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pce12147-sup-0001-fs1.pdf269K

Figure S1. Alignment of RPA1 subunits from Arabidopsis thaliana (At), Oryza sativa (Os), Medicago truncatula (Mt), Ricinus communis (Rc) and Glycine max (Gm) used for phylogenetic analysis. Alignment is based on the amino acid sequence of each predicted protein. Predicted pseudogenes from soybean were excluded from the analysis. Sequences were aligned using Pileup in GCG (Accelrys Inc., San Diego, CA, USA). Extraneous sequence was trimmed to include the most conserved regions.

pce12147-sup-0002-fs2.pdf30K

Figure S2. Alignment of RPA2 subunits from Arabidopsis thaliana, Oryza sativa, Medicago truncatula, Ricinus communis and Glycine max used for phylogenetic analysis. Alignment is based on the amino acid sequence of each predicted protein. Sequences were aligned using Pileup in GCG (Accelrys Inc., San Diego, CA, USA). Extraneous sequence was trimmed to include the most conserved regions.

pce12147-sup-0003-fs3.pdf25K

Figure S3. Alignment of RPA3 subunits Arabidopsis thaliana (At), Oryza sativa (Os), Medicago truncatula (Mt), Ricinus communis (Rc), and Glycine max (Gm) used for phylogenetic analysis. Alignment is based on the amino acid sequence of each predicted protein. Sequences were aligned using Pileup in GCG (Accelrys Inc., San Diego, CA, USA). Extraneous sequence was trimmed to include the most conserved regions.

pce12147-sup-0004-fs4.pdf471K

Figure S4. RPA homologs are broadly expressed during soybean development. To examine the expression patterns of RPA homologs, we took advantage of the soybean RNA-Seq atlases described by Severin et al. (2010a, http://soybase.org/soyseq/) and Libault et al. (2010a, 2010b, http://soykb.org/). The Glyma identifiers of all eighteen soybean RPA homologs were used as queries; however, only 13 RPAs were expressed. A. Expression of RPA homologs in the Severin et al. (2010a) atlas, focused largely on above ground tissues. B. Expression of RPA homologs in the Libault et al. (2010a,b) atlases focused largely on below ground tissues. Genes are coded the same colour in each panel.

pce12147-sup-0005-fs5.pdf94K

Figure S5. VIGS constructs GmRPA3S and GmRPA3AS reduce expression GmRPA3c and GmRPA3d relative to vector only controls. To confirm silencing of GmRPA3c and GmRPA3d, gene-specific primers specific to the 3′ UTR of each gene were developed for qPCR of VIGS plants 21 days after VIGS treatment. RNA was pooled from three biological replicates of vector, GmRPA3AS and GmRPA3S treated Isoclark plants grown in iron sufficient and deficient conditions, 21 days after treatment. Replicates from different iron conditions were combined to give six replicates per vector. Silencing vector GmRPA3AS and GmRPA3S reduced GmRPA3c expression by an average of 12.0 and 7.3 fold, respectively. In contrast, GmRPA3d expression was reduced 3.3 and 2.0 fold, respectively. Each data point is the average of six replicates ± standard error. Statistically significant reduction in expression relative to vector controls is indicated by an asterisk (P < 0.05).

pce12147-sup-0006-ts1.xlsx44K

Table S1. Primer sequences for GmRPA homologs. RT-PCR primers were designed for all RPA homologs using the program Primer 3 (Rozen & Skaletsky 2000). Primers were designed using the Primer 3 defaults, specifying an amplicon size (125–175 bp). Primers were designed based on coding sequences of RPA homologs (http://www.phytozome.net, Table 1). RPA coding sequences were compared to each other using BLASTN (Altschul et al. 1997, E < 10E-30), and only unique sequences were used in primer design in order to distinguish between homeologs located in duplicated genomic regions. Primers were tested on Clark and Isoclark total RNA harvested from an iron-insufficient bucket at 14 days post iron stress.

pce12147-sup-0007-ts2.xlsx58K

Table S2. Relative gene expression values of GmRPA homologs over three time points in iron efficient line Clark. Relative gene expression was determined by qPCR of the gene of interest in iron sufficient or deficient conditions. Relative gene expression values are presented as a ratio to the value at the same time point in iron sufficient conditions, averaged over three biological replicates, then log2 transformed. Values above zero indicate greater expression in iron insufficient conditions, while values below zero indicate lesser expression in iron insufficient conditions. Log-transformed data was analysed for standard deviation and standard error. Differences in relative quantity were analysed with anova (Chambers et al. 1992) and then Tukey's Honestly Significant Difference test (Yandell 1997) for pairwise comparisons, with a significance cut-off of 0.05. For fold change comparisons relative to 1 hps, standard error was calculated using the equation SEFC = SQRT ((SD1 + SD2)/N) (SEFC = Standard error of fold change comparison relative to time point 1, SD1 is the standard deviation of time point 1, SD2 is the standard deviation of time point 2 and N is the total number of samples compared.

pce12147-sup-0008-ts3.xlsx60K

Table S3. Relative gene expression values of GmRPA homologs over three time points in iron inefficient line Isoclark. Relative gene expression was determined by qPCR of the gene of interest in iron sufficient or deficient conditions. Relative gene expression values are presented as a ratio to the value at the same time point in iron sufficient conditions, averaged over three biological replicates, then log transformed (base 2). Values above zero indicate greater expression in iron insufficient conditions, while values below zero indicate lesser expression in iron insufficient conditions. Log-transformed data were analysed for standard deviation and standard error. Differences in relative quantity were analysed with anova (Chambers et al. 1992) and then Tukey's Honestly Significant Difference test (Yandell 1997) for pairwise comparisons, with a significance cut-off of 0.05. For fold change comparisons relative to 1 hps, standard error was calculated using the equation SEFC = SQRT ((SD1 + SD2)/N) (SEFC = Standard error of fold change comparison relative to time point 1, SD1 is the standard deviation of time point 1, SD2 is the standard deviation of time point 2 and N is the total number of samples compared.

pce12147-sup-0009-ts4.xlsx47K

Table S4. Identification of significantly overrepresented transcription factor binding sites in the promoters of differentially expressed RPA homologs. Clover (Frith et al. 2004) was used in conjunction with the TRANSFAC transcription factor database (Matys et al. 2006) to identify transcription factor binding sites overrepresented in the promoters of the differentially expressed RPA homologs when compared to all promoters in the soybean genome. Analysis was limited to the plant transcription factors present in TRANSFAC. Promoter sequences were defined as 1000 base pairs upstream of the transcription start site.

pce12147-sup-0010-ts5.xlsx27K

Table S5. Genes involved in DNA replication are repressed in response to iron stress in the iron efficient line Clark. To determine the role of RPA during iron deficiency, we examined the expression of soybean homologs of known Arabidopsis replication and repair proteins under iron deficient conditions. Best reciprocal BLASTP was used to identify soybean proteins with homology to known DNA replication (Shultz et al. 2007) or repair (Singh et al. 2010) proteins. Identified soybean proteins were then queried against the O'Rourke et al. (2009) and Peiffer et al. (2012) data sets.

pce12147-sup-0011-ts6.xlsx455K

Table S6. Annotation of genes significantly (FDR < 0.05) differentially expressed between GmRPA3c silenced and empty vector treated plants.

pce12147-sup-0012-ts7.xlsx62K

Table S7. Genes significantly differentially expressed in response to iron deficiency in empty vector treated plants (FDR < 0.1), but not in GmRPA3 silenced plants (FDR > 0.1).

pce12147-sup-0013-ts8.xlsx45K

Table S8. Genes significantly differentially expressed in response to iron deficiency in GmRPA3 silenced plants (FDR <0.1), but not in empty vector treated plants (FDR > 0.1).

pce12147-sup-0014-ts9.xlsx55K

Table S9. GO terms significantly overrepresented among genes induced by GmRPA3 silencing. Overrepresented GO terms were identified using the Ontologizer 2.0 (Bauer et al. 2008) software using the Parent-Child-Union and Westfall-Young-Single-Step Corrections and 1000 replicates. All identified GO terms are indicated.

pce12147-sup-0015-ts10.xlsx56K

Table S10. GO terms significantly overrepresented among genes repressed by GmRPA3 silencing. Overrepresented GO terms were identified using the Ontologizer 2.0 software (Bauer et al. 2008) using the Parent-Child-Union and Westfall-Young-Single-Step Corrections and 1000 replicates. All identified GO terms are indicated.

pce12147-sup-0016-ts11.xlsx77K

Table S11. Annotation of transcription factors significantly (FDR < 0.05) differentially expressed between GmRPA3c silenced and empty vector treated plants.

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