secondary metabolites that can be considered suitable IS for
the characterization of polar and semipolar extract from many
plant species. Furthermore formononetin is restricted to a
limited number of plants.
5. When plant extracts are prepared for the transportomics assay
(Subheading 3.4, step 4), add the internal standard (IS) to
characterize the extract by HPLC-PDA-HRMS and not when
performing transport assays.
6. The volume of plant extract suggested to perform the transport
assays is 10–20 μL in 650 μL of reaction mixture. The final
metabolite concentration in the reaction mixture should be
similar to their estimated cellular concentration (or, if not
detectable in LC-PDA-HRMS, up to tenfold higher). This
step needs to be carefully optimized by the operator.
7. If the apocarotenoids present in your extract are glycosylated,
don’t add formic acid. This avoids deglycosylation of the
respective apocarotenoids.
8. Use LC-MS grade solvents for the sample extraction and chromatography separation.
9. Some apocarotenoids can stick to the nitrocellulose filter. Make
a first trial by loading the reaction mixture in the absence of
ATP on nitrocellulose filter and proceed with washes. If the
substrate sticks to the filter try with other types, such as acetate
cellulose filters.
Acknowledgments
Work in GG’s lab was partially supported by the European Union’s
Horizon 2020 programme, project “Newcotiana”, Grant Agreement 760331, and by a grant from the Lazio Region, project
“ProBioZaff”.
References
1. Havaux M (2014) Carotenoid oxidation products as stress signals in plants. Plant J 79
(4):597–606. https://doi.org/10.1111/tpj.
12386
2. McQuinn RP, Giovannoni JJ, Pogson BJ
(2015) More than meets the eye: from carotenoid biosynthesis, to new insights into apocarotenoid signaling. Curr Opin Plant Biol
27:172–179. https://doi.org/10.1016/j.pbi.
2015.06.020
3. Hou X, Rivers J, Leon P, McQuinn RP, Pogson
BJ (2016) Synthesis and function of Apocarotenoid signals in plants. Trends Plant Sci 21
(9):792–803.
https://doi.org/10.1016/j.
tplants.2016.06.001
4. Harrison EH, Quadro L (2018) Apocarotenoids: emerging roles in mammals. Annu Rev
Nutr 38:153–172. https://doi.org/10.1146/
annurev-nutr-082117-051841
5. Kang J, Park J, Choi H, Burla B,
Kretzschmar T, Lee Y, Martinoia E (2011)
Plant ABC transporters. The Arabidopsis
Book:e0153
6. Martinoia E, Meyer S, De Angeli A, Nagy R
(2012) Vacuolar transporters in their physiological context. Annu Rev Plant Biol
98
Olivia Costantina Demurtas et al.
the characterization of polar and semipolar extract from many
plant species. Furthermore formononetin is restricted to a
limited number of plants.
5. When plant extracts are prepared for the transportomics assay
(Subheading 3.4, step 4), add the internal standard (IS) to
characterize the extract by HPLC-PDA-HRMS and not when
performing transport assays.
6. The volume of plant extract suggested to perform the transport
assays is 10–20 μL in 650 μL of reaction mixture. The final
metabolite concentration in the reaction mixture should be
similar to their estimated cellular concentration (or, if not
detectable in LC-PDA-HRMS, up to tenfold higher). This
step needs to be carefully optimized by the operator.
7. If the apocarotenoids present in your extract are glycosylated,
don’t add formic acid. This avoids deglycosylation of the
respective apocarotenoids.
8. Use LC-MS grade solvents for the sample extraction and chromatography separation.
9. Some apocarotenoids can stick to the nitrocellulose filter. Make
a first trial by loading the reaction mixture in the absence of
ATP on nitrocellulose filter and proceed with washes. If the
substrate sticks to the filter try with other types, such as acetate
cellulose filters.
Acknowledgments
Work in GG’s lab was partially supported by the European Union’s
Horizon 2020 programme, project “Newcotiana”, Grant Agreement 760331, and by a grant from the Lazio Region, project
“ProBioZaff”.
References
1. Havaux M (2014) Carotenoid oxidation products as stress signals in plants. Plant J 79
(4):597–606. https://doi.org/10.1111/tpj.
12386
2. McQuinn RP, Giovannoni JJ, Pogson BJ
(2015) More than meets the eye: from carotenoid biosynthesis, to new insights into apocarotenoid signaling. Curr Opin Plant Biol
27:172–179. https://doi.org/10.1016/j.pbi.
2015.06.020
3. Hou X, Rivers J, Leon P, McQuinn RP, Pogson
BJ (2016) Synthesis and function of Apocarotenoid signals in plants. Trends Plant Sci 21
(9):792–803.
https://doi.org/10.1016/j.
tplants.2016.06.001
4. Harrison EH, Quadro L (2018) Apocarotenoids: emerging roles in mammals. Annu Rev
Nutr 38:153–172. https://doi.org/10.1146/
annurev-nutr-082117-051841
5. Kang J, Park J, Choi H, Burla B,
Kretzschmar T, Lee Y, Martinoia E (2011)
Plant ABC transporters. The Arabidopsis
Book:e0153
6. Martinoia E, Meyer S, De Angeli A, Nagy R
(2012) Vacuolar transporters in their physiological context. Annu Rev Plant Biol
98
Olivia Costantina Demurtas et al.