Figure 1.
Schematic view of plastidic methylerythritol 4-phosphate (MEP) pathway providing the precursors for secologanin (monoterpene) synthesis.
Enzymes analyzed in the present study are in bold face (DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, deoxyxylulose 5-phosphate reductoisomerase; HDS, hydroxymethylbutenyl diphosphate synthase). GAP, glyceraldehyde 3-phosphate; DXP, deoxyxylulose 5-phosphate; MEP, methylerythritol 4-phosphate; MEcPP, methylerythritol 2,4-cyclodiphosphate; HMBPP, hydroxymethylbutenyl diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; GGPP, geranylgeranyl diphosphate. ABA, abscisic acid. The step branching to thiamine from DXP is indicated. Furthermore, inhibition of DXS and DXR by 5-ketoclomazone (formed in planta from clomazone) and fosmidomycin, respectively, is highlighted. Dashed arrows indicate multiple steps.
Figure 2.
Tissue-specific expression of MEP pathway genes in young leaves, mature leaves and roots of 6-week-old non-flowering C. roseus plants, at protein and transcript level, respectively.
Soil-grown periwinkle (Catharanthus roseus) plants were cultivated in a growth chamber at 25°C with 14 hrs light period (170 µmol m−2 s−1) and 22°C during dark period. (A) DXS, DXR, and HDS proteins were detected by immunoblot with polyclonal antisera raised against recombinant CrDXS2A, DXR and HDS proteins. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein. (B) transcript amounts for DXS isoforms (1, 2A & 2B), DXR, and HDS were determined by qPCR (the experiment was performed 3 times, values from a representative experiment are presented ± SD), relative to the geometric mean of multiple reference genes according to Vandesompele et al. [60].
Figure 3.
Effect of paraquat treatment on the expression of MEP pathway genes at protein and transcript level, respectively, in leaf discs obtained from mature leaves of 6-week-old C. roseus plants.
Leaf discs (5 mm diameter) were collected from fully expanded leaves and floated on distilled water in the presence or absence of 0.5 µM paraquat for the indicated time intervals at 25°C under continuous light (270 µmol m−2 s−1). (A) DXS, DXR and HDS proteins were detected by immunoblot. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein.(B) transcript amounts for DXS (isoforms 1, 2A & 2B), DXR and HDS were determined by qPCR relative to the geometric mean of multiple reference genes according to Vandesompele et al. [60]. The experiment was performed 3 times; values from a representative experiment are presented ± SD.
Figure 4.
Activation of DXS isoforms' promoters (CrDXS1p, CrDXS2Ap and CrDXS2Bp) by the transcription factor ORCA3, analyzed via transient expression of promoter-luciferase fusions in leaves of 6-week old C. roseus plants.
Results are expressed as fold change of promoter activation, based on four independent assays. The promoter activity was determined by the ratio of luciferase (LUC) activity to renilla expression. Dual CaMV 35S promoter was employed as a positive control showing high LUC activity, while promoterless LUC control (pGreenII 0800-LUC vector) reflected the LUC background value. DXS promoter sequences were fused to the firefly luciferase and transiently co-transformed with ORCA3 or without (mock control). Agrobacteria-mediated transient transformation was carried via leaf infiltration of 6-week-old soil-grown C. roseus plants. Two days after transformation, infiltrated leaves were harvested and subjected to dual-luciferase reporter assay. Error bars represent the standard deviation of four independent experiments for promoter activity analysis. The asterisks represent significant difference (** p<0.001, * p<0.1 by student's t-test).
Figure 5.
Time course of in vivo clomazone treatment on the expression of MEP pathway genes, and subsequent degradation of DXS and DXR proteins in isolated chloroplasts.
For each plant, two pairs of mature leaves were injected with a 50 µM clomazone solution (or water for control) until the entire leaf blades were fully soaked, using a 1 ml needleless syringe applied to the lower epidermis. For each time point, young leaves were pooled from three independent plants and processed for MEP pathway protein and transcript analysis, respectively. (A) DXS, DXR and HDS proteins were detected by immunoblot. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein. (B) transcript amounts for DXS isoforms (1, 2A & 2B), DXR and HDS were determined by qPCR relative to the geometric mean of multiple reference genes according to Vandesompele et al.[60]. The experiment was performed 3 times, values from a representative experiment are presented ± SD. (C) chloroplasts were isolated from young leaves of 6-week-old soil-grown C. rosues control and 50 µM clomazone-treated plants (see (A) this Figure) 78 hours after treatment. Chloroplasts were incubated for 1 h in the light (100 µmol m−2 s−1) at 25°C in the presence of 5 mM ATP. Aliquots were taken at 0, 15, 30 and 60 minutes and used for protein extraction. DXS and DXR proteins were detected by immunoblot. Note that to obtain similar signal intensity at time point 0, the loading amount of protein from control samples (chloroplast isolated from water infiltrated plants) was twice that of clomazone samples.
Figure 6.
Time course of in vivo fosmidomycin treatment on the expression of MEP pathway genes in young leaves of 6-week-old C. roseus plants at protein and transcript level, respectively.
For each plant, two pairs of mature leaves were injected with a 50 µM fosmidomycin solution. Application procedure and time course of sampling was as described for clomazone treatment (see Figure 5). (A) DXS, DXR and HDS proteins were detected by immunoblot. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein. (B), transcript amounts for DXS isoforms (1, 2A & 2B), DXR, and HDS were determined by qPCR, relative to the geometric mean of multiple reference genes according to Vandesompele et al. [60]. The experiment was performed 3 times, values from a representative experiment are presented ± SD.
Figure 7.
Effect of fosmidomycin on the expression of DXS protein and DXS isoform (1, 3) transcripts in 8-day-old seedlings of Arabidopsis thaliana.
A. thaliana Ler seedlings were cultivated for 8 days at 22°C in petri dishes on filter paper as described by Guevara-Garcia et al. [23]. Light period was 16 hrs (230 µmol m−2 s−1). Thereafter, 5 ml of a 100 µM fosmidomycin solution (or water as control) were added. After the indicated time intervals, seedlings were harvested and processed for MEP pathway protein and transcript analysis, respectively. (A) DXS, DXR, and HDS proteins were detected by immunoblot with polyclonal antisera raised against recombinant C. roseus DXS2A, DXR and HDS proteins. Note that exposure time was five times longer for Arabidopsis samples than for C. roseus samples. (B) transcript amounts for DXS isoforms (1& 3) were determined by qPCR. Relative transcript levels were calculated by normalizing to actin as reference gene. Means of triplicate values from a representative experiment are presented ± SD.
Figure 8.
Time course of in vivo cycloheximide treatment as compared to simultaneous treatment with fosmidomycin or clomazone, respectively, and subsequent turnover of DXS and DXR proteins in isolated chloroplasts.
(A) for each plant, two pairs of mature leaves were injected with 100 µM cycloheximide solution alone, or in combination with 50 µM fosmidomycin or 50 µM clomazone, respectively. For each time point, young leaves were collected from three independent plants and processed for immunoblot analysis of DXS, DXR and HDS, respectively. (B) chloroplasts from young leaves of 6-week-old soil-grown C. rosues plants, treated with 50 µM fosmidomycin, 50 µM clomazone, or water (control) were isolated 78 hours after leaf injection (see (A) this Fig.). Chloroplasts were incubated for 1 h in the light (100 µmol m−2 s−1) at 25°C in the presence of 5 mM ATP and fosmidomycin or clomazone (50 µM). Aliquots were taken at 0, 15, 30 and 60 minutes and used for protein extraction. DXS and DXR proteins were detected by immunoblot. To obtain similar signal intensity at time point 0, the loading amount of protein from control samples (chloroplast isolated from water infiltrated plants) was twice that of fosmidomycin or clomazone samples (see also Fig. 5C).