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Fructan dynamics in the underground organs of Chresta exsucca (Asteraceae), a dry season flowering species

ABSTRACT

Climatic seasonality has an influence on the phenology of native Cerrado plants. Herbs and subshrubs tend to flower in the rainy season, although some species of these habits flower in the dry season. Reserve carbohydrates, stored in the underground organs, are used to support phases of high energy-demand, but also may protect plants from damage during periods of environmental limitation. In this study we evaluated variation in fructan levels in the underground organs of field-grown plants of Chresta exsucca among different phenological phases. Chresta exsucca flowers in the dry season and possesses a diffuse underground system, which stores inulin-type fructans. Resprouting was continual during the sampling period. Oligosaccharide content was always higher than polysaccharide content, except during senescence, the only phase with an oligosaccharide: polysaccharide ratio < 1. Fructan accumulation occurred during vegetative growth until flowering. Fructan mobilization was prominent during resprouting until the beginning of vegetative growth. Fructans stored in the underground organs of C. exsucca serve to fulfill the energetic demands of development and maintenance of this complex structure. In this way, fructans are essential to the persistence of this species in the environment of the Cerrado by ensuring reproduction in harsh conditions, such as drought.

Keywords:
Cerrado; drought; inulin; non-structural carbohydrate; phenology

Introduction

Cerrado occupies nearly 23 % of the territory of Brazil, and its vegetation comprises several phytophysiognomies that are spread throughout a wide geographical area (Ratter et al. 1997Ratter JA, Ribeiro JF, Bridgewater S. 1997. The Brazilian cerrado vegetation and threats to its biodiversity. Annals of Botany 80: 223-230.). Cerrado phytophysiognomies constitute a gradient of forms, from grasslands to woodlands, which are determined by abiotic factors, mainly soil fertility, depth of the water table and fire frequency (Franco et al. 2014Franco AC, Rossatto DR, Silva LCR, Ferreira CS. 2014. Cerrado vegetation and global change: the role of functional types, resource availability and disturbance in regulating plant community responses to rising CO2 levels and climate warming. Theoretical and Experimental Plant Physiology 26: 19-38.). Rainfall seasonality is another crucial determinant of Cerrado phytophysiognomies. The rainy season occurs from October to April with average monthly rainfall ranging from 150 to 500 mm. In the dry season, from May to September, rainfall is considerably reduced to 0-50 mm per month (Silva et al. 2008Silva FAM, Assad ED, Evangelista BA. 2008. Caracterização climática do Bioma Cerrado. In: Sano SM, Almeida SP, Ribeiro JF. (eds.) Cerrado Ecologia e Flora. Planaltina, Embrapa. p. 69-88.), resulting in soil water deficit, particularly for herbs and subshrubs (Rossatto et al. 2013Rossatto D, Sternberg LSL, Franco AC. 2013. The partitioning of water uptake between growth forms in a Neotropical savanna: do herbs exploit a third water source niche? Plant Biology 15: 84-92.).

Though herbs and subshrubs are affected by seasonal drought, they predominate in the open physiognomies of the Cerrado, and are much more diverse than trees (Ratter et al. 1997Ratter JA, Ribeiro JF, Bridgewater S. 1997. The Brazilian cerrado vegetation and threats to its biodiversity. Annals of Botany 80: 223-230.). Herbs and subshrubs also have a high proportion of underground phytomass (Haridasan 2000Haridasan M. 2000. Nutrição mineral de plantas nativas do cerrado. Revista Brasileira de Fisiologia Vegetal 12: 54-64.), very often represented by several morphological types of perennial organs with buds and reserve compounds (Appezzato-da-Glória et al. 2008Appezzato-da-Glória B, Cury G, Soares MKM, Rocha R, Hayashi AH. 2008. Underground systems of Asteraceae species from the Brazilian Cerrado. The Journal of the Torrey Botanical Society 135: 103-113.). Such underground organs enable regrowth following seasonal drought or fire.

Seasonality may also influence the phenological cycle of herbaceous plants of the Cerrado. Herbs and subshrubs typically sprout with the first rains, after a variable period of dormancy, and gradually develop as the rainy season progresses (Mantovani & Martins 1988Mantovani W, Martins FR. 1988. Variações fenológicas das espécies do cerrado da Reserva Biológica de Moji Guaçu, Estado de São Paulo. Acta Botanica Brasilica 11: 101-112.). Reproduction occurs during higher water availability (Batalha & Martins 2004Batalha MA, Martins FR. 2004. Reproductive phenology of the cerrado plant community in Emas National Park (central Brazil). Australian Journal of Botany 52: 149-161.). Aerial shoots senesce after reproduction, at the beginning of dry season, and only the underground organs of the plants remain, dormant in the environment (Mantovani & Martins 1988Mantovani W, Martins FR. 1988. Variações fenológicas das espécies do cerrado da Reserva Biológica de Moji Guaçu, Estado de São Paulo. Acta Botanica Brasilica 11: 101-112.). This pattern is exemplified by the Asteraceae species Chrysolaena obovata (previously named Vernonia herbacea), Aldama discolor (previously named Viguiera discolor), Ichthyothere terminalis and by the Amaranthaceae species Gomphrena macrocephala and Gomphrena marginata (Carvalho & Dietrich 1993Carvalho MAM, Dietrich SMC. 1993. Variation in fructan content in the underground organs of Vernonia herbacea (Vell.) Rusby at different phenological phases. New Phytologist 123: 735-740.; Isejima & Figueiredo-Ribeiro 1993Isejima EM, Figueiredo-Ribeiro RCL. 1993. Fructan variations in tuberous roots of Viguiera discolor Baker (Asteraceae): the influence of phenology. Plant and Cell Physiology 34: 723-727.; Vieira & Figueiredo-Ribeiro 1993Vieira CCJ, Figueiredo-Ribeiro RCL. 1993. Fructose-containing carbohydrates in the tuberous root of Gomphrena macrocephala St.-Hil.(Amaranthaceae) at different phenological phases. Plant, Cell & Environment 16: 919-928.; Silva et al. 2013Silva FGD, Cangussu LMB, Paula SLA, Melo GA, Silva EA. 2013. Seasonal changes in fructan accumulation in the underground organs of Gomphrena marginata Seub. (Amaranthaceae) under rock-field conditions. Theoretical and Experimental Plant Physiology 25: 46-55.; Almeida et al. 2017Almeida LV, Ferri PH, Seraphin JC, Moraes MG. 2017. Seasonal changes of fructans in dimorphic roots of Ichthyothere terminalis (Spreng.) Blake (Asteraceae) growing in Cerrado. Science of The Total Environment 598: 404-412.). All of these species accumulate fructans in their underground organs.

Fructans are fructose-based polymers and are present as a reserve compound in 15 % of all Angiosperms, including the derived Asteraceae (Hendry 1993Hendry GAF. 1993. Evolutionary origins and natural functions of fructans - a climatological, biogeographic and mechanistic appraisal. New Phytologist 123: 3-14.). In this family, fructans are linear molecules with β(2,1) linkages between fructosyl units and a terminal glucose residue, constituting the inulin-type. Edelman & Jefford (1968Edelman J, Jefford T. 1968. The mechanisim of fructosan metabolism in higher plants as exemplified in Helianthus tuberosus. New Phytologist 67: 517-531.) proposed a model for inulin synthesis in Heliantus tuberosus tubers, which starts with the fructosyl transfer between two sucrose molecules by the enzyme sucrose:sucrose 1 fructosyltranferase (1-SST), producing the trisaccharide 1-kestose and free glucose. Subsequently, the enzyme fructan:fructan 1-fructosyltransferase (1-FFT) transfers fructosyl units between fructan molecules, producing one fructan molecule with a high degree of polymerization (DP), and another with a low DP. Inulin hydrolysis is the function of fructan exohydrolase (1-FEH), which removes terminal fructosyl units.

Inulin stored in underground organs is used to resume growth during resprouting or defoliation (Raccuia & Melili 2010Raccuia SA, Melilli MG. 2010. Seasonal dynamics of biomass, inulin, and water-soluble sugars in roots of Cynara cardunculus L. Field Crops Research 116: 147-153.), since these processes require carbon in the absence of aerial organs, which precludes carbon accumulation by photosynthesis. Additionally, fructans are believed to protect plants against environmental constraints, such as cold and drought, since fructan-accumulating plants are common in the flora of temperate and arid zones, with seasonal frost and drought, respectively (Hendry 1993Hendry GAF. 1993. Evolutionary origins and natural functions of fructans - a climatological, biogeographic and mechanistic appraisal. New Phytologist 123: 3-14.). This protective role can be achieved by membrane stabilization (Hincha et al. 2007Hincha DK, Livingston DP, Premakumar R, et al. 2007. Fructans from oat and rye: composition and effects on membrane stability during drying. Biochimica et Biophysica Acta 1768: 1611-1619.) and by antioxidant properties (Peshev et al. 2013Peshev D, Vergauwen R, Moglia A, Hideg É, Ende W. 2013. Towards understanding vacuolar antioxidant mechanisms: a role for fructans? Journal of Experimental Botany 64: 1025-1038.).

Changes in fructan metabolism have been observed throughout the seasonal growth of Cerrado herbs with the typical phenological cycle. Generally, herbs accumulate carbohydrates during vegetative growth, which are later used for reproduction and resprouting in the rainy season (Carvalho & Dietrich 1993Carvalho MAM, Dietrich SMC. 1993. Variation in fructan content in the underground organs of Vernonia herbacea (Vell.) Rusby at different phenological phases. New Phytologist 123: 735-740.; Isejima & Figueiredo-Ribeiro 1993Isejima EM, Figueiredo-Ribeiro RCL. 1993. Fructan variations in tuberous roots of Viguiera discolor Baker (Asteraceae): the influence of phenology. Plant and Cell Physiology 34: 723-727.; Vieira & Figueiredo-Ribeiro 1993Vieira CCJ, Figueiredo-Ribeiro RCL. 1993. Fructose-containing carbohydrates in the tuberous root of Gomphrena macrocephala St.-Hil.(Amaranthaceae) at different phenological phases. Plant, Cell & Environment 16: 919-928.; Almeida et al. 2017Almeida LV, Ferri PH, Seraphin JC, Moraes MG. 2017. Seasonal changes of fructans in dimorphic roots of Ichthyothere terminalis (Spreng.) Blake (Asteraceae) growing in Cerrado. Science of The Total Environment 598: 404-412.). However, there are variations in this pattern. Phenological notes from a floristic study of the tribe Vernonieae (Asteraceae) reported that some species differ in their period of reproduction by flowering in the dry season, as exemplified by species of the genus Chresta (Moreira & Teles 2014Moreira GL, Teles AM. 2014. A tribo Vernonieae Cass. (Asteraceae) na Serra Dourada, Goiás, Brasil. Iheringia Série Botânica 69: 357-385.). This raises the question of how reserve dynamics occurs in dry season flowering species. We hypothesize that fructan dynamics is mainly governed by phenology, since phenological phases have distinct energy requirements. Thus, the main objective of this study was to evaluate fructan variation among different phenological phases in underground organs of field-grown plants of Chresta exsucca, a species with reproduction in the dry season.

Materials and methods

Study site

The study was carried out in Parque Nacional da Chapada dos Veadeiros, Alto Paraíso de Goiás, Goiás, Brazil (13°51'-14°10'S 47°25'-47°42'W; 800- 1,650 m). This site is known for its high levels of biodiversity richness and endemism (Munhoz & Felfili 2006Munhoz CBR, Felfili JM. 2006. Floristics of the herbaceous and subshrub layer of a moist grassland in the Cerrado biosphere reserve (Alto Paraíso de Goiás), Brazil. Edinburgh Journal of Botany 63: 343-354.), and contains a variety of Cerrado phytophysiognomies, of which rocky outcrops and open grasslands predominate (Lenza et al. 2011Lenza E, Pinto JRR, Pinto AS, Maracahipes L, Bruziguessi EP. 2011. Comparação da vegetação arbustivo-arbórea de uma área de cerrado rupestre na Chapada dos Veadeiros, Goiás, e áreas de cerrado sentido restrito do Bioma Cerrado. Revista Brasileira de Botânica 34: 247-259.). Meteorological data for the period from October 2015 to September 2016 were obtained from the meteorological station closest to the study site, located in Posse, Goiás, Brazil (www.inmet.gov.br) (Fig. 1).

Figure 1
Monthly means of maximum (diamonds), average (squares) and minimum (triangles) temperatures (A), and rainfall (bars) and relative air humidity (circles) (B) from October 2015 to September 2016 in Posse, Goiás, Brasil, (www.inmet.gov.br). Resprouting (RE), vegetative growth (VG), flower buds (FB), flower opening (FO), fruiting (FR) and senescence of the aerial organs (SE).

Plant material

Chresta exsucca DC. is an herbaceous species that is 0.6 - 2.0 m high, syncephalous, and with lilac flowers and cypsela-type fruits (MacLeish 1985MacLeish NF. 1985. Revision of Chresta and Pycnocephalum (Compositae: Vernonieae). Systematic Botany 10: 459-470.). A voucher specimen was deposited in the herbarium of the Universidade Federal de Goiás (UFG 50894). Individuals of C. exsucca were evaluated for identification of their phenological phase. Four distinct plants, separated by more than 10 m, with the same phenological phase were sampled in October 2015, December 2015, February 2016, May 2016, and July 2016. Thickened underground organs were washed in distilled water, cut and weighed. Aliquots of fresh material (1.5 g) were boiled in aqueous ethanol (80 %) for 15 min for enzyme inactivation, and then kept in ethanol until arriving at the laboratory where the following extraction procedures were performed. Water content (WC) was determined by oven drying (50 °C) aliquots of fresh material (1.0 g) to constant dry mass (DM), and expressed as a percentage of fresh mass. These analyses were performed for four distinct individuals for each phenological phase: resprouting (RE), vegetative growth (VG), flower buds (FB), flower opening (FO), fruiting (FR) and senescence (SE).

Extraction and analysis of soluble carbohydrates

In the laboratory, samples stored in aqueous ethanol were homogenized and filtered through nylon cloth. Residues were re-extracted twice in aqueous ethanol (80 %) at 80 °C for 15 min. All the ethanolic filtrates were pooled and consisted mainly of mono-, di- and oligosaccharides, hereafter referred to as the oligosaccharide fraction. Residues were extracted twice in distilled water (60 °C) for 30 min. The aqueous filtrates, consisting of predominantly polysaccharides, were collected and hereafter referred to as polysaccharide fractions (Carvalho et al. 1998Carvalho MAM, Pinto MM, Figueiredo-Ribeiro RCL. 1998. Inulin production by Vernonia herbacea as influenced by mineral fertilization and time of harvest. Revista Brasileira de Botânica 21: 275-280.). Both fractions were vacuum-concentrated in a rotary evaporator (39 °C) and analyzed independently.

Soluble carbohydrate of the oligosaccharide and polysaccharide fractions was quantified by the phenol-sulfuric method using glucose as standard at 490 nm (Dubois et al. 1956Dubois M, Gilles KA, Hamilton JK, Rebers P, Smith F. 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28: 350-356.). Total soluble carbohydrate was obtained by the sum of the soluble carbohydrate of both fractions. All quantifications were done in triplicate for each of the four biological replicates.

The oligosaccharide and polysaccharide fractions were deionized using ion exchange resins (Amberlite IRA 120, cationic and Amberlite IRA 410, anionic), eluted with 10 volumes of ultrapure water (18 MΩ). The purified fractions had their pH checked, and neutralized if necessary, prior to being concentrated in a rotary evaporator and solubilized in ultrapure water. Free and combined fructose content was determined in each purified fraction using anthrone reagent with fructose as a standard at 620 nm (Jermyn 1956Jermyn M. 1956. A new method for determining ketohexoses in the presence of aldohexoses. Nature 177: 38-39.). Fractions were adjusted to the final concentration of 400 μg. mL-1 of fructose equivalents.

The purified fractions were filtered through membranes (0.45 μm) and analyzed by high-performance anion exchange chromatography with integrated pulsed amperometric detection (HPAEC/IPAD) in a CarboPac PA100 column (4 x 250 mm), with the corresponding guard column, coupled to a ICS 5000 chromatograph (Dionex). Mobile phase was a gradient of sodium acetate (500 mM) in sodium hydroxide (100 mM) with the following schedule: 0-10 min: 5 mM; 10.1 - 35min, 5 - 50 mM; 35.1 - 40 min, 50 - 375 mM; 40.1 - 45min, 500 mM, eluted at 1 mL.min-1 (Silva et al. 2015Silva TM, Vilhalva DA, Moraes MG, Figueiredo-Ribeiro RCL. 2015. Anatomy and fructan distribution in vegetative organs of Dimerostemma vestitum (Asteraceae) from the campos rupestres. Anais da Academia Brasileira de Ciências 87: 797-812.). Chromatograms were prepared in the same scale of time and detector response. Extracts obtained from Helianthus tuberosus L. (Asteraceae) tubers were used as standard for comparison and identification of soluble carbohydrate. Qualitative analysis was performed by comparison of the relative area of a certain sugar peak among the different phenological phases, since the detector signal decreased with elution time. Quantification of glucose, fructose and sucrose was performed by the external standard method using authentic standards in Chromeleon 6.8 software (Dionex). Means obtained for each sugar in both fractions were expressed as a percentage in 400 μg. mL-1 of fructose equivalents.

Statistical analysis

Water content, oligosaccharide and polysaccharide levels, and the oligosaccharide:polysaccharide ratio, plus glucose, fructose, sucrose and fructans with DP ≥ 3, of samples collected during different phenological phases were submitted to Kruskal-Wallis analysis of variance, followed by Dunn´s test for multiple comparisons (p < 0.05). This test was chosen since these data did not meet the requirements for parametric analysis. All statistical analyses were conducted in BioEstat v. 5.3.

Results

Chresta exsucca has a diffuse underground system with thickened organs with orthogravitropic and diagravitropic growth. Resprouting plants were found throughout the study period, but were more intense in the rainy season, followed by vegetative growth. The development of reproductive organs occurred in the dry season, with the development of flower buds, flower opening and fruiting. Senescence of the shoots occurred in the rainy season (Fig. 2). Plants resprouted before the full senescence of the old aerial shoots.

Figure 2
Individuals of Chresta exsucca growing in Parque Nacional da Chapada dos Veadeiros, GO, Brazil at different phenological phases: resprouting (A), vegetative growth (B), flower buds (C), flower opening (D), fruiting (E) and senescence of the aerial organs (F).

Water and carbohydrate content

The water content of the underground organs of C. exsucca was lower in resprouting plants (rainy season) and during flower opening (dry season), compared to plants in vegetative growth (rainy season). Medians ranged from 52.9 % in flower opening to 67.37 % in vegetative growth (Fig. 3A). Total soluble carbohydrate (oligo- + polysaccharides) in the underground organs ranged 6 - 23 % (Fig. 3B). The oligosaccharide levels detected in plants with flower buds and fruiting were higher than those found in resprouting and senescence (Fig. 3C). Polysaccharide contents during senescence and in plants with flower buds were significantly higher than in plants undergoing flower opening (Fig. 3D). Oligosaccharide content was always higher than polysaccharide content, except during senescence, as demonstrated by the oligosaccharide:polysaccharide ratio, which was always greater than 1. The oligosaccharide:polysaccharide ratio during flower opening differed from that found during senescence (Fig. 3E), the only phase with values less than 1.

Figure 3
Box-plot showing water (A), total soluble carbohydrate (B), oligosaccharide (C), polysaccharide (D) content, and oligosaccharide:polysaccharide ratio (E) of underground organs of field-grown Chresta exsucca plants in different phenological phases. Resprouting (RE), vegetative growth (VG), flower buds (FB), flower opening (FO), fruiting (FR) and senescence of the aerial organs (SE), total soluble carbohydrate (TSC). Dashed line in E indicates oligosaccharide:polysaccharide ratio = 1. Different letters indicate different medians (n = 4, p < 0.05).

The percentage of glucose and sucrose during vegetative growth was higher than that found in fruiting and senescence. The opposite trend was observed for DP ( 3 sugars. Fructose percentage was also higher during vegetative growth compared to plants with flower buds and in senescence (Tab. 1).

Table 1
Percentages of glucose, fructose, sucrose and fructans with degree of polimerization ≥ 3 in total free and combined fructose in underground organs of field-grown Chresta exsucca plants. Different letters indicate different medians (n = 4, p < 0.05) comparing phenological phases for each sugar (columns).

Qualitative analysis of carbohydrates

Thickened underground organs of C. exsucca store inulin-type fructans. In all analyzed phases, glucose, fructose, sucrose, 1-kestose (DP3) and nystose (DP4) peaks were detected (Fig. 4). During resprouting, chromatograms showed oligosaccharides with higher glucose and fructose peaks than during the other phases. In the oligosaccharide profile, peaks with DP > 3 were low, while in the polysaccharide fraction DP > 3 peaks were pronounced. During vegetative growth, low DP sugar peaks remained high in both fractions, and high DP fructans in the polysaccharide fraction were substantially lower than in the resprouting chromatograms. The oligosaccharide chromatograms for plants developing floral buds showed lower glucose, fructose and sucrose peaks compared to the previous phases. In this phase the oligosaccharides of the inulin series peaks were well-defined, compared to the other phases. Some non-identified peaks, probably of the inulo-n-ose series were also present. In the polysaccharide fractions, low-DP sugar peaks were low, while high-DP fructans were well-defined and reached DP > 50. In the subsequent phase, flower opening, glucose and fructose peaks were as high as in resprouting and vegetative growth with well-defined DP > 3 sugar peaks in both fractions, including some non-identified peaks. At fruiting, low DP sugars and fructan peaks were present in both fractions, but were lower than in the previous phase, and there were no non-identified peaks. In senescence, glucose and fructose peaks were lower than in the other phases, oligosaccharide peaks were well defined and high-DP fructan peaks were higher than in the other phases, reaching DP > 50.

Figure 4
HPAEC/IPAD profiles of oligosaccharides and polysaccharides of underground organs of Chresta exsucca in different phenological phases. Glucose (G), fructose (F), sucrose (S), 1-kestose (1-K), degree of polymerization (DP). Arrows indicate unidentified peaks.

Discussion

Chresta exsucca has a diffuse underground system with numerous superficial parts with diagravitropic growth such that one individual can occupy a large area. Appezzato-da-Glória et al. (2008Appezzato-da-Glória B, Cury G, Soares MKM, Rocha R, Hayashi AH. 2008. Underground systems of Asteraceae species from the Brazilian Cerrado. The Journal of the Torrey Botanical Society 135: 103-113.) described a bud-forming diffuse underground system of radicular structure for the congener C. sphaerocephala. The aerial stems of a single individual of this species can be found up to 10 m distant from each other. This feature seems to be similar in C. exsucca, however, anatomical studies are necessary to confirm if they have cauline or root origin. The development of diffuse systems spread over a large area could enable the exploitation of areas of different soils, which could increase the possibility of obtaining water and minerals and the subsequent distribution of these resources among ramets. Evidence of physiological integration among ramets was observed in experimentally drought-stressed Populus balsamifera, where irrigated ramets supplied water for the connected water-deprived ramets (Adonsou et al. 2016Adonsou KE, DesRochers A, Tremblay F. 2016. Physiological integration of connected balsam poplar ramets. Tree Physiology 36: 797-806.).

Diffuse underground systems may bear buds (Appezzato-da-Glória et al. 2008Appezzato-da-Glória B, Cury G, Soares MKM, Rocha R, Hayashi AH. 2008. Underground systems of Asteraceae species from the Brazilian Cerrado. The Journal of the Torrey Botanical Society 135: 103-113.) and contribute to clonal reproduction, which is an important feature in ecosystems with limited resources and/or that experience climatic limitations (Couteron et al. 2014Couteron P, Anthelme F, Clerc M, Escaff D, Fernandez-Oto C, Tlidi M. 2014. Plant clonal morphologies and spatial patterns as self-organized responses to resource-limited environments. Philosophical Transactions of the Royal Society 372: 20140102 doi:10.1098/rsta.2014.0102
https://doi.org/doi:10.1098/rsta.2014.01...
). As diffuse systems grow to occupy a large space, they can come to represent a strong sink for photoassimilates, not only due to the demands of growth but also for storage, which, in the case of C. exsucca, occurs mainly as fructans (Fig. 4).

Water content

Water content detected in the underground organs of field grown C. exsucca during flower opening and resprouting was lower than that during vegetative growth. In the underground organs of I. terminalis, water content was found influenced mainly by soil water availability (Almeida et al. 2017Almeida LV, Ferri PH, Seraphin JC, Moraes MG. 2017. Seasonal changes of fructans in dimorphic roots of Ichthyothere terminalis (Spreng.) Blake (Asteraceae) growing in Cerrado. Science of The Total Environment 598: 404-412.). In general, water content of the sampled underground organs was lower than that reported for other species. For example, rhizophores of irrigated C. obovata plants had 85 % water content (Garcia et al. 2011Garcia PM, Asega AF, Silva EA, Carvalho MAM. 2011. Effect of drought and re-watering on fructan metabolism in Vernonia herbacea (Vell.) Rusby. Plant Physiology and Biochemistry 49: 664-670.), while the tuberous roots of irrigated A. discolor had 80 % (Oliveira et al. 2013Oliveira VF, Silva EA, Zaidan LBP, Carvalho MAM. 2013. Effects of elevated CO2 concentration and water deficit on fructan metabolism in Viguiera discolor Baker. Plant Biology 15: 471-482.). Dimorphic thickened roots of field-grown I. terminalis plants had 61-85 %, with the highest values being during the rainy season (Almeida et al. 2017Almeida LV, Ferri PH, Seraphin JC, Moraes MG. 2017. Seasonal changes of fructans in dimorphic roots of Ichthyothere terminalis (Spreng.) Blake (Asteraceae) growing in Cerrado. Science of The Total Environment 598: 404-412.). Our results suggest that water content in underground organs may also be affected by phenological shifts and developmental stage, since plants in resprouting and in vegetative growth were collected at the same time.

Water content of underground organs varies among species of Asteraceae, as well as fructan content (Tertuliano & Figueiredo-Ribeiro 1993Tertuliano MF, Figueiredo-Ribeiro RCL. 1993. Distribution of fructose polymers in herbaceous species of Asteraceae from the cerrado. New Phytologist 123: 741-749.), because storage of resources depends on the relative distribution of tissues in a specific organ (Moraes et al. 2013Moraes MG, Chatterton NJ, Harrison PA, Filgueiras TS, Figueiredo-Ribeiro RCL. 2013. Diversity of non-structural carbohydrates in grasses (Poaceae) from Brazil. Grass and Forage Science 68: 165-177. ). During dry periods, water maintenance in vegetative organs depends on the interaction of different factors including the presence of osmoprotectants that regulate osmotic adjustment (Singh et al. 2015Singh M, Kumar J, Singh S, Singh VP, Prasad SM. 2015. Roles of osmoprotectants in improving salinity and drought tolerance in plants: a review. Reviews in Environmental Science and Bio/Technology 14: 407-426.). Soluble carbohydrates are significant osmotic-active compounds and fructans stand out because they are reserve carbohydrates, protect membranes and proteins and removing reactive forms of oxygen (Peshev et al. 2013Peshev D, Vergauwen R, Moglia A, Hideg É, Ende W. 2013. Towards understanding vacuolar antioxidant mechanisms: a role for fructans? Journal of Experimental Botany 64: 1025-1038.), which are features that contribute for plant preservation during environmental constraints.

Phenology

The timing of phenological shifts in C. exsucca differs from that of other species of Asteraceae from the Cerrado, as exemplified by C. obovata (Carvalho & Dietrich 1993Carvalho MAM, Dietrich SMC. 1993. Variation in fructan content in the underground organs of Vernonia herbacea (Vell.) Rusby at different phenological phases. New Phytologist 123: 735-740.; Rigui et al. 2015Rigui AP, Gaspar M, Oliveira VF, Purgatto E, Carvalho MAM. 2015. Endogenous hormone concentrations correlate with fructan metabolism throughout the phenological cycle in Chrysolaena obovata. Annals of Botany 115: 1163-1175.), A. discolor (Isejima & Figueiredo-Ribeiro 1993Isejima EM, Figueiredo-Ribeiro RCL. 1993. Fructan variations in tuberous roots of Viguiera discolor Baker (Asteraceae): the influence of phenology. Plant and Cell Physiology 34: 723-727.; Itaya et al. 1999Itaya NM, Figueiredo-Ribeiro RCL, Buckeridge MS. 1999. Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae). Brazilian Journal of Medical and Biological Research 32: 435-442.) and I. terminalis (Almeida et al. 2017Almeida LV, Ferri PH, Seraphin JC, Moraes MG. 2017. Seasonal changes of fructans in dimorphic roots of Ichthyothere terminalis (Spreng.) Blake (Asteraceae) growing in Cerrado. Science of The Total Environment 598: 404-412.). These species have a well-defined phenological cycle with seasonal development, characterized by resprouting in the beginning of the rainy season, senescence and abscission of the aerial organs at the end of the rainy season, and dormancy of the underground organs in the dry season. In the Cerrado, a great proportion of the herbaceous species flower with increased rainfall (Batalha & Martins 2004Batalha MA, Martins FR. 2004. Reproductive phenology of the cerrado plant community in Emas National Park (central Brazil). Australian Journal of Botany 52: 149-161.). However, in C. exsucca flowering occurred during a 3-4 month period in the dry season and senescence of the aerial organs in the rainy season. Furthermore, resprouting was continual throughout the year. These differences suggest the occurrence of distinct strategies to regulate water content and metabolism of reserve compounds, since the use of these resources may be high due to intense resprouting and flowering in the dry season.

Since C. exsucca has a short period without aerial organs, relative to other herbaceous species, the extended presence of leaves indicates the potential for the synthesis of photoassimilates, even in the dry season. Studies on photosynthesis of herbaceous species of the Cerrado are scarce, but native trees are known to have several photosynthetic strategies for the rainy and dry seasons. Some species reduce the rate of carbon assimilation in the dry season, while others maintain rates unchanged. There are also tree species with reduced carbon assimilation rates even in the rainy season (Palhares et al. 2010Palhares D, Franco AC, Zaidan LBP. 2010. Respostas fotossintéticas de plantas do cerrado nas estações seca e chuvosa. Revista Brasileira de Biociências 8: 213-220.). This suggests that herbs and shrubs may have several photosynthetic strategies for responding to climatic seasonality.

Soluble carbohydrates

Soluble carbohydrates, predominantly as inulin-type fructans (Fig. 4), are present in 5 to 33 % of the dry mass of the underground organs of C. exsucca (Fig. 3B). These carbohydrates are the main reserve compounds for species of Asteraceae (Hendry 1993Hendry GAF. 1993. Evolutionary origins and natural functions of fructans - a climatological, biogeographic and mechanistic appraisal. New Phytologist 123: 3-14.). Carbohydrate storage occurs when photoassimilate production exceeds the demand for growth and maintenance of metabolism (Pollock 1986Pollock CJ. 1986. Fructans and the metabolism of sucrose in vascular plants. New Phytologist 104: 1-24. ), which varies according to phenological phase and shifts in environmental factors. Phenological shifts modify source-sink relationships in fructan accumulators. In phases of high energy demand, such as regrowth and flowering, higher oligosaccharide levels are expected, while in phases with intense sugar translocation for storage organs, such as the senescence of aerial organs, polysaccharide content is higher. Such a phenological shift was also found for C. obovata (Carvalho & Dietrich 1993Carvalho MAM, Dietrich SMC. 1993. Variation in fructan content in the underground organs of Vernonia herbacea (Vell.) Rusby at different phenological phases. New Phytologist 123: 735-740.; Portes & Carvalho 2006Portes MT, Carvalho MAM. 2006. Spatial distribution of fructans and fructan metabolizing enzymes in rhizophores of Vernonia herbacea (Vell.) Rusby (Asteraceae) in different developmental phases. Plant Science 170: 624-633.), Cichorium intybus (Ende & Laere 1996aEnde W, Laere A. 1996a. Fructan synthesizing and degrading activities in chicory roots (Cichorium intybus L.) during field-growth, storage and forcing. Journal of Plant Physiology 149: 43-50.), A. discolor (Itaya et al. 1999Itaya NM, Figueiredo-Ribeiro RCL, Buckeridge MS. 1999. Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae). Brazilian Journal of Medical and Biological Research 32: 435-442.), Taraxacum officinale (Wilson et al. 2001Wilson RG, Kachman SD, Martin AR. 2001. Seasonal changes in glucose, fructose, sucrose, and fructans in the roots of dandelion. Weed Science 49: 150-155. ) and I. terminalis (Almeida et al. 2017Almeida LV, Ferri PH, Seraphin JC, Moraes MG. 2017. Seasonal changes of fructans in dimorphic roots of Ichthyothere terminalis (Spreng.) Blake (Asteraceae) growing in Cerrado. Science of The Total Environment 598: 404-412.).

Variation in fructan level among phenological phases of C. exsucca was similar to that reported for other species of Asteraceae. Higher oligosaccharide levels occurred in vegetative growth until fruiting (Fig. 3C). The chromatograms in these phases confirm this observation (Fig. 4). However, in resprouting, vegetative growth and flower opening, the peaks of glucose, fructose and low DP sugar were higher, as occurred in A. discolor, in which oligosaccharides of up to DP4 were predominant during sprouting and flowering (Itaya et al. 1999Itaya NM, Figueiredo-Ribeiro RCL, Buckeridge MS. 1999. Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae). Brazilian Journal of Medical and Biological Research 32: 435-442.). The distribution of fructan molecules with varied DP is the result of the activity of fructan metabolizing enzymes. Higher free fructose peaks suggest intense 1-FEH activity or side 1-FFT activity transferring free fructose to water (Ende et al. 1996Ende W, Mintiens A, Speleers H, Onuoha AA, Laere A. 1996. The metabolism of fructans in roots of Cichorium intybus during growth, storage and forcing. New Phytologist 132: 555-563. ; Roover et al. 1999Roover J, Laere A, Ende W. 1999. Effect of defoliation on fructan pattern and fructan metabolizing enzymes in young chicory plants (Cichorium intybus). Physiologia Plantarum 106: 158-163.). Generally, 1-FEH has low activity, but it can be stimulated by defoliation, sprouting, flowering and environmental factors such as cold (Roover et al. 1999Roover J, Laere A, Ende W. 1999. Effect of defoliation on fructan pattern and fructan metabolizing enzymes in young chicory plants (Cichorium intybus). Physiologia Plantarum 106: 158-163.; Vergauwen et al. 2000Vergauwen R, Ende W, Laere A. 2000. The role of fructan in flowering of Campanula rapunculoides. Journal of Experimental Botany 51: 1261-1266. ; Asega & Carvalho 2004Asega AF, Carvalho MAM. 2004. Fructan metabolising enzymes in rhizophores of Vernonia herbacea upon excision of aerial organs. Plant Physiology and Biochemistry 42: 313-319. ; Asega et al. 2011Asega AF, Nascimento JRO, Carvalho MAM. 2011. Increased expression of fructan 1-exohydrolase in rhizophores of Vernonia herbacea during sprouting and exposure to low temperature. Journal of Plant Physiology 168: 558-565.). The field-grown plants in uncontrolled environmental conditions studied here were thus subjected to a set of factors and phenological demands that resulted in the obtained fructan profiles. Since the monosaccharide peaks of plants with flower buds and fruiting were not as high as in resprouting, vegetative growth and flower opening (Fig. 4), photoassimilate production may have a greater contribution to supporting the energetic requirements of these phases, without the need of intensify fructan hydrolysis in the underground organs, even at the beginning of the dry season.

During senescence of aerial organs, the oligosaccharide:polysaccharide ratio was < 1, which is the result of the prevalence of polysaccharides relative to oligosaccharides during this phase (Fig. 3C-E), as supported by the chromatogram profile (Fig. 4). This indicates intense fructan synthesis and polymerization, which are catalyzed by 1-SST and 1-FFT, respectively. Increased 1-SST activity is induced by high sucrose levels (Ende & Laere 1996bEnde W, Laere A. 1996b. De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots ( Cichorium intybus L.). Planta 200: 335-342.), which could be a result of photoassimilate translocation from the senescing shoots towards underground organs, as is known to occur in Chrysolaena obovata (Rigui et al. 2015Rigui AP, Gaspar M, Oliveira VF, Purgatto E, Carvalho MAM. 2015. Endogenous hormone concentrations correlate with fructan metabolism throughout the phenological cycle in Chrysolaena obovata. Annals of Botany 115: 1163-1175.). In all other phases, oligosaccharide levels were higher than polysaccharide levels, as expressed by a oligosaccharide:polysaccharide ratio > 1 (Fig. 3C- E), indicating the constant use of photoassimilates to sustain plant development, especially flowering in the dry season. When the oligosaccharide:polysaccharide ratio is < 1, the sink activity of the underground organs may be intense for carbon storage. In addition to phenology, environmental factors may also influence this ratio, as noticed in water-stressed plants of C. obovata, which exhibited an increase in the oligosaccharide:polysaccharide ratio (Garcia et al. 2011Garcia PM, Asega AF, Silva EA, Carvalho MAM. 2011. Effect of drought and re-watering on fructan metabolism in Vernonia herbacea (Vell.) Rusby. Plant Physiology and Biochemistry 49: 664-670.; 2015Garcia PMA, Hayashi AH, Silva EA, Figueiredo-Ribeiro RCL, Carvalho MAM. 2015. Structural and metabolic changes in rhizophores of the Cerrado species Chrysolaena obovata (Less.) Dematt. as influenced by drought and re-watering. Frontiers in Plant Science 6: 721 doi: 10.3389/fpls.2015.00721
https://doi.org/doi: 10.3389/fpls.2015.0...
).

In this work we verified that phenology has a strong influence on fructan dynamics in the underground organs of field-grown C. exsucca plants. Fructan storage supplies the energetic demands for the development and maintenance of the complex structure of the underground organs of C. exsucca, which is essential for the persistence of this species in the environment of the Cerrado, and to ensure reproduction in harsh conditions, such as drought.

Acknowledgements

Camila S. Santos thanks FAPEG for a research grant (Process 17671, 04/2015). The authors also thank the Instituto Chico Mendes de Biodiversidade for permission to collect in Parque Nacional da Chapada dos Veadeiros, and Rogério Neves Ribeiro for species identification.

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Publication Dates

  • Publication in this collection
    06 Nov 2017
  • Date of issue
    Jan-Mar 2018

History

  • Received
    30 May 2017
  • Accepted
    11 Sept 2017
Sociedade Botânica do Brasil SCLN 307 - Bloco B - Sala 218 - Ed. Constrol Center Asa Norte CEP: 70746-520 Brasília/DF. - Alta Floresta - MT - Brazil
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