Figure 1.
Investigation of IPMI SSU transcript levels.
(A) Semi-quantitative RT-PCR analysis of Col-0 wild type and three different lines (#9, #11 and #25) expressing amiR-SSU1-B. This analysis was done with three technical and two biological replicates. Analyzed transcripts are indicated on the right hand side of the image. The input of equal amounts of cDNA was tested with a PCR detecting UBC9 transcripts. Relative levels of the PCR products were normalized to UBC9 mRNAs and are given below the images with respect to wild type. (B) Real Time qRT-PCR of IPMI SSU1 mRNA levels in the same knockdown lines.
Figure 2.
Macroscopic characterization of amiR-SSU1-B knockdown plants.
In images (A) to (C) and (E) to (K) Col-0 wild type is shown on the left side while amiR-SSU1-B line #9 is on the right side. In image (D) Col-0 wild type is shown in the upper part, whereas amiR-SSU1-B line #9 is shown in the lower part. (A) to (C) show 8, 21 and 35 day-old plants, respectively. (D) Rosette leaves of 35 day-old plants. (E) Primary inflorescences, (F) flowers, (G) petals, (H) sepals, (I) stamen, (J) and gynecium of 42 day-old plants. (K) shows mature siliques. Thick scale bars correspond to 1 cm, thin scale bars to 0.1 cm.
Figure 3.
Microscopic examination of amiR-SSU1-B leaves.
Images show cross sections through leaves of Col-0 wild type (A, C, E) and amiR-SSU1-B line #9 (B, D, F). Scale bars correspond to 20 µm (A to D), or 2 µm (E and F).
Figure 4.
Supplementation experiments with branched-chain amino acids.
(A to C) Col-0 wild-type (left parts of the images) and amiR-SSU1-B seedlings (line #9, right hand parts) were germinated and grown for 10 days on MS medium without exogenous amino acids (A, mock), in the presence of 0.2 mM Leu (B) and in the presence of 0.2 mM BCAA (C). (D to F) amiR-SSU1-B plants (line #9) were grown for 35 days on soil. Plants were watered without amino acids (D), with 2 mM Leu (E) or 2 mM BCAA (F).
Table 1.
Amino acid content in rosette leaves of amiR-SSU1-B plants.
Table 2.
Glucosinolate profile of rosette leaves of amiR-SSU1-B plants.
Table 3.
Diverse metabolites in rosette leaves of amiR-SSU1-B plants.
Table 4.
Phytohormone content in rosette leaves of amiR-SSU1-B and Col-0.
Table 5.
Accumulation of isopropylmalate and glucosinolate intermediates in different sections of rosette leaves of ipmi ssu2-1/ipmi ssu3-1 and amiR-SSU1-B knockdown plants.
Figure 5.
Semi-quantitative RT-PCR analysis of the IPMI SSU and IPMI LSU1 mRNA levels.
The transcripts of these genes were examined in different parts of the Col-0 wild-type leaves. Analyzed transcripts are indicated on the right hand side of the image. The use of equal amounts of cDNA was tested with UBC9 transcripts. This analysis was done with three technical and three biological replicates. Relative levels of the PCR products are given below the images with respect to levels in whole leaves.
Figure 6.
Histochemical GUS staining of plant tissues expressing the β-glucuronidase gene.
The glucuronidase gene was expressed under the control of the IPMI SSU1 to 3 promoters (indicated in the top). (A and B) 8 and 15 day-old seedlings; (C) leaves, (D) sections and cross section (positions indicated by white dashed lines) of the inflorescence axis. White arrows indicate staining at the basis of branches of cauline leaves. (E) flowers, arrows indicate staining in the anthers, which are enlarged in the insets.
Figure 7.
Complementation analysis of Leu-auxotrophic E. coli strains.
Complementation capabilities of the different IPMI subunits were tested with the Leu-auxotrophic E. coli strains CV524 (ΔleuD, A, D) and CV522 (ΔleuC, B, C, E, F) on minimal medium with 1 mM IPTG and 2 mM Leu (D, E, F) at 37°C. The auxotrophic strains were transformed either with empty pUC19, leuC-leuD, or the different combinations of IPMI subunits leuC-SSU1/2/3, leuC*/LSU/LSU*-leuD or LSU*-SSU1/2/3. Untransformed bacteria were used as controls CV524 (ΔleuD) and CV522 (ΔleuC).