BACKGROUND
cyanogenic glycosides  are bioactive specialized metabolites found in more than  <dig>  plant species distributed in  <dig> different plant families of angiosperms, gymnosperms and ferns  <cit>  with a large proportion found among of our cultivated plants  <cit> .
examples of cyanogenic crop plants include barley   <cit> , wheat   <cit> , sorghum  moench)  <cit> , cassava   <cit> , almond   <cit> , cherry   <cit>  and apple   <cit> .
upon tissue disruption, the cnglcs stored in the tissues of cyanogenic plants, are brought in contact with endogenous β-glucosidases  resulting in hydrolysis and release of the cyanohydrin aglycone, which spontaneously or catalyzed by an α-hydroxynitrile lyase  dissociates to release hydrogen cyanide  and a keto compound.
this pathway is often referred to as the bioactivation pathway and the process as cyanogenesis  <cit> .
the toxic effect of hcn is caused by its ability to inhibit metalloenzymes and in particular cytochrome c oxidase, the key enzyme in the respiratory electron transport chain in mitochondria  <cit> .
cnglc storage in plants and the release of hcn upon tissue disruption may serve as a deterrent against generalist herbivores  <cit> .
in addition to herbivore defense, cnglcs may serve a multiplicity of other biological functions  <cit> .
in the rubber tree , linamarin accumulates in the mature seed  <cit> .
upon germination, the cnglc is converted into the cyanogenic diglucoside  linustatin and transported to the cotyledon where it is turned over  <cit> .
a similar situation has been observed in bitter almonds where prunasin is biosynthesized in the tegument of the almond fruit throughout development and eventually transported into the developing cotyledons of the kernel where it is converted into the cndglc amygdalin  <cit> .
upon germination, the amygdalin stored in the cotyledon of the bitter almond variants is most likely catabolized as observed for amygdalin accumulated in seeds of black cherry   <cit> .
in sorghum, dhurrin derived cndglcs are found in guttation droplets and are similarly hypothesized to act as transport forms  <cit> .
three cndglcs which differ from each other in the configuration of the glucosidic linkage between the primary and secondary glucose molecule have been found  <cit> , but their specific roles as possible transporters remain unknown.
in young sorghum seedlings, the cnglc dhurrin rapidly accumulated following germination after which biosynthesis decreased and turnover increased to result in a reduced concentration of dhurrin  <cit> .
this turnover of cnglcs was earlier thought to occur via the bio-activation pathway with the hcn released being incorporated into β-cyanoalanine by β-cyanoalanine synthase  and finally converted by nitrilases belonging to the nit <dig> family to asparagine and to aspartate with concomitant release of ammonia  <cit> .
however, evidence for the occurrence of alternative turnover pathways that bypasses the release of the toxic intermediates hcn and β-cyanoalanine has been proposed  <cit> .
in the first alternative pathway suggested for sorghum, dhurrin was hypothesized converted to p-hydroxyphenylacetonitrile  via an unknown novel bgd/protein co-factor complex  <cit> .
no evidence has subsequently been found to support the presence of such a protein complex.
instead, more recent results pointed to the formation of pohpcn through the action of glutathione s-transferases  supported by genome wide association analysis of a sorghum population with variable leaf dhurrin levels, where a gst was shown to be associated with dhurrin concentration in leaves  <cit> .
the pohpcn was subsequently hydrolyzed to p-hydroxyphenylacetic acid  and ammonia by the action of a heteromeric enzyme complex composed of the enzymes nit4a and nit4b <dig>  <cit> .
finally, the pohpaac was glucosylated to produce p-glucosyloxyphenylacetic acid  which has been shown to accumulate in vegetative tissue of different sorghum cultivars as the plants got older and the dhurrin content decreased  <cit> .
the second proposed endogenous turnover pathway included a unifying set of proposed turnover products in sorghum, almond and cassava where the nitrile group of the cnglc is converted into an amide, a carboxylic acid and finally completely lost in a decarboxylation reaction  <cit> .
for clarity, these possible intermediates are referred to as cnglc amide, cnglc acid and cnglc anitrile in this manuscript.
for investigating the turnover of cnglcs in cyanogenic plants, the developing grains of sorghum were selected as an experimental model system.
developing seeds are terminal sink organs and do not have net export of nutrients and amino acids  <cit>  and therefore offer an attractive system for study of endogenous turnover processes.
the mature sorghum grain contains only minute amounts or no dhurrin at all  <cit> .
in this study, we report that dhurrin is formed in the initial stages of grain development and turned over in the course of grain maturation.
the grain was found not to possess dhurrinase  activity signifying the operation of one or more endogenous turnover pathways not involving the release of toxic hcn.
high concentrations of condensed tannins  were formed throughout development and accumulated in the mature grain.
for the investigation of endogenous turnover of dhurrin, time-resolved transcriptomic analyses guided the identification of novel gene candidates encoding enzymes belonging to the families of gsts, the nitrilase superfamily and udp-glycosyltransferases .
based on the expression profiles of the genes supposedly involved in endogenous dhurrin turnover and the dhurrin metabolites formed, we propose a dual role for dhurrin in the developing grain of sorghum.
RESULTS
metabolite profiling of developing sorghum grain
immediately following anthesis, the total content of dhurrin in the grain was low and barely detectable ).
dhurrin content then rapidly increased reaching a maximum 25 days after anthesis   .
after this maximum, a similarly rapid decrease occurred which resulted in complete disappearance of dhurrin at maturity,  <dig> daa .
in the same time period, the dhurrin content of the husk remained constant at  <dig>  nmol/husk .
accordingly, the concentration of dhurrin was approximately  <dig> times higher in the grain compared to the husk right after anthesis and  <dig> times higher at the maximum dhurrin concentration.fig.
<dig> concentrations of dhurrin and proanthocyanidins in developing grains.
the concentration of dhurrin and proanthocyanidins  measured pr.
grain  and pr.
mg grain tissue  at different days after anthesis .
the dhurrin concentration is measured in nmol and the pa concentration as absorbance.
the colored circles represent the actual measurements in the three replicas and the solid line the trend line calculated using local polynomial regression fitting, with the area surrounding the average line representing the 95% confidence interval.
each measurement represents the average of  <dig> grains with one of the five grains and husks displayed in the table
the presence of dhurrin turnover products in the extracts of the sorghum grain from anthesis to maturation was analyzed by lc-ms/ms.
dhurrin amide, dhurrin acid, pglcpaac and the dhurrin glucosides a, b and c were all found in the grain extracts.
a previous study by pičmanová et al.
<cit>  detected the presence of caffeoyl-dhurrin  and caffeoyl-dhurrin-acid  in sorghum leaf extracts.
these compounds were not found in the sorghum grain.
the appearance of the putative dhurrin turnover products in the sorghum grain from anthesis to maturity in relation to the total content and concentration of dhurrin is shown in fig.
<dig>  in whole grains, dhurrin amide peaked around  <dig> daa, i.e.
immediately before the concentration of dhurrin started to increase.
in contrast, the related compound dhurrin acid , began to accumulate around  <dig> daa, i.e.
at the time point where the dhurrin content started to decrease.
in relative terms, the dhurrin acid reached 34% of the total dhurrin concentration , while dhurrin amide only reached 9%.
similarly, the content of pglcpaac reached 8% of the total dhurrin content.
the dhurrin glucosides were observed to reach their maximum content at  <dig> daa, i.e.
a week after the dhurrin content started to decrease and reached 6% of the total dhurrin content.fig.
<dig> the accumulation of putative dhurrin turnover products.
the concentration of dhurrin and putative turnover products measured as intensity pr.
grain  and intensity pr.
mg grain tissue  throughout development.
the colored circles represent the actual measurements in the three replicates and the solid line the trend line calculated using local polynomial regression fitting.
the area surrounding the average line representing the 95% confidence interval.
the different colors corresponds to the different metabolites listed in the box below the graphs
proanthocyanidin content in the sorghum grain and husk from anthesis to maturity
a high concentration of proanthocyanidins  in developing and mature sorghum grains could increase herbivore resistance and compensate for the lack of dhurrin at maturity  <cit> .
the concentration of pas throughout grain development was therefore investigated.
as part of the maturation process, the grain changed color from an initial green to a deep dark/brown color.
the browning process was initiated  <dig> daa  and was correlated with formation of pas .
in the sorghum grain, the content of pa increased from a low initial level to a maximum content per grain at  <dig> daa.
from  <dig> to  <dig> daa, the content decreased to approximately half the maximum value.
when measured per mg grain tissue, the variations in concentration of pas was less pronounced with only a slight increased concentration around  <dig> daa followed by a small decrease towards maturation.
cyanide potential of seeds
the total content of a cnglc in plant tissue is often referred to as the cyanide potential .
despite incubation for  <dig>  h following maceration, only a small fraction of the total amount of dhurrin present in the samples as determined by lc-ms/ms analysis was hydrolyzed in the native samples .
the amount of dhurrin hydrolyzed is positively correlated with the total estimated amount of dhurrin but does not exceed the amount undergoing non-enzymatic hydrolysis in the same time frame .
addition of almond bgd to homogenates of the initial samples , increased the amount of hcn released and was positively correlated to the total dhurrin content although complete hydrolysis of the dhurrin present was not observed.
from  <dig> to  <dig> daa, the addition of bgd did not increase the amount of hcn released.
spiking of the samples with 5 nmol dhurrin increased the release of hcn slightly, but did not result in an additional 5 nmol hcn compared to the hcn released from the samples containing tissue plus almond bgd.
increasing the concentration of bgd from  <dig>  to 5u/ml only increased the hcn release relative to the total dhurrin content in the samples spiked with additional dhurrin .fig.
<dig> cyanide potential in grains.
nmol hydrogen cyanide  released from macerated tissue at different developmental stages .
the hcn release was measured from macerated tissue in buffer alone , tissue in buffer plus  <dig>  u/ml almond β-glucosidase  and tissue in buffer plus  <dig>  u/ml almond β-glucosidase and an additional 5 nmol dhurrin .
for each sample/bar, the total possible release of hcn from dhurrin is indicated by the striped bar stacked on top of the measured hcn release.
the total possible release of hcn is calculated from the equation describing the trendline for dhurrin in fig.
1b.
the hcn release was measured a second time with macerated tissue from  <dig> daa with  <dig> u/ml almond β-glucosidase instead of  <dig>  u/ml .
as a control, an assay measuring hcn release from varying concentrations of dhurrin  in buffer was also performed
time-resolved transcriptome analysis of developing grain
to investigate the expression profiles of genes encoding enzymes involved in dhurrin biosynthesis and putative turnover processes, transcriptome analysis of the developing grain was performed.
more than  <dig> million 100 bp paired-end reads were generated resulting in an average of  <dig> million reads per library.
of these reads, an average of 87% were mapped to the phytozome sorghum transcriptome v <dig> , with 55% being uniquely mapped and 31% non-specifically mapped.
of the predicted  <dig>  protein-coding gene loci in sorghum,  <dig>  loci  were detected as expressed during sorghum grain development.
among these transcripts, a large fraction showed an expression below a single fragment per kilobase of transcript per million mapped reads  .
with these values considered as the background level,  <dig>  genes  were registered as not being expressed.
the results obtained from the transcriptome study were verified by quantitative pcr  using specific primers towards the gene cas  .
functional genomics and identification of interesting genes was initially guided by categorizing the expressed genes according to their expression pattern throughout grain development.
all expression profiles were normalized  and clustered using hierarchical clustering with wards method  <cit>  .
the expression patterns of the cluster groups containing the biosynthetic genes  79a <dig>  and ugt85b <dig>  in 8– <dig> and cyp71e <dig>  in 12–1) as well as nitrilase  4b <dig>  in 10– <dig> is shown along with the concentration of dhurrin in fig.
<dig>  these six cluster groups shared an overall similarity .
the remaining  <dig> cluster group profiles were contained in three larger groups  which are presented in additional file  <dig>  it is to be noted that the nadph-dependent cytochrome p <dig> oxidoreductase  isoforms crucial for the dhurrin biosynthesis were not contained in these clusters, but did exhibit maximum gene expression before the dhurrin concentration peak and belonged to the same overall group as the biosynthetic genes .
this difference in the expression profiles is more easily observed in fig.
<dig> fig.
<dig> clustering analysis of transcript expression profiles.
the hierarchical clustering is divided into  <dig> groups, which was further subdivided into  <dig> subgroups .
only the cluster groups containing the biosynthetic genes  is displayed on the figure.
the complete overview of all cluster groups can be found in additional file  <dig>  the number of genes in each cluster is listed in parenthesis next the cluster number.
the grey field represents the span of all the expression profiles in a single cluster.
the dashed line represents the concentration of dhurrin pr.
mg tissue through the grain development in relation to the expression profiles
fig.
<dig> expression profiles linked to the biosynthetic- and bioactivation pathway.
the biosynthetic pathway involves the enzymes cyp79a <dig>  cyp71e <dig> and ugt85b <dig> with the formation of dhurrin from l-tyrosine.
the bioactivation pathway hydrolyzes dhurrin to release hcn via the enzymes dhr <dig> and  <dig> and hnl.
the detoxification pathway converts the hcn to aspartate + nh <dig> and/or asparagine via cas and the nit4a/b <dig> and nit4a/b <dig> heterodimers.
the expression profiles for the specific enzyme involved in the reactions in the biosynthetic- or bioactivation pathway are colored in the same color as the enzyme boxes.
in the box representing the por expression, the a, b and c represents the three different isoforms found in sorghum.
expression is displayed in fragments per kilobase of exon per million reads mapped .
the dashed line in each transcription box represents the dhurrin concentration throughout development
in the bio-activation pathway, the two dhr genes known to be present in the sorghum genome were not expressed, while the hnl transcript reached maximum abundance a few days after the dhurrin concentration peak and then decreased in conjunction with dhurrin.
in contrast, the two genes encoding the two subsequent enzymes cas and nit4b <dig> in the detoxification pathway showed maximum expression at anthesis with a steady decline towards grain maturation.
a similar trend was seen for 1-aminocyclopropane-1-carboxylic acid  oxidase.
this enzyme represents the final enzymatic step in ethylene biosynthesis and releases stoichiometric amounts of hcn, which is detoxified, via the same cas detoxification pathway  <cit> .
the transcripts for nit4a and nit4b <dig> displayed a third type of expression profile with an almost linearly increased expression from anthesis to grain maturation .
for many of the genes displayed, the standard deviations of their expression profiles were relatively low.
the two genes cyp71e <dig> and ugt85b <dig> in the dhurrin biosynthesis were exceptions and displayed relatively large standard deviations at  <dig> daa .
despite these large standard deviations, the three genes in the dhurrin biosynthesis displayed a relatively fixed ratio between the transcript abundances from  <dig> daa, with cyp79a <dig> being the lowest expressed and ugt85b <dig> the highest expressed gene, with cyp71e <dig> having an intermediate expression.
from western blot analysis using available antibodies against cyp79a <dig> and cyp71e <dig>  a similar distribution was found with cyp71e <dig> having the highest concentration .
however, the protein ratio was on average 4-fold lower compared to the rna ratio.
in terms of maximum abundance, cyp71e <dig> and ugt85b <dig> displayed large values of several hundred fpkm as opposed to the por isoforms, hnl, cas, nit4b <dig>  nit4b <dig> and acc oxidase, which all displayed transcript abundances below  <dig> fpkm .
glutathione s-transferases in s. bicolor
a total of  <dig> genes encoding putative gsts were identified in the s. bicolor genome by blastp searches using a selection of gst protein sequences from arabidopsis thaliana  <cit> , barley  <cit> , rice   <cit> , black poplar   <cit> , physcomitrella patens  <cit>  and a selection of  <dig> other plant, animal, fungi, and bacteria species  <cit> .
a conserved domain search at the national center for biotechnology information  database and a pfam search at the european bioinformatics institute database revealed that  <dig> of the selected sorghum protein sequences did not contain any gst domains whereas four only contained the c-terminal domain.
these genes were retraced to the new phytozome genome v <dig> , to test if they were annotated incorrectly in the old database.
the retracing did not change the sequences and these enzymes are likely catalytically inactive and were therefore deleted from the collection.
two sorghum sequences only contained an n-terminal domain .
retracing these genes revealed that sobic.005g <dig> is not annotated in the new database, while sobic.009g <dig> is replaced by sobic.009g <dig>  domain search revealed that this gene has both n- and c-terminal domains.
both these genes were included in the phylogenetic- and transcript expression analyses.
all  <dig> sorghum gst genes were classified into  <dig> distinct classes via an ncbi conserved domain search and a phylogenetic analysis using the list of  <dig> full-length gst described by liu et al.
<cit>  .
the phylogenetic tree revealed that sorghum contained eleven gst classes : microsomal prostaglandin e-synthase  , zeta , glutathionyl-hydroquinone reductases  , lambda , dehydroascorbate reductases  including sobic.009g <dig> , tetrachloro-hydroquinone dehalogenases  , elongation factor 1b gamma  , theta , phi , metaxin  and tau .
in general, the positions of the individual genes in the outer branches of the tree have good support with high bootstrap values, but the relation between the different gst classes in the tree were not very well supported.
among the  <dig> gsts, the tau and phi genes were by far the most numerous representing 77% of the total number of genes.fig.
<dig> phylogenetic analysis of sorghum bicolor glutathione-s transferases.
the phylogenetic tree represents the  <dig> gsts found in sorghum.
the tree is constructed with phyml using  <dig> bootstrapping replicates.
the bootstrapping values are displayed in percentage and only values above  <dig> are displayed.
the prokaryotic glutaredoxin , was used as an outgroup to root the tree.
the two gene names marked in red color represent one gene  where only the n-terminal gst domain was detected and a gene , with a new gene name in phytozome database version  <dig> .
the colored circles represent the cluster groups displayed in fig.
<dig>  uncolored circles represent genes not expressed in the grain during development.
the triangular shapes on the outside of the phylogenetic tree represent the gst genes potentially involved in dhurrin turnover.
their specific functions are listed in the bottom right legend
a previous genome-wide survey, identified  <dig> gsts in sorghum  <cit> .
their division of the gsts into the different classes was based on phylogenetic analysis of only the n-terminal parts of the proteins although the division was classically based on the entire sequences.
as an example, they identified only two lambda gsts  <cit> , none of which were identical to the four identified in the present study.
furthermore, the sequences classified as lambda gsts by chi et al.
<cit>  have a serine residue in the active site rather than the catalytic cysteine characteristic of lambda gsts and were therefore incorrectly classified as a consequence of the n-terminal based phylogenetic analysis.
in our analyses, these two genes were categorized as tau gsts.
the validity of our approach was confirmed by identical numbers and gene ids of cys gsts  to those reported by lallement et al.
<cit> .
our analysis also revealed a new gst class  not previously described in sorghum.
expression analysis of gsts in s. bicolor
based on the transcriptome analyses of the sorghum grain from anthesis to maturation, the expression patterns of the  <dig> sorghum gsts were assigned to different cluster groups.
this was done to guide the identification of possible gene candidates for involvement in the putative turnover pathway.
of the  <dig> gst genes found,  <dig> were not expressed during the development of the grain.
group clustering of the remaining  <dig> genes  into six groups revealed that the genes were primarily distributed among clusters  <dig>   <dig> and  <dig>  from the dendrogram , it was possible to determine the relatedness between the cluster groups.
cluster group 1– <dig> were related and all displayed expression profiles that have the highest abundance after the concentration of dhurrin peaked .
in contrast, the two related cluster groups  <dig> and  <dig> displayed the highest abundance prior to the dhurrin peak , while cluster group  <dig> displayed an intermediate expression profile with high abundance both before and after the dhurrin peak .fig.
<dig> expression profiles of gsts in the course of sorghum grain development.
all  <dig> expressed gsts found in the phylogenetic analysis were normalized to z-score and clustered using hierarchical clustering with wards method .
the hierarchical clustering was divided into  <dig> groups, which was further subdivided into  <dig> subgroups  .
the lighter colored line represents the first subgroup and the darker colored line the last subgroup in a cluster.
the number of genes in each cluster is listed in parenthesis next to the cluster number.
the grey field represents the span of all the expression profiles in a single cluster.
the dashed lines represent the concentration of dhurrin  and pglcpaac through grain development in relation to the expression profiles of the different gsts.
glutathionylating gsts from cluster 1– <dig> plotted in fpkm .
deglutathionylating gsts from cluster 1– <dig> plotted in fpkm .
gst identified by hayes et al.
along with similar genes in similar cluster group  <dig> .
highly expressed genes in panel b–d have colored lines referring to the genes listed next to panel a. gray lines are genes only expressed at low abundance
the gsts found in cluster group 1– <dig> were of most interest as their expression followed that of nit4a and nit4b <dig> and increased over time, at least until after the dhurrin concentration had peaked.
in gst enzymes that catalyze glutathionylation, a serine or tyrosine is the catalytic residue, whereas deglutathionylating gsts harbor a catalytic cysteine  <cit> .
the classes zeta, tchqd, ef1bγ, theta, phi, metaxin and tau belong to the first group, while the classes dhar, lambda, ghr and mpges belong to the second group of gsts.
the expression profiles of glutathionylating and deglutathionylating gsts from cluster 1– <dig> are shown in fig.
7b and c, respectively, with the most abundant members displayed in colored lines.
the five most abundant genes in fig.
7b included four tau class gsts  and a phi class gst .
among the gst genes from cluster 1– <dig>  the ones containing a cysteine residue were depicted in fig.
7c.
of these genes, the most abundant ones were displayed in colored lines.
these genes included two lambda class gsts  and a ghr class gst .
hayes et al.
<cit>  suggested that the phi class gst gene sobic.001g <dig>  lying close to the gene cluster containing the dhurrin biosynthetic genes   <cit> , could be involved in dhurrin turnover in the tissue.
this gene was found in cluster group  <dig>  a cluster group, which shared a similar initial expression profile to the biosynthetic genes .
the genes from cluster group  <dig> are plotted in fig.
7d.
the two most abundant genes beside the gst described by hayes et al.
<cit>  include a phi class gst  and an ef1bγ class gst .
when the expression profiles of all  <dig> gst genes were compared to the average expression profile of nit4a and nit4b <dig> using pearson correlation, the glutathionylating gsts in fig.
7b are ranked as the 7th , 9th , 12th , 13th  and 22nd  most similar .
in the deglutathionylating group in fig.
7c, sobic.002g <dig> was found to be the gst most similar to the two nit expressions with sobic.003g <dig> ranked as 16th and sobic.001g <dig> as 25th.
in comparison the three deglutathionylating gsts in fig.
7d  were only ranked as 35th, 38th and 47th, respectively .
nitrilase activity in a novel turnover pathway
the dhurrin turnover products, dhurrin amide and dhurrin acid  are envisioned to be produced by the action of either a nitrilase or by the combined action of a nitrile hydratase and an amidase.
to find putative nitrilase candidate genes in the sorghum genome, it was blasted using protein sequences ascribed to each of the  <dig> branches of the nitrilase superfamily, which include amidases and cyano hydratases but not nitrile hydratases  <cit> .
to classify the ten genes discovered, they were aligned against the known nitrilase superfamily sequences and compared to the consensus sequences around the ekc catalytic triad  <cit>  .
the ten genes were categorized as four branch- <dig> nitrilases , a single branch- <dig> β-ureidoproprionase , a branch- <dig> glutamine-dependent nad synthetase , three branch- <dig> nit   and a single branch-11 n-carbamyl putrescine amidohydrolase .
nine of the ten genes were expressed during grain development .
three of these nine genes belong to the well-characterized nit <dig> family  involved in the detoxification pathway and one of the two putative turnover pathways.
these three genes were therefore not included .
the six genes were divided into three low abundance genes  and three high abundance genes .fig.
<dig> expression profiles of the sorghum nitrilase superfamily genes in the course of grain development.
the expression profiles of the six nitrilase superfamily genes expressed in the course of grain development shown in fpkm.
the colors of the profiles corresponds to the gene names listed in the box below the plot.
the three dashed curves represent dhurrin , dhurrin amide  and dhurrin amide .
the nit <dig> genes are excluded from this figure, but shown in figs.
<dig> and 11
udp-glycosyltransferase expression and possible involvement in metabolite formation
the concentration of pglcpaac was observed to accumulate to maximum levels before the dhurrin concentration of the grain reached its maximum .
to identify potential ugt candidates capable of glucosylating pohpaac, a total of  <dig> genes encoding putative ugts were identified in the sorghum genome by blast searches using  <dig> a. thaliana ugt protein sequences  <cit>  and two sequences from malus domestica and glycine max representing the novel o group ugt  <cit>  .
alignment and domain analyses revealed that  <dig> sequences were not recognized as ugts by the domain analyses.
additionally, two sequences were missing the first conserved residues of the pspg motif located near the c-terminal which is involved in binding the nucleotide sugar donor  <cit> .
these sequences were excluded from further analyses.
the remaining  <dig> genes were subjected to phylogenetic analyses along with the previous reference sequences   <cit> .
the relationship between the individual genes is generally supported by high bootstrap values, while the relationship between the different ugt classes has lower bootstrap support.
all  <dig> ugt groups previously defined  <cit>  were represented in the phylogenetic tree.
ten sequences were assigned to group a,  <dig> to group b,  <dig> to group c,  <dig> to group d,  <dig> to group e,  <dig> to group f,  <dig> to group g,  <dig> to group h,  <dig> to group i,  <dig> to group j,  <dig> to group k,  <dig> to group l,  <dig> to group m,  <dig> to group n,  <dig> to group o and  <dig> to group p .fig.
<dig> phylogenetic analysis of sorghum udp-glycosyltransferases.
the phylogenetic tree represents the  <dig> ugts found in sorghum.
the tree is constructed with phyml using  <dig> bootstrapping replicates.
the bootstrapping values are displayed in percentage and only values above  <dig> are displayed.
the phylogenetic tree is divided into separate groups of ugts from a-m plus o. the colored circles represent the cluster groups displayed in fig.
<dig>  uncolored circles represent genes not expressed in the grain.
in the bottom left legend, the number of expressed genes in each cluster group is listed.
the triangular shapes on the outside of the phylogenetic tree represent the ugt genes potentially involved in dhurrin and flavonoid metabolism.
their specific names are listed in the bottom right legend
to narrow down the number of possible gene candidates, the expression data from the transcriptome study were used to cluster the ugt genes according to their expression patterns .
of the  <dig> ugt genes found,  <dig> were not expressed in the grain in the course of development.
the remaining  <dig> genes were clustered into six distinct cluster groups, which were further subdivided into  <dig> subgroups .
the expression profiles of cluster group  <dig> and  <dig> were related  and both increased towards the end of grain development .
in comparison, cluster group  <dig> and  <dig> had expression profiles, which peaked over a broader range from  <dig> to  <dig> daa.
cluster group  <dig> and  <dig> peaked in the early development from  <dig> to  <dig> daa.fig.
<dig> expression profiles of sorghum ugts.
all  <dig> expressed ugts found in the phylogenetic analysis were normalized to z-score and clustered using hierarchical clustering with wards method .
the hierarchical clustering was divided into  <dig> groups, which was further subdivided into  <dig> subgroups  .
the lighter color represents the first subgroup and the darker color the last subgroup in a cluster.
the number of genes in each cluster is listed in parenthesis next the cluster number.
the grey field represents the span of all the expression profiles in a single cluster.
the dashed curves represent the concentration of pglcpaac  and the three dhurrin glucosides  pr.
mg tissue in the course of grain development in relation to the expression profiles.
genes in cluster  <dig> plotted in fpkm values of interest to the conversion of the glucosylation of pohpaac .
genes from cluster  <dig> plotted in fpkm values representing good candidates for the formation of the three cndglcs .
ugts contained in phylogenetic group g  .
the colors in b–d refers to the gene names listed next to panel a
because pglcpaac reached maximum levels at the early phase of grain development, the gene encoding this ugt was expected to have a high initial expression.
cluster group 1– <dig>  4– <dig> and  <dig> meet these criteria .
only one  of the five genes from these cluster groups was expressed throughout development , albeit at a low abundance compared to the earlier described genes.
this gene belonged to the group d ugts.
the other four all had time points where they were not expressed and were only expressed in low abundance .
the dhurrin glucosides accumulated late in the course of grain development, when the dhurrin content was decreasing .
genes encoding the potential ugt candidates capable of catalyzing glucosylation of dhurrin were therefore expected to display maximum expression later than the maximum expression time point for ugt85b <dig> .
genes matching this trend were found in cluster groups 3– <dig>  4– <dig> and 4– <dig> .
combined this selection contained  <dig> genes, of which five were expressed with a maximum abundance above  <dig> fpkm   and belonged to group l, h, e, e and d, respectively .
none of the genes expressed at high abundance from the previous two selections  belonged to the group g ugts.
the genes in group g were of particular interest, because ugt85b <dig> involved in the biosynthesis of dhurrin resided in this class.
as dhurrin is most likely the substrate for the formation of the dhurrin glucoside, it is possible that a ugt capable of glucosylation of dhurrin would share sequence similarities with ugt85b <dig>  all genes from this group were therefore displayed together in fig.
10d to reveal ugt gene candidates not discovered via the cluster analysis.
of particular interest were three gene candidates  expressed at high abundance and correlated with the concentration of the dhurrin glucoside during early development .
gene expression profiles of genes involved in the dhurrin catabolism
all the expression profiles of the putative gene candidates described in the previous sections have been incorporated with their possible positions within the putative turnover pathways hypothesized by jenrich et al.
<cit>  and pičmanová et al.
<cit>  showing the catabolism of dhurrin .
only the most promising gene candidates involved in the catabolism are displayed in fig.
<dig>  of the gst genes selected as potentially involved in conversion of dhurrin to pohpcn, three of the glutathionylating ones and one deglutathionylating gst have expression values above  <dig> fpkm, while the two other gst genes and nit4a and nit4b <dig> have a maximum abundance above <dig> fpkm.
in comparison, the ugt gene candidate and the putative nitrilase/amidase genes involved in the second turnover pathway display a lower maximum abundances below  <dig> fpkm.fig.
<dig> metabolite accumulation and expression of genes in the two putative dhurrin turnover pathways.
the transcript boxes are simplified versions of the earlier figures: gst <dig> , gst <dig> , nit4a/b <dig> , ugt <dig> , ugt <dig>  and nitrilase/amidase .
the dhurrin acid and amide can in principle be formed via two different routes: from dhurrin or directly from pohmn, *which then requires an additional glycosylation step in order to get the dhurrin acid and amide
transcriptome analysis of the genes involved in the flavonoid pathway
to investigate the transcriptional regulation of the biosynthesis genes involved in the relatively unknown flavonoid pathway in sorghum and to support the possible role of pas as defense compounds in the grain , the sorghum genome was blasted with a selection of protein sequences involved in the above metabolic pathways in different plant species.
all the matching sorghum genes were aligned with species characteristic amino acid sequences and a phylogenetic analysis conducted for all genes found for each of the individual enzymatic steps .
the sorghum genes most closely related to monocot sequences and in particular to maize were extracted from the analysis and their expression profiles plotted in the flavonoid biochemical pathway .
the gene encoding the initial enzyme chalcone synthase  in the flavonoid metabolism had previously been identified in sorghum  <cit> .
this enzyme converts the two substrates p-coumaroyl-coa and malonyl-coa into naringenin-chalcone.
eight isoforms of the enzyme have previously been identified, but several other gene candidates exist in the genome .
only two of these identified genes  were expressed in the grain in high transcript abundance and both peaked before the second concentration peak in pas between  <dig> and  <dig> daa.
five sequences were found for the second enzyme, chalcone isomerase  which converts naringenin-chalcone to naringenin .
one of these genes had been identified earlier   <cit> .
another transcript  was expressed in equally high abundance to the identified gene, but this gene resided in the clade with dicotyledons and is a less likely candidate.
the b-phenyl ring of naringenin can be hydroxylated at the 3’ or at the 3’- and 5’ positions to produce eriodictyol or dihydrotricetin, respectively.
these hydroxylations are performed by flavonoid 3’-hydroxylase  and flavonoid 3’,5’-hydroxylase .
in sorghum, four putative f3’h and 16 f3’5’h  were identified.
of the four f3’h genes,  <dig> had previously been identified in sorghum   <cit> .
none of the 16 f3’5’h genes had previously been described, but sobic.007g <dig>  and sobic.001g <dig>  were closely related to maize and barley f3’5’h sequences.
two of the f3’h genes  might be of importance to the hydroxylation of the flavonoids destined for pas as they peaked in correlation with the pas.
for the f3’5’h genes, only the gene sobic.001g <dig>  displayed an expression profile of interest, but this was not closely related to any of the known monocot f3’5’h sequences .
the naringenin, eriodictyol and dihydrotricetin followed the same basic pathway and ended up as the three different catechins, which upon conjugation form the pas.
in the following description of the steps leading up to the formation of the catechins, only the one originating from naringenin is mentioned as the enzymes involved were similar.
from naringenin, it is possible to form three different compounds.
flavone synthase ii  converts naringenin to the flavone apigenin.
the gene sobic.002g <dig>  had previously been identified  <cit>  and displayed an increasing abundance of transcript from  <dig> daa towards maturation.
the concentration of flavones has not previously been measured in the grain, but the transcript level co-occurred with the appearance of the grain coat pigmentation, which started around  <dig> daa and increased towards maturation .
a second gene  could be of interest because of its relatedness to the maize fnsii gene grmzm2g <dig> and shared a similar expression profile to the characterized sorghum fnsii gene.
the final transcript , resided in the same monocotyledon clade, and although it was not very closely related to other known transcripts, it shared an expression profile correlated with that of chs and chi .
naringenin is further converted to dihydrokaempferol by flavanone 3-hydroxylase .
two genes sobic.006g <dig>  and sobic.006g <dig>  encoding f3h had earlier been identified in sorghum  <cit>  and in the current study displayed profiles correlated with the pas .
dihydrokaempferol can be converted to flavonols by flavonol synthases .
two genes were found, but only one was expressed and at a very low abundance .
following the pathway of pas, dihydrokaempferol is converted to leucopelargonidin by dihydroflavonol reductase .
of the four genes encoding dfr previously identified in sorghum , three were expressed, sobic.003g <dig> , sobic.003g <dig>  and sobic.004g <dig>    <cit> .
only the two transcripts dfr <dig> and dfr <dig> displayed expression profiles correlated with the concentration of pas.
the leucopelargonidin produced is further converted to pelargonidin by anthocyanidin synthase .
the ans encoding gene  had previously been identified in sorghum  <cit>  and is the best candidate due to a high initial abundance.
six other putative ans sequences were also found, but they only shared an average amino acid similarity to sbans of 33 ± 1% and were positioned in a separate clade in the monocot clade .
pelargonidin can be glucosylated by flavonoid-3-o-glucosyltransferase  and flavonoid-5-o-glucosyltransferase  to produce anthocyanins.
no 3- or 5gt genes had previously been identified in sorghum, but eight putative genes were found in our analysis, with six being expressed in the grain .
the two genes sobic.010g <dig>  and sobic.002g <dig> were positioned close to maize and barley 3gt, in the clade containing all other 3gt genes and belonged to group f ugts .
only sobic.010g <dig> was expressed in the grain and displayed an expression profile that correlated with the pigment formation in the grain coat.
two of the other genes  were likely 5gts as they group together with other 5gt genes .
they both belong to group l ugts  and had an increase in transcript abundance towards the end of grain maturation.
for the formation of pas, pelargonidin is further converted to -epiafzelechin by anthocyanidin reductase .
eleven anr genes were discovered , with eight expressed and four of these having a relevant transcript expression .
the epicatechin produced from pelargonidin by anr have a cis orientation of the b-phenyl ring.
a second enzyme, the leucoanthocyanidin reductase  is capable of producing catechins, which have a trans orientation of the b-phenyl ring.
lar enzymes are found in barley and rice  <cit> , but our phylogenetic analysis did not identify any such genes in sorghum .
the last enzymatic step resulting in the formation of pas has still not been elucidated, but laccases  have previously been determined to play a role in polymerization of pas in several plant species  <cit> .
in sorghum,  <dig> lac encoding genes were found of which  <dig> were not expressed in the grain .
of the remaining eight genes, two  had high transcript abundances, but only sobic.001g <dig> displayed a profile correlated with the pa concentration.
discussion
dhurrin biosynthesis and accumulation in the developing grain
the content of cnglcs within a plant species varies ontogenetically, phenologically, and chronologically.
although many exceptions can be found, the concentration of cnglcs is in general highest in seedlings and decreases with plant age  <cit> .
cnglcs may accumulate in any part of a plant and newly formed tissues are typically more cyanogenic compared to older tissues .
forage and grain sorghum plants are known to be cyanogenic due to their content of the cnglc dhurrin  <cit> .
the dhurrin content of the mature sorghum grain has been shown to be negligible or zero  <cit> .
in the current study, it was discovered that dhurrin accumulate in the immature grain .
the dhurrin content of the husk, which is the maternal tissue component of the grain, was close to zero throughout the entire grain development and thus not correlated with the amount of dhurrin in the grain.
from the transcriptome analysis, it is also evident that the genes encoding the enzymes involved in dhurrin biosynthesis in vegetative tissue  were expressed in high abundances immediately before the concentration of dhurrin peaks .
this substantiated that dhurrin is de novo biosynthesized in the grain from tyrosine via the pathway discovered in etiolated seedlings of sorghum  <cit> .
similar to sorghum, the mature seeds of lotus japonicus, cassava  and barley  contain no or only negligible amounts of cnglcs  <cit> .
in bitter almonds, the cyanogenic monoglucoside prunasin is biosynthesized in the tegument of the almond fruit throughout development to reach a maximum hcnp of 16 nmol/mg  <cit> .
at the end of maturation, prunasin is transported to the developing cotyledons of the kernel where it is converted and stored as amygdalin, which reaches a maximum hcnp of 89 nmol/mg  <cit> .
in mature seeds of rubber tree , the cotyledon is similarly high in cnglcs with a hcnp of 132 nmol/mg tissue  <cit> .
from these examples, it is clear that the sorghum grain only contains low amounts of cnglcs.
earlier research into the biosynthesis of dhurrin concluded that the biosynthesis of dhurrin in the vegetative tissue is largely controlled at the transcriptional level  <cit> .
this earlier finding is confirmed by our results, where large standard deviations in the transcript levels of cyp71e <dig> and ugt85b <dig> coincided with the large deviations in the dhurrin concentration at day  <dig> .
furthermore, these large standard deviations in the gene transcript abundance was only seen at day  <dig> and was therefore not attributable to technical variation or a general biological variation observed for every time point during grain development.
despite these variations, the ratio between the transcript abundances of cyp79a <dig>  cyp71e <dig> and ugt85b <dig> were relatively fixed and showed an incremental increase of transcript from the first  to the last enzyme in the biosynthesis  .
the same tendency was seen in the ratio between proteins  and could be a safety feature to ensure that none of the toxic intermediates ) from cyp79a <dig> and cyp71e <dig> are released during biosynthesis, but converted by the downstream processes to dhurrin.
sorghum grain is not cyanogenic
cyanogenesis is the classical defense mechanism assigned to cnglcs and is based on their hydrolysis by bgd and hnl resulting in the release of hcn and ketones  <cit> .
the specific bgds hydrolyzing dhurrin are assigned as dhrs in sorghum  <cit> .
the absence of dhr activity in the developing grain was demonstrated by the observed lack of hydrogen cyanide release from grain macerates .
furthermore, the lack of an expected quantitative hcn release upon addition of almond bgd must reflect the presence of endogenous inhibitors in the grain such as the high amounts of pas   <cit> .
the missing hydrolysis of dhurrin in grain homogenates was also substantiated by the complete absence of transcripts encoding dhr.
although transcripts encoding the enzymes hnl, cas and nit4b <dig> were all found to be expressed in the grain, their expression profiles indicate that they are not directly involved in dhurrin turnover in the grain .
instead, the expression profile of hnl in relation to the dhurrin concentration curve and the lack of dhrs indicate that it might serve as a scavenger of pohmn, released by chemical degradation of dhurrin during turnover.
the expression profiles of cas and nit4b <dig> correlate with acc oxidase and indicate that these enzymes are primarily involved in detoxification of hcn from ethylene production and not cyanogenesis.
in other cyanogenic plants, the endogenous bgds are extremely active and will rapidly hydrolyze any freely available cnglcs.
in the past, this prevented the use of cnglcs as the key substrates in experiments with such extracts aimed at detecting the possible operation of endogenous turnover pathways.
the lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study cnglc synthesis as well as endogenous turnover.
involvement of two alternative pathways in the turnover of dhurrin
the observed disappearance of dhurrin in the course of grain maturation, the absence of bgd activity and the lack of correlation between cas and nit4b <dig> expression with the dhurrin disappearance must reflect the operation of one or more dhurrin turnover pathways that do not proceed via hcn release.
the operation of such pathways has previously been proposed  <cit> .
interestingly, the expression profiles of the genes encoding the nit4a/nit4b <dig> heterodimer indicate that they are involved in a continuous turnover of dhurrin throughout grain development and not only when the concentration of dhurrin decreases at  <dig> daa .
this is supported by the finding that the glucosylated nit4a/nit4b <dig> product, pglcpaac, accumulates in the early stages of grain development while the dhurrin content is still increasing .
a similar result was presented by adewusi  <cit> , who showed that dhurrin turnover proceeded in young plants while the total amount of dhurrin was still increasing.
although the transcript abundances indicate that the nitrilases might be involved in dhurrin turnover, no traces of pohpcn and pohpaac were found in the grains indicating that they might be transient intermediates.
however, the applied lc-ms method does not effectively detect the free pohpaac.
it is therefore possible that in the developing grain, this precursor of pglcpaac is either accumulated in its free form instead of being glucosylated or incorporated into other compounds.
with the lack of dhr activity and transcript in the sorghum grain, the hypothesis that conversion of dhurrin to the nitrilase substrate pohpcn should be mediated by this enzyme in concert with e.g.
a protein co-factor must be rejected.
we instead propose the involvement of gsts as supported by hayes et al.
<cit>  and have identified a number of candidate gst genes.
the transcriptome data suggested the involvement of both glutathionylating and deglutathionylating gsts in this metabolic transformation.
the expression profile of one highly expressed deglutathionylating lambda gst  was ranked as the gst most correlated with the expression of nit4a and nit4b <dig>  and sobic.001g <dig> and sobic.001g <dig> highly expressed glutathionylating gsts were likewise strongly correlated .
these high similarities to the expression patterns of nit4a and nit4b <dig> are consistent with their possible involvement in continuous turnover of dhurrin.
the phi class gst  and the other gsts in cluster group  <dig> , did not show a favorable expression pattern and had a low similarity to the nit expression profiles and are therefore not as obvious gene candidates for the glutathionylation as the gst mentioned above.
future expression of the best gene candidates and characterization of the enzymes will reveal if they are indeed involved in dhurrin turnover.
in vegetative tissues, pglcpaac accumulates as the sorghum plants get older and dhurrin is turned over  <cit> .
the time restricted production of this compound in grain, guided identification of potential ugt candidates for the glucosylation of pohpaac into pglcpaac.
our transcriptome analyses demonstrated that  <dig> ugt encoding genes were expressed in the developing grain .
in comparison,  <dig> of the  <dig> genes encoding ugts in flax  are expressed in the developing seeds  <cit> .
from the phylogenetic- and hierarchical clustering analyses only a single ugt  gene candidate  was found with an expression profile correlated with the concentration of pglcpaac .
in a. thaliana, group d ugts is known to glucosylate a range of different aglycones including terpenoids, flavonoids, benzoates and brassinosteroids and it is therefore possible that this sorghum ugt is responsible for production of pglcpaac.
besides the pathway just discussed, a second putative turnover pathway involving the dhurrin amide and dhurrin acid probably exists  <cit> .
the formation of these two dhurrin derivatives may be envisioned from two possible precursors, either dhurrin or directly from pohmn .
in both scenarios the nitrile-function can be converted into a carboxylic acid group by the direct action of a nitrilase or via the amide by the combined action of a nitrile hydratase and an amidase  <cit> .
interestingly, a nitrile hydratase capable of producing an amide from a nitrile has never been characterized in any plant species, although such enzymatic activities have been measured in several species including sorghum  <cit> .
only the cyano hydratases, which are both structurally and mechanistically different from the nitrile hydratases, have been characterized in plants  <cit> .
in bacteria, the formation of indole-3-acetamide from indole-3-acetonitrile occurs via the action of a nitrile hydratase  <cit> .
a blastp search of the sorghum proteome using known bacterial nitrile hydratase protein sequences gave no significant hits .
the bacterial nitrile hydratases are metallo-containing enzymes and thus both structurally and mechanistically different from the nitrilase superfamily enzymes from plants, e.g.
nit4a, nit4b <dig> and nit4b <dig>  enzymes in this family are the best candidates for the production of the amide and acid derivatives  <cit> .
only one new gene candidate was found in this family .
the abundance of this gene makes it unlikely, as a candidate for the formation of dhurrin acid, but it could however be involved in the formation of the dhurrin amide.
similarly, the glutamine-dependent nad synthetase  could be involved in the dhurrin amide formation as this type of enzyme hydrolyze amides to produce the corresponding acid and ammonia  <cit> .
the last two low abundance nitrilases  belong to the branch  <dig> nitrilases with unknown functions and has the potential to be involved.
for the gene sobic.002g <dig> protein homologs with high sequence similarity  were found in acyanogenic monocots, whereas the sequence similarity for the genes sobic.008g <dig> and sobic.006g <dig> were lower  .
the last two genes are therefore better gene candidates for the formation of the dhurrin amide, but needs to be cloned and expressed before any conclusions can be drawn.
all the high abundance genes had a maximum abundance peaking after the dhurrin amide and are therefore probably not involved in its formation, but could still be involved in conversion of this compound into dhurrin acid.
however, none of them showed expression profiles correlated with the accumulation of dhurrin acid and protein homologs with high similarities were found in other acyanogenic plants  .
as sobic.001g <dig> has the lowest sequence similarity, this would be the first gene to clone and express for activity.
in conclusion, only dhurrin acid accumulated with a profile directly matching the degradation of its presumed precursor dhurrin.
this was in contrast to the previous results in studies using vegetative sorghum tissues where the level of pglcpaac continued to increase as dhurrin was turned over  <cit> .
the content of dhurrin acid was not analyzed by jenrich et al.
<cit> , but in the two other cases, the content of this compound also increased with age.
our current metabolite analyses indicated that in developing sorghum grain, the turnover pathway leading to pglcpaac formation was dominating during dhurrin accumulation, whereas the pathway producing dhurrin acid was dominating at the later stages.
a dual function for dhurrin in the developing grain
although the hcnp of the developing sorghum grain is low compared to seeds and fruits of other cyanogenic plants, they still contain a potentially lethal content of dhurrin.
reports indicate that 1–3 mg of cyanide per kilogram body mass is lethal to most vertebrates  <cit> .
this means that only 15– <dig> grains each containing 1 μg dhurrin could be lethal to a small  <dig> g bird.
this may be the reason that the african bird quelea quelea avoids feeding on developing grains of sorghum  <cit> .
the lack of sorghum bgds can be compensated for by presence of bgds in the saliva and gut of the birds and other herbivores, which would activate the toxicity of dhurrin.
this remains a hypothesis, as we were not able to demonstrate a stoichiometric hcn release from dhurrin upon addition of almond bgd to sorghum grain homogenates indicating the presence of inhibitors, which may or may not inhibit the release upon consumption by birds.
the deterrent effect of dhurrin may be augmented by the presence of high amounts of pas, which have previously been shown to deter birds and other grain predators  <cit> .
our analysis did not quantify the pa concentration, as no standards were available, but research show that sorghum grains have a very high pa abundance compared to other food plants  <cit> .
in other cyanogenic species, cnglcs and pas may function in a similar trade-off strategy, where they complement or even compensate each other  <cit> .
the chaparral shrub, heteromeles arbutifolia is one such example  <cit> .
in the immature fruit, the pulp accumulates high amounts of pas and cnglcs.
once the fruits mature and turn red, a rapid turnover of pas occur in the pulp and the cnglcs are transported to the seed.
the fruit is thus safe for birds to eat and the seed will be dispersed and remain protected by cnglcs until germination.
in sorghum, it is tempting to speculate that the combined effects of dhurrin and pas protect the more attractive immature sorghum grain while the pas alone are sufficient to protect the mature grain against herbivores.
similar to the dhurrin biosynthetic gene transcripts, transcripts involved in the flavonoid pathway peaked prior to the concentration maximum of the pas at  <dig> daa .
few reports are available on the expression of all the flavonoid genes in course of plant ontogeny and of these, the majority only compare the differential expression between tissue types and treatments .
in the flowers of cotton , expression of the genes in the flavonoid pathway responsible for the production of anthocyanins correlated with the concentration of anthocyanin, while anr and fls <dig> showed different expression profiles  <cit> .
for sorghum, the genes chs, chi, f3’h, fnsii, f3h, dfr, ans have previously been described in other publications, but none of them have been characterized.
the research presented here add additional gene candidates to the aforementioned range of enzymes, but also pinpoints some genes which are likely to the primary ones involved in the flavonoid biosynthesis in the developing grain.
furthermore, novel gene candidates including the 3gt/5gt, anr and lac were also identified which were previously unknown in sorghum and could help the understanding of the pa biosynthesis.
the dramatic turnover of dhurrin in the developing sorghum grain is initiated at the time point where the pa content peaked.
at this developmental stage the pas are envisioned to protect the sorghum grain towards herbivory.
the function of dhurrin as a defense compound may then be shifted towards a function as a nitrogen resource.
this dual function could be a cost-effective measure during the energy demanding grain-filling period, saving the plant from simultaneous production of both defensive compounds and storage proteins  <cit> .
furthermore, the pas do not contain nitrogen and can thus be biosynthesized without the need for substrate containing nitrogen, which is needed for storage protein production.
the accumulation of dhurrin means that nitrogen is still being assimilated in the grain, albeit in a form unavailable to herbivores.
as the grain matures and becomes less attractive due to lower water content and higher concentrations of pas, the nitrogen in dhurrin can be remobilized as ammonia and utilized to produce storage proteins.
in the developing sorghum grain, storage protein in the form of protein bodies first occurs in the endosperm  <dig> daa and continue to increase in concentration towards maturation  <cit> .
assimilation of nitrogen from the soil in cereals is often insufficient to supply the developing grain and the majority of the nitrogen in the grain originates from leaf tissue remobilization  <cit> .
it is therefore possible that the accumulation and remobilization of dhurrin in the early developing sorghum grain supplies it with a more direct and readily available source of nitrogen for the formation of protein bodies in the later stages.
to supply endosperm cells with nitrogen, the dhurrin glucosides could function as a transport form of dhurrin from the site of biosynthesis at the er membranes to the vacuoles for storage  <cit> .
for the glucosylation of dhurrin, eight potential ugt gene candidates were discovered .
although the expression profiles of two of the genes  correlated with the concentration of dhurrin diglucosides, only in-vitro assays can determine the precise substrate specificity of these enzymes.
as three different structural configurations of the cndglcs exist, it is possible that three different ugts also have to be found among the eight gene candidates.
in most plants, the formations of the different glycosides are catalyzed by distinct ugts.
this is the case for the production of anthocyanins in the flavonoid pathway, where the 5-o and 3-o glycosylations are performed by two different enzymes   <cit> .
to remobilize the nitrogen in dhurrin, the dhurrin glucosides are likely hydrolyzed and metabolized via the turnover pathways described .
the results obtained in this study suggest that the pathway producing pohpaac continuously metabolize dhurrin, to potentially act as a nitrogen buffer and supply the growing grain with reduced nitrogen.
the metabolite accumulation in the secondary pathway suggests that the dhurrin amide and acid might have different substrate sources.
the late accumulation of dhurrin acid indicates that this compound most likely is a turnover product from the degradation of dhurrin.
on the contrary, the early accumulation of the dhurrin amide indicates that this might originate directly from pohmn as a result of improper metabolon formation and could function as an additional nitrogen source.
when the production of protein bodies starts  <dig> daa, all available nitrogen sources are remobilized, including the dhurrin amide.
as pglcpaac and dhurrin acid have already released their nitrogen, the turnover of these compounds may not be as urgent as the glucose moieties they contain are not a limiting source.
they are therefore degraded at a slower rate and only decreases to zero at the time of maturation .
CONCLUSIONS
we have used the developing grains of sorghum bicolor  moench to study the metabolism of dhurrin.
metabolite profiling using lc-ms/ms revealed that dhurrin is accumulated in high amounts during the early phases of grain development but is absent in the mature grain.
despite the high amount of dhurrin in the early developmental phase, the grains were acyanogenic and only released minute amounts hydrogen cyanide upon tissue maceration.
this result was confirmed by the complete absence of transcripts encoding the dhurrin specific β-glucosidases .
although the accumulated dhurrin may still have a secondary protective role and be toxic to potential grain predators via herbivore β-glucosidases, the primary function of dhurrin is probably as a nitrogen store.
the loss of the protective functions of dhurrin could be replaced by the high amounts of proanthocyanidins accumulated in the grain via the flavonoid pathway.
several new gene candidates for this pathway were discovered in this study, including 3gt/5gt, anr and lac.
with the missing activity of the dhurrinases, the turnover of dhurrin occurs via two alternative turnover pathways for which intermediates were confirmed by metabolite profiling.
for the turnover of dhurrin via dhurrin amide and dhurrin acid, several novel gene candidates encoding enzymes from the nitrilase super family were discovered.
although their expression profiles are not ideal in relation to metabolite accumulation, their possible modes of action still prompt for further investigation.
on the contrary, cluster analyses revealed strong candidates for the glucosylation of dhurrin to dhurrin glucoside, glucosylation of pohpaac acid and potential gsts for sequential conversion of dhurrin to pohpcn.
by combining all the acquired results, we can conclude that one turnover pathway is likely to be involved in continuous turnover of dhurrin, while the other is primarily involved in turnover at later stages of grain development.
the combined effects of these two putative turnover pathways could supply the developing grain with nitrogen to fuel the production of storage protein.

 cyanogenic glycosides are bioactive specialized metabolites found in more than plant species distributed in different plant families of angiosperms, gymnosperms and ferns with a large proportion found among of our cultivated plants. examples of cyanogenic crop plants include barley, wheat, sorghum moench), cassava, almond, cherry and apple. upon tissue disruption, the cnglcs stored in the tissues of cyanogenic plants, are brought in contact with endogenous β-glucosidases resulting in hydrolysis and release of the cyanohydrin aglycone, which spontaneously or catalyzed by an α-hydroxynitrile lyase dissociates to release hydrogen cyanide and a keto compound. this pathway is often referred to as the bioactivation pathway and the process as cyanogenesis. the toxic effect of hcn is caused by its ability to inhibit metalloenzymes and in particular cytochrome c oxidase, the key enzyme in the respiratory electron transport chain in mitochondria. cnglc storage in plants and the release of hcn upon tissue disruption may serve as a deterrent against generalist herbivores. in addition to herbivore defense, cnglcs may serve a multiplicity of other biological functions. in the rubber tree, linamarin accumulates in the mature seed. upon germination, the cnglc is converted into the cyanogenic diglucoside linustatin and transported to the cotyledon where it is turned over. a similar situation has been observed in bitter almonds where prunasin is biosynthesized in the tegument of the almond fruit throughout development and eventually transported into the developing cotyledons of the kernel where it is converted into the cndglc amygdalin. upon germination, the amygdalin stored in the cotyledon of the bitter almond variants is most likely catabolized as observed for amygdalin accumulated in seeds of black cherry. in sorghum, dhurrin derived cndglcs are found in guttation droplets and are similarly hypothesized to act as transport forms. three cndglcs which differ from each other in the configuration of the glucosidic linkage between the primary and secondary glucose molecule have been found, but their specific roles as possible transporters remain unknown. in young sorghum seedlings, the cnglc dhurrin rapidly accumulated following germination after which biosynthesis decreased and turnover increased to result in a reduced concentration of dhurrin. this turnover of cnglcs was earlier thought to occur via the bio-activation pathway with the hcn released being incorporated into β-cyanoalanine by β-cyanoalanine synthase and finally converted by nitrilases belonging to the nit family to asparagine and to aspartate with concomitant release of ammonia. however, evidence for the occurrence of alternative turnover pathways that bypasses the release of the toxic intermediates hcn and β-cyanoalanine has been proposed. in the first alternative pathway suggested for sorghum, dhurrin was hypothesized converted to p-hydroxyphenylacetonitrile via an unknown novel bgd/protein co-factor complex. no evidence has subsequently been found to support the presence of such a protein complex. instead, more recent results pointed to the formation of pohpcn through the action of glutathione s-transferases supported by genome wide association analysis of a sorghum population with variable leaf dhurrin levels, where a gst was shown to be associated with dhurrin concentration in leaves. the pohpcn was subsequently hydrolyzed to p-hydroxyphenylacetic acid and ammonia by the action of a heteromeric enzyme complex composed of the enzymes nit4a and nit4b. finally, the pohpaac was glucosylated to produce p-glucosyloxyphenylacetic acid which has been shown to accumulate in vegetative tissue of different sorghum cultivars as the plants got older and the dhurrin content decreased. the second proposed endogenous turnover pathway included a unifying set of proposed turnover products in sorghum, almond and cassava where the nitrile group of the cnglc is converted into an amide, a carboxylic acid and finally completely lost in a decarboxylation reaction. for clarity, these possible intermediates are referred to as cnglc amide, cnglc acid and cnglc anitrile in this manuscript. for investigating the turnover of cnglcs in cyanogenic plants, the developing grains of sorghum were selected as an experimental model system. developing seeds are terminal sink organs and do not have net export of nutrients and amino acids and therefore offer an attractive system for study of endogenous turnover processes. the mature sorghum grain contains only minute amounts or no dhurrin at all. in this study, we report that dhurrin is formed in the initial stages of grain development and turned over in the course of grain maturation. the grain was found not to possess dhurrinase activity signifying the operation of one or more endogenous turnover pathways not involving the release of toxic hcn. high concentrations of condensed tannins were formed throughout development and accumulated in the mature grain. for the investigation of endogenous turnover of dhurrin, time-resolved transcriptomic analyses guided the identification of novel gene candidates encoding enzymes belonging to the families of gsts, the nitrilase superfamily and udp-glycosyltransferases. based on the expression profiles of the genes supposedly involved in endogenous dhurrin turnover and the dhurrin metabolites formed, we propose a dual role for dhurrin in the developing grain of sorghum.  metabolite profiling of developing sorghum grain immediately following anthesis, the total content of dhurrin in the grain was low and barely detectable ). dhurrin content then rapidly increased reaching a maximum 25 days after anthesis. after this maximum, a similarly rapid decrease occurred which resulted in complete disappearance of dhurrin at maturity, daa. in the same time period, the dhurrin content of the husk remained constant at  nmol/husk. accordingly, the concentration of dhurrin was approximately times higher in the grain compared to the husk right after anthesis and times higher at the maximum dhurrin concentration.fig. concentrations of dhurrin and proanthocyanidins in developing grains. the concentration of dhurrin and proanthocyanidins measured pr. grain and pr. mg grain tissue at different days after anthesis. the dhurrin concentration is measured in nmol and the pa concentration as absorbance. the colored circles represent the actual measurements in the three replicas and the solid line the trend line calculated using local polynomial regression fitting, with the area surrounding the average line representing the 95% confidence interval. each measurement represents the average of grains with one of the five grains and husks displayed in the table the presence of dhurrin turnover products in the extracts of the sorghum grain from anthesis to maturation was analyzed by lc-ms/ms. dhurrin amide, dhurrin acid, pglcpaac and the dhurrin glucosides a, b and c were all found in the grain extracts. a previous study by pičmanová et al. detected the presence of caffeoyl-dhurrin and caffeoyl-dhurrin-acid in sorghum leaf extracts. these compounds were not found in the sorghum grain. the appearance of the putative dhurrin turnover products in the sorghum grain from anthesis to maturity in relation to the total content and concentration of dhurrin is shown in fig.  in whole grains, dhurrin amide peaked around daa, i.e. immediately before the concentration of dhurrin started to increase. in contrast, the related compound dhurrin acid, began to accumulate around daa, i.e. at the time point where the dhurrin content started to decrease. in relative terms, the dhurrin acid reached 34% of the total dhurrin concentration, while dhurrin amide only reached 9%. similarly, the content of pglcpaac reached 8% of the total dhurrin content. the dhurrin glucosides were observed to reach their maximum content at daa, i.e. a week after the dhurrin content started to decrease and reached 6% of the total dhurrin content.fig. the accumulation of putative dhurrin turnover products. the concentration of dhurrin and putative turnover products measured as intensity pr. grain and intensity pr. mg grain tissue throughout development. the colored circles represent the actual measurements in the three replicates and the solid line the trend line calculated using local polynomial regression fitting. the area surrounding the average line representing the 95% confidence interval. the different colors corresponds to the different metabolites listed in the box below the graphs proanthocyanidin content in the sorghum grain and husk from anthesis to maturity a high concentration of proanthocyanidins in developing and mature sorghum grains could increase herbivore resistance and compensate for the lack of dhurrin at maturity. the concentration of pas throughout grain development was therefore investigated. as part of the maturation process, the grain changed color from an initial green to a deep dark/brown color. the browning process was initiated daa and was correlated with formation of pas. in the sorghum grain, the content of pa increased from a low initial level to a maximum content per grain at daa. from to daa, the content decreased to approximately half the maximum value. when measured per mg grain tissue, the variations in concentration of pas was less pronounced with only a slight increased concentration around daa followed by a small decrease towards maturation. cyanide potential of seeds the total content of a cnglc in plant tissue is often referred to as the cyanide potential. despite incubation for  h following maceration, only a small fraction of the total amount of dhurrin present in the samples as determined by lc-ms/ms analysis was hydrolyzed in the native samples. the amount of dhurrin hydrolyzed is positively correlated with the total estimated amount of dhurrin but does not exceed the amount undergoing non-enzymatic hydrolysis in the same time frame. addition of almond bgd to homogenates of the initial samples, increased the amount of hcn released and was positively correlated to the total dhurrin content although complete hydrolysis of the dhurrin present was not observed. from to daa, the addition of bgd did not increase the amount of hcn released. spiking of the samples with 5 nmol dhurrin increased the release of hcn slightly, but did not result in an additional 5 nmol hcn compared to the hcn released from the samples containing tissue plus almond bgd. increasing the concentration of bgd from to 5u/ml only increased the hcn release relative to the total dhurrin content in the samples spiked with additional dhurrin.fig. cyanide potential in grains. nmol hydrogen cyanide released from macerated tissue at different developmental stages. the hcn release was measured from macerated tissue in buffer alone, tissue in buffer plus u/ml almond β-glucosidase and tissue in buffer plus u/ml almond β-glucosidase and an additional 5 nmol dhurrin. for each sample/bar, the total possible release of hcn from dhurrin is indicated by the striped bar stacked on top of the measured hcn release. the total possible release of hcn is calculated from the equation describing the trendline for dhurrin in fig. 1b. the hcn release was measured a second time with macerated tissue from daa with u/ml almond β-glucosidase instead of u/ml. as a control, an assay measuring hcn release from varying concentrations of dhurrin in buffer was also performed time-resolved transcriptome analysis of developing grain to investigate the expression profiles of genes encoding enzymes involved in dhurrin biosynthesis and putative turnover processes, transcriptome analysis of the developing grain was performed. more than million 100 bp paired-end reads were generated resulting in an average of million reads per library. of these reads, an average of 87% were mapped to the phytozome sorghum transcriptome v, with 55% being uniquely mapped and 31% non-specifically mapped. of the predicted protein-coding gene loci in sorghum, loci were detected as expressed during sorghum grain development. among these transcripts, a large fraction showed an expression below a single fragment per kilobase of transcript per million mapped reads. with these values considered as the background level, genes were registered as not being expressed. the results obtained from the transcriptome study were verified by quantitative pcr using specific primers towards the gene cas. functional genomics and identification of interesting genes was initially guided by categorizing the expressed genes according to their expression pattern throughout grain development. all expression profiles were normalized and clustered using hierarchical clustering with wards method. the expression patterns of the cluster groups containing the biosynthetic genes 79a and ugt85b in 8– and cyp71e in 12–1) as well as nitrilase 4b in 10– is shown along with the concentration of dhurrin in fig.  these six cluster groups shared an overall similarity. the remaining cluster group profiles were contained in three larger groups which are presented in additional file it is to be noted that the nadph-dependent cytochrome p oxidoreductase isoforms crucial for the dhurrin biosynthesis were not contained in these clusters, but did exhibit maximum gene expression before the dhurrin concentration peak and belonged to the same overall group as the biosynthetic genes. this difference in the expression profiles is more easily observed in fig.  fig. clustering analysis of transcript expression profiles. the hierarchical clustering is divided into groups, which was further subdivided into subgroups. only the cluster groups containing the biosynthetic genes is displayed on the figure. the complete overview of all cluster groups can be found in additional file the number of genes in each cluster is listed in parenthesis next the cluster number. the grey field represents the span of all the expression profiles in a single cluster. the dashed line represents the concentration of dhurrin pr. mg tissue through the grain development in relation to the expression profiles fig. expression profiles linked to the biosynthetic- and bioactivation pathway. the biosynthetic pathway involves the enzymes cyp79a cyp71e and ugt85b with the formation of dhurrin from l-tyrosine. the bioactivation pathway hydrolyzes dhurrin to release hcn via the enzymes dhr and and hnl. the detoxification pathway converts the hcn to aspartate + nh and/or asparagine via cas and the nit4a/b and nit4a/b heterodimers. the expression profiles for the specific enzyme involved in the reactions in the biosynthetic- or bioactivation pathway are colored in the same color as the enzyme boxes. in the box representing the por expression, the a, b and c represents the three different isoforms found in sorghum. expression is displayed in fragments per kilobase of exon per million reads mapped. the dashed line in each transcription box represents the dhurrin concentration throughout development in the bio-activation pathway, the two dhr genes known to be present in the sorghum genome were not expressed, while the hnl transcript reached maximum abundance a few days after the dhurrin concentration peak and then decreased in conjunction with dhurrin. in contrast, the two genes encoding the two subsequent enzymes cas and nit4b in the detoxification pathway showed maximum expression at anthesis with a steady decline towards grain maturation. a similar trend was seen for 1-aminocyclopropane-1-carboxylic acid oxidase. this enzyme represents the final enzymatic step in ethylene biosynthesis and releases stoichiometric amounts of hcn, which is detoxified, via the same cas detoxification pathway. the transcripts for nit4a and nit4b displayed a third type of expression profile with an almost linearly increased expression from anthesis to grain maturation. for many of the genes displayed, the standard deviations of their expression profiles were relatively low. the two genes cyp71e and ugt85b in the dhurrin biosynthesis were exceptions and displayed relatively large standard deviations at daa. despite these large standard deviations, the three genes in the dhurrin biosynthesis displayed a relatively fixed ratio between the transcript abundances from daa, with cyp79a being the lowest expressed and ugt85b the highest expressed gene, with cyp71e having an intermediate expression. from western blot analysis using available antibodies against cyp79a and cyp71e a similar distribution was found with cyp71e having the highest concentration. however, the protein ratio was on average 4-fold lower compared to the rna ratio. in terms of maximum abundance, cyp71e and ugt85b displayed large values of several hundred fpkm as opposed to the por isoforms, hnl, cas, nit4b nit4b and acc oxidase, which all displayed transcript abundances below fpkm. glutathione s-transferases in s. bicolor a total of genes encoding putative gsts were identified in the s. bicolor genome by blastp searches using a selection of gst protein sequences from arabidopsis thaliana, barley, rice, black poplar, physcomitrella patens and a selection of other plant, animal, fungi, and bacteria species. a conserved domain search at the national center for biotechnology information database and a pfam search at the european bioinformatics institute database revealed that of the selected sorghum protein sequences did not contain any gst domains whereas four only contained the c-terminal domain. these genes were retraced to the new phytozome genome v, to test if they were annotated incorrectly in the old database. the retracing did not change the sequences and these enzymes are likely catalytically inactive and were therefore deleted from the collection. two sorghum sequences only contained an n-terminal domain. retracing these genes revealed that sobic.005g is not annotated in the new database, while sobic.009g is replaced by sobic.009g domain search revealed that this gene has both n- and c-terminal domains. both these genes were included in the phylogenetic- and transcript expression analyses. all sorghum gst genes were classified into distinct classes via an ncbi conserved domain search and a phylogenetic analysis using the list of full-length gst described by liu et al.. the phylogenetic tree revealed that sorghum contained eleven gst classes: microsomal prostaglandin e-synthase, zeta, glutathionyl-hydroquinone reductases, lambda, dehydroascorbate reductases including sobic.009g, tetrachloro-hydroquinone dehalogenases, elongation factor 1b gamma, theta, phi, metaxin and tau. in general, the positions of the individual genes in the outer branches of the tree have good support with high bootstrap values, but the relation between the different gst classes in the tree were not very well supported. among the gsts, the tau and phi genes were by far the most numerous representing 77% of the total number of genes.fig. phylogenetic analysis of sorghum bicolor glutathione-s transferases. the phylogenetic tree represents the gsts found in sorghum. the tree is constructed with phyml using bootstrapping replicates. the bootstrapping values are displayed in percentage and only values above are displayed. the prokaryotic glutaredoxin, was used as an outgroup to root the tree. the two gene names marked in red color represent one gene where only the n-terminal gst domain was detected and a gene, with a new gene name in phytozome database version. the colored circles represent the cluster groups displayed in fig.  uncolored circles represent genes not expressed in the grain during development. the triangular shapes on the outside of the phylogenetic tree represent the gst genes potentially involved in dhurrin turnover. their specific functions are listed in the bottom right legend a previous genome-wide survey, identified gsts in sorghum. their division of the gsts into the different classes was based on phylogenetic analysis of only the n-terminal parts of the proteins although the division was classically based on the entire sequences. as an example, they identified only two lambda gsts, none of which were identical to the four identified in the present study. furthermore, the sequences classified as lambda gsts by chi et al. have a serine residue in the active site rather than the catalytic cysteine characteristic of lambda gsts and were therefore incorrectly classified as a consequence of the n-terminal based phylogenetic analysis. in our analyses, these two genes were categorized as tau gsts. the validity of our approach was confirmed by identical numbers and gene ids of cys gsts to those reported by lallement et al.. our analysis also revealed a new gst class not previously described in sorghum. expression analysis of gsts in s. bicolor based on the transcriptome analyses of the sorghum grain from anthesis to maturation, the expression patterns of the sorghum gsts were assigned to different cluster groups. this was done to guide the identification of possible gene candidates for involvement in the putative turnover pathway. of the gst genes found, were not expressed during the development of the grain. group clustering of the remaining genes into six groups revealed that the genes were primarily distributed among clusters and from the dendrogram, it was possible to determine the relatedness between the cluster groups. cluster group 1– were related and all displayed expression profiles that have the highest abundance after the concentration of dhurrin peaked. in contrast, the two related cluster groups and displayed the highest abundance prior to the dhurrin peak, while cluster group displayed an intermediate expression profile with high abundance both before and after the dhurrin peak.fig. expression profiles of gsts in the course of sorghum grain development. all expressed gsts found in the phylogenetic analysis were normalized to z-score and clustered using hierarchical clustering with wards method. the hierarchical clustering was divided into groups, which was further subdivided into subgroups. the lighter colored line represents the first subgroup and the darker colored line the last subgroup in a cluster. the number of genes in each cluster is listed in parenthesis next to the cluster number. the grey field represents the span of all the expression profiles in a single cluster. the dashed lines represent the concentration of dhurrin and pglcpaac through grain development in relation to the expression profiles of the different gsts. glutathionylating gsts from cluster 1– plotted in fpkm. deglutathionylating gsts from cluster 1– plotted in fpkm. gst identified by hayes et al. along with similar genes in similar cluster group. highly expressed genes in panel b–d have colored lines referring to the genes listed next to panel a. gray lines are genes only expressed at low abundance the gsts found in cluster group 1– were of most interest as their expression followed that of nit4a and nit4b and increased over time, at least until after the dhurrin concentration had peaked. in gst enzymes that catalyze glutathionylation, a serine or tyrosine is the catalytic residue, whereas deglutathionylating gsts harbor a catalytic cysteine. the classes zeta, tchqd, ef1bγ, theta, phi, metaxin and tau belong to the first group, while the classes dhar, lambda, ghr and mpges belong to the second group of gsts. the expression profiles of glutathionylating and deglutathionylating gsts from cluster 1– are shown in fig. 7b and c, respectively, with the most abundant members displayed in colored lines. the five most abundant genes in fig. 7b included four tau class gsts and a phi class gst. among the gst genes from cluster 1– the ones containing a cysteine residue were depicted in fig. 7c. of these genes, the most abundant ones were displayed in colored lines. these genes included two lambda class gsts and a ghr class gst. hayes et al. suggested that the phi class gst gene sobic.001g lying close to the gene cluster containing the dhurrin biosynthetic genes, could be involved in dhurrin turnover in the tissue. this gene was found in cluster group a cluster group, which shared a similar initial expression profile to the biosynthetic genes. the genes from cluster group are plotted in fig. 7d. the two most abundant genes beside the gst described by hayes et al. include a phi class gst and an ef1bγ class gst. when the expression profiles of all gst genes were compared to the average expression profile of nit4a and nit4b using pearson correlation, the glutathionylating gsts in fig. 7b are ranked as the 7th, 9th, 12th, 13th and 22nd most similar. in the deglutathionylating group in fig. 7c, sobic.002g was found to be the gst most similar to the two nit expressions with sobic.003g ranked as 16th and sobic.001g as 25th. in comparison the three deglutathionylating gsts in fig. 7d were only ranked as 35th, 38th and 47th, respectively. nitrilase activity in a novel turnover pathway the dhurrin turnover products, dhurrin amide and dhurrin acid are envisioned to be produced by the action of either a nitrilase or by the combined action of a nitrile hydratase and an amidase. to find putative nitrilase candidate genes in the sorghum genome, it was blasted using protein sequences ascribed to each of the branches of the nitrilase superfamily, which include amidases and cyano hydratases but not nitrile hydratases. to classify the ten genes discovered, they were aligned against the known nitrilase superfamily sequences and compared to the consensus sequences around the ekc catalytic triad. the ten genes were categorized as four branch- nitrilases, a single branch- β-ureidoproprionase, a branch- glutamine-dependent nad synthetase, three branch- nit and a single branch-11 n-carbamyl putrescine amidohydrolase. nine of the ten genes were expressed during grain development. three of these nine genes belong to the well-characterized nit family involved in the detoxification pathway and one of the two putative turnover pathways. these three genes were therefore not included. the six genes were divided into three low abundance genes and three high abundance genes.fig. expression profiles of the sorghum nitrilase superfamily genes in the course of grain development. the expression profiles of the six nitrilase superfamily genes expressed in the course of grain development shown in fpkm. the colors of the profiles corresponds to the gene names listed in the box below the plot. the three dashed curves represent dhurrin, dhurrin amide and dhurrin amide. the nit genes are excluded from this figure, but shown in figs.  and 11 udp-glycosyltransferase expression and possible involvement in metabolite formation the concentration of pglcpaac was observed to accumulate to maximum levels before the dhurrin concentration of the grain reached its maximum. to identify potential ugt candidates capable of glucosylating pohpaac, a total of genes encoding putative ugts were identified in the sorghum genome by blast searches using a. thaliana ugt protein sequences and two sequences from malus domestica and glycine max representing the novel o group ugt. alignment and domain analyses revealed that sequences were not recognized as ugts by the domain analyses. additionally, two sequences were missing the first conserved residues of the pspg motif located near the c-terminal which is involved in binding the nucleotide sugar donor. these sequences were excluded from further analyses. the remaining genes were subjected to phylogenetic analyses along with the previous reference sequences. the relationship between the individual genes is generally supported by high bootstrap values, while the relationship between the different ugt classes has lower bootstrap support. all ugt groups previously defined were represented in the phylogenetic tree. ten sequences were assigned to group a, to group b, to group c, to group d, to group e, to group f, to group g, to group h, to group i, to group j, to group k, to group l, to group m, to group n, to group o and to group p.fig. phylogenetic analysis of sorghum udp-glycosyltransferases. the phylogenetic tree represents the ugts found in sorghum. the tree is constructed with phyml using bootstrapping replicates. the bootstrapping values are displayed in percentage and only values above are displayed. the phylogenetic tree is divided into separate groups of ugts from a-m plus o. the colored circles represent the cluster groups displayed in fig.  uncolored circles represent genes not expressed in the grain. in the bottom left legend, the number of expressed genes in each cluster group is listed. the triangular shapes on the outside of the phylogenetic tree represent the ugt genes potentially involved in dhurrin and flavonoid metabolism. their specific names are listed in the bottom right legend to narrow down the number of possible gene candidates, the expression data from the transcriptome study were used to cluster the ugt genes according to their expression patterns. of the ugt genes found, were not expressed in the grain in the course of development. the remaining genes were clustered into six distinct cluster groups, which were further subdivided into subgroups. the expression profiles of cluster group and were related and both increased towards the end of grain development. in comparison, cluster group and had expression profiles, which peaked over a broader range from to daa. cluster group and peaked in the early development from to daa.fig. expression profiles of sorghum ugts. all expressed ugts found in the phylogenetic analysis were normalized to z-score and clustered using hierarchical clustering with wards method. the hierarchical clustering was divided into groups, which was further subdivided into subgroups. the lighter color represents the first subgroup and the darker color the last subgroup in a cluster. the number of genes in each cluster is listed in parenthesis next the cluster number. the grey field represents the span of all the expression profiles in a single cluster. the dashed curves represent the concentration of pglcpaac and the three dhurrin glucosides pr. mg tissue in the course of grain development in relation to the expression profiles. genes in cluster plotted in fpkm values of interest to the conversion of the glucosylation of pohpaac. genes from cluster plotted in fpkm values representing good candidates for the formation of the three cndglcs. ugts contained in phylogenetic group g. the colors in b–d refers to the gene names listed next to panel a because pglcpaac reached maximum levels at the early phase of grain development, the gene encoding this ugt was expected to have a high initial expression. cluster group 1– 4– and meet these criteria. only one of the five genes from these cluster groups was expressed throughout development, albeit at a low abundance compared to the earlier described genes. this gene belonged to the group d ugts. the other four all had time points where they were not expressed and were only expressed in low abundance. the dhurrin glucosides accumulated late in the course of grain development, when the dhurrin content was decreasing. genes encoding the potential ugt candidates capable of catalyzing glucosylation of dhurrin were therefore expected to display maximum expression later than the maximum expression time point for ugt85b. genes matching this trend were found in cluster groups 3– 4– and 4–. combined this selection contained genes, of which five were expressed with a maximum abundance above fpkm and belonged to group l, h, e, e and d, respectively. none of the genes expressed at high abundance from the previous two selections belonged to the group g ugts. the genes in group g were of particular interest, because ugt85b involved in the biosynthesis of dhurrin resided in this class. as dhurrin is most likely the substrate for the formation of the dhurrin glucoside, it is possible that a ugt capable of glucosylation of dhurrin would share sequence similarities with ugt85b all genes from this group were therefore displayed together in fig. 10d to reveal ugt gene candidates not discovered via the cluster analysis. of particular interest were three gene candidates expressed at high abundance and correlated with the concentration of the dhurrin glucoside during early development. gene expression profiles of genes involved in the dhurrin catabolism all the expression profiles of the putative gene candidates described in the previous sections have been incorporated with their possible positions within the putative turnover pathways hypothesized by jenrich et al. and pičmanová et al. showing the catabolism of dhurrin. only the most promising gene candidates involved in the catabolism are displayed in fig.  of the gst genes selected as potentially involved in conversion of dhurrin to pohpcn, three of the glutathionylating ones and one deglutathionylating gst have expression values above fpkm, while the two other gst genes and nit4a and nit4b have a maximum abundance above fpkm. in comparison, the ugt gene candidate and the putative nitrilase/amidase genes involved in the second turnover pathway display a lower maximum abundances below fpkm.fig. metabolite accumulation and expression of genes in the two putative dhurrin turnover pathways. the transcript boxes are simplified versions of the earlier figures: gst, gst, nit4a/b, ugt, ugt and nitrilase/amidase. the dhurrin acid and amide can in principle be formed via two different routes: from dhurrin or directly from pohmn, *which then requires an additional glycosylation step in order to get the dhurrin acid and amide transcriptome analysis of the genes involved in the flavonoid pathway to investigate the transcriptional regulation of the biosynthesis genes involved in the relatively unknown flavonoid pathway in sorghum and to support the possible role of pas as defense compounds in the grain, the sorghum genome was blasted with a selection of protein sequences involved in the above metabolic pathways in different plant species. all the matching sorghum genes were aligned with species characteristic amino acid sequences and a phylogenetic analysis conducted for all genes found for each of the individual enzymatic steps. the sorghum genes most closely related to monocot sequences and in particular to maize were extracted from the analysis and their expression profiles plotted in the flavonoid biochemical pathway. the gene encoding the initial enzyme chalcone synthase in the flavonoid metabolism had previously been identified in sorghum. this enzyme converts the two substrates p-coumaroyl-coa and malonyl-coa into naringenin-chalcone. eight isoforms of the enzyme have previously been identified, but several other gene candidates exist in the genome. only two of these identified genes were expressed in the grain in high transcript abundance and both peaked before the second concentration peak in pas between and daa. five sequences were found for the second enzyme, chalcone isomerase which converts naringenin-chalcone to naringenin. one of these genes had been identified earlier. another transcript was expressed in equally high abundance to the identified gene, but this gene resided in the clade with dicotyledons and is a less likely candidate. the b-phenyl ring of naringenin can be hydroxylated at the 3’ or at the 3’- and 5’ positions to produce eriodictyol or dihydrotricetin, respectively. these hydroxylations are performed by flavonoid 3’-hydroxylase and flavonoid 3’,5’-hydroxylase. in sorghum, four putative f3’h and 16 f3’5’h were identified. of the four f3’h genes, had previously been identified in sorghum. none of the 16 f3’5’h genes had previously been described, but sobic.007g and sobic.001g were closely related to maize and barley f3’5’h sequences. two of the f3’h genes might be of importance to the hydroxylation of the flavonoids destined for pas as they peaked in correlation with the pas. for the f3’5’h genes, only the gene sobic.001g displayed an expression profile of interest, but this was not closely related to any of the known monocot f3’5’h sequences. the naringenin, eriodictyol and dihydrotricetin followed the same basic pathway and ended up as the three different catechins, which upon conjugation form the pas. in the following description of the steps leading up to the formation of the catechins, only the one originating from naringenin is mentioned as the enzymes involved were similar. from naringenin, it is possible to form three different compounds. flavone synthase ii converts naringenin to the flavone apigenin. the gene sobic.002g had previously been identified and displayed an increasing abundance of transcript from daa towards maturation. the concentration of flavones has not previously been measured in the grain, but the transcript level co-occurred with the appearance of the grain coat pigmentation, which started around daa and increased towards maturation. a second gene could be of interest because of its relatedness to the maize fnsii gene grmzm2g and shared a similar expression profile to the characterized sorghum fnsii gene. the final transcript, resided in the same monocotyledon clade, and although it was not very closely related to other known transcripts, it shared an expression profile correlated with that of chs and chi. naringenin is further converted to dihydrokaempferol by flavanone 3-hydroxylase. two genes sobic.006g and sobic.006g encoding f3h had earlier been identified in sorghum and in the current study displayed profiles correlated with the pas. dihydrokaempferol can be converted to flavonols by flavonol synthases. two genes were found, but only one was expressed and at a very low abundance. following the pathway of pas, dihydrokaempferol is converted to leucopelargonidin by dihydroflavonol reductase. of the four genes encoding dfr previously identified in sorghum, three were expressed, sobic.003g, sobic.003g and sobic.004g. only the two transcripts dfr and dfr displayed expression profiles correlated with the concentration of pas. the leucopelargonidin produced is further converted to pelargonidin by anthocyanidin synthase. the ans encoding gene had previously been identified in sorghum and is the best candidate due to a high initial abundance. six other putative ans sequences were also found, but they only shared an average amino acid similarity to sbans of 33 ± 1% and were positioned in a separate clade in the monocot clade. pelargonidin can be glucosylated by flavonoid-3-o-glucosyltransferase and flavonoid-5-o-glucosyltransferase to produce anthocyanins. no 3- or 5gt genes had previously been identified in sorghum, but eight putative genes were found in our analysis, with six being expressed in the grain. the two genes sobic.010g and sobic.002g were positioned close to maize and barley 3gt, in the clade containing all other 3gt genes and belonged to group f ugts. only sobic.010g was expressed in the grain and displayed an expression profile that correlated with the pigment formation in the grain coat. two of the other genes were likely 5gts as they group together with other 5gt genes. they both belong to group l ugts and had an increase in transcript abundance towards the end of grain maturation. for the formation of pas, pelargonidin is further converted to -epiafzelechin by anthocyanidin reductase. eleven anr genes were discovered, with eight expressed and four of these having a relevant transcript expression. the epicatechin produced from pelargonidin by anr have a cis orientation of the b-phenyl ring. a second enzyme, the leucoanthocyanidin reductase is capable of producing catechins, which have a trans orientation of the b-phenyl ring. lar enzymes are found in barley and rice, but our phylogenetic analysis did not identify any such genes in sorghum. the last enzymatic step resulting in the formation of pas has still not been elucidated, but laccases have previously been determined to play a role in polymerization of pas in several plant species. in sorghum, lac encoding genes were found of which were not expressed in the grain. of the remaining eight genes, two had high transcript abundances, but only sobic.001g displayed a profile correlated with the pa concentration. discussion dhurrin biosynthesis and accumulation in the developing grain the content of cnglcs within a plant species varies ontogenetically, phenologically, and chronologically. although many exceptions can be found, the concentration of cnglcs is in general highest in seedlings and decreases with plant age. cnglcs may accumulate in any part of a plant and newly formed tissues are typically more cyanogenic compared to older tissues. forage and grain sorghum plants are known to be cyanogenic due to their content of the cnglc dhurrin. the dhurrin content of the mature sorghum grain has been shown to be negligible or zero. in the current study, it was discovered that dhurrin accumulate in the immature grain. the dhurrin content of the husk, which is the maternal tissue component of the grain, was close to zero throughout the entire grain development and thus not correlated with the amount of dhurrin in the grain. from the transcriptome analysis, it is also evident that the genes encoding the enzymes involved in dhurrin biosynthesis in vegetative tissue were expressed in high abundances immediately before the concentration of dhurrin peaks. this substantiated that dhurrin is de novo biosynthesized in the grain from tyrosine via the pathway discovered in etiolated seedlings of sorghum. similar to sorghum, the mature seeds of lotus japonicus, cassava and barley contain no or only negligible amounts of cnglcs. in bitter almonds, the cyanogenic monoglucoside prunasin is biosynthesized in the tegument of the almond fruit throughout development to reach a maximum hcnp of 16 nmol/mg. at the end of maturation, prunasin is transported to the developing cotyledons of the kernel where it is converted and stored as amygdalin, which reaches a maximum hcnp of 89 nmol/mg. in mature seeds of rubber tree, the cotyledon is similarly high in cnglcs with a hcnp of 132 nmol/mg tissue. from these examples, it is clear that the sorghum grain only contains low amounts of cnglcs. earlier research into the biosynthesis of dhurrin concluded that the biosynthesis of dhurrin in the vegetative tissue is largely controlled at the transcriptional level. this earlier finding is confirmed by our results, where large standard deviations in the transcript levels of cyp71e and ugt85b coincided with the large deviations in the dhurrin concentration at day. furthermore, these large standard deviations in the gene transcript abundance was only seen at day and was therefore not attributable to technical variation or a general biological variation observed for every time point during grain development. despite these variations, the ratio between the transcript abundances of cyp79a cyp71e and ugt85b were relatively fixed and showed an incremental increase of transcript from the first to the last enzyme in the biosynthesis. the same tendency was seen in the ratio between proteins and could be a safety feature to ensure that none of the toxic intermediates ) from cyp79a and cyp71e are released during biosynthesis, but converted by the downstream processes to dhurrin. sorghum grain is not cyanogenic cyanogenesis is the classical defense mechanism assigned to cnglcs and is based on their hydrolysis by bgd and hnl resulting in the release of hcn and ketones. the specific bgds hydrolyzing dhurrin are assigned as dhrs in sorghum. the absence of dhr activity in the developing grain was demonstrated by the observed lack of hydrogen cyanide release from grain macerates. furthermore, the lack of an expected quantitative hcn release upon addition of almond bgd must reflect the presence of endogenous inhibitors in the grain such as the high amounts of pas. the missing hydrolysis of dhurrin in grain homogenates was also substantiated by the complete absence of transcripts encoding dhr. although transcripts encoding the enzymes hnl, cas and nit4b were all found to be expressed in the grain, their expression profiles indicate that they are not directly involved in dhurrin turnover in the grain. instead, the expression profile of hnl in relation to the dhurrin concentration curve and the lack of dhrs indicate that it might serve as a scavenger of pohmn, released by chemical degradation of dhurrin during turnover. the expression profiles of cas and nit4b correlate with acc oxidase and indicate that these enzymes are primarily involved in detoxification of hcn from ethylene production and not cyanogenesis. in other cyanogenic plants, the endogenous bgds are extremely active and will rapidly hydrolyze any freely available cnglcs. in the past, this prevented the use of cnglcs as the key substrates in experiments with such extracts aimed at detecting the possible operation of endogenous turnover pathways. the lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study cnglc synthesis as well as endogenous turnover. involvement of two alternative pathways in the turnover of dhurrin the observed disappearance of dhurrin in the course of grain maturation, the absence of bgd activity and the lack of correlation between cas and nit4b expression with the dhurrin disappearance must reflect the operation of one or more dhurrin turnover pathways that do not proceed via hcn release. the operation of such pathways has previously been proposed. interestingly, the expression profiles of the genes encoding the nit4a/nit4b heterodimer indicate that they are involved in a continuous turnover of dhurrin throughout grain development and not only when the concentration of dhurrin decreases at daa. this is supported by the finding that the glucosylated nit4a/nit4b product, pglcpaac, accumulates in the early stages of grain development while the dhurrin content is still increasing. a similar result was presented by adewusi, who showed that dhurrin turnover proceeded in young plants while the total amount of dhurrin was still increasing. although the transcript abundances indicate that the nitrilases might be involved in dhurrin turnover, no traces of pohpcn and pohpaac were found in the grains indicating that they might be transient intermediates. however, the applied lc-ms method does not effectively detect the free pohpaac. it is therefore possible that in the developing grain, this precursor of pglcpaac is either accumulated in its free form instead of being glucosylated or incorporated into other compounds. with the lack of dhr activity and transcript in the sorghum grain, the hypothesis that conversion of dhurrin to the nitrilase substrate pohpcn should be mediated by this enzyme in concert with e.g. a protein co-factor must be rejected. we instead propose the involvement of gsts as supported by hayes et al. and have identified a number of candidate gst genes. the transcriptome data suggested the involvement of both glutathionylating and deglutathionylating gsts in this metabolic transformation. the expression profile of one highly expressed deglutathionylating lambda gst was ranked as the gst most correlated with the expression of nit4a and nit4b and sobic.001g and sobic.001g highly expressed glutathionylating gsts were likewise strongly correlated. these high similarities to the expression patterns of nit4a and nit4b are consistent with their possible involvement in continuous turnover of dhurrin. the phi class gst and the other gsts in cluster group, did not show a favorable expression pattern and had a low similarity to the nit expression profiles and are therefore not as obvious gene candidates for the glutathionylation as the gst mentioned above. future expression of the best gene candidates and characterization of the enzymes will reveal if they are indeed involved in dhurrin turnover. in vegetative tissues, pglcpaac accumulates as the sorghum plants get older and dhurrin is turned over. the time restricted production of this compound in grain, guided identification of potential ugt candidates for the glucosylation of pohpaac into pglcpaac. our transcriptome analyses demonstrated that ugt encoding genes were expressed in the developing grain. in comparison, of the genes encoding ugts in flax are expressed in the developing seeds. from the phylogenetic- and hierarchical clustering analyses only a single ugt gene candidate was found with an expression profile correlated with the concentration of pglcpaac. in a. thaliana, group d ugts is known to glucosylate a range of different aglycones including terpenoids, flavonoids, benzoates and brassinosteroids and it is therefore possible that this sorghum ugt is responsible for production of pglcpaac. besides the pathway just discussed, a second putative turnover pathway involving the dhurrin amide and dhurrin acid probably exists. the formation of these two dhurrin derivatives may be envisioned from two possible precursors, either dhurrin or directly from pohmn. in both scenarios the nitrile-function can be converted into a carboxylic acid group by the direct action of a nitrilase or via the amide by the combined action of a nitrile hydratase and an amidase. interestingly, a nitrile hydratase capable of producing an amide from a nitrile has never been characterized in any plant species, although such enzymatic activities have been measured in several species including sorghum. only the cyano hydratases, which are both structurally and mechanistically different from the nitrile hydratases, have been characterized in plants. in bacteria, the formation of indole-3-acetamide from indole-3-acetonitrile occurs via the action of a nitrile hydratase. a blastp search of the sorghum proteome using known bacterial nitrile hydratase protein sequences gave no significant hits. the bacterial nitrile hydratases are metallo-containing enzymes and thus both structurally and mechanistically different from the nitrilase superfamily enzymes from plants, e.g. nit4a, nit4b and nit4b enzymes in this family are the best candidates for the production of the amide and acid derivatives. only one new gene candidate was found in this family. the abundance of this gene makes it unlikely, as a candidate for the formation of dhurrin acid, but it could however be involved in the formation of the dhurrin amide. similarly, the glutamine-dependent nad synthetase could be involved in the dhurrin amide formation as this type of enzyme hydrolyze amides to produce the corresponding acid and ammonia. the last two low abundance nitrilases belong to the branch nitrilases with unknown functions and has the potential to be involved. for the gene sobic.002g protein homologs with high sequence similarity were found in acyanogenic monocots, whereas the sequence similarity for the genes sobic.008g and sobic.006g were lower. the last two genes are therefore better gene candidates for the formation of the dhurrin amide, but needs to be cloned and expressed before any conclusions can be drawn. all the high abundance genes had a maximum abundance peaking after the dhurrin amide and are therefore probably not involved in its formation, but could still be involved in conversion of this compound into dhurrin acid. however, none of them showed expression profiles correlated with the accumulation of dhurrin acid and protein homologs with high similarities were found in other acyanogenic plants. as sobic.001g has the lowest sequence similarity, this would be the first gene to clone and express for activity. in conclusion, only dhurrin acid accumulated with a profile directly matching the degradation of its presumed precursor dhurrin. this was in contrast to the previous results in studies using vegetative sorghum tissues where the level of pglcpaac continued to increase as dhurrin was turned over. the content of dhurrin acid was not analyzed by jenrich et al., but in the two other cases, the content of this compound also increased with age. our current metabolite analyses indicated that in developing sorghum grain, the turnover pathway leading to pglcpaac formation was dominating during dhurrin accumulation, whereas the pathway producing dhurrin acid was dominating at the later stages. a dual function for dhurrin in the developing grain although the hcnp of the developing sorghum grain is low compared to seeds and fruits of other cyanogenic plants, they still contain a potentially lethal content of dhurrin. reports indicate that 1–3 mg of cyanide per kilogram body mass is lethal to most vertebrates. this means that only 15– grains each containing 1 μg dhurrin could be lethal to a small g bird. this may be the reason that the african bird quelea quelea avoids feeding on developing grains of sorghum. the lack of sorghum bgds can be compensated for by presence of bgds in the saliva and gut of the birds and other herbivores, which would activate the toxicity of dhurrin. this remains a hypothesis, as we were not able to demonstrate a stoichiometric hcn release from dhurrin upon addition of almond bgd to sorghum grain homogenates indicating the presence of inhibitors, which may or may not inhibit the release upon consumption by birds. the deterrent effect of dhurrin may be augmented by the presence of high amounts of pas, which have previously been shown to deter birds and other grain predators. our analysis did not quantify the pa concentration, as no standards were available, but research show that sorghum grains have a very high pa abundance compared to other food plants. in other cyanogenic species, cnglcs and pas may function in a similar trade-off strategy, where they complement or even compensate each other. the chaparral shrub, heteromeles arbutifolia is one such example. in the immature fruit, the pulp accumulates high amounts of pas and cnglcs. once the fruits mature and turn red, a rapid turnover of pas occur in the pulp and the cnglcs are transported to the seed. the fruit is thus safe for birds to eat and the seed will be dispersed and remain protected by cnglcs until germination. in sorghum, it is tempting to speculate that the combined effects of dhurrin and pas protect the more attractive immature sorghum grain while the pas alone are sufficient to protect the mature grain against herbivores. similar to the dhurrin biosynthetic gene transcripts, transcripts involved in the flavonoid pathway peaked prior to the concentration maximum of the pas at daa. few reports are available on the expression of all the flavonoid genes in course of plant ontogeny and of these, the majority only compare the differential expression between tissue types and treatments. in the flowers of cotton, expression of the genes in the flavonoid pathway responsible for the production of anthocyanins correlated with the concentration of anthocyanin, while anr and fls showed different expression profiles. for sorghum, the genes chs, chi, f3’h, fnsii, f3h, dfr, ans have previously been described in other publications, but none of them have been characterized. the research presented here add additional gene candidates to the aforementioned range of enzymes, but also pinpoints some genes which are likely to the primary ones involved in the flavonoid biosynthesis in the developing grain. furthermore, novel gene candidates including the 3gt/5gt, anr and lac were also identified which were previously unknown in sorghum and could help the understanding of the pa biosynthesis. the dramatic turnover of dhurrin in the developing sorghum grain is initiated at the time point where the pa content peaked. at this developmental stage the pas are envisioned to protect the sorghum grain towards herbivory. the function of dhurrin as a defense compound may then be shifted towards a function as a nitrogen resource. this dual function could be a cost-effective measure during the energy demanding grain-filling period, saving the plant from simultaneous production of both defensive compounds and storage proteins. furthermore, the pas do not contain nitrogen and can thus be biosynthesized without the need for substrate containing nitrogen, which is needed for storage protein production. the accumulation of dhurrin means that nitrogen is still being assimilated in the grain, albeit in a form unavailable to herbivores. as the grain matures and becomes less attractive due to lower water content and higher concentrations of pas, the nitrogen in dhurrin can be remobilized as ammonia and utilized to produce storage proteins. in the developing sorghum grain, storage protein in the form of protein bodies first occurs in the endosperm daa and continue to increase in concentration towards maturation. assimilation of nitrogen from the soil in cereals is often insufficient to supply the developing grain and the majority of the nitrogen in the grain originates from leaf tissue remobilization. it is therefore possible that the accumulation and remobilization of dhurrin in the early developing sorghum grain supplies it with a more direct and readily available source of nitrogen for the formation of protein bodies in the later stages. to supply endosperm cells with nitrogen, the dhurrin glucosides could function as a transport form of dhurrin from the site of biosynthesis at the er membranes to the vacuoles for storage. for the glucosylation of dhurrin, eight potential ugt gene candidates were discovered. although the expression profiles of two of the genes correlated with the concentration of dhurrin diglucosides, only in-vitro assays can determine the precise substrate specificity of these enzymes. as three different structural configurations of the cndglcs exist, it is possible that three different ugts also have to be found among the eight gene candidates. in most plants, the formations of the different glycosides are catalyzed by distinct ugts. this is the case for the production of anthocyanins in the flavonoid pathway, where the 5-o and 3-o glycosylations are performed by two different enzymes. to remobilize the nitrogen in dhurrin, the dhurrin glucosides are likely hydrolyzed and metabolized via the turnover pathways described. the results obtained in this study suggest that the pathway producing pohpaac continuously metabolize dhurrin, to potentially act as a nitrogen buffer and supply the growing grain with reduced nitrogen. the metabolite accumulation in the secondary pathway suggests that the dhurrin amide and acid might have different substrate sources. the late accumulation of dhurrin acid indicates that this compound most likely is a turnover product from the degradation of dhurrin. on the contrary, the early accumulation of the dhurrin amide indicates that this might originate directly from pohmn as a result of improper metabolon formation and could function as an additional nitrogen source. when the production of protein bodies starts daa, all available nitrogen sources are remobilized, including the dhurrin amide. as pglcpaac and dhurrin acid have already released their nitrogen, the turnover of these compounds may not be as urgent as the glucose moieties they contain are not a limiting source. they are therefore degraded at a slower rate and only decreases to zero at the time of maturation.  we have used the developing grains of sorghum bicolor moench to study the metabolism of dhurrin. metabolite profiling using lc-ms/ms revealed that dhurrin is accumulated in high amounts during the early phases of grain development but is absent in the mature grain. despite the high amount of dhurrin in the early developmental phase, the grains were acyanogenic and only released minute amounts hydrogen cyanide upon tissue maceration. this result was confirmed by the complete absence of transcripts encoding the dhurrin specific β-glucosidases. although the accumulated dhurrin may still have a secondary protective role and be toxic to potential grain predators via herbivore β-glucosidases, the primary function of dhurrin is probably as a nitrogen store. the loss of the protective functions of dhurrin could be replaced by the high amounts of proanthocyanidins accumulated in the grain via the flavonoid pathway. several new gene candidates for this pathway were discovered in this study, including 3gt/5gt, anr and lac. with the missing activity of the dhurrinases, the turnover of dhurrin occurs via two alternative turnover pathways for which intermediates were confirmed by metabolite profiling. for the turnover of dhurrin via dhurrin amide and dhurrin acid, several novel gene candidates encoding enzymes from the nitrilase super family were discovered. although their expression profiles are not ideal in relation to metabolite accumulation, their possible modes of action still prompt for further investigation. on the contrary, cluster analyses revealed strong candidates for the glucosylation of dhurrin to dhurrin glucoside, glucosylation of pohpaac acid and potential gsts for sequential conversion of dhurrin to pohpcn. by combining all the acquired results, we can conclude that one turnover pathway is likely to be involved in continuous turnover of dhurrin, while the other is primarily involved in turnover at later stages of grain development. the combined effects of these two putative turnover pathways could supply the developing grain with nitrogen to fuel the production of storage protein. 
dhurrin accumulation correlated with the transcript abundance of the three genes involved in biosynthesis. despite the accumulation of dhurrin, the grains were acyanogenic as demonstrated by the lack of hydrogen cyanide release from macerated grain tissue and by the absence of transcripts encoding dhurrinases. dhurrin was found to accumulate in the early phase of grain development reaching maximum amounts 25 days after pollination. the results presented in this article reveal the existence of two endogenous dhurrin turnover pathways in sorghum, identify genes putatively involved in these transformations and show that dhurrin in addition to its insect deterrent properties may serve as a storage form of reduced nitrogen. by combining cluster- and phylogenetic analyses with the metabolite profiling, potential gene candidates of glutathione s-transferases, nitrilases and glycosyl transferases involved in these pathways were identified. cluster- and phylogenetic analyses coupled with metabolite profiling, identified gene candidates involved in proanthocyanidin biosynthesis in sorghum. evidence for the operation of two such pathways was obtained by metabolite profiling and time-resolved transcriptome analysis. sorghumendogenous turnovertranscriptome analysisudp-glycosyltransferaseglutathione s-transferaseproanthocyanidinshttp://dx.doi.org/ /100008398villum fonden00007523møller birger lindberg http://dx.doi.org/ /501100000781european research councilerc-2012-adg_20120314møller birger lindberg ucph excellence programme for interdisciplinary research500657møller birger lindberg issue-copyright-statement© the author 2016 the lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study cnglc synthesis as well as endogenous turnover. during the subsequent maturation period, the dhurrin content was turned over, resulting in only negligible residual dhurrin amounts in the mature grain. with the missing activity of dhurrinases, the decrease in dhurrin content in the course of grain maturation represents the operation of hitherto uncharacterized endogenous dhurrin turnover pathways. in the course of sorghum grain maturation, proanthocyanidins replace dhurrin as a defense compound. in this study, the biosynthesis and endogenous turnover of dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses. the absence of dhurrin in the mature grain was replaced by a high content of proanthocyanidins. crucial for this function is the endogenous turnover of dhurrin for which putative pathways have been suggested but not confirmed.
 the important cereal crop sorghum bicolor moench biosynthesize and accumulate the defensive compound dhurrin during development. previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. crucial for this function is the endogenous turnover of dhurrin for which putative pathways have been suggested but not confirmed.  in this study, the biosynthesis and endogenous turnover of dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses. dhurrin was found to accumulate in the early phase of grain development reaching maximum amounts 25 days after pollination. during the subsequent maturation period, the dhurrin content was turned over, resulting in only negligible residual dhurrin amounts in the mature grain. dhurrin accumulation correlated with the transcript abundance of the three genes involved in biosynthesis. despite the accumulation of dhurrin, the grains were acyanogenic as demonstrated by the lack of hydrogen cyanide release from macerated grain tissue and by the absence of transcripts encoding dhurrinases. with the missing activity of dhurrinases, the decrease in dhurrin content in the course of grain maturation represents the operation of hitherto uncharacterized endogenous dhurrin turnover pathways. evidence for the operation of two such pathways was obtained by metabolite profiling and time-resolved transcriptome analysis. by combining cluster- and phylogenetic analyses with the metabolite profiling, potential gene candidates of glutathione s-transferases, nitrilases and glycosyl transferases involved in these pathways were identified. the absence of dhurrin in the mature grain was replaced by a high content of proanthocyanidins. cluster- and phylogenetic analyses coupled with metabolite profiling, identified gene candidates involved in proanthocyanidin biosynthesis in sorghum.  the results presented in this article reveal the existence of two endogenous dhurrin turnover pathways in sorghum, identify genes putatively involved in these transformations and show that dhurrin in addition to its insect deterrent properties may serve as a storage form of reduced nitrogen. in the course of sorghum grain maturation, proanthocyanidins replace dhurrin as a defense compound. the lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study cnglc synthesis as well as endogenous turnover. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords sorghumendogenous turnovertranscriptome analysisudp-glycosyltransferaseglutathione s-transferaseproanthocyanidinshttp://dx.doi.org/ /100008398villum fonden00007523møller birger lindberg http://dx.doi.org/ /501100000781european research councilerc-2012-adg_20120314møller birger lindberg ucph excellence programme for interdisciplinary research500657møller birger lindberg issue-copyright-statement© the author 2016 