Figure S1. Molecular identification of the bio4-1 mutant. (A) Schematic picture of T-DNA insertion in bio4-1. The T-DNA is inserted in the 5′UTR 864 bp upstream of the putative translation start ATG of At5g04620 (genomic DNA and T-DNA not drawn to scale). The arrow indicates the predicted coding sequence of At5g04620. (B) Northern blots of 20 μg total RNA from wild type (WT) and bio4-1 hybridized with the DIG-labeled probes indicated on the left. Membranes were reprobed with a ribosomal probe to ensure equal loading. (C) RT-PCR analysis of At5g04620 expression. Equal amounts of RNA extracted from four-week-old wild-type (WT) and bio4-1 mutant plants were used. The levels of actin2 were assayed as controls. (D) Complementation of bio4-1 by D-biotin addition Seeds were germinated in MS/2 medium. Subsequently one-week-old wild-type (WT) and bio4-1 mutant seedlings were transplanted to MS/2 plates containing different concentrations of D-biotin as indicated and pictures were taken after an additional 8-day growth period. (E) Genetic complementation of bio4-1 with a genomic clone of the BIO4 locus. Complemented plants are bigger than bio4-1 mutants and lack their characteristic cell death regions on leaves. As shown by RNA gel blot analysis, BIO4 expression is restored in the complemented plants. The ethidium bromide-stained rRNA is shown for loading control.

Figure S2. Hormone levels in the bio4-1 mutant. (A) Free salicylic acid  (SA; gray column) and total salicylate (black column). (B) Jasmonate content. (C) Ethylene evolution. Data presented here were from fully expanded leaves of 4-week-old soil-grown plants under a light intensity of 40 µmol m-2 sec-1 with a 12 h photoperiod. The values represent the average of the five replicates ±SE. FW, fresh weight of tissue.

Figure S3. Epistatic analysis of lesion formation and chlorophyll content in A. thaliana leaves. (A) Representative Col-0, bio4-1, NahG, bio4-1/nahG, npr1-1, bio4-1/npr1-1, coi1-1, bio4-1/coi1-1 plants were grown for three weeks under soil growth conditions. Red arrows indicate visible cell death area. Inlets show DAB staining indicating H2O2 precipitation. (B) Total content of chlorophyll. Chlorophyll was extracted from five-week old wild-type, bio4-1, npr1-1, bio4-1 /npr1-1, NahG, and bio4-1 /NahG plants. The values are mean of three replicates ±SE. The experiments were repeated once with similar results. FW, fresh weight of tissue.

Table S1. Significantly upregulated genes (P < 0.05; 2fold) in bio4-1 compared to wt. Upper part (A) shows probe sets also significantly upregulated in bio4-1/bio4-1 complemented with D-biotin. Lower part (B) lists probe sets only significantly and 2 fold upregulated in bio4-1 compared to wt.

Table S2. Significantly downregulated genes (P < 0.05; 2-fold) in bio4-1 compared to wt. Upper part (A) shows probe sets also significantly downregulated in bio4-1/bio4-1 complemented with D-biotin. Lower part (B) lists probe sets only significantly and 2-fold downregulated in bio4-1 compared to wt.

Table S3. Overrepresented Gene-Ontology (GO) attributes of biotin dependent genes using the GO Term Enrichment tool. (A) Overrepresented Attributes (P < 0.01) in upregulated genes (bio4-1/wt corrected by bio4-1/bio4-1 plus biotin) (B) Overrepresented Attributes (P < 0.01) in downregulated genes (bio4-1/wt corrected by bio4-1/bio4-1 plus biotin)

Appendix S1. Supplemental Experimental Procedures. Experimental procedures used for genetic analysis, hormone and chlorophylle quantification, protein extraction and western blot analysis.

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