Supplemental Data. Benstein et al. (2013). Plant Cell /tpc

Transcription

1 Supplemental Figure 1. Purification of the heterologously expressed PGDH1, PGDH2 and PGDH3 enzymes by Ni-NTA affinity chromatography. Protein extracts (2 µl) of different fractions (lane 1 = total extract, lane 2 = flowthrough, lane 3 = wash fraction 1, lane 4 = wash fraction 2, lanes 5 8 = eluate fraction) were separated on SDS-polyacrylamide gels and stained either by Coomassie protein stain or blotted and detected by western blot analysis using anti- His antibodies.

2 Supplemental Figure 2. Biochemical characterization of PGDH isoenzymes. K m values of PGDH1, PGDH2 and PGDH3 for 3-PGA (left panel) and NAD + (right panel) were determined by non-linear regression based on the Michaelis-Menten equation. Measurements were performed at two different ph conditions; ph 8.1 (closed squares) and ph 7.2 (open squares). Data represent the mean of seven to ten independent measurements, Error bars indicate the SD.

3 Supplemental Figure 3. Phenotype of pgdh2 1 and pgdh2 2 homozygous knockout mutants. (A) Shoot phenotype of Col-0, and the pgdh2 1 and pgdh2 2 mutants grown under standard greenhouse conditions. (B C) Two allelic T-DNA insertion lines for the PGDH2 gene were isolated by PCR using gene-specific (pgdh2 1: 480 and 479; pgdh2 2: 478 and 479) and T- DNA-specific primers (538). For both allelic mutants, the T-DNA insertion was localized in the second intron of the PGDH2 gene.

4 (D) RT-PCR to determine residual transcript of PGDH2 gene in pgdh2 1 and pgdh2 2 mutants. Equal amounts of total RNA isolated from Col-0, pgdh2-1 and pgdh2-2 were used for cdna synthesis. Resulting cdna was used as template in a standard 28-cycle PCR with primers 480 and 479 for PGDH2 and 525 and 526 for Actin. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. Actin was used as a positive control.

5 Supplemental Figure 4. Biochemical characterization of pgdh2 1 and pgdh2 2 mutants. (A) Total PGDH activity determined in Col-0, pgdh2 1 and pgdh2 2 mutants. Bars represent the mean of four independent measurements, error bars indicate the SD. (B) Root length in cm of Col-0, and pgdh2 1 and pgdh2 2 mutants. Bars represent the mean of twenty four independent measurements, error bars indicate the SD. (C) Content of major and minor abundant amino acids in leaves of Col-0, and pgdh2 1 and pgdh2 2 mutants. Bars represent the mean of four independent measurements, error bars indicate the SD. (D) Content of major and minor abundant amino acids in roots of Col-0, and pgdh2 1 and pgdh2 2 mutants. Bars represent the mean of four independent measurements, error bars indicate the SD.

6 Supplemental Figure 5. Embryo-lethal phenotype of psp 1 and psp 2 mutants. (A) Two allelic T-DNA insertion lines for the PSP gene were isolated by PCR using gene-specific (psp 1: 430 and 411; psp 2: 532 and 533) and a T-DNA-specific primer (psp 1: 538; psp 2: 473). In mutants, the T-DNA insertion was either localized in the fourth intron (psp 1) or in the promoter region (psp 2) of the PSP gene. (B) Seed establishment in mature siliques of psp 1/+ and psp 2/+. Asterisks indicate the position of bright seeds among green seeds within siliques of heterozygous lines. (C) Bright and green seeds were extracted from siliques of heterozygous mutant lines, cleared and analyzed by differential interference contrast microscopy. Arrows mark the position of the embryo within in the seed.

7 (D) Embryos of bright (b) and green (g) seeds from heterozygous lines psp 1 were extracted for the isolation of genomic DNA. Embryo zygosity was determined by PCR using gene-specific and T-DNA-specific primers as described above.

8 Supplemental Figure 6. Phenotype of PGDH1-silenced (#133.7, #133.15) and EV control plants. (A) Germination and seedling establishment of control and PGDH1 silenced plants on ½ strength MS plates without sucrose in the presence or absence of 100 µm serine

9 (B) Growth of control and PGDH1 silenced plants on vertical ½ strength MS plates without sucrose in the absence and presence of either 10 µm tryptophan or 10 µm serine (C) Phenotype of soil-grown ProDR5:GUS lines transformed with Pro35S:miRNA173_PGDH1 construct (#139) compared to the nontransformed ProDR5:GUS marker line (DR5). Plants were carefully removed from soil by immersion in water before image capture. Root systems appear dark because of adhering soil particles.

10 Supplemental Data. Benstein et al. (2013). Plant Cell /tpc Supplemental Figure 7. Histochemical staining of GUS activity in germinating seedlings expressing ProPGDH1:GUS, ProPGDH2:GUS and ProPGDH3:GUS constructs. Seeds were germinated on plates containing ½ MS medium without sucrose and GUS activity was analyzed in seedlings, two (A, F, K), three (B, G, L), four (C, H, M), six (D, I, N) and ten (E, J, O) days after imbibition (d.a.i.).

11 Supplemental Figure 8. Biotic stress induces PS pathway gene expression. (A) Expression data obtained from the genvestigator database. (B) GUS activity in plants expressing the uida gene under the control of the respective PS pathway promoters were infected with Botrytis cinera spores. Five days after infection GUS activity was analyzed by staining. The experiments were repeated twice. Arrows indicate either the site of infection or mock treatment.

12 Supplemental Figure 9. Metabolic alterations in roots of PGDH1-silenced plants and control. Alteration in amino acid concentration in transgenic lines (#133.7, #133.15) and EV control plants. Bars represent the means of six to eight replicates, and error bars indicate the SD. Significant differences between control and transgenic lines are indicated by asterisks (**P 0.05; *P 0.1).

13 Supplemental Figure 10. Histochemical staining of GUS activity in plants expressing ProPSAT1:GUS, ProPSAT2:GUS and ProPSP:GUS constructs. (a, i, q) Mature rosette (b, j, r) Mature rosette leaf (c, k, s) Flowers (d, l, t) Seedlings three days after imbibition (e, m, u) Root (f, n, v) Root section at the branch side (g, o, w) Root tip (h, p, x) Embryo

14 Supplemental Table 1. List of genes co-expressed with PGDH1 using the ATTED database. The Mutual Rank (MR) describes a measure for gene co-expression derived from the geometric average of the Pearson Correlation Coefficient (PCC) rank. Increasing numbers of MR indicate decreasing probability of co-expression. For detailed information see

15 Supplemental Table 2. List of primers used in this study.