Original articleFructan metabolising enzymes in rhizophores of Vernonia herbacea upon excision of aerial organs
Introduction
Fructans are fructose polymers that occur as storage compounds in about 15% of flowering plants [12], including the economically important and highly evolved families Asteraceae and Poaceae [21]. Different fructan types can be found, depending on the prevailing glycosidic linkages: inulin is β-(2-1)-linked, levan is β-(2-6)-linked and graminan is both β-(2-1)- and β-(2-6)-linked. Inulin type fructan is commonly found in the Asteraceae whereas the remaining types are widespread in other plant groups presenting the fructan syndrome.
In addition to their role as reserve carbohydrates, fructans act in the maintenance of the sucrose gradient between phloem and sink tissues, allowing phloem unloading to proceed [23]. Fructans also seem to be involved in osmoregulation and in the resistance of plants to drought and cold stress [12], [17], [28]. In fact, the presence of fructans in Asteraceae has been documented for floras exhibiting seasonal growth patterns, in temperate regions [29], and in a tropical restricted area of the Brazilian cerrado [7], [33] in which plants are subjected to periods of intensive drought in the winter.
The currently accepted model for fructan biosynthesis is based upon studies on tubers of Helianthus tuberosus [6], an Asteraceae from temperate regions that accumulates inulin type fructans. As originally described by these authors and later confirmed elsewhere by other authors, two distinct fructosyltransferases are responsible for the biosynthesis of inulin: sucrose: sucrose fructosyltransferase (1-SST), which catalyses the production of the trisaccharide 1-kestose and glucose from sucrose [18], [35] and fructan: fructan fructosyltransferase (1-FFT), which transfers fructosyl residues between fructan molecules [16]. According to this model, inulin degradation is catalysed by fructan exohydrolase (1-FEH), which releases free fructose from the terminal non-reducing fructosyl residue, and 1-FFT, by the transfer of fructosyl units from molecules of high molecular mass.
Vernonia herbacea (Vell.) Rusby is a perennial herb from the Brazilian cerrado, which accumulates fructan of the inulin type in the underground reserve organs (rhizophores). Carvalho and Dietrich [2] carried out a study on the fructan contents and composition during the phenological cycle of V. herbacea, and reported that changes in these carbohydrates included a decrease in fructan during sprouting of buds and flowering in spring. According to the authors, this decrease indicated fructan mobilisation in the rhizophore to provide substrate for growth. Further, a period of intensive vegetative growth and increase in fructan contents occurred during summer, followed by senescence of aerial organs in autumn and dormancy of the rhizophores in winter.
Preliminary studies showed that 1-FEH in rhizophores of V. herbacea presented high activity only during sprouting in spring while in the other phases of the phenological cycle its activity was barely detectable. Similar results were observed for other Asteraceae as reported for Taraxacum officinale [31], Cichorium intybus [34] and H. tuberosus [19].
According to Marx et al. [19] the in vivo activity of 1-FEH in sprouting tubers of H. tuberosus rises because the enzyme is less inhibited than in dormant tubers, possibly due to a lower vacuolar sucrose concentration in the sprouting tissue and because higher temperatures allow for higher metabolic rates. The enhanced rate of hydrolysis of the stored fructan would result in the high concentration of fructose observed in sprouting tubers.
In yacon (Polymnia sonchifolia), a large increase in 1-FEH activity and a decrease in fructan levels were observed in the underground reserve organs after strong typhoon winds caused serious damage to the photosynthetic apparatus of the plants [8]. De Roover et al. [5] detected an increase in 1-FEH activity and a decrease in 1-SST activity in defoliated young chicory plants (C. intybus). As the changes in the activities of 1-FEH and 1-SST were always in opposite direction, the authors suggested that the genes encoding for these enzymes are regulated by the same effector, in opposite ways. They suggested, for instance, that a sudden increase in sucrose influx could upregulate the 1-SST gene and downregulate the 1-FEH gene.
In grasses, the increase in FEH activity and fructan mobilisation has been associated to defoliation by grazing or mowing, as described for timothy (Phleum pratense) [22], Lolium perenne [23], [26], [30] and orchardgrass (Dactylis glomerata) [38].
This paper reports the activities of 1-SST, 1-FFT, 1-FEH and invertase in rhizophores of V. herbacea after shoot excision and during re-growth of new shoots. As activity of FEH was naturally detected only during sprouting, in spring, we were particularly interested to know if this enzyme could be induced in other phases of the phenological cycle and how it relates to fructan mobilisation during re-growth of aerial organs. We were also interested to know if the mechanism of fructan mobilisation in V. herbacea, a tropical species, is similar to that reported for other fructan accumulating species from temperate regions.
Section snippets
Results
Sprouting of new shoots began 9 days after shoot excision. By the 13th day, all plants presented new sprouts.
Discussion
According to earlier studies, 1-FEH activity in rhizophores of V. herbacea is high only in spring, during sprouting, remaining very low in the other stages of the phenological cycle (data not shown).
Results presented here demonstrate that the activity of 1-FEH in the rhizophores is induced after excision of the aerial organs from adult plants, during the growing season, in the summer. The increase in 1-FEH activity detected after day 13 followed sprouting of buds quite closely and occurred
Plant material
The experiment was conducted in the summer, with 2-year-old plants of V. herbacea (Vell.) Rusby in the vegetative stage, cultivated in individual pots. Aerial organs were cut at the soil level at the beginning of the experiment (day 0) and the plants were kept outdoors. Rhizophore samples for carbohydrate analyses (2 g) and enzymatic activities (20 g) were taken from three plants just before excision of the aerial organs (day 0) and at days 3, 6, 9, 13, 17, 20, 23, 27 and 32 after cutting.
Acknowledgements
This work was supported by the State of São Paulo Research Foundation (FAPESP) within the BIOTA/FAPESP, The Biodiversity Virtual Institute Program. The authors thank Dr Norio Shiomi (Rakuno Gakuen University, Japan) for kindly providing pure samples of nystose and 1-kestose and Drs. Rita de Cássia L.F. Ribeiro and Lilian B.P. Zaidan for critical review of the manuscript. M.A.M. Carvalho thanks CNPq for an awarded fellowship and A.F. Asega thanks FAPESP and CNPq/PIBIC for fellowships.
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