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Subcellular distribution of raffinose oligosaccharides and other metabolites in summer and winter leaves of Ajuga reptans (Lamiaceae)

Abstract

Main conclusion

In Ajuga reptans, raffinose oligosaccharides accumulated during winter. Stachyose, verbascose, and higher RFO oligomers were exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma.

Abstract

The evergreen labiate Ajuga reptans L. can grow at low temperature. The carbohydrate metabolism changes during the cold phase, e.g., raffinose family oligosaccharides (RFOs) accumulate. Additionally, A. reptans translocates RFOs in the phloem. In the present study, subcellular concentrations of metabolites were studied in summer and winter leaves of A. reptans to gain further insight into regulatory instances involved in the cold acclimation process and into the function of RFOs. Subcellular metabolite concentrations were determined by non-aqueous fractionation. Volumes of the subcellular compartments of summer and winter leaves were analyzed by morphometric measurements. The metabolite content varied strongly between summer and winter leaves. Soluble metabolites increased up to tenfold during winter whereas the starch content was decreased. In winter leaves, the subcellular distribution showed a shift of carbohydrates from cytoplasm to vacuole and chloroplast. Despite this, the metabolite concentration was higher in all compartments in winter leaves compared to summer leaves because of the much higher total metabolite content in winter leaves. The different oligosaccharides did show different compartmentations. Stachyose, verbascose, and higher RFO oligomers were almost exclusively found in the vacuole whereas one-fourth of raffinose was localized in the stroma. Apparently, the subcellular distribution of the RFOs differs because they fulfill different functions in plant metabolism during winter. Raffinose might function in protecting chloroplast membranes during freezing, whereas higher RFO oligomers may exert protective effects on vacuolar membranes. In addition, the high content of RFOs in winter leaves may also result from reduced consumption of assimilates.

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Abbreviations

FW:

Fresh weight

HPLC:

High-performance liquid chromatography

RFO:

Raffinose family oligosaccharides

References

  • Adhikari J, Bhaduri TJ, DasGupta S, Majumder AL (1987) Chloroplast as a locale of L-myo-inositol-1-phosphate synthase. Plant Physiol 85:611–614

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Bachmann M, Keller F (1995) Metabolism of the raffinose family oligosaccharides in leaves of Ajuga reptans L.—inter- and intracellular compartmentation. Plant Physiol 109:991–998

    CAS  PubMed Central  PubMed  Google Scholar 

  • Bachmann M, Matile P, Keller F (1994) Metabolism of the raffinose family oligosaccharides in leaves of Ajuga reptans L.—cold acclimation, translocation, and sink to source transition: discovery of chain elongation enzyme. Plant Physiol 105:1335–1345

    CAS  PubMed Central  PubMed  Google Scholar 

  • Moore BD, Palmquist DE, Seemann JR (1997) lnfluence of plant growth at high CO2 concentrations on leaf content of rinolose-1,5-bisphosphate carboxylase/oxygenase and intracellular distribution of soluble carbohydrates in tobacco, snapdragon, and parsley. Plant Physiol 115:241–248

    CAS  PubMed Central  PubMed  Google Scholar 

  • Egert A, Keller F, Peters S (2013) Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase (RS5, At5g40390). BMC Plant Biol 13:218

    PubMed Central  PubMed  Article  Google Scholar 

  • Espinoza C, Degenkolbe T, Caldana C, Zuther E, Leisse A, Willmitzer L, Hincha DK, Hannah MA (2010) Interaction with diurnal and circadian regulation results in dynamic metabolic and transcriptional changes during cold acclimation in Arabidopsis. PLoS ONE 5:e14101

    PubMed Central  PubMed  Article  Google Scholar 

  • Farré EM, Tiessen A, Roessner U, Geigenberger P, Trethewey RN, Willmitzer L (2001) Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method. Plant Physiol 127:685–700

    PubMed Central  PubMed  Article  Google Scholar 

  • Gamalei YV (1991) Phloem loading and its development related to plant evolution from trees to herbs. Trees 5:50–64

    Article  Google Scholar 

  • Gerhardt R, Heldt HW (1984) Measurements of subcellular metabolite levels by fractionation of freeze-stopped material in nonaqueous media. Plant Physiol 75:542–547

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Gerrits N, Turk SCHJ, van Dun KPM, Hulleman SHD, Visser RGF, Weisbeek PJ, Smeekens SCM (2001) Sucrose metabolism in plastids. Plant Physiol 125:926–934

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol 124:1854–1865

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Heineke D, Wildenberger K, Sonnewald U, Willmitzer L, Heldt HW (1994) Accumulation of hexoses in leaf vacuoles: studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol, vacuole or apoplasm. Planta 194:29–33

    CAS  Article  Google Scholar 

  • Hincha DK, Sonnewald U, Willmitzer L, Schmitt J (1996) The role of sugar accumulation in leaf frost hardiness: investigations with transgenic tobacco expressing a bacterial pyrophosphatase or a yeast invertase gene. J Plant Physiol 147:604–610

    CAS  Article  Google Scholar 

  • Hincha DK, Zuther E, Heyer AG (2003) The preservation of liposomes by raffinose family oligosaccharides during drying in mediated by effects on fusion and lipid phase transitions. Biochim Biophys Acta 1612:172–177

    CAS  PubMed  Article  Google Scholar 

  • Hoffmann-Thoma G, van Bel AJE, Ehlers K (2001) Ultrastructure of minor-vein phloem and assimilate export in summer and winter leaves of the symplasmically loading evergreens Ajuga reptans L., Aucuba japonica Thunb., and Hedera helix L. Planta 169:231–242

    Article  Google Scholar 

  • Holthaus U, Schmitz K (1991) Distribution and immunolocalization of stachyose synthase in Cucumis melo L. Planta 162:283–288

    Google Scholar 

  • Iftime D, Hannah MA, Peterbauer T, Heyer AG (2011) Stachyose in the cytosol does not influence freezing tolerance of transgenic Arabidopsis expressing stachyose synthase from adzuki bean. Plant Sci 180:24–30

    CAS  PubMed  Article  Google Scholar 

  • Inan Haab C, Keller F (2002) Purification and characterization of the raffinose oligosaccharide chain elongation enzyme, galactan:galactan galactosyltransferase (GGT) from Ajuga reptans leaves. Physiol Plant 114:361–371

    Article  Google Scholar 

  • Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137–138

    Google Scholar 

  • Klie S, Krueger S, Krall L, Giavalisco P, Flügge UI, Willmitzer L, Steinhauser D (2011) Analysis of the compartmentalized metabolome—a validation of the non-aqueous fractionation technique. Front Plant Sci 2:Art 55

    Article  Google Scholar 

  • Klotke J, Kopka J, Gatzke N, Heyer AG (2004) Impact of soluble sugar concentrations on the acquisition of freezing tolerance in accessions of Arabidopsis thaliana with contrasting cold adaptation—evidence for a role of raffinose in cold acclimation. Plant Cell Environ 27:1395–1404

    CAS  Article  Google Scholar 

  • Knaupp M, Mishra KB, Nedbal L, Heyer AG (2011) Evidence for a role of raffinose in stabilizing photosystem II during freeze–thaw cycles. Planta 234:477–486

    CAS  PubMed  Article  Google Scholar 

  • Knop C, Voitsekhovskaja O, Lohaus G (2001) Sucrose transporters in two members of the Scrophulariaceae with different types of transport sugar. Planta 213:80–91

    CAS  PubMed  Article  Google Scholar 

  • Koster K, Lynch D (1992) Solute accumulation and compartmentation during the cold acclimation of Puma rye. Plant Physiol 98:108–113

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Krueger S, Niehl A, Lopez Martin MC, Steinhauser D, Donath A, Hildebrandt T, Romero LC, Hoefgen R, Gotor C, Hesse H (2009) Analysis of cytosolic and plastidic serine acetyltransferase mutants and subcellular metabolite distributions suggests interplay of the cellular compartments for cysteine biosynthesis in Arabidopsis. Plant Cell Environ 32:349–367

    CAS  PubMed  Article  Google Scholar 

  • Krueger S, Giavaisco P, Krall L, Steinhauser MC, Büssis D, Usadel B, Flügge UI, Fernie AR, Willmitzer L, Steinhauser D (2011) A topological map of the compartmentalized Arabidopsis thaliana leaf metabolome. PLoS ONE 6(3):e17806

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Krueger S, Steinhauser D, Lisec J, Giavaisco P (2014) Analysis of subcellular metabolite distributions within Arabidopsis thaliana leaf tissue: a primer for subcellular metabolomics. Arabidopsis protocols. Methods Mol Biol 1062:575–596

    PubMed  Article  Google Scholar 

  • Lohaus G, Hussmann M, Schneider H, Zhu JJ, Sattelmacher B (2000) Solute balance of a maize (Zea mays L) source leaf as affected by salt treatment with special emphasis on phloem re-translocation and ion leaching. J Exp Bot 51:1721–1732

    CAS  PubMed  Article  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  • Lunn JE (2007) Compartmentation in plant metabolism. J Exp Bot 58:35–47

    CAS  PubMed  Article  Google Scholar 

  • Martinoia E, Rentsch D (1994) Malate compartmentation: responses to a complex metabolism. Annu Rev Plant Physiol Plant Mol Biol 45:447–467

    CAS  Article  Google Scholar 

  • Martinoia E, Flügge UI, Kaiser G, Heber U, Heldt HW (1985) Energy-dependent uptake of malate into vacuoles isolated from barley mesophyll protoplasts. Biochem Biophys Acta 806:311–319

    CAS  Google Scholar 

  • Nadwodnik J, Lohaus G (2008) Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens. Planta 227:1079–1089

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Nägele T, Heyer AG (2013) Approximating subcellular organisation of carbohydrate metabolism during cold acclimation in different natural accessions of Arabidopsis thaliana. New Phytol 198:777–787

    PubMed  Article  Google Scholar 

  • Öner-Sieben S, Lohaus G (2014) Apoplastic and symplastic phloem loading in Quercus robur and Fraxinus excelsior. J Exp Bot 65:1905–1916

    PubMed Central  PubMed  Article  Google Scholar 

  • Peterbauer T, Richter A (2001) Biochemistry and physiology of raffinose family oligosaccharides and galactosyl cyclitols in seeds. Seed Sci Res 11:185–197

    CAS  Google Scholar 

  • Peters S, Keller F (2009) Frost tolerance in excised leaves of the common bugle (Ajuga reptans L.) correlates positively with the concentrations of raffinose family oligosaccharides (RFOs). Plant Cell Environ 32:1099–1107

    CAS  PubMed  Article  Google Scholar 

  • Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Riens B, Lohaus G, Heineke D, Heldt HW (1991) Amino acid and sucrose content determined in the cytosolic, chloroplastic and vacuolar compartments and in the phloem sap of spinach leaves. Plant Physiol 97:227–233

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Riens B, Lohaus G, Winter H, Heldt HW (1994) Production and diurnal utilization of assimilates in leaves of spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.). Planta 192:497–501

    CAS  Article  Google Scholar 

  • Rohde P, Hincha DK, Heyer AG (2004) Heterosis in the freezing tolerance of crosses between two Arabidopsis thaliana accessions (Columbia-0 and C24) that will differences in non-acclimated and acclimated freezing tolerance. Plant J 38:790–799

    CAS  PubMed  Article  Google Scholar 

  • Santarius KA, Milde H (1977) Sugar compartmentation in frost-hardy and partially dehardened cabbage leaf cells. Planta 136:163–166

    CAS  PubMed  Article  Google Scholar 

  • Scarth GW, Levitt J (1937) The frost hardening mechanism of plant cells. Plant Physiol 12:51–78

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Schneider T, Keller F (2009) Raffinose in chloroplasts is synthesized in the cytosol and transported across the chloroplast envelope. Plant Cell Physiol 50:2174–2182

    CAS  PubMed  Article  Google Scholar 

  • Shahba MA, Qian YL, Hughes HG, Koski AJ, Christensen D (2003) Relationships of soluble carbohydrates and freeze tolerance in saltgrass. Crop Sci 43:2148–2153

    CAS  Article  Google Scholar 

  • Sprenger N, Keller F (2000) Allocation of raffinose family oligosaccharides to transport and storage in Ajuga reptans: the roles of two distinct galactinol synthases. Plant J 21:249–258

    CAS  PubMed  Article  Google Scholar 

  • Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:32–43

    Article  Google Scholar 

  • Stitt M, Hurry V (2002) A plant for all seasons: alterations in photosynthetic carbon metabolism during cold acclimation in Arabidopsis. Curr Opin Plant Biol 5:199–206

    CAS  PubMed  Article  Google Scholar 

  • Strand Å, Hurry V, Gustafsson P, Gardeström P (1997) Development of Arabidopsis thaliana leaves at low temperatures releases the suppression of photosynthesis and photosynthetic gene expression despite the accumulation of soluble carbohydrates. Plant J 12:605–614

    CAS  PubMed  Article  Google Scholar 

  • Strand Å, Hurry V, Henkes S, Huner N, Gustafsson P, Gardeström P, Stitt M (1999) Acclimation of Arabidopsis leaves developing at low temperatures increasing cytoplasmic volume accompanies increased activities of enzymes in the calvin cycle and in the sucrose-biosynthesis pathway. Plant Physiol 119:1387–1397

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Tapernoux-Lüthi EM, Böhm A, Keller F (2002) Cloning, functional expression, and characterization of the raffinose oligosaccharide chain elongation enzyme, galactan:galactan galactosyltransferase (GGT), from common bugle leaves. Physiol Plant 134:1377–1387

    Article  Google Scholar 

  • Turgeon R, Beebe DU, Gowan E (1993) The intermediary cell: minor-vein anatomy and raffinose oligosaccharide synthesis in the Scrophulariaceae. Planta 191:446–456

    CAS  Article  Google Scholar 

  • Uemura M, Steponkus PL (2003) Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves. Plant Cell Environ 26:1083–1096

    CAS  Article  Google Scholar 

  • Valluru R, Lammens W, Claupein W, Van den Ende W (2008) Freezing tolerance by vesicle-mediated fructan transport. Trends Plant Sci 13:409–414

    CAS  PubMed  Article  Google Scholar 

  • Vargas WA, Pontis HG, Salerno GL (2008) New insights on sucrose metabolism: evidence for an active A/N-Inv in chloroplasts uncovers a novel component in the intracellular carbon trafficking. Planta 227:795–807

    CAS  PubMed  Article  Google Scholar 

  • Voitsekhovskaja OV, Koroleva OA, Batashev DR, Knop C, Tomos D, Gamalei YV, Heldt HW, Lohaus G (2006) Phloem loading in two Scrophulariaceae species what can drive symplastic flow via plasmodesmata? Plant Physiol 140:383–395

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Voitsekhovskaja OV, Rudashevskaya EL, Demchenko KN, Pakhomova MV, Batashev DR, Gamalei YV, Lohaus G, Pawlowski K (2009) Evidence for functional heterogeneity of sieve element-companion cell complexes in minor vein phloem of Alonsoa meridionalis. J Exp Bot 60:1873–1883

    CAS  PubMed  Article  Google Scholar 

  • Winter H, Robinson DG, Heldt HW (1993) Subcellular volumes and metabolite concentrations in barley leaves. Planta 191:180–190

    CAS  Article  Google Scholar 

  • Winter H, Robinson DG, Heldt HW (1994) Subcellular volumes and metabolite concentrations in spinach leaves. Planta 193:530–535

    CAS  Article  Google Scholar 

  • Zuther E, Büchel K, Hundertmark M, Stitt M, Hincha DK, Heyer AG (2004) The role of raffinose in the cold acclimation response of Arabidopsis thaliana. FEBS Lett 576:169–173

    CAS  PubMed  Article  Google Scholar 

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Acknowledgments

The authors would like to thank Tim Kreutzer for technical assistance, Sarah Rau for help with analyzing the electron micrographs, Elisabeth Wesbuer for her excellent electron-microscopical assistance and Kira Tiedge for the critical reading of the manuscript. Comments from the anonymous reviewer have greatly helped to improve the previous version of the present manuscript.

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The authors declare that they have no conflict of interest.

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Correspondence to Gertrud Lohaus.

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Findling, S., Zanger, K., Krueger, S. et al. Subcellular distribution of raffinose oligosaccharides and other metabolites in summer and winter leaves of Ajuga reptans (Lamiaceae). Planta 241, 229–241 (2015). https://doi.org/10.1007/s00425-014-2183-2

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  • DOI: https://doi.org/10.1007/s00425-014-2183-2

Keywords

  • Ajuga
  • Cold acclimation
  • Non-aqueous fractionation
  • Raffinose oligosaccharides
  • Subcellular metabolite concentration