Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism
Abstract Background Better understanding of the physiological and metabolic status of plants can only be obtained when metabolic fluxes are accurately assessed in a growing plant. Steady state 13C-MFA has been established as a routine method for analysis of fluxes in plant primary metabolism. Howeve...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2019-05-01
|
| Series: | Plant Methods |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13007-019-0434-8 |
| _version_ | 1819047345468211200 |
|---|---|
| author | Somnath Koley Manish L. Raorane Björn H. Junker |
| author_facet | Somnath Koley Manish L. Raorane Björn H. Junker |
| author_sort | Somnath Koley |
| collection | DOAJ |
| description | Abstract Background Better understanding of the physiological and metabolic status of plants can only be obtained when metabolic fluxes are accurately assessed in a growing plant. Steady state 13C-MFA has been established as a routine method for analysis of fluxes in plant primary metabolism. However, the experimental system needs to be improved for continuous carbon enrichment from labelled sugars into metabolites for longer periods until complex secondary metabolism reaches steady state. Results We developed an in vitro plant culture strategy by using peppermint as a model plant with minimizing unlabelled carbon fixation where growing shoot tip was strongly dependent on labelled glucose for their carbon necessity. We optimized the light condition and detected the satisfactory phenotypical growth under the lower light intensity. Total volatile terpenes were also highest at the same light. Analysis of label incorporation into pulegone monoterpene after continuous U-13C6 glucose feeding revealed nearly 100% 13C, even at 15 days after first leaf visibility (DALV). Label enrichment was gradually scrambled with increasing light intensity and leaf age. This study was validated by showing similar levels of label enrichment in proteinogenic amino acids. The efficiency of this method was also verified in oregano. Conclusions Our shoot tip culture depicted a method in achieving long term, stable and a high percentage of label accumulation in secondary metabolites within a fully functional growing plant system. It recommends the potential application for the investigations of various facets of plant metabolism by steady state 13C-MFA. The system also provides a greater potential to study sink leaf metabolism. Overall, we propose a system to accurately describe complex metabolic phenotypes in a growing plant. |
| first_indexed | 2024-12-21T10:58:53Z |
| format | Article |
| id | doaj.art-0d9dc1ff90c14d59911c2331694fa3da |
| institution | Directory Open Access Journal |
| issn | 1746-4811 |
| language | English |
| last_indexed | 2024-12-21T10:58:53Z |
| publishDate | 2019-05-01 |
| publisher | BMC |
| record_format | Article |
| series | Plant Methods |
| spelling | doaj.art-0d9dc1ff90c14d59911c2331694fa3da2022-12-21T19:06:24ZengBMCPlant Methods1746-48112019-05-0115111510.1186/s13007-019-0434-8Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolismSomnath Koley0Manish L. Raorane1Björn H. Junker2Institute of Pharmacy, Martin Luther UniversityInstitute of Pharmacy, Martin Luther UniversityInstitute of Pharmacy, Martin Luther UniversityAbstract Background Better understanding of the physiological and metabolic status of plants can only be obtained when metabolic fluxes are accurately assessed in a growing plant. Steady state 13C-MFA has been established as a routine method for analysis of fluxes in plant primary metabolism. However, the experimental system needs to be improved for continuous carbon enrichment from labelled sugars into metabolites for longer periods until complex secondary metabolism reaches steady state. Results We developed an in vitro plant culture strategy by using peppermint as a model plant with minimizing unlabelled carbon fixation where growing shoot tip was strongly dependent on labelled glucose for their carbon necessity. We optimized the light condition and detected the satisfactory phenotypical growth under the lower light intensity. Total volatile terpenes were also highest at the same light. Analysis of label incorporation into pulegone monoterpene after continuous U-13C6 glucose feeding revealed nearly 100% 13C, even at 15 days after first leaf visibility (DALV). Label enrichment was gradually scrambled with increasing light intensity and leaf age. This study was validated by showing similar levels of label enrichment in proteinogenic amino acids. The efficiency of this method was also verified in oregano. Conclusions Our shoot tip culture depicted a method in achieving long term, stable and a high percentage of label accumulation in secondary metabolites within a fully functional growing plant system. It recommends the potential application for the investigations of various facets of plant metabolism by steady state 13C-MFA. The system also provides a greater potential to study sink leaf metabolism. Overall, we propose a system to accurately describe complex metabolic phenotypes in a growing plant.http://link.springer.com/article/10.1186/s13007-019-0434-8Sink leavesMentha piperitaShoot cultureSecondary metabolismMetabolic flux analysis |
| spellingShingle | Somnath Koley Manish L. Raorane Björn H. Junker Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism Plant Methods Sink leaves Mentha piperita Shoot culture Secondary metabolism Metabolic flux analysis |
| title | Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism |
| title_full | Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism |
| title_fullStr | Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism |
| title_full_unstemmed | Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism |
| title_short | Shoot tip culture: a step towards 13C metabolite flux analysis of sink leaf metabolism |
| title_sort | shoot tip culture a step towards 13c metabolite flux analysis of sink leaf metabolism |
| topic | Sink leaves Mentha piperita Shoot culture Secondary metabolism Metabolic flux analysis |
| url | http://link.springer.com/article/10.1186/s13007-019-0434-8 |
| work_keys_str_mv | AT somnathkoley shoottipcultureasteptowards13cmetabolitefluxanalysisofsinkleafmetabolism AT manishlraorane shoottipcultureasteptowards13cmetabolitefluxanalysisofsinkleafmetabolism AT bjornhjunker shoottipcultureasteptowards13cmetabolitefluxanalysisofsinkleafmetabolism |