Maria Angela Machado de Carvalho

Elevated CO2 atmosphere promotes plant growth and inulin production in the cerrado species Vernonia herbacea

Carbon allocation in biomass is an important response of plants to the increasing atmospheric [CO2]. The effects of elevated [CO2] are scarcely reported in fructan-accumulating plants and even less in tropical wild species storing this type of carbohydrate. In the present study, the effects of high [CO2] atmosphere was evaluated on growth, biomass allocation and fructan metabolism in Vernonia herbacea (Vell.) Rusby, an Asteraceae from the Brazilian cerrado, which accumulates inulin-type fructans in the underground organs (rhizophores). Plants were cultivated for 120 days in open-top chambers (OTCs) under ambient (~380 μmol mol–1), and elevated (~760 μmol mol–1) [CO2]. Plant growth, photosynthesis, fructan contents, and the activities of fructan metabolising enzymes were analysed in the rhizophores at Time 0 and 15, 30, 60, 90 and 120 days. Plants under elevated [CO2] presented increases in height (40%), photosynthesis (63%) and biomass of aerial (32%) and underground (47%) organs when compared with control plants. Under elevated [CO2] plants also presented higher 1-SST, 1-FFT and invertase activities and lower 1-FEH activity. Although fructan concentration remained unchanged, fructan productivity was higher in plants maintained under elevated [CO2], due to their higher rhizophore biomass. This is the first report on the effects of elevated [CO2] on a plant species bearing underground organs that accumulate fructans. Our results indicate that plants of V. herbacea can benefit from elevated atmospheric [CO2] by increasing growth and carbon allocation for the production of inulin, and may contribute to predict a future scenario for the impact of this atmospheric condition on the herbaceous vegetation of the cerrado.

Soluções nutritivas para cultivo e produção de frutanos em plantas de Vernonia herbacea

The limited growth of rhizophores of Vernonia herbacea in Hoagland solution demanded the definition of a nutrient solution for plants of V. herbacea, aiming at the increase of the rhizophore biomass and fructan production. This solution, named Vernonia, is comprised of Ca(NO 3 ) 2 .4H 2 O 2.5 mmol L -1 , KNO 3 2.3 mmol L -1 , KH 2 PO 4 0.52 mmol L -1 , Mg(NO 3 ) 2 .6H 2 O 1.7 mmol L -1 and Na 2 SO 4 1.3 mmol L -1 . Its effect on plants was compared to that of Hoagland solution, both with different ionic strengths, 50%, 100% and 200%. The effect of the solutions on plant growth and fructan content was evaluated twice in a six-month period. Plants did not survive up to two months, when cultivated in 200% Hoagland solution. The 50% Vernonia solution was the most effective for rhizophore biomass increase and fructan production per plant. Growth of aerial organs was promoted in 100% Hoagland and Vernonia solutions. Compared to Hoagland, Vernonia solution contains less macronutrients, which confirms the hypothesis that plants adapted to the oligotrophic soils of the cerrado, as V. herbacea, demand less mineral nutrients to achieve full growth.

Growth, photosynthate partitioning and fructan accumulation in plants of Vernonia herbacea (Vell.) Rusby under two nitrogen levels

The effect of two nitrogen concentrations on fructan accumulation and plant growth was analysed in plants of Vernonia herbacea during a year period. Plants of this species accumulate inulin-type fructans in the underground reserve organs (rhizophores). The plants were cultivated in glasshouse conditions and received weekly nutrient solutions containing 1.3 mmol.L-1 NO3- (N-limited) or 10.7 mmol.L-1 NO3- (N-sufficient). Plants treated with N-sufficient solution presented an increase in total growth with higher biomass, more but smaller leaves resulting in higher total leaf area, higher net assimilation rate, specific leaf mass and higher biomass allocation to aerial organs. In addition, these plants presented higher contents of reducing sugars and lower fructan contents. In contrast, N-limited plants showed reduced total growth with lower biomass, higher biomass allocation to underground organs and higher fructan contents. Therefore, although N-limited plants presented a higher fructan concentration, the amount of fructan produced per plant was similar in both N treatments.

Structural and metabolic changes in rhizophores of the Cerrado species Chrysolaena obovata (Less.) Dematt. as influenced by drought and re-watering.

The high fructan contents in underground organs of Cerrado species, high water solubility, and fast metabolism of these compounds highlight their role as carbon storage and as an adaptive feature in plants under drought. In this study, we showed that anatomical structure, in association with soluble compounds and metabolism of inulin-type fructans were modified in rhizophores of Crysolaena obovata submitted to water suppression and recovery after re-watering. Plants were subjected to daily watering (control), suppression of watering for 22 days (water suppression) and suppression of watering followed by re-watering after 10 days (re-watered). Plants were collected at time 0 and after 3, 7, 10, 12, 17, and 22 days of treatment. In addition to changes in fructan metabolism, high proline content was detected in drought stressed plants, contributing to osmoregulation and recovery after water status reestablishment. Under water suppression, total inulin was reduced from approx. 60 to 40%, mainly due to exohydrolase activity. Concurrently, the activity of fructosyltransferases promoted the production of short chain inulin, which could contribute to the increase in osmotic potential. After re-watering, most parameters analyzed were similar to those of control plants, indicating the resumption of regular metabolism, after water absorption. Inulin sphero-crystals accumulated in parenchymatic cells of the cortex, vascular tissues and pith were reduced under drought and accompanied anatomical changes, starting from day 10. At 22 days of drought, the cortical and vascular tissues were collapsed, and inulin sphero-crystals and inulin content were reduced. The localization of inulin sphero-crystals in vascular tissues of C. obovata, as well as the decrease of total inulin and the increase in oligo:polysaccharide ratio in water stressed plants is consistent with the role of fructans in protecting plants against drought.

Frutanos em calos de Smallanthus sonchifolius (Poepp.) H. Rob

Smallanthus sonchifolius is an Asteraceae native from the Andes Mountains that accumulates inulin-type fructans in the underground reserve organs, the tuberous roots and the rhizophores. Yellow callus was obtained from tuberous roots, giving origin, spontaneously to red callus. Fructans and their metabolizing enzymes were analyzed in calli of both types, showing similar fructan contents in both lineages. However, synthesizing activity was higher in red callus, while hydrolyzing activity was higher in yellow callus. HPAEC/ PAD analyses revealed the presence of fructo-oligosaccharides in both calli with degrees of polymerization lower than those found in plants cultured ex vitro, indicating that this characteristic was maintained in undifferentiated cells. The presence of fructans and the differences between the activities of fructosyltransferases and hydrolases found in red and yellow calli indicate this material as suitable for studies of fructan metabolism under controlled conditions.

Morpho-anatomy and fructans in the underground system of Apopyros warmingii and Ichthyothere terminalis (Asteraceae) from the cerrado rupestre1

Abstract The cerrado rupestre is a physiognomy of the Cerrado biome with a prominent ground layer. It is characterized by rocky outcrops; acidic, nutrient-poor soil; and water shortage due to climatic seasonality. Species of this biome have several mechanisms to survive these adverse conditions. This study aimed to investigate morpho-anatomical characteristics and soluble carbohydrates in underground systems of Apopyros warmingii and Ichthyothere terminalis (Asteraceae), and to evaluate the potentially adaptive roles of these structures under the environmental stressors of the cerrado rupestre. Samples of underground organs were fixed, dehydrated in graded ethylic ethanol, embedded in historesin, and sectioned. Qualitative and quantitative analyses of soluble carbohydrates were performed in distinct portions of the underground systems. Thickened underground systems of both species are composed of stem and root portions with buds in the proximal region. Vascular and cortical parenchymas are abundant and accumulate inulin, which can be visualized as spherocrystals. Secretory structures have lipophilic secretion. High fructan amounts in the parenchyma indicate their use to support resprouting. These carbohydrates may be mobilized to meet the increased demands of phenology and abiotic factors. Secretory structures and sclereids in both species and raphides in A. warmingii may assist the protection of the underground system against herbivores.

Effects of different carbohydrate sources on fructan metabolism in plants of Chrysolaena obovata grown in vitro

Chrysolaena obovata (Less.) Dematt., previously named Vernonia herbacea, is an Asteraceae native to the Cerrado which accumulates about 80% of the rhizophore dry mass as inulin-type fructans. Considering its high inulin production and the wide application of fructans, a protocol for C. obovata in vitro culture was recently established. Carbohydrates are essential for in vitro growth and development of plants and can also act as signaling molecules involved in cellular adjustments and metabolic regulation. This work aimed to evaluate the effect of different sources of carbohydrate on fructan metabolism in plants grown in vitro. For this purpose, C. obovata plants cultivated in vitro were submitted to carbon deprivation and transferred to MS medium supplemented with sucrose, glucose or fructose. Following, their fructan composition and activity and expression of genes encoding enzymes for fructan synthesis (1-SST and 1-FFT) and degradation (1-FEH) were evaluated. For qRT-PCR analysis partial cDNA sequences corresponding to two different C. obovata genes, 1-SST and 1-FFT, were isolated. As expected, C. obovata sequences showed highest sequence identity to other Asteraceae 1-SST and 1-FFT, than to Poaceae related proteins. A carbon deficit treatment stimulated the transcription of the gene 1-FEH and inhibited 1-SST and 1-FFT and carbohydrate supplementation promoted reversal of the expression profile of these genes. With the exception of 1-FFT, a positive correlation between enzyme activity and gene expression was observed. The overall results indicate that sucrose, fructose and glucose act similarly on fructan metabolism and that 1-FEH and 1-SST are transcriptionally regulated by sugar in this species. Cultivation of plants in increasing sucrose concentrations stimulated synthesis and inhibited fructan mobilization, and induced a distinct pattern of enzyme activity for 1-SST and 1-FFT, indicating the existence of a mechanism for differential regulation between them.

Aspectos fisiológicos, anatômicos e ultra-estruturais do rizoma de Costus arabicus L. (Costaceae) sob condições de déficit hídrico

Costus arabicus L. is an herbaceous species, native to the forest that accumulates starch in the rhizome as the main reserve carbohydrate. Water stress is one the most important environmental factor that regulates plant growth and development, leading to changes in physiological, biochemical and anatomical traits. The aim of this study was to evaluate the effect of water deficit on the anatomy, carbohydrate concentration and ultrastructure of starch grains of the rhizome of Costus arabicus L. through the following water regimes: plants irrigated daily and every seven (7d) and 15 days (15d). Anatomical and ultrastructural traits of the rhizome, relative water content in leaves (RWC), leaf water potential (Ψw), osmotic potential of the rhizome (Ψo), total soluble sugars (TSS), reducing sugars (RS) and starch of the rhizome were evaluated. We observed significant reductions in Ψw and Ψs in 15d plants, but with small variations in the RWC. The anatomical analysis showed that the rhizome is covered by epidermis or stratified cork and there is a delimitation between the cortical and vascular regions. We detected the presence of numerous starch grains and idioblasts with phenolic contents in the parenchyma of the two regions. Changes in the anatomy of the rhizomes were only observed in plants irrigated every 15 days, in which cells of covering tissue and the outer cortical layers became more flattened and sinuous walls; however, no ultrastructural differences were observed on the starch grains. The water deficit imposed was sufficient to generate physiological, biochemical and anatomical changes in plants of C. arabicus L. Such alterations suggest the occurence of an avoidance mechanism and that anatomical changes were dependent of the intensity and duration of water deficit.

Fructan production in Vernonia herbacea (Vell.) Rusby is related to adequate nitrogen supply and period of cultivation

Estudos realizados anteriormente mostraram que plantas de Vernonia herbacea cultivadas durante um ano sob uma condicao limitante de nitrogenio tiveram seu crescimento reduzido e apresentaram maiores teores de frutanos em comparacao a plantas que receberam suprimento adequado de nitrogenio. Entretanto, a producao total de frutanos foi similar nos dois grupos de plantas devido a maior biomassa dos orgaos subterrâneos de reserva das plantas N-suficientes. O objetivo do presente estudo foi avaliar se o cultivo sob uma condicao adequada de nitrogenio, que promove o crescimento das plantas, seguido de uma condicao estressante de crescimento imposta por suprimento limitante de nitrogenio, promoveria a producao de frutanos. As plantas receberam durante um ano solucao N-suficiente (10,7 mmol L1 N-NO3). Nos seis meses subsequentes, metade das plantas passou a receber solucao N-limitante (1,3 mmol L1 N-NO3), permanecendo as demais no tratamento N-suficiente. O crescimento, a fotossintese e os carboidratos soluveis foram analisados no dia da transferencia (dia 0) e aos 30, 60, 90 e 180 dias. Aos 30 dias as plantas transferidas para N-limitado mostraram aumento significativo do crescimento e diminuicao da concentracao de frutanos, em resposta a condicao nutricional estressante. No entanto, no periodo seguinte (60 dias) o crescimento foi reduzido e a concentracao de frutanos aumentou em relacao as plantas mantidas em N-suficiente, confirmando a relacao inversa entre suprimento de nitrogenio e conteudo de frutanos. Apos 180 dias, embora a concentracao de frutanos em N-limitado tenha sido significativamente mais elevada, com uma producao de frutanos de 6,0 g planta-1, o maior ganho de biomassa de rizoforos observado nas plantas mantidas sob N-suficiente durante 18 meses, levou a uma producao de frutanos de 8,3 g planta1, compensando assim a menor concentracao de frutanos observada nessas plantas.

Development, characterization and cross-amplification of microsatellite markers for Chrysolaena obovata, an important Asteraceae from Brazilian Cerrado

Chrysolaena obovata (Less.) Dematt. (=Vernonia herbacea (Vell.) Rusby) is an Asteraceae species widely spread in the Brazilian Cerrado. The underground organs of this perennial herb, known as rhizophores, store up to 80% of its dry mass as inulin-type fructans (Carvalho and Dietrich 1993). Fructans are known as storage carbohydrates, able to confer tolerance to drought and low temperatures (Carvalho et al. 2007). Inulin is also recognized as a functional food ingredient, due to its beneficial roles in human health (Ritsema and Smeekens 2003). The Cerrado biome occupies 21% of the Brazilian territory, hosts a high biodiversity and has been classified as one of the world’s hot spots (Simon et al. 2009). Because of its intensive exploitation, the Cerrado area was drastically reduced leading to intense fragmentation of this biome (Sano et al. 2007). This fragmentation may result in loss of genetic variability due to genetic drift, inbreeding and reduced gene flow (Zucchi et al. 2003). Thus, the characterization of genetic structure and population dynamics of Cerrado species is crucial for the development of management strategies and conservation programmes.