possible chemical formula C 11 H 13 NO 6 (Fig. 2f, see Note 16).
Assaying the standard compound nicotinate D-ribonucleoside
revealed that it shares the same MS/MS fragmentation pattern
as our metabolite (Fig. 2g, see Note 16), albeit with a slight
retention-time shift, suggesting that our metabolite is structurally highly similar to nicotinate d-ribonucleoside.
C. A significant association (LOD ¼ 5.46), only appearing in the
control-condition GWAS, was detected between gamma-Lglutamyl-L-cysteine and a locus on chromosome 5 (Fig. 3a,
see Note 17) harboring two candidate genes: CYDS2 (cysteine
synthase D2, AT5G28020) and DES1 (L-cysteine desulfhydrase
1, AT5G28030). Both correlations between the metabolite
feature and the two genes were supported by network analysis
in darkness-related conditions (Fig. 3b, see Notes 17 and 18).
We confirmed the metabolites as gamma-L-glutamyl-L-cysteine
using isotope labeling (Fig. 3c, see Note 17) and MS/MS
fragmentation analysis with a standard (Fig. 3d, see Note 14).
DES1 was reported to catalyze the desulfuration of cysteine to
sulfide [36], whereas the function of CYSD2 remains elusive to
date. Our results provide genetic evidence that the associations
between gamma-L-glutamyl-L-cysteine and CYDS2 and DES1
are involved in cysteine metabolism (Fig. 3e, see Note 17).
4 Notes
1. Arabidopsis Hapmap population is a collection of
350 A. thaliana accessions. The population was developed
from a global collection of 5810 accessions [19]. This population has been genotyped for 248,584 bi-allelic single-nucleotide polymorphisms (SNPs). The SNPs were reduced to a set of
214,051 SNPs. For GWA analysis, SNPs with a minor allele
frequency (MAF) higher than 0.05 were used for mapping
(Yarive, Si).
2. Segregating populations, such as recombinant inbred lines
(RIL) and introgression lines (IL), are obtained from a cross
(F1) of two parental accessions, through repeated selfing (RIL)
or backcrossing of the initial hybrid with one of its parents
(IL) followed by a selfing until a homozygous state is reached.
Alternate alleles at loci in homozygous lines with distinct
genetic basis in such structured populations potentially influence the trait of interest and allow to map the genomic loci
responsible for the observed intra- or interspecific variation
3. Toxic chemicals should be handled in a fume hood with gloves.
4. In order to minimize the technical sources of variation, several
samples are taken per plant, in addition to multiple independent replicates per parental genotypes; all the samples are
402
Si Wu et al.
Assaying the standard compound nicotinate D-ribonucleoside
revealed that it shares the same MS/MS fragmentation pattern
as our metabolite (Fig. 2g, see Note 16), albeit with a slight
retention-time shift, suggesting that our metabolite is structurally highly similar to nicotinate d-ribonucleoside.
C. A significant association (LOD ¼ 5.46), only appearing in the
control-condition GWAS, was detected between gamma-Lglutamyl-L-cysteine and a locus on chromosome 5 (Fig. 3a,
see Note 17) harboring two candidate genes: CYDS2 (cysteine
synthase D2, AT5G28020) and DES1 (L-cysteine desulfhydrase
1, AT5G28030). Both correlations between the metabolite
feature and the two genes were supported by network analysis
in darkness-related conditions (Fig. 3b, see Notes 17 and 18).
We confirmed the metabolites as gamma-L-glutamyl-L-cysteine
using isotope labeling (Fig. 3c, see Note 17) and MS/MS
fragmentation analysis with a standard (Fig. 3d, see Note 14).
DES1 was reported to catalyze the desulfuration of cysteine to
sulfide [36], whereas the function of CYSD2 remains elusive to
date. Our results provide genetic evidence that the associations
between gamma-L-glutamyl-L-cysteine and CYDS2 and DES1
are involved in cysteine metabolism (Fig. 3e, see Note 17).
4 Notes
1. Arabidopsis Hapmap population is a collection of
350 A. thaliana accessions. The population was developed
from a global collection of 5810 accessions [19]. This population has been genotyped for 248,584 bi-allelic single-nucleotide polymorphisms (SNPs). The SNPs were reduced to a set of
214,051 SNPs. For GWA analysis, SNPs with a minor allele
frequency (MAF) higher than 0.05 were used for mapping
(Yarive, Si).
2. Segregating populations, such as recombinant inbred lines
(RIL) and introgression lines (IL), are obtained from a cross
(F1) of two parental accessions, through repeated selfing (RIL)
or backcrossing of the initial hybrid with one of its parents
(IL) followed by a selfing until a homozygous state is reached.
Alternate alleles at loci in homozygous lines with distinct
genetic basis in such structured populations potentially influence the trait of interest and allow to map the genomic loci
responsible for the observed intra- or interspecific variation
3. Toxic chemicals should be handled in a fume hood with gloves.
4. In order to minimize the technical sources of variation, several
samples are taken per plant, in addition to multiple independent replicates per parental genotypes; all the samples are
402
Si Wu et al.