Abstract
Steviol glycosides (SGs) and gibberellins (GAs) share the same molecular basis. However, the coordination of their respective biosynthetic pathways is very intriguing. Thus, the present study aimed to investigate the role of plant growth regulators, gibberellic acid (GA3), chlorocholine chloride (CCC), and paclobutrazol (PBZ), on the metabolism of Stevia rebaudiana and identify possible ameliorates of the evaluated parameters when CCC and PBZ-treated plants were subsequently treated with GA3. For this, explants were cultured in the absence or presence of 2 mg L−1 GA3, CCC, or PBZ (Step 1). After 20 days, half explants incubated with CCC and PBZ were treated with 2 mg L−1 GA3 and the other half, as well as the rest of the explants, were sub-cultured in their respective initial conditions for 20 days (Step 2). GA3-treated plants showed increased stevioside and phenolic compounds content, as well as a downregulation of most of the SGs/GAs biosynthesis-related genes, with a more pronounced effect upstream of steviol. Following this trend, CCC downregulated some MEP pathway genes, including SrDXS, SrDXR, SrCDPS, and SrKS, and upregulated SrUGT6G1. PBZ also upregulated SrUGT76G1 and inhibited five genes of the MEP pathway and all genes coding for kaurenoid pathway enzymes. The obtained results highlight the capability of GA3 to reverse the negative effects of PBZ on the pattern of many transcripts and to additionally increase the stevioside content to levels comparable to those found in field-grown plants.
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Key message
A feedback loop in in vitro cultured stevia plants in response to GA3, CCC and PBZ was demonstrated. GA3-treated plants showed increases in stevioside and phenolic compound contents.
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Abbreviations
- ANOVA:
-
Analysis of variance
- CCC:
-
Chlorocholine chloride
- CDPS:
-
Copalyl diphosphate synthase
- CMK:
-
4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase
- CMS:
-
4-Diphosphocytidyl-2-C-methyl-D-erythritol synthase
- DMAPP:
-
Dimethylallyl diphosphate
- DXR:
-
Deoxyxylulose-5-phosphate reductase
- DXS:
-
Deoxyxylulose-5-phosphate synthase
- FLA:
-
Total soluble flavonoids
- GAs:
-
Gibberellins
- GA3 :
-
Gibberellic acid
- GA2ox:
-
Gibberellin 2-oxidase
- GA20ox:
-
Gibberellin 20-oxidase
- GA3ox:
-
Gibberellin 3-oxidase
- GGDP:
-
Geranylgeranyl diphosphate
- GGDPS:
-
Geranylgeranyl diphosphate synthase
- HDR:
-
1-Hydroxy-2-methyl-2(E)-butenyl-4-diphosphate reductase
- HDS:
-
1-Hydroxy-2-methyl-2(E)–butenyl-4-diphosphate synthase
- IPP:
-
Isopentenyl diphosphate
- KAH:
-
Kaurenoic acid 13-hydroxylase
- KAO:
-
Kaurenoic acid oxidase
- KO:
-
Kaurene oxidase
- KS:
-
Kaurene synthase
- MCS:
-
4-Diphosphocytidyl-2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase
- MEP:
-
2-C-methyl-D-erythritol-4 phosphate
- MVA:
-
Mevalonate
- PBZ:
-
Paclobutrazol
- PGRs:
-
Plant growth regulators
- Reb-A:
-
Rebaudioside-A
- Reb-D:
-
Rebaudioside-D
- Reb-M:
-
Rebaudioside-M
- SGs:
-
Steviol glycosides
- STEV:
-
Stevioside
- TPC:
-
Total soluble phenol content
- UGT73E1:
-
UDP glucosyltransferase-73E1
- UGT74G1:
-
UDP glucosyltransferase-74G1
- UGT76G1:
-
UDP glucosyltransferase-76G1
- UGT85C2:
-
UDP glucosyltransferase-85C2
- UGT91D2:
-
UDP glucosyltransferase-91D2
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Acknowledgements
The authors gratefully acknowledge the CNPq (Conselho Nacional de Desenvolvimento Cientifíco e Tecnológico) for their financial support and research fellowship EJBB and VJB, as well as the FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul). This study was financed in part by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil—Finance Code 001. This work was partially carried out at IBV (Instituto de Biotecnología Vegetal), UPCT (Spain).
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Conceived and designed the experiments: SRL and EJBB. Performed the experiments: SRL, CM, LA and MNA. Analysed the data: SRL, MNA and EJBB. Wrote the paper: SRL, AAC and EJBB. Corrected the manuscript: MAF, AAC, VJB and EJBB.
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Communicated by Mohammad Faisal.
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Supplementary Information
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11240_2021_2059_MOESM1_ESM.pdf
Supplementary Figure 1 Schematic representation of the experimental conditions. Stevia mother plants grown in greenhouse (A), in vitro establishment (B and C) and multiplication (D) before applying treatments (E) (PDF 108 kb)
11240_2021_2059_MOESM2_ESM.pdf
Supplementary Figure 2 Agarose gel electrophoresis (1%) of PCR products amplified from Stevia rebaudiana under the effect of GA3 and its inhibitors PBZ and CCC after 40 days of in vitro growth. (A and B) Specificity of primer pairs used for RT-qPCR reactions. Lane 1 is 100pb DNA ladder; 2: DXS; 3: DXR; 4: CMS; 5: CMK; 6: MCS; 7: HDS; 8: HDR; 9: GGDPS; 10: CDPS; 11: KS; 12: KO; 13: KAH; 14: UGT85C2; 15: UGT74G1; 16: UGT76G1; 17: UGT73E1; 18: UGT91D2; 19: UBQ and C: Negative control (PDF 413 kb)
11240_2021_2059_MOESM3_ESM.pdf
Supplementary Figure 3 Specificity of the primers used for the RT-qPCR reactions in Stevia rebaudiana under the effect of GA3 and its inhibitors PBZ and CCC after 40 days of in vitro growth (PDF 261 kb)
11240_2021_2059_MOESM4_ESM.pdf
Supplementary Figure 4 Effect of GA3 and its inhibitors PBZ and CCC on growth and development of Stevia rebaudiana after 40 days of in vitro growth (PDF 857 kb)
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Lucho, S.R., do Amaral, M.N., Milech, C. et al. Gibberellin reverses the negative effect of paclobutrazol but not of chlorocholine chloride on the expression of SGs/GAs biosynthesis-related genes and increases the levels of relevant metabolites in Stevia rebaudiana. Plant Cell Tiss Organ Cult 146, 171–184 (2021). https://doi.org/10.1007/s11240-021-02059-6
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DOI: https://doi.org/10.1007/s11240-021-02059-6