BACKGROUND
the synthesis of bioactive compounds for adaptation to abiotic stress conditions and for defence against herbivores and pathogen infections is a fundamental survival strategy of plants.
the biosynthesis of phytoalexins, which contain an indole moiety substituted with additional ring systems or side chains, often containing sulphur and nitrogen, is characteristic for cruciferous plants  <cit> .
the individual structures are very diverse even among different brassica cultivars.
in arabidopsis thaliana, a variety of compounds are synthesized from the intermediate indole-3-acetonitrile  in response to pathogen infection or heavy metal stress  <cit>  with camalexin as the most prominent metabolite.
the camalexin biosynthetic pathway from tryptophan and glutathione and its role in defence against a number of fungal pathogens has been investigated in detail  <cit> .
phytoalexin biosynthesis is induced upon pathogen infection, but also under harsh abiotic conditions, such as high dosages of heavy metal ions or uv light, which lead to the generation of reactive oxygen species and ultimately to programmed cell death.
for studies on plant metabolism, abiotic stress treatments provide the advantage that no interference of pathogen metabolism, which is often strain specific  <cit> , has to be taken into account.
eutrema salsugineum has been established recently as an alternative model system for crucifers in addition to arabidopsis, because of its high tolerance of various abiotic stresses  <cit> .
the e. salsugineum genome sequence  <cit> , as well as a reference transcriptome,  <cit>  are available and additional transcriptomics data were published recently  <cit> .
e. salsugineum is also referred to as thellungiella salsuginea.
the ecotype shandong analysed in this study was initially assigned as t. halophila and this species name was used in a number of publications  <cit> .
consequently, gene and transcript sequences isolated from shandong ecotype have been deposited under the species names t. halophila, t. salsuginea and e. salsugineum.
according to work by koch and german  <cit> , the species name t. salsuginea is acceptable, but e. salsugineum, which we refer to in this manuscript, is preferred.
within the brassicaceae, eutrema and arabidopsis are rather distantly related and their last common ancestor is estimated to have lived  <dig> million years ago  <cit> .
still, large stretches of syntenic regions were identified in the genomes, allowing clear assignment of putative orthologs  <cit> .
at the protein level, for the number of best hit pairs between eutrema and arabidopsis a peak at 85% amino acid sequence identity was determined  <cit> .
eutrema and arabidopsis have developed a diversified spectrum of defence compounds, such as glucosinolates  <cit>  and indolic phytoalexins.
in arabidopsis, these phytoalexins are predominantly synthesized from the intermediate indole-3-acetaldoxime  <cit> , while the characteristic eutrema phytoalexins are most likely derivatives of 1-methoxy-indole glucosinolate  <cit> .
the identification of biosynthetic genes for presumably glucosinolate-derived  and glucosinolate-independent  phytoalexins will build the basis for metabolic engineering studies of indolic phytoalexins and for establishment of a model for phytoalexin evolution in the brassicaceae.
in this work, we analysed the transcriptional reprogramming of e. salsugineum in response to abiotic stress conditions, which lead to the accumulation of phytoalexins.
we show that genes of tryptophan and indole glucosinolate biosynthesis and modification are highly upregulated providing candidates for phytoalexin biosynthesis.
also the eutrema ortholog of wrky <dig>  a key regulator of arabidopsis phytoalexin induction, was highly upregulated, even though known wrky <dig> target genes, such as cyp71b <dig>  <cit>  are apparently missing in e. salsugineum.
RESULTS
induction of phytoalexin biosynthesis in response to uv light and silver nitrate spraying
the biosynthesis of phytoalexins by brassicaceae species is induced by pathogen infection, but also specific abiotic stress treatments, such as high dosages of heavy metals and uv light.
applying abiotic stressors provides the advantage of a high degree of experimental reproducibility and excludes the modulation of plant defence reactions and metabolism by the pathogen.
induction of phytoalexin biosynthesis by the heavy metal salt cucl and uv treatment was previously established by pedras and coworkers  <cit> .
here, wasalexin induction was confirmed for 10-week old e. salsugineum  leaves in response to uvc light, silver nitrate application, and botrytis cinerea infection .
in arabidopsis, expression of camalexin biosynthetic genes is coregulated with expression of asa <dig>  encoding the committing enzyme of tryptophan biosynthesis.
we therefore assumed that also in e. salsugineum tryptophan biosynthesis is upregulated under phytoalexin inducing conditions, which we later confirmed .
quantitative rt-pcr was used to determine the induction kinetics of esasa <dig> .
for both treatments, transcript levels were highly elevated  <dig>  h and 10 h after the onset of induction.
therefore, for transcriptomics analysis 8 h induction was selected.figure  <dig> rt-qpcr analysis.
time course of expression after treatment with uv light  and agno3
.
esasa <dig> , esigmt <dig> , espen <dig> , esbglu18- <dig> , esbglu18- <dig> , and eswrky <dig> , were analysed.
the expression levels, relative to the mean for 0 h, were determined by rt-qpcr, normalized to the geometric mean of three reference genes .
values are means of three independent experiments ± se.
the eutrema transcriptome in response to uv light and heavy metal stress
rna was isolated from non-treated leaves and from leaves treated with either agno <dig> or uv light.
cdna libraries were prepared and approximately  <dig> mio to  <dig> mio 50 bp reads per library were obtained by illumina sequencing.
reads were mapped to the jgi genome  <cit> .
for each cdna library, approx.
75% of total transcript models were covered  and a large overlap between treatments was observed .
transcript models were analysed for read-counts in the different samples and annotated for best hit in the arabidopsis thaliana genome .table  <dig>
rnaseq metrics and alignments
n.i.
uv
agno
3
b.c.
reads were mapped to the jgi genome ,  <dig> reference transcripts ; uncounted/counted: number of unmapped/mapped reads; uniquely: number of uniquely mapped reads; non-specific: number of reads with multiple locations in the reference.
similarly, we have analysed the transcriptome 48 h after infection of plants with b. cinerea .
<dig> transcripts were identified as more than 2-fold upregulated with respect to untreated leaves.
of this set, 56% and 61% were also upregulated more than 2-fold after uv and agno <dig> treatment, respectively, indicating overlapping responses to the abiotic and biotic stressors.
however, as transcriptional changes in response to uv light and agno <dig> were much more pronounced, we focussed on these treatments for further analysis.
microarray analysis of four biological replicates was conducted with agilent arrays based on the design by lee et al.
<cit> .
statistically robust differential regulation was observed for the majority of transcripts .
of a total of  <dig> oligonucleotide probes, signal intensities of  <dig>  and  <dig>  probes were significantly  elevated, while signal intensities of  <dig>  and  <dig>  probes were significantly reduced in response to uv light and agno <dig>  respectively.
these array data were compared with the rnaseq data, which in addition provide information about absolute expression levels.
a correlation analysis with the log <dig> fold-change values obtained by the two methods in response to uv and agno <dig> is shown in additional file 5: figure s <dig>
we matched rnaseq and array data based on the comparison of array probe and transcript model sequences and omitted those probes from further analysis for which no match was found.
duplicated genes with highly homologous sequences were sometimes indistinguishable on array level .
here, the more highly abundant transcript from the rnaseq analysis was chosen for the matched dataset.
log <dig> fold-change values based on rnaseq and array analyses were correlated .
for further analysis, we worked with a set of  <dig>  genes, for which both array and rnaseq data are available .
correlations of log <dig> fold-change values in response to uv and agno <dig> treatment obtained by microarray hybridization are shown in figure  <dig>  for a large proportion of these transcripts , significant changes in abundance were detected in response to uv or agno <dig> treatment .
<dig>  transcripts were upregulated,  <dig>  downregulated in response to both treatments, indicating substantial overlap in metabolic and regulatory responses.figure  <dig> global analysis of transcriptomics data.
the set of  <dig>  genes, for which rnaseq and array data could be matched, was analysed for significant  transcriptional changes  in response to uv light and agno <dig>  a large overlap in response to the two stressors was observed.
figure  <dig> shows a mapman  <cit>  representation of log2-fold transcriptional changes, in response to uv light  and agno <dig> , based on array data.
strongly repressed processes include photosynthesis and starch synthesis.
the tricarboxylic acid cycle, providing precursors of aromatic amino acid and the biosynthesis of cell wall precursors are induced on the level of transcript abundance, consistent with plant defence reactions.figure  <dig> mapman visualisation of transcript abundance changes for metabolic genes.
metabolism overview for microarray data.
a: uv versus not induced .
b: agno <dig> versus n.i.. red indicates downregulated, blue upregulated genes.
the colour code indicates log2-fold changes in expression.
transcriptional changes induced upon both uv and heavy metal stress
transcripts that were strongly and consistently upregulated in response to both uv light and agno <dig> include a number of genes that encode enzymes involved in biosynthesis or modification of hormones and signalling compounds.
this indicated that reprogramming the hormone balance is one of the key elements in the adaptation of eutrema to high dosages of uv light or heavy metals.
genes upregulated most strongly in response to both stressors include essot <dig> and, based on ngs data, esst2a/essot <dig> .
the corresponding arabidopsis orthologs encode a sulfotransferase, which sulphonates salicylic acid, thereby positively regulating salicylic acid accumulation  <cit> , and a sulfotransferase, which sulphonates hydroxyjasmonic acid  <cit> .
sot <dig> is also strongly induced in a. thaliana seedlings in response to uvb light  <cit> .
furthermore, we observed that genes encoding eutrema orthologs of 1-amino-cyclopropane-1-carboxylate synthase  <dig>  and cis-zeatin o-β-d-glucosyltransferase   <cit>  were highly upregulated in response to both uv light and agno <dig>  other induced processes are senescence and regulation of cell death.
here, examples of highly upregulated genes include the eutrema orthologs of atdlah  <cit>  and atbap <dig>  an inhibitor of programmed cell death  <cit> .
we observed significant transcriptional reprogramming of phenylpropanoid metabolism.
genes of the core phenylpropanoid biosynthetic pathway, i.e.
e. salsugineum orthologs putatively encoding phenylalanine ammonia-lyase  <dig> and  <dig>  cinnamate-4-hydroxylase, cinnamoyl coa reductase, and cinnamyl alcohol dehydrogenase were upregulated in response to uv and agno <dig>  the e. salsugineum ortholog of tt <dig>  encoding naringenine chalcone synthase, was strongly downregulated.
interestingly, in arabidopsis strong tt <dig> upregulation was observed in response to uv light  <cit> .
whether this is due to experimental differences, such as plant age or uv wavelength or reflects a species-specific difference in adaptation with respect to the phenylpropanoids that are synthesized remains to be investigated.
further, fundamental changes in the transcript abundance of genes encoding enzymes involved in the biosynthesis of defence-related secondary metabolites were observed, which are discussed in detail below.
a number of genes have been functionally associated with the halophytic lifestyle of e. salsugineum.
these include the sodium transporter eshkt <dig>  <cit>  and eserf <dig>  <cit> , which are also strongly and significantly upregulated under both agno <dig> and uv treatment .
arabidopsis erf <dig> is an integrator of different abiotic and biotic stress responses  <cit> .
for other genes associated with salt tolerance, such as sos <dig> and iron superoxide dismutase this was not observed  <cit> .
we have surveyed transcriptional changes in response to agno <dig> and uv in e. salsugineum for similarity to changes in response to drought or cold  <cit> .
there was a clear overlap among downregulated genes, which are mainly related to photosynthesis.
a common pattern among upregulated genes was not observed .
apparently, the responses of e. salsugineum to drought/cold and to uv/heavy metal stresses differ substantially.
the effect of silver treatment on the arabidopsis transcriptome was investigated previously by kaveh and coworkers  <cit> .
the number of significantly upregulated genes was much lower than in our work on eutrema, probably due to differences in the experimental setup.
only for a few genes, the corresponding orthologs were identified in both studies, including the orthologs of the β-glucosidase genes  <dig> and  <dig>
recently, genes were identified in a. thaliana which are upregulated in response to both b. cinerea infection and oxidative stress  <cit> .
for  <dig> out of these  <dig> transcripts, corresponding e. salsuginea orthologs were identified here.
strikingly, for a large fraction of these genes , including e.g.
escyp79b <dig> and escyp83b <dig> , we observed upregulation by both uv and agno <dig> treatments .
possibly, all these processes lead to the generation of reactive oxygen species, inducing transcriptional reactions that are largely conserved between arabidopsis and eutrema.
tryptophan biosynthetic genes
in brassicaceae, tryptophan is a precursor of indole glucosinolates and indolic phytoalexins  <cit> , which constitute the major tryptophan sinks.
as cellular tryptophan concentrations are low in arabidopsis leaves, tryptophan biosynthesis is strongly coregulated with the biosynthesis of camalexin  <cit> .
here, we observed significant and strong increases in transcript levels associated with the tryptophan biosynthetic pathway in response to uv light and agno <dig> .
this includes genes encoding tryptophan synthase β  type  <dig> isoforms, while the ortholog of tsbtype <dig>  of which the biological function is unknown  <cit> , is significantly downregulated in response to uv light.table  <dig>
analysis of transcript abundance changes of genes associated with the biosynthesis of defence-related metabolites
transcript id
best ath hit
gene symbol
annotation
uv
ag
+
fold change log <dig>
fdr-p-value test
fold change log <dig>
fdr-p-value test
rnaseq unique reads
n.i.
uv
agno3
tryptophan biosynthesis
biosynthesis of aliphatic glucosinolates
indole and general glucosinolate biosynthesis
phenylpropanoid biosynthesis
glucosinolate biosynthesis and modification
members of the order brassicales synthesize glucosinolates from non-polar amino acids as major defence compounds against herbivores and pathogens.
in arabidopsis thaliana, almost exclusively methionine-derived aliphatic and tryptophan-derived indole glucosinolates are found.
their biosynthetic pathways are known in great detail  <cit> .
in eutrema salsugineum shandong, the short chain aliphatic allyl-2-phenylethyl-, 3-methylsulfinylpropyl-, and 3-methylthiopropylglucosinolate, the very-long-chain aliphatic 10-methylsulfinyldecylglucosinolate, as well as 3-indolylmethyl- and 1-methoxy-3-indolylmethylglucosinolate were identified as major compounds  <cit>  .
according to labelling experiments, 1-methoxy-3-indolylmethylglucosinolate is likely to be a biosynthetic intermediate of the phytoalexins 1-methoxybrassinin and wasalexin a and b  <cit> .
for all defined steps of the core aliphatic and indole glucosinolate biosynthetic pathways, putative orthologs of the genes encoding the corresponding enzymes were found in eutrema salsugineum, based on homology and synteny to a. thaliana.
some additional duplication events or losses of tandem copies were detected.
in contrast to the tandem duplicates cyp79f <dig> and cyp79f <dig> in a. thaliana, only one copy, designated as escyp79f <dig> was found in e. salsugineum, suggesting that this single gene is essential for the biosynthesis of aliphatic glucosinolates.
a putative cyp79a <dig>  <cit>  ortholog was found, which is expressed at very low levels  consistent with the apparent absence of phenylalanine-derived glucosinolates  <cit> .
e. salsugineum contains three cyp79b genes due to a recent duplication of cyp79b <dig> leading to two transcripts hybridizing to the same array probe and generating proteins with  <dig> % identity of their amino acid sequences.
these two duplicates strongly differ in expression level based on rnaseq data .
in response to uv light and agno <dig>  the core genes of indole glucosinolate biosynthesis are strongly upregulated, consistent with the proposed role of 1-methoxy-3-indolylmethylglucosinolate as precursor of the characteristic eutrema phytoalexins .
also, the ortholog of myb51/hig <dig>  encoding a master regulator of indole glucosinolate biosynthesis in arabidopsis  <cit> , is consistently induced.
strikingly, in response to these stressors, transcripts encoding indole glucosinolate biosynthetic genes, such as escyp83b <dig> and esggp <dig> are among the most highly abundant, according to our rnaseq data, indicating an important metabolic response.
in arabidopsis, a time course experiment has been performed for uv response  <cit> .
we surveyed these data for the responses of orthologs of e. salsugineum genes we analysed by rt-qpcr .
moderate upregulation with respect to 0 h, peaking at 3 h for atasa <dig>  and atpen <dig> , and at 6 h for atigmt <dig>  and atbglu <dig>  was observed.
more generally, we surveyed these data for core indole glucosinolate biosynthetic genes and again observed only modest transcript induction 6 h after uv treatment .
in contrast, the camalexin biosynthetic genes cyp71b <dig> and cyp71a <dig> were induced approximately 121-fold and 66-fold, respectively  <cit> .
these differential responses are consistent with the proposed phytoalexin biosynthetic pathways in the two species.
in arabidopsis, unmodified indole glucosinolate is methoxylated in response to pathogen infection, involving members of the cyp81f family and indole glucosinolate methyl transferases   <cit> .
e. salsugineum contains five cyp81f members, due to an additional gene copy in the cyp81f1/3/ <dig> cluster.
for three of these genes, microarray and rnaseq data were obtained and matched.
based on its expression pattern, escyp81f <dig>  is a candidate gene for catalysing n-hydroxylation of 3-indolylmethylglucosinolate in the biosynthesis of eutrema phytoalexins.
escyp81f <dig>  was induced by agno <dig> but not by uv light.
also, esigmt <dig>  highly expressed in response to stress treatment , is a candidate for involvement in the biosynthesis of n-methoxylated indolic compounds.
in response to pathogen infection, in arabidopsis indole glucosinolates are degraded to bioactive compounds by the β-glucosidase pen <dig>   <cit> .
we hypothesize that β-glucosidases are also involved in the biosynthesis of eutrema phytoalexins.
a number of β-glucosidase-encoding genes were significantly upregulated in response to agno <dig> and uv challenge , including espen <dig> , esbglu18- <dig> , and esbglu18- <dig> .
the time course of induction of these genes was monitored by quantitative rt-pcr and strong induction responses to agno <dig> and uv treatment were confirmed .
in conclusion, the eutrema orthologs of pen <dig>  and bglu <dig> are candidates for an involvement in phytoalexin biosynthesis.table  <dig>
analysis of transcript abundance changes of genes encoding β-glucosidases
transcript id
best ath hit
gene symbol
uv
ag
+
fold change log <dig>
fdr-p-value test
fold change log <dig>
fdr-p-value test
rnaseq unique reads
n.i.
uv
agno3
in response to uv light and agno <dig>  most genes involved in aliphatic glucosinolate biosynthesis were strongly downregulated, with the exception of the putative orthologs of flavin-containing monooxygenase  genes encoding glucosinolate s-oxygenases , probably resulting in a metabolic shift towards indolic and oxidized aliphatic glucosinolates.
based on homology and chromosomal position thhalv10008073m is orthologous to atsot17/atst5c , encoding a sulfotransferase with a preference for aliphatic desulfoglucosinolates as substrates  <cit> .
here, we observed strong transcriptional upregulation of essot <dig>  in response to uv irradiation and agno <dig> treatment, similar to genes involved in indole glucosinolate biosynthesis.
we speculate that in the two species the two orthologs have acquired different substrate specificities and that the eutrema gene functions in indole glucosinolate biosynthesis.
the two other confirmed desulfoglucosinolate sulfotransferases atsot18/atst5b and atsot16/atst5a have probably retained their function in aliphatic and indole glucosinolate biosynthesis, respectively.
wrky transcription factors
in arabidopsis, wrky transcription factors play an essential role in the regulation of phytoalexin responses.
our data show that also in eutrema several wrky genes are upregulated, including the orthologs of wrky <dig>  wrky <dig>  wrky <dig>  wrky <dig>  wrky <dig> and wrky <dig> .
wrky <dig> and wrky <dig> are central regulators of indole glucosinolate modification in response to pathogens  <cit> .
wrky <dig> is associated with both senescence- and defence-related processes  <cit>  and wrky <dig>  besides its role in phosphate acquisition  <cit> , is also linked to senescence and pathogen defence  <cit> .
wrky <dig> plays a role in modulation of salicylate- and jasmonate signalling in defence  <cit> .
in summary, these transcriptional changes indicate that also in eutrema wrkys are crucial for induced metabolic defence.table  <dig>
analysis of transcript abundance changes of genes encoding wrky transcription factors
transcript id
best ath hit
wrky
uv
ag
+
fold change log <dig>
fdr-p-value test
fold change log <dig>
fdr-p-value test
rnaseq unique reads
n.i.
uv
agno3
eswrky <dig> complements camalexin deficiency in an arabidopsis wrky mutant
in arabidopsis, wrky <dig> is an essential regulator of camalexin biosynthesis and directly binds to the promoter of cyp71b <dig>   <cit> .
accordingly, its expression is induced by pathogen-associated molecular patterns  and it is important for resistance against necrotrophic fungal pathogens  <cit> .
camalexin has not been detected in eutrema and it does not contain a clear ortholog of cyp71b <dig>  the closest cyp71b <dig> homolog in e. salsugineum shares only  <dig> % identical amino acids.
nevertheless, eswrky <dig> is strongly upregulated upon phytoalexin inducing conditions .
we investigated whether eswrky <dig> can functionally replace atwrky <dig> as a positive regulator of camalexin biosynthesis and expressed eswrky <dig> in the arabidopsis wrky33- <dig> mutant  <cit> .
while in wrky <dig> leaves camalexin levels were significantly reduced in relation to wild type, wild type levels were restored in the complementing line .
this indicates that even though eutrema does not synthesize camalexin, eswrky <dig> can act as a positive regulator of camalexin biosynthesis in arabidopsis.figure  <dig> complementation of camalexin deficiency in arabidopsis wrky <dig> knockout mutant by eswrky <dig> expression.
all plants were induced by uv light and analysed 20 h after the onset of induction.
mean and standard deviation is depicted.
different letters above the bars indicate significantly different amounts of camalexin in the respective samples, as determined by one-way anova ; n =  <dig>
CONCLUSIONS
in e. salsugineum, uv irradiation or heavy metal application resulted in substantial transcriptional reprogramming consistent with the induction of defence responses.
photosynthesis and starch synthesis were transcriptionally downregulated, while processes providing precursors for aromatic defence metabolites and cell wall compounds were transcriptionally induced.
strikingly, a shift in expression is observed from orthologs of genes for the biosynthesis of aliphatic glucosinolates, probably functioning primarily in insect defence, to orthologs of genes for the biosynthesis of indole glucosinolates, serving as precursors of phytoalexins.
wrky <dig> is an essential regulator of the camalexin biosynthetic gene cyp71b <dig>   <cit> , for which there is probably no functional homolog in e. salsugineum, consistent with the absence of camalexin in this species  <cit> .
nevertheless, there is a putative eutrema wrky <dig> ortholog, which is strongly upregulated under phytoalexin inducing conditions.
eswrky <dig> was functionally tested and shown to complement camalexin deficiency in an atwrky <dig> mutant.
we hypothesize that regulatory mechanisms for phytoalexin induction are conserved among members of the brassicaceae, while the individual chemical structures have strongly diversified.

 the synthesis of bioactive compounds for adaptation to abiotic stress conditions and for defence against herbivores and pathogen infections is a fundamental survival strategy of plants. the biosynthesis of phytoalexins, which contain an indole moiety substituted with additional ring systems or side chains, often containing sulphur and nitrogen, is characteristic for cruciferous plants. the individual structures are very diverse even among different brassica cultivars. in arabidopsis thaliana, a variety of compounds are synthesized from the intermediate indole-3-acetonitrile in response to pathogen infection or heavy metal stress with camalexin as the most prominent metabolite. the camalexin biosynthetic pathway from tryptophan and glutathione and its role in defence against a number of fungal pathogens has been investigated in detail. phytoalexin biosynthesis is induced upon pathogen infection, but also under harsh abiotic conditions, such as high dosages of heavy metal ions or uv light, which lead to the generation of reactive oxygen species and ultimately to programmed cell death. for studies on plant metabolism, abiotic stress treatments provide the advantage that no interference of pathogen metabolism, which is often strain specific, has to be taken into account. eutrema salsugineum has been established recently as an alternative model system for crucifers in addition to arabidopsis, because of its high tolerance of various abiotic stresses. the e. salsugineum genome sequence, as well as a reference transcriptome, are available and additional transcriptomics data were published recently. e. salsugineum is also referred to as thellungiella salsuginea. the ecotype shandong analysed in this study was initially assigned as t. halophila and this species name was used in a number of publications. consequently, gene and transcript sequences isolated from shandong ecotype have been deposited under the species names t. halophila, t. salsuginea and e. salsugineum. according to work by koch and german, the species name t. salsuginea is acceptable, but e. salsugineum, which we refer to in this manuscript, is preferred. within the brassicaceae, eutrema and arabidopsis are rather distantly related and their last common ancestor is estimated to have lived million years ago. still, large stretches of syntenic regions were identified in the genomes, allowing clear assignment of putative orthologs. at the protein level, for the number of best hit pairs between eutrema and arabidopsis a peak at 85% amino acid sequence identity was determined. eutrema and arabidopsis have developed a diversified spectrum of defence compounds, such as glucosinolates and indolic phytoalexins. in arabidopsis, these phytoalexins are predominantly synthesized from the intermediate indole-3-acetaldoxime, while the characteristic eutrema phytoalexins are most likely derivatives of 1-methoxy-indole glucosinolate. the identification of biosynthetic genes for presumably glucosinolate-derived and glucosinolate-independent phytoalexins will build the basis for metabolic engineering studies of indolic phytoalexins and for establishment of a model for phytoalexin evolution in the brassicaceae. in this work, we analysed the transcriptional reprogramming of e. salsugineum in response to abiotic stress conditions, which lead to the accumulation of phytoalexins. we show that genes of tryptophan and indole glucosinolate biosynthesis and modification are highly upregulated providing candidates for phytoalexin biosynthesis. also the eutrema ortholog of wrky a key regulator of arabidopsis phytoalexin induction, was highly upregulated, even though known wrky target genes, such as cyp71b are apparently missing in e. salsugineum.  induction of phytoalexin biosynthesis in response to uv light and silver nitrate spraying the biosynthesis of phytoalexins by brassicaceae species is induced by pathogen infection, but also specific abiotic stress treatments, such as high dosages of heavy metals and uv light. applying abiotic stressors provides the advantage of a high degree of experimental reproducibility and excludes the modulation of plant defence reactions and metabolism by the pathogen. induction of phytoalexin biosynthesis by the heavy metal salt cucl and uv treatment was previously established by pedras and coworkers. here, wasalexin induction was confirmed for 10-week old e. salsugineum leaves in response to uvc light, silver nitrate application, and botrytis cinerea infection. in arabidopsis, expression of camalexin biosynthetic genes is coregulated with expression of asa encoding the committing enzyme of tryptophan biosynthesis. we therefore assumed that also in e. salsugineum tryptophan biosynthesis is upregulated under phytoalexin inducing conditions, which we later confirmed. quantitative rt-pcr was used to determine the induction kinetics of esasa. for both treatments, transcript levels were highly elevated  h and 10 h after the onset of induction. therefore, for transcriptomics analysis 8 h induction was selected.figure rt-qpcr analysis. time course of expression after treatment with uv light and agno3. esasa, esigmt, espen, esbglu18-, esbglu18-, and eswrky, were analysed. the expression levels, relative to the mean for 0 h, were determined by rt-qpcr, normalized to the geometric mean of three reference genes. values are means of three independent experiments ± se. the eutrema transcriptome in response to uv light and heavy metal stress rna was isolated from non-treated leaves and from leaves treated with either agno or uv light. cdna libraries were prepared and approximately mio to mio 50 bp reads per library were obtained by illumina sequencing. reads were mapped to the jgi genome. for each cdna library, approx. 75% of total transcript models were covered and a large overlap between treatments was observed. transcript models were analysed for read-counts in the different samples and annotated for best hit in the arabidopsis thaliana genome.table rnaseq metrics and alignments n.i. uv agno 3 b.c. reads were mapped to the jgi genome, reference transcripts; uncounted/counted: number of unmapped/mapped reads; uniquely: number of uniquely mapped reads; non-specific: number of reads with multiple locations in the reference. similarly, we have analysed the transcriptome 48 h after infection of plants with b. cinerea. transcripts were identified as more than 2-fold upregulated with respect to untreated leaves. of this set, 56% and 61% were also upregulated more than 2-fold after uv and agno treatment, respectively, indicating overlapping responses to the abiotic and biotic stressors. however, as transcriptional changes in response to uv light and agno were much more pronounced, we focussed on these treatments for further analysis. microarray analysis of four biological replicates was conducted with agilent arrays based on the design by lee et al.. statistically robust differential regulation was observed for the majority of transcripts. of a total of oligonucleotide probes, signal intensities of and probes were significantly elevated, while signal intensities of and probes were significantly reduced in response to uv light and agno respectively. these array data were compared with the rnaseq data, which in addition provide information about absolute expression levels. a correlation analysis with the log fold-change values obtained by the two methods in response to uv and agno is shown in additional file 5: figure s we matched rnaseq and array data based on the comparison of array probe and transcript model sequences and omitted those probes from further analysis for which no match was found. duplicated genes with highly homologous sequences were sometimes indistinguishable on array level. here, the more highly abundant transcript from the rnaseq analysis was chosen for the matched dataset. log fold-change values based on rnaseq and array analyses were correlated. for further analysis, we worked with a set of genes, for which both array and rnaseq data are available. correlations of log fold-change values in response to uv and agno treatment obtained by microarray hybridization are shown in figure  for a large proportion of these transcripts, significant changes in abundance were detected in response to uv or agno treatment. transcripts were upregulated, downregulated in response to both treatments, indicating substantial overlap in metabolic and regulatory responses.figure global analysis of transcriptomics data. the set of genes, for which rnaseq and array data could be matched, was analysed for significant transcriptional changes in response to uv light and agno a large overlap in response to the two stressors was observed. figure  shows a mapman representation of log2-fold transcriptional changes, in response to uv light and agno, based on array data. strongly repressed processes include photosynthesis and starch synthesis. the tricarboxylic acid cycle, providing precursors of aromatic amino acid and the biosynthesis of cell wall precursors are induced on the level of transcript abundance, consistent with plant defence reactions.figure mapman visualisation of transcript abundance changes for metabolic genes. metabolism overview for microarray data. a: uv versus not induced. b: agno versus n.i.. red indicates downregulated, blue upregulated genes. the colour code indicates log2-fold changes in expression. transcriptional changes induced upon both uv and heavy metal stress transcripts that were strongly and consistently upregulated in response to both uv light and agno include a number of genes that encode enzymes involved in biosynthesis or modification of hormones and signalling compounds. this indicated that reprogramming the hormone balance is one of the key elements in the adaptation of eutrema to high dosages of uv light or heavy metals. genes upregulated most strongly in response to both stressors include essot and, based on ngs data, esst2a/essot. the corresponding arabidopsis orthologs encode a sulfotransferase, which sulphonates salicylic acid, thereby positively regulating salicylic acid accumulation, and a sulfotransferase, which sulphonates hydroxyjasmonic acid. sot is also strongly induced in a. thaliana seedlings in response to uvb light. furthermore, we observed that genes encoding eutrema orthologs of 1-amino-cyclopropane-1-carboxylate synthase and cis-zeatin o-β-d-glucosyltransferase were highly upregulated in response to both uv light and agno other induced processes are senescence and regulation of cell death. here, examples of highly upregulated genes include the eutrema orthologs of atdlah and atbap an inhibitor of programmed cell death. we observed significant transcriptional reprogramming of phenylpropanoid metabolism. genes of the core phenylpropanoid biosynthetic pathway, i.e. e. salsugineum orthologs putatively encoding phenylalanine ammonia-lyase and cinnamate-4-hydroxylase, cinnamoyl coa reductase, and cinnamyl alcohol dehydrogenase were upregulated in response to uv and agno the e. salsugineum ortholog of tt encoding naringenine chalcone synthase, was strongly downregulated. interestingly, in arabidopsis strong tt upregulation was observed in response to uv light. whether this is due to experimental differences, such as plant age or uv wavelength or reflects a species-specific difference in adaptation with respect to the phenylpropanoids that are synthesized remains to be investigated. further, fundamental changes in the transcript abundance of genes encoding enzymes involved in the biosynthesis of defence-related secondary metabolites were observed, which are discussed in detail below. a number of genes have been functionally associated with the halophytic lifestyle of e. salsugineum. these include the sodium transporter eshkt and eserf, which are also strongly and significantly upregulated under both agno and uv treatment. arabidopsis erf is an integrator of different abiotic and biotic stress responses. for other genes associated with salt tolerance, such as sos and iron superoxide dismutase this was not observed. we have surveyed transcriptional changes in response to agno and uv in e. salsugineum for similarity to changes in response to drought or cold. there was a clear overlap among downregulated genes, which are mainly related to photosynthesis. a common pattern among upregulated genes was not observed. apparently, the responses of e. salsugineum to drought/cold and to uv/heavy metal stresses differ substantially. the effect of silver treatment on the arabidopsis transcriptome was investigated previously by kaveh and coworkers. the number of significantly upregulated genes was much lower than in our work on eutrema, probably due to differences in the experimental setup. only for a few genes, the corresponding orthologs were identified in both studies, including the orthologs of the β-glucosidase genes and recently, genes were identified in a. thaliana which are upregulated in response to both b. cinerea infection and oxidative stress. for out of these transcripts, corresponding e. salsuginea orthologs were identified here. strikingly, for a large fraction of these genes, including e.g. escyp79b and escyp83b, we observed upregulation by both uv and agno treatments. possibly, all these processes lead to the generation of reactive oxygen species, inducing transcriptional reactions that are largely conserved between arabidopsis and eutrema. tryptophan biosynthetic genes in brassicaceae, tryptophan is a precursor of indole glucosinolates and indolic phytoalexins, which constitute the major tryptophan sinks. as cellular tryptophan concentrations are low in arabidopsis leaves, tryptophan biosynthesis is strongly coregulated with the biosynthesis of camalexin. here, we observed significant and strong increases in transcript levels associated with the tryptophan biosynthetic pathway in response to uv light and agno. this includes genes encoding tryptophan synthase β type isoforms, while the ortholog of tsbtype of which the biological function is unknown, is significantly downregulated in response to uv light.table analysis of transcript abundance changes of genes associated with the biosynthesis of defence-related metabolites transcript id best ath hit gene symbol annotation uv ag + fold change log fdr-p-value test fold change log fdr-p-value test rnaseq unique reads n.i. uv agno3 tryptophan biosynthesis biosynthesis of aliphatic glucosinolates indole and general glucosinolate biosynthesis phenylpropanoid biosynthesis glucosinolate biosynthesis and modification members of the order brassicales synthesize glucosinolates from non-polar amino acids as major defence compounds against herbivores and pathogens. in arabidopsis thaliana, almost exclusively methionine-derived aliphatic and tryptophan-derived indole glucosinolates are found. their biosynthetic pathways are known in great detail. in eutrema salsugineum shandong, the short chain aliphatic allyl-2-phenylethyl-, 3-methylsulfinylpropyl-, and 3-methylthiopropylglucosinolate, the very-long-chain aliphatic 10-methylsulfinyldecylglucosinolate, as well as 3-indolylmethyl- and 1-methoxy-3-indolylmethylglucosinolate were identified as major compounds. according to labelling experiments, 1-methoxy-3-indolylmethylglucosinolate is likely to be a biosynthetic intermediate of the phytoalexins 1-methoxybrassinin and wasalexin a and b. for all defined steps of the core aliphatic and indole glucosinolate biosynthetic pathways, putative orthologs of the genes encoding the corresponding enzymes were found in eutrema salsugineum, based on homology and synteny to a. thaliana. some additional duplication events or losses of tandem copies were detected. in contrast to the tandem duplicates cyp79f and cyp79f in a. thaliana, only one copy, designated as escyp79f was found in e. salsugineum, suggesting that this single gene is essential for the biosynthesis of aliphatic glucosinolates. a putative cyp79a ortholog was found, which is expressed at very low levels consistent with the apparent absence of phenylalanine-derived glucosinolates. e. salsugineum contains three cyp79b genes due to a recent duplication of cyp79b leading to two transcripts hybridizing to the same array probe and generating proteins with % identity of their amino acid sequences. these two duplicates strongly differ in expression level based on rnaseq data. in response to uv light and agno the core genes of indole glucosinolate biosynthesis are strongly upregulated, consistent with the proposed role of 1-methoxy-3-indolylmethylglucosinolate as precursor of the characteristic eutrema phytoalexins. also, the ortholog of myb51/hig encoding a master regulator of indole glucosinolate biosynthesis in arabidopsis, is consistently induced. strikingly, in response to these stressors, transcripts encoding indole glucosinolate biosynthetic genes, such as escyp83b and esggp are among the most highly abundant, according to our rnaseq data, indicating an important metabolic response. in arabidopsis, a time course experiment has been performed for uv response. we surveyed these data for the responses of orthologs of e. salsugineum genes we analysed by rt-qpcr. moderate upregulation with respect to 0 h, peaking at 3 h for atasa and atpen, and at 6 h for atigmt and atbglu was observed. more generally, we surveyed these data for core indole glucosinolate biosynthetic genes and again observed only modest transcript induction 6 h after uv treatment. in contrast, the camalexin biosynthetic genes cyp71b and cyp71a were induced approximately 121-fold and 66-fold, respectively. these differential responses are consistent with the proposed phytoalexin biosynthetic pathways in the two species. in arabidopsis, unmodified indole glucosinolate is methoxylated in response to pathogen infection, involving members of the cyp81f family and indole glucosinolate methyl transferases. e. salsugineum contains five cyp81f members, due to an additional gene copy in the cyp81f1/3/ cluster. for three of these genes, microarray and rnaseq data were obtained and matched. based on its expression pattern, escyp81f is a candidate gene for catalysing n-hydroxylation of 3-indolylmethylglucosinolate in the biosynthesis of eutrema phytoalexins. escyp81f was induced by agno but not by uv light. also, esigmt highly expressed in response to stress treatment, is a candidate for involvement in the biosynthesis of n-methoxylated indolic compounds. in response to pathogen infection, in arabidopsis indole glucosinolates are degraded to bioactive compounds by the β-glucosidase pen. we hypothesize that β-glucosidases are also involved in the biosynthesis of eutrema phytoalexins. a number of β-glucosidase-encoding genes were significantly upregulated in response to agno and uv challenge, including espen, esbglu18-, and esbglu18-. the time course of induction of these genes was monitored by quantitative rt-pcr and strong induction responses to agno and uv treatment were confirmed. in conclusion, the eutrema orthologs of pen and bglu are candidates for an involvement in phytoalexin biosynthesis.table analysis of transcript abundance changes of genes encoding β-glucosidases transcript id best ath hit gene symbol uv ag + fold change log fdr-p-value test fold change log fdr-p-value test rnaseq unique reads n.i. uv agno3 in response to uv light and agno most genes involved in aliphatic glucosinolate biosynthesis were strongly downregulated, with the exception of the putative orthologs of flavin-containing monooxygenase genes encoding glucosinolate s-oxygenases, probably resulting in a metabolic shift towards indolic and oxidized aliphatic glucosinolates. based on homology and chromosomal position thhalv10008073m is orthologous to atsot17/atst5c, encoding a sulfotransferase with a preference for aliphatic desulfoglucosinolates as substrates. here, we observed strong transcriptional upregulation of essot in response to uv irradiation and agno treatment, similar to genes involved in indole glucosinolate biosynthesis. we speculate that in the two species the two orthologs have acquired different substrate specificities and that the eutrema gene functions in indole glucosinolate biosynthesis. the two other confirmed desulfoglucosinolate sulfotransferases atsot18/atst5b and atsot16/atst5a have probably retained their function in aliphatic and indole glucosinolate biosynthesis, respectively. wrky transcription factors in arabidopsis, wrky transcription factors play an essential role in the regulation of phytoalexin responses. our data show that also in eutrema several wrky genes are upregulated, including the orthologs of wrky wrky wrky wrky wrky and wrky. wrky and wrky are central regulators of indole glucosinolate modification in response to pathogens. wrky is associated with both senescence- and defence-related processes and wrky besides its role in phosphate acquisition, is also linked to senescence and pathogen defence. wrky plays a role in modulation of salicylate- and jasmonate signalling in defence. in summary, these transcriptional changes indicate that also in eutrema wrkys are crucial for induced metabolic defence.table analysis of transcript abundance changes of genes encoding wrky transcription factors transcript id best ath hit wrky uv ag + fold change log fdr-p-value test fold change log fdr-p-value test rnaseq unique reads n.i. uv agno3 eswrky complements camalexin deficiency in an arabidopsis wrky mutant in arabidopsis, wrky is an essential regulator of camalexin biosynthesis and directly binds to the promoter of cyp71b. accordingly, its expression is induced by pathogen-associated molecular patterns and it is important for resistance against necrotrophic fungal pathogens. camalexin has not been detected in eutrema and it does not contain a clear ortholog of cyp71b the closest cyp71b homolog in e. salsugineum shares only % identical amino acids. nevertheless, eswrky is strongly upregulated upon phytoalexin inducing conditions. we investigated whether eswrky can functionally replace atwrky as a positive regulator of camalexin biosynthesis and expressed eswrky in the arabidopsis wrky33- mutant. while in wrky leaves camalexin levels were significantly reduced in relation to wild type, wild type levels were restored in the complementing line. this indicates that even though eutrema does not synthesize camalexin, eswrky can act as a positive regulator of camalexin biosynthesis in arabidopsis.figure complementation of camalexin deficiency in arabidopsis wrky knockout mutant by eswrky expression. all plants were induced by uv light and analysed 20 h after the onset of induction. mean and standard deviation is depicted. different letters above the bars indicate significantly different amounts of camalexin in the respective samples, as determined by one-way anova; n =   in e. salsugineum, uv irradiation or heavy metal application resulted in substantial transcriptional reprogramming consistent with the induction of defence responses. photosynthesis and starch synthesis were transcriptionally downregulated, while processes providing precursors for aromatic defence metabolites and cell wall compounds were transcriptionally induced. strikingly, a shift in expression is observed from orthologs of genes for the biosynthesis of aliphatic glucosinolates, probably functioning primarily in insect defence, to orthologs of genes for the biosynthesis of indole glucosinolates, serving as precursors of phytoalexins. wrky is an essential regulator of the camalexin biosynthetic gene cyp71b, for which there is probably no functional homolog in e. salsugineum, consistent with the absence of camalexin in this species. nevertheless, there is a putative eutrema wrky ortholog, which is strongly upregulated under phytoalexin inducing conditions. eswrky was functionally tested and shown to complement camalexin deficiency in an atwrky mutant. we hypothesize that regulatory mechanisms for phytoalexin induction are conserved among members of the brassicaceae, while the individual chemical structures have strongly diversified. 
cruciferous plants synthesize a large variety of tryptophan-derived phytoalexins in response to pathogen infection, uv irradiation, or high dosages of heavy metals. while core genes of the biosynthesis of aliphatic glucosinolates were repressed, tryptophan and indole glucosinolate biosynthetic genes, as well as defence-related wrky transcription factors, were consistently upregulated. the transcriptional response of e. salsugineum to uv irradiation and agno was monitored by rnaseq and microarray analysis. in e. salsugineum, uv irradiation or heavy metal application resulted in substantial transcriptional reprogramming. the putative eutrema wrky ortholog was functionally tested and shown to complement camalexin deficiency in atwrky mutant. the major phytoalexins of eutrema salsugineum, which has recently been established as an extremophile model plant, are probably derivatives of indole glucosinolates, in contrast to arabidopsis, which synthesizes characteristic camalexin from the glucosinolate precursor indole-3-acetaldoxime. most transcripts were significantly differentially regulated and a large overlap between the two treatments was observed. eutrema salsugineumthellungiella salsugineatranscriptomicsglucosinolate biosynthesisphytoalexinissue-copyright-statement© the author 2015 consistently induced genes of indole glucosinolate biosynthesis and modification will serve as candidate genes for the biosynthesis of eutrema-specific phytoalexins.
 cruciferous plants synthesize a large variety of tryptophan-derived phytoalexins in response to pathogen infection, uv irradiation, or high dosages of heavy metals. the major phytoalexins of eutrema salsugineum, which has recently been established as an extremophile model plant, are probably derivatives of indole glucosinolates, in contrast to arabidopsis, which synthesizes characteristic camalexin from the glucosinolate precursor indole-3-acetaldoxime.  the transcriptional response of e. salsugineum to uv irradiation and agno was monitored by rnaseq and microarray analysis. most transcripts were significantly differentially regulated and a large overlap between the two treatments was observed. while core genes of the biosynthesis of aliphatic glucosinolates were repressed, tryptophan and indole glucosinolate biosynthetic genes, as well as defence-related wrky transcription factors, were consistently upregulated. the putative eutrema wrky ortholog was functionally tested and shown to complement camalexin deficiency in atwrky mutant.  in e. salsugineum, uv irradiation or heavy metal application resulted in substantial transcriptional reprogramming. consistently induced genes of indole glucosinolate biosynthesis and modification will serve as candidate genes for the biosynthesis of eutrema-specific phytoalexins. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords eutrema salsugineumthellungiella salsugineatranscriptomicsglucosinolate biosynthesisphytoalexinissue-copyright-statement© the author 2015 