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. consent for publication not applicable. ethics approval and consent to participate not applicable.
abiotic stresses are serious threats to plant growth, productivity and result in crop loss worldwide, reducing average yields of most major crops. although abiotic stresses might elicit different plant responses, most induce the accumulation of reactive oxygen species in plant cells leads to oxidative damage. l-ascorbic acid is known as an antioxidant and h2o2-scavenger that defends plants against abiotic stresses. therefore, increasing the vitamin c content is important for improving abiotic stresses tolerance and nutrition quality in crops production. here, we show that the expression of atoxr gene is response to multiple abiotic stresses in both the leaves and roots of arabidopsis. atoxr-overexpressing transgenic arabidopsis plants enhance the tolerance to abiotic stresses. in this study, our results show that atoxr responds to multiple abiotic stresses. overexpressing atoxr improves tolerance to abiotic stresses and increase vitamin c content in arabidopsis thaliana. abiotic stressesl-ascorbic acid hydrogen peroxide transgenic plantsheilongjiang province government postdoctoral science foundationlbh-z15003bu yuanyuan heilongjiang province foundation for returneeslc201405bu yuanyuan the program for changjiang scholars and innovative research team in universityirt13053liu shenkui china postdoctoral science foundation 2016m590272bu yuanyuan issue-copyright-statement© the author 2016 atoxr protein was localized to the endoplasmic reticulum in yeast and arabidopsis cells by co-localization analysis with er specific dye. in addition, vitamin c is also an important component of human nutrition that has to be obtained from different foods. overexpression of atoxr gene resulted in asa accumulation and decreased h2o content in transgenic plants.
 abiotic stresses are serious threats to plant growth, productivity and result in crop loss worldwide, reducing average yields of most major crops. although abiotic stresses might elicit different plant responses, most induce the accumulation of reactive oxygen species in plant cells leads to oxidative damage. l-ascorbic acid is known as an antioxidant and h2o2-scavenger that defends plants against abiotic stresses. in addition, vitamin c is also an important component of human nutrition that has to be obtained from different foods. therefore, increasing the vitamin c content is important for improving abiotic stresses tolerance and nutrition quality in crops production.  here, we show that the expression of atoxr gene is response to multiple abiotic stresses in both the leaves and roots of arabidopsis. atoxr protein was localized to the endoplasmic reticulum in yeast and arabidopsis cells by co-localization analysis with er specific dye. atoxr-overexpressing transgenic arabidopsis plants enhance the tolerance to abiotic stresses. overexpression of atoxr gene resulted in asa accumulation and decreased h2o content in transgenic plants.  in this study, our results show that atoxr responds to multiple abiotic stresses. overexpressing atoxr improves tolerance to abiotic stresses and increase vitamin c content in arabidopsis thaliana. atoxr will be useful for the improvement of important crop plants through moleculer breeding. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords abiotic stressesl-ascorbic acid hydrogen peroxide transgenic plantsheilongjiang province government postdoctoral science foundationlbh-z15003bu yuanyuan heilongjiang province foundation for returneeslc201405bu yuanyuan the program for changjiang scholars and innovative research team in universityirt13053liu shenkui china postdoctoral science foundation 2016m590272bu yuanyuan issue-copyright-statement© the author 2016 