Science of the Total Environment 598 (2017) 404–412

Seasonal changes of fructans in dimorphic roots of Ichthyothere terminalis (Spreng.)

Blake (Asteraceae) growing in Cerrado
Lorrayne Veloso de Almeida a, Pedro Henrique Ferri b, José Carlos Seraphin c, Moemy Gomes de
Moraes a,⁎
a Programa de Pós Graduação em Biodiversidade Vegetal, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Av.
Esperança s/n, Campus Samambaia, Goiânia, GO 74690-900, Brazil b Instituto de Química, Universidade Federal de Goiás,
Goiânia, GO 74690-900, Brazil c Instituto de Matemática e Estatística, Universidade Federal de Goiás, Goiânia, GO 74690-900,
Brazil
HIGHLIGHTS
• Rainfall seasonality influences phenolo- gy of herbaceous species in Cerrado.
• Ichthyothere terminalis is an herb with fructan-storing dimorphic roots.
• Orthogravitropic and diagravitropic roots have similar fructan oscillations.
• I. terminalis has a fructan pool that can be used in environmental constrains.
• Rainfall reduction strongly influenced carbohydrate levels in both roots.
⁎ Corresponding author at: Universidade Federal de Goiás, Instituto de Ciências Biológicas, Av. Esperança, s/n, 74690-900
Goiânia, GO, Brazil.
E-mail address: moemy@ufg.br (M.G. de Moraes).
GRAPHICALABSTRACT
a r t i c l e i n f o a b s t r a c t Article history: Received 10 February 2017 Received in revised form 12 April 2017 Accepted 13
April 2017 Available online 25 April 2017
Editor: Elena Paoletti
Cerrado is a floristically rich savanna in Brazil, whose vegetation consists of a physiognomic mosaic, influenced by rainfall
seasonality. In the dry season rainfall is substantially lower and reduces soil water supply, mainly for herbs and subshrubs.
Climatic seasonal variations may well define phenological shifts and induce fluctuations of plant reserve pools. Some Cerrado
native species have thickened underground organs that bear buds and store reserves, as adaptive features to enable plant survival
following environmental stresses. Asteraceae species accumulate fructans in storage organs, which are not only reserve, but also
protecting compounds against the ef- fects of cold and drought. Ichthyothere terminalis is one Asteraceae species abundant in
cerrado rupestre, with un-
Keywords:
derground organs consisting of thickened orthogravitropic and diagravitropic roots.
The objectives of this study Inulin
were to analyze how abiotic environmental factors and plant phenology influence
fructan dynamics in field Cerrado rupestre
grown plants, and verify if fructan metabolism differs in both root types for one
year. I. terminalis accumulates Drought
inulin-type fructans in 10–40% of the dry mass in both root types. Fructan
dynamics have similar patterns de- Phenology Underground organ
scribed for other Asteraceae species, exhibiting a proportional increase of polysaccharides with the senescence of the aerial
organs. Multivariate analyzes showed that, as rainfall decreased, environmental factors had a stron- ger influence on metabolite
levels than phenological shifts in both root types. Only slight differences were found in fructan dynamics between
orthogravitropic and diagravitropic roots, suggesting they may have similar fructan metabolism regulation. However, these small
differences may reflect distinct microclimatic conditions in both root types and also represent the influence of sink strength.
© 2017 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scitotenv.2017.04.100 0048-9697/© 2017 Elsevier B.V. All rights reserved.
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1. Introduction
Cerrado is a floristically rich savanna located in the central region of Brazil. Its vegetation is composed of a mosaic of
physiognomies, ranging from open grasslands to woodlands, with intermediary forms between them (Ratter et al., 1997). One of
the main environmental determinants of the Cerrado vegetation is rainfall seasonality. Rainfall occurs mainly in spring-summer
(October–March), when monthly average ranges from 150 to 500 mm. However, in autumn-winter (April–September) monthly
rainfall is reduced to between 0 and 50 mm (Silva et al., 2008), which depletes soil-water supplies, mainly for herbaceous spe-
cies (Rossatto et al., 2013). Besides climatic seasonality, fire incidence is another key feature of the Cerrado, which are more
frequent in open physiognomies than in woodlands, and occur mainly in the dry season (Pereira-Júnior et al., 2014). These
restrictions affect herba- ceous-shrubby plants, in which most of the species lose the aerial or- gans in the dry season and resprout
during the rainy season (Mantovani and Martins, 1988).
Frequent and unpredictable changes in environmental factors re- quire traits that allow plant survival. Carbohydrate storage
may buffers plants against temporal changes in external as well as in internal growth conditions (Suzuki and Stuefer, 1999).
Variations in environmental fac- tors throughout the year may induce seasonal fluctuations of plant re- serves, including
non-structural carbohydrates (NSC). NSC pools vary according to phenological patterns and the source-sink balance (Würth et
al., 2005). Therefore, the presence of well-developed peren- nial underground organs, with plenty reserves and viable buds are
im- portant adaptive traits of Cerrado-native plants that assure survival after long periods of water shortage and fire occurrence
(Appezzato-da-Glória et al., 2008; Mantovani and Martins, 1988; Ratter et al., 1997).
Several Cerrado herbaceous species have different types of under- ground organs, with distinctive morphological characteristics,
propor- tion of storage tissues, and the type of stored compounds (Moraes et al., 2016). Among them, several Asteraceae species
have thickened bud-bearing underground organs, which accumulate fructans as the major reserve (Appezzato-da-Glória et al.,
2008; Figueiredo-Ribeiro, 1993). Fructans are fructose polymers, occurring in nearly 15% of the Angiosperms, including the
derived families Poaceae and Asteraceae (Hendry, 1993). These carbohydrates are prominent plant NSC, surpassed only by starch
and sucrose (Hendry and Wallace, 1993). They can be found at high levels in storage organs, reaching up to 80% of the dry mass
of certain species, such as Helianthus tuberosus L. (Edelman and Jefford, 1968), Cynara cardunculus L. (Raccuia and Melilli,
2010) and Chrysolaena obovata (Less.) Dematt. (previously named Vernonia herbacea (Vell.) Rusby) (Carvalho and Dietrich,
1993). These and other Asteraceae species accumulate inulin-type fructans, which have β(2,1)-linked fructosyl units on sucrose
(Hendry, 1993). In- ulin synthesis starts with the transfer of one fructosyl unit from a donor to an acceptor sucrose, catalyzed by
the enzyme sucrose:sucrose 1- fructosyltransferase (1-SST), producing the trisaccharide 1-kestose. Inu- lin polymerization results
from the action of the enzyme fructan:fructan 1-fructosyltransferase (1-FFT), which transfers fructosyl units from a donor to an
acceptor fructan molecule with degree of polymerization (DP) ≥ 3 (Edelman and Jefford, 1968; Van den Ende, 2013). The activity
of these enzymes results in fructan molecules with unequal length (Vijn and Smeekens, 1999). Inulin catabolism occurs with the
removal of ter- minal fructosyl units from fructans, catalyzed by the enzyme fructan 1- exohydrolase (1-FEH) (Van den Ende et
al., 2000).
As storage compounds, fructans support high energy demanding developmental phases, such as sprouting and reproduction
(Mellado-Mojica and López, 2012; Raccuia and Melilli, 2010). Further- more, they are essential for plant recovery after
disturbances, such as fire occurrence, that cause damage to aerial parts (Asega and Carvalho, 2004; De Roover et al., 1999;
Oliveira et al., 2012). In addition to its re- serve function, fructan metabolism is associated with resistance to cold
405 L.V. de Almeida et al. / Science of the Total Environment 598 (2017) 404–412
and drought, through osmotic regulation, membrane protection and scav- enging of free radicals (Garcia et al., 2011; Hincha et
al., 2007; Hisano et al., 2004; Peshev et al., 2013), protecting underground organs during dry seasons.
Variations in total fructose contents and in fructan molecular mass were described in distinct phenological phases for
Cerrado-native spe- cies of Asteraceae and Amaranthaceae growing in the subtropical region (Carvalho and Dietrich, 1993;
Isejima and Figueiredo-Ribeiro, 1993; Silva et al., 2013; Vieira and Figueiredo-Ribeiro, 1993). All these studies showed high
oligosaccharide levels during sprouting and an increase of polysaccharides in the dry season, following the senescence of the
aerial parts. Recently, the same pattern was shown for the temperate Helianthus tuberosus L. (Krivorotova and Sereikaite, 2014)
and for the Mediterranean Cynara cardunculus L. (Raccuia and Melilli, 2010). How- ever studies on seasonal variations of
fructans in species growing in the Cerrado core with distinct types of underground organs are still scarce. Ichthyothere terminalis
(Spreng.) Blake (Asteraceae–Millerieae) is abundant in cerrado rupestre, a Cerrado phytophysiognomy where plants grow on
 rocky outcrops, with acidic and nutrient-poor soil, scarce water and frequent fires (Abdalla et al., 2016; Ribeiro and Walter,
2008). Its thickened underground system is dimorphic and constituted of a proximal region located close to the soil surface, with
several buds from which aerial stems originate. Below this portion, the main axis (root origin) is orthogravitropic (growing
vertically oriented). The me- dian region is soft and has a fleshy texture, with both short thin and long thickened diagravitropic
roots, growing alongside the soil surface. All regions of this underground system accumulate inulin-type fructans (Abdalla et al.,
2016).
In this study, we collected orthogravitropic and diagravitropic roots of field-grown I. terminalis plants for 12 months, which
comprised rainy and dry seasons, as well as different phenological phases. Our objectives were to analyze how abiotic
environmental factors and plant phenology influence fructan dynamics in field grown plants, and to verify if fructan metabolism
differs in the two root types during the year. As dimorphic roots allow plants to optimize water absorption in different substrate
regions between seasons (Oliveira et al., 2016), we hypothesize that seasonality of environmental factors would affect fructan
concentra- tions in both root types in different ways.
2. Materials and methods
2.1. Study site
This study was carried out in a preserved area in Cerrado core at Reserva Biológica “Prof. José Ângelo Rizzo” of the
Universidade Federal de Goiás, Mossâmedes, Goiás, Brazil (16°22′–15°48′S and 50°44′– 49°55′W). This area includes a gradient
of Cerrado physiognomies, such as rocky outcrops savanna, known as cerrado rupestre. The altitude varies from 700 to 1080 m
(Almeida et al., 2015) and the climate is Köppen's Aw (Silva et al., 2008).
Underground organs of adult plants of I. terminalis were collected monthly, from February 2012 to January 2013. The voucher
was depos- ited in the Herbarium of the Universidade Federal de Goiás (UFG, 47501).
The climatic data of this period from the closest meteorological sta- tion (Goiás, GO, A014) were accessed in “Instituto
Nacional de Meteorologia” homepage (www.inmet.gov.br) (Fig. 1). Adult plants were marked with wooden sticks to facilitate
finding the underground organs after abscission of aerial parts during the dry season. The most recent fire that occurred in the
study area was in August 2010, seven- teen months before the beginning of plant collection.
2.2. Plant material
Underground organs of four plants of I. terminalis with similar aerial developmental stages were collected each month as
mentioned above.
The median region of thickened orthogravitropic and diagravitropic roots were chosen for this study because inulin spherocrystals
are wide- ly distributed in these tissues (Abdalla et al., 2016).
Roots were washed immediately after harvest to remove soil par- ticles. Healthy and viable parts from the median regions of
the two root types were pooled independently. Samples (2 g) of each part were cut in small pieces and immediately boiled in
aqueous ethanol (80%) for enzyme denaturation and stored (−20 °C) for subsequent soluble carbohydrate extraction. The ethanol
with dissolved carbo- hydrates was kept and incorporated to the crude extract. Aliquots (1 g) were oven dried (60 °C) to constant
weight for dry mass (DM) determination. Water content (WC) was determined as a per- centage of the fresh mass.
2.3. Soluble carbohydrate extraction and analyses
Samples boiled in ethanol were homogenized and the suspension filtered. The filtrate was reserved and the plant residue was
re-extracted twice with boiling ethanol (80 °C) for 5 min. The residue was then ex- tracted twice with distilled water at 60 °C for
30 min (modified from Pollock and Jones, 1979). In all steps filtrates were separated from resi- dues by vacuum filtering through
filter paper. Crude extracts, which consisted of the pooled soluble phases, were vacuum-concentrated in a rotary evaporator at 37
°C. Aliquots of the crude extracts (1 mL) were fractionated into fructo-oligosaccharides (FOS) and fructo-poly- saccharides
(FPS) by cold precipitation by adding to the extracts three volumes of ethanol (100%), stored overnight at −20 °C and centrifuga-
tion at 177 ×g for 10 min at 5 °C. The supernatants were composed of mainly hexoses, sucrose and low molecular weight sugars
while the precipitates contained the FPS fractions (Abdalla et al., 2016; Carvalho et al., 1998).
406 L.V. de Almeida et al. / Science of the Total Environment 598 (2017) 404–412
Fig. 1. Monthly means of climatic parameters registered from February 2012 to January 2013 in Serra Dourada, Mossâmedes,
Goiás, Brazil, according to the meteorological station closest to the study site. Maximum (circles), mean (filled diamonds) and
minimum temperatures (squares) (A). Rainfall (bars) and relative air humidity (circle) (B).
Water soluble carbohydrates (WSC) in the crude extracts were quantified by a colorimetric assay with phenol-sulfuric acid
using glucose as a standard (Dubois et al., 1956). Total fructose (TF) was determined in crude extracts, FOS and in FPS fractions
using a kestose-specific assay with anthrone reagent and fructose as stan- dard (Jermyn, 1956).
Crude extracts were deionized using ion exchange resins (Amberlite cationic - IRA 120 and anionic - IRA 410, VETEC,
Duque de Caxias, Brazil). Following pH neutralization with ammonium hydroxide, the extracts were vacuum-dried at 37 °C and
solubilized in ultrapure water (18.2 MΩ) to the final concentration of 400 μg mL−1 of fruc- tose equivalents. Deionized extracts
were filtered through 0.45 μm membranes prior the analysis by high performance anion exchange chromatography with
integrated pulsed amperometric detection (HPAEC/IPAD) on a Dionex system ICS-5000, using a CarboPac PA100 column (4 ×
250 mm). Soluble neutral carbohydrates were separated by a gradient of sodium acetate (500 mM) in sodium hy- droxide (100
mM), as described by Silva et al. (2015). Extracts of C. obovata rhizophores were used as a reference for inulin series (Carvalho
and Dietrich, 1993).
2.4. Statistical analysis
Average multiple comparisons were performed by Kruskal- Wallis one-way ANOVA on ranks, with Dunn's post hoc test (p b
0·05).
To perform multivariate analysis, roots metabolite contents (re- sponse matrix, 96 × 6), as well as the monthly climatic
averages (five parameters) of the sampling time and the corresponding plant phenological stages, as categorical variable
(explanatory ma- trix, 96 × 6) were submitted to the canonical redundancy analysis (RDA). RDA revealed an ordination of the
response data constrained by explanatory variables, which accounts for the patterns of the only explained variation between data
sets. Unrestricted Monte Carlo permutation tests (999 permutations) were performed to assess the significance of canonical axes,
showing the relationships be- tween metabolite variables and the selected climatic and phenolog- ical predictors (Lepš and
Šmilauer, 2003). In all the analyses, the variance inflation factor of the variable (VIF) with Bonferroni's cor- rection was used to
determine the selection of the explanatory vari- ables. VIF-values ≥ 10 were considered as multicollinears (Lepš and Šmilauer,
2003).
The hierarchical cluster analysis (HCA) was used aiming to detect the natural sample grouping from the RDA analyses and
their intra and intergroup relations. Euclidean distance was used as similarity index and the hierarchical grouping was done
according to the variance minimization method (Ward, 1963).
 Total variation partitioning of response data was obtained by partial RDAs (pRDAs), using the explanatory matrix reordered in
two sets of se- lected variables: phenological (senescence, sprouting and reproduction) and environmental (average monthly
minimum temperature and rainfall precipitation) data. This method resulted in distinct variation fractions with or without overlap
between both sets of variables (Borcard et al., 1992; Legendre and Legendre, 2003).
In addition to the previous techniques, principal response curve (PRC) was applied to investigate the effects of the response
variables and their alterations in time (Ter Braak and Šmilauer, 2012; Van den Brink and Ter Braak, 1999). In PRC, sampling
months were used as categorical covariable and the interaction between sampling time and treatment (roots) were used as
explanatory variables. The anal- ysis produces a diagram which shows the time gradient in x-axis and the first or the second
canonical axes of metabolite differences of the treatment in relation to a control, here attributed to orthogravitropic roots, in y-axis
(Moser et al., 2007). Monte Carlo permutation tests were performed to evaluate if the PRC explains a significant part of the
variance of the treatment in relation to control in all the time
series (999 permutations), as well as to verify if the treatment results in one metabolite significantly altered in each sampling time
(499 permutations).
Before multivariate analyses, data were pre-processed: the response matrix was centered in the mean and standardized (all
variables have
407 L.V. de Almeida et al. / Science of the Total Environment 598 (2017) 404–412
Fig. 2. Box plot diagrams showing water content (A, B), total water soluble carbohydrates (WSC) (C, D), total fructose (E, F),
fructo-oligosaccharides (FOS) (G, H), fructo-polysaccharides (FPS) (I, J) in orthogravitropic (A, C, E, G, I) and diagravitropic
roots (B, D, F, H, J) of field-grown I. terminalis. Crosses represent medians (n = 4). Phenological phases were: Reproduction (R),
senescence of the aerial organs (Se), dormancy (D), sprouting (Sp) and vegetative growth (VG).
means equal to zero and variances equal to one), so that they have the same importance in the multivariate analysis. The
explanatory matrix was also centered and standardized. The multivariate analyses were conducted in Canoco (Canonical
Community Ordination, version 5.0, Biometrics, The Netherlands, 2012).
3. Results and discussion
3.1. Phenology and water content
I. terminalis has a developmental cycle with vegetative growth and reproduction in the rainy season and senescence of aerial
parts and dor- mancy of the underground organs in the dry season. In the present study, in February 2012, most of I. terminalis
population in the Biological Reserve was at reproduction, exhibiting inflorescences and fruits until March 2012 (end of the rainy
season). Senescence of aerial parts began in April 2012, and was followed by dormancy of underground or- gans. Buds
resprouted and originated new aerial stems at the end of September, restarting the developmental cycle. Flowering began in
January 2013.
The phenological cycle of Cerrado herbaceous species is highly influ- enced by rainfall, as in other seasonal neotropical
environments (Chambers et al., 2013; Mantovani and Martins, 1988). In the Cerrado, rainfall is positively correlated with the
number of flowering herba- ceous species (Batalha and Martins, 2004). However, in several Cerrado species some phenological
events are synchronized by occurrence of fire (Coutinho, 1990). In I. terminalis flowering was observed 1to 2 months after a fire
event (Abdalla et al., 2016 and field observations). During the present study, I. terminalis plants were not subjected to fire, since
the previous fire occurred in August 2010.
Changes in water content of the underground organs in I. terminalis were influenced by rainfall precipitation. Water content in
roots ranged from 61 to 85%, with lower levels in dry season and higher levels at the onset of rainfall (Fig. 2A, B). Higher water
contents were found in No- vember and December in orthogravitropic roots, and in March and De- cember (p b 0.05) in
diagravitropic roots. Lower levels were detected in May, July and August (p b 0.05) for orthogravitropic roots and in August (p b
0.05) for diagravitropic roots. In September and October, even with low rainfall, water content in roots started to increase. Both
root types showed similar oscillations in water content, except in March, when it increased in diagravitropic roots. The difference
between seasons is probably due to variations in soil moisture as pointed out by Dimitrakopoulos and Bemmerzouk (2003).
3.2. Water soluble carbohydrate dynamics
In orthogravitropic roots, WSC levels were higher in October, reaching 393.38 mg g−1 DM, during vegetative growth, and
lower in February, May and June (p b 0.05) (Fig. 2C). In diagravitropic roots, WSC ranged from 123.73 mg g−1 DM in August to
229.92 mg g−1 DM in October (Fig. 2D). Both root types appeared to retain at least 10% of their dry mass in WSC throughout
the year. Resprouting species, such as I. terminalis, have increased amounts of NSC stored in underground organs to support
resprouting (Clarke et al., 2013). Stored carbohy- drates allow plants to develop, even during phases when photosynthet- ic aerial
organs are absent (Landhäusser and Lieffers, 2003). Carbohydrate storage occurs when assimilation exceeds demands for
maintenance and growth (Chapin et al., 1990). In I. terminalis, water sol- uble carbohydrates oscillated in the analyzed period,
indicating alterna- tion of phases with high carbohydrate production and phases of carbohydrate mobilization and use (Fig. 2).
Inulin-type fructans are the main water soluble carbohydrates accu- mulated in underground organs of I. terminalis (Abdalla et
al., 2016). In this study, fructans were the major component (35–80%) of the WSC pool (Fig. 2 E, F), present as a linear
homologous series that co-eluted with the inulin series from C. obovata rhizophores. Fructan storage in underground organs of
Asteraceae from Cerrado is an adaptive feature to environmental constrains (Carvalho et al., 2007). Fructans support the
high-energy demanding phenological phases, such as sprouting and flowering (Carvalho et al., 2007; Edelman and Jefford, 1968;
Silva et al., 2013).
408 L.V. de Almeida et al. / Science of the Total Environment 598 (2017) 404–412
The highest total fructose concentrations were in the beginning of the rainy season. Interestingly, in April, at the beginning of
aerial organs senescence, total fructose levels increased in both root types, especially the FPS fraction (Fig. 2I, J). This could be
due to sugar mobilization to the underground organs, since a large fraction of carbon compounds and mineral nutrients are
translocated to the underground organs during leaf senescence (Lapointe, 2001). These carbohydrates seem to be the energy
source to promote rapid vegetative development and reproduc- tion after constraints, such as prolonged drought periods and fires.
Throughout the developmental phases, the fructan profile displayed a balance between synthesis and mobilization in both root
types, ev- idenced by the variation in FOS and FPS proportions (Fig. 2G, H, I, J). In high-demanding phenological phases, such
as sprouting, vegeta- tive growth and reproduction, FOS contents increased. However, taking into account the ratio FOS:FPS
(Fig. 3A, B), FPS prevailed from April to October/November (orthogravitropic/diagravitropic), when FOS:FPS was ≤1. Inversely,
in December, just before inflores- cence development, a sharp increase occurred and the ratio remained N1 during reproduction.
Chromatograms showed glucose, fructose, sucrose, and inulin-type fructans present throughout all the analyzed months (Fig.
4). Monosac- charide peaks increased at the end of dormancy and remained higher during sprouting and vegetative growth until
the onset of reproduction in January 2013. At the end of the vegetative growth (December 2012), fructan peaks were the lowest
among all the analyzed phases, and sucrose and 1-kestose peaks predominated. By the end of the reproduc- tion in March, and
during senescence and dormancy, these peaks were lower while fructo-oligo- and fructo-polysaccharide peaks were higher,
especially in orthogravitropic roots, and DP reached values N 40 at the end of dormancy (August 2012). Intermediary
non-identified peaks, probably of the reducing inulo-n-ose series, were observed in both
Fig. 3. Box plot showing fructo-oligosaccharides:fructo-polysaccharides (FOS:FPS) ratio in orthogravitropic (A) and
diagravitropic (B) roots of field grown I. terminalis. Crosses represent medians (n = 4). Dotted lines indicate FOS:FPS = 1.
Phenological phases were: Reproduction (R), senescence of the aerial organs (Se), dormancy (D), sprouting (Sp) and vegetative
growth (VG).
root types in all phenological phases, except in senescence. This series lacks the terminal glucose and is found in several
Asteraceae species, generally in periods of intense inulin hydrolysis (Gupta and Kaur, 2000; Carvalho et al., 1997; Ueno et al.,
2011; Van Arkel et al., 2012).
3.3. Relationships between climate, phenology and fructans
In phenological studies, it is relatively common to detect correlations between climatic variables and a particular phenological
response; however multiple climatic factors are expected to co-vary (Forrest and Miller-Rushing, 2010). Thus multivariate
analyses are frequently used to detect interactions among several factors simultaneously.
The variability patterns of I. terminalis samples were evaluated by the RDA of the root metabolites levels conditioned to
climatic parame- ters, as well as the plant phenological phases. At the end of response data modelling by RDAs, two climatic
parameters were obtained (min- imum temperature and rainfall precipitation) in addition to three plant phenological phases
(senescence, sprouting and reproduction) as ex- planatory variables (Fig. 5).
RDA results indicated that correlations between both data matrices were higher in the first two canonical axes (R1 = 0.703 and
R2 = 0.453) and with variance inflation factors considered low (VIF b 2.3), suggesting no multicollinearity among the variables in
multivariate re- gression models (Lepš and Šmilauer, 2003). Monte Carlo permutation
409 L.V. de Almeida et al. / Science of the Total Environment 598 (2017) 404–412
Fig. 4. HPAEC/IPAD profiles of soluble carbohydrates extracted from dimorphic roots of field-grown I. terminalis during an
annual developmental cycle. Arrows indicate inulo-n-ose peaks. Phenological phases were: Reproduction (R), senescence of the
aerial organs (Se), dormancy (D), sprouting (Sp) and vegetative growth (VG). Glucose (G), fructose (F), sucrose (S), 1- kestose
(1-K).
tests (999 permutations) showed highly significant results for the first two canonical axes (RDA1: 20.9% of the explained
variation, F = 23.8, p = 0.001; RDA2: 5.6%; F = 6.8, p = 0.008), indicating that variation patterns of the original matrices did not
arise by chance. The sum of the canonical axes was also significant (sum = 0.2860; F = 7.2; p = 0.001), so that 28.6% of the total
variance of the metabolites was retained by selected explanatory variables.
According to RDA triplot distribution, the increase in RDA1 axis is as- sociated, especially, to a decline in metabolites as
rainfall decreases, co- inciding with dry and cold winter, independently of the sampled root type. These conditions are mainly
related to samples harvested in the period of the aerial organs senescence and suggest a seasonal influence on RDA1. The results
support the recognition of rainfall as one of the main drivers for phenological shifts in tropical South American seasonal
environments (Chambers et al., 2013), since these changes may be in- fluenced by the assimilation and allocation of reserves
(Rathcke and Lacey, 1985).
Differently, an increase on RDA2 is related to higher levels of total water soluble carbohydrates, FPS and total fructose, which
occurred mainly in plants in senescence and sprouting phases, independently of the sampled root. Higher FOS levels, water
content and the increase in FOS:FPS ratio are related to reproduction, coincidently with months with higher rainfall precipitation.
Thus, while RDA2 shows mainly the phenological variations of the samples, especially in senescence,
sprouting and reproduction, RDA1 described, mainly, the variation of the metabolites in I. terminalis roots, as a response to water
shortage.
In this way, three natural groups of samples were suggested by RDA, which were corroborated by the HCA, using the RDA's
scores as vari- ables. Fig. 6 shows the similarities between individual roots in terms of Euclidean distances, in which samples
collected during the dry period (April–September), in senescence and sprouting phases constituted class 1. The samples collected
between October and December, with higher minimum temperatures provided class 2, while samples in re- production, during
January and March, composed class 3.
Although RDA/HCA contributed significantly to the general compre- hension of data, suggesting a strong influence of the
seasonal effect followed by a lower contribution of the phenological phases in I. terminalis, little can be deduced about the
importance of each one to
Fig. 6. Dendrogram of similarity representing the hierarchical clustering of I. terminalis samples based on RDA scores, using
Euclidean distance and variance minimization technique. The samples are represented by the month of collection and
orthogravitropic (O) or diagravitropic (D) roots.
410 L.V. de Almeida et al. / Science of the Total Environment 598 (2017) 404–412
Fig. 5. RDA triplot showing the distribution of I. terminalis samples according to root metabolites (small arrows) explained by
phenological stage (senescence, sprouting and reproduction) and climate (rainfall and minimum temperature). The centroids of
the phenological stages are represented by triangles, while the climatic variables are represented by large arrows.
the total explained variance by the canonical axes, that is, those involv- ing response and explanatory matrices.
One method developed by Borcard et al. (1992) and improved by Peres-Neto et al. (2006) allows the partitioning of the
explained vari- ance, using partial RDAs (pRDAs). The procedures assess the relative im- portance of constraining matrix after
adjusting the variability of other data sets which are regarded as covariate. In this study, variation partitioning was performed on
two separate sets, one containing the plant phenological phase (senescence, sprouting and reproduction) and the other containing
environmental data (selected climatic vari- ables) as explanatory matrices (Table 1).
Results indicated that the total explained variation in metabolites re- sponse matrix (28.6%; fraction [a + b + c], Table 1) can
be partitioned by the two descriptor sets, resulting in a model whose residue was 71.4% ([d]). Among explained variation's
fractions with purer contribu- tion, without overlapping, the environmental set stood out (21.0%; [a]), and phenology represented
7.4% ([b]) of the total variance, all highly significant (p ≤ 0.002). These results are in accordance with RDA analy- sis, whose
variances explained by the two first canonical axes (RDA1: 20.9%; RDA2: 5.6%) were attributed to the environmental effects and
the phenological influence, respectively. It can also be noticed that the total contribution of the environmental set corresponds to
21.2% ([a + c]; p = 0.001), while the phenological predictor ([b + c]; p = 0.011) explains 7.6% of the total variability. The fraction
constituted of the overlapping of descriptors sets effects showed a small contribution (0.2%; [c]).
One alternative to better understand the partitioning of the total ex- plained variation can be obtained by the visualization of
Venn's diagram (Fig. 7). The results of the total variation partitioning indicated that ap- proximately three-quarters of the
explained variation can be modeled by climatic variables, that is, the variation in I. terminalis root metabo- lites are mainly
determined by the environment.
Aiming to evaluate if the differences between metabolite levels in roots are altered along sampling time, PRC was performed
between orthogravitropic (control) and diagravitropic roots (treatment) for the 12 month sampling period (Fig. 8). The results
indicated that 95% of the total variation of the data can be explained by treatment effect, 81.7% of which are shown in the first
PRC diagram (F = 11.6, p = 0.023; 999 permutations). The differences among the sampling
months contributed to the majority of total variance (50.5%), and the remaining part (31.6%) can be attributed to the differences
among samples.
The variables that mostly influenced the first PRC axis were the FPS and WSC levels, followed by FOS and total fructose
levels, with higher weights. The FOS:FPS ratio and water did not show any contribution to the differentiation between samples.
The second PRC axis was not significant and is not presented. When the data were restricted to each time period, a discrete
difference (0.054 b p b 0.082) was observed in the period between July and September, time that coincides with dor- mancy and
sprouting phases. In the beginning of this period, the metab- olites had a sharp decrease in their levels in diagravitropic roots
(treatment), followed by an increase at the end relative to the control. Phenology influences spatial differences in fructan
metabolism, with in- creased activity of fructan hydrolytic enzymes during sprouting near the proximal and bud-bearing region
(Portes and Carvalho, 2006). These differences may indicate that in phases with high energetic demand, both root types, despite
having similar fructan dynamics, exhibit a fructan metabolism modulated by local and temporal changes attending to the
source-sink relationships.
4. Conclusions
During a 12 month period without a drastic disturbance, such as fire, naturally field-grown plants of I. terminalis stored
approximately 10– 40% of inulin-type fructans in dimorphic roots. This means that orthogravitropic and diagravitropic roots
share the same function as storage organ. Fructan dynamics have similar patterns described for other Asteraceae species, with the
proportional increase of polysaccha- rides with the senescence of the aerial organs, as exampled byC. obovata
Table 1 Variance partitioning summary of I. terminalis through partial RDAs.
Effects and variables
F Pa
Total effect Environmental and
phenological
Covariables Fraction of
variation
Explained variation (%)
[a + b + c] 28.6 7.2 0.001
Partial effects Environmental Phenological [a] 21.0 13.3 0.001 Environmental [a + c] 21.2 12.5 0.001 Phenological
Environmental [b] 7.4 3.1 0.002 Phenological [b + c] 7.6 2.5 0.011 Joint effects Environmental and
phenological
[c] 0.2
Residuals [d] 71.4
a Based on the Monte Carlo permutation test (999 permutations). Phenological pre- dictor: senescence, sprouting and
reproduction; environmental predictor: average monthly rainfall and minimum temperature.
Fig. 7. Venn diagram of total variation partitioning in metabolites from I. terminalis roots which is attributable to two sets of
explanatory variables: phenological phases and environmental predictors. Percentages refer to explained variation by each
fraction of variation, according to Table 1. Unexplained variance corresponds to residuals.
411 L.V. de Almeida et al. / Science of the Total Environment 598 (2017) 404–412
and Gomphrena species (Carvalho and Dietrich, 1993; Silva et al., 2013; Vieira and Figueiredo-Ribeiro, 1993). However, in this
study, multivari- ate analyses showed that environmental factors, such as rainfall de- crease, had a stronger influence on
metabolite levels in both root types than phenological shifts. Future experiments with controlled con- ditions and water
withholding are necessary to confirm this finding. Only slight differences were found in fructan dynamics between
orthogravitropic and diagravitropic roots, thus they may have similar fructan metabolism regulation. These differences may reflect
distinct microclimatic conditions in both regions and also represent the influ- ence of sink-strength in carbon mobilization. Thus,
both root types can be analyzed in futures studies concerning fructan metabolism in this promising fructan accumulator species.
Acknowledgments
This study was supported by FAPEG (PRONEX AUX PESQ 007/2009). L.V. Almeida is grateful to CAPES, Brasília, Brasil
for a study grant. P.H. Ferri is a CNPq research fellow. The authors are grateful to Dr. Maria Angela M. Carvalho for critical and
English review and to Cinara F. Abraão for lab support.
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