Maria Angela M. Carvalho

Effect of drought and re-watering on fructan metabolism in Vernonia herbacea (Vell.) Rusby.

Vernonia herbacea (Vell.) Rusby, a native species from the Brazilian Cerrado, accumulates about 80% of fructans in the rhizophores, the underground reserve organs. Besides their role as reserve, fructans have been recognized as protective compounds against drought. This physiological function attributed to fructans seems consistent with the wide occurrence of these carbohydrates in the cerrado, a biome that undergoes seasonal drought. The aim of this work was to analyze fructan composition and the activities of the enzymes involved in fructan synthesis, sucrose:sucrose 1-frutosyltransferase (1-SST) and fructan:fructan 1-frutosyltransferase (1-FFT), and depolymerization, fructan 1-exohydrolase (1-FEH) in plants submitted to water suppression. The plants were divided into 3 groups receiving 3 treatments: daily watering (control), water suppression for 23 days (WS) and re-watering after 15 days (RW). Samples were taken at the beginning of the experiment (Time 0) and after 3, 7, 11, 15, 17 and 23 days of water suppression. 1-SST and 1-FFT activities increased at the beginning of the water restriction period, coinciding with a decrease in 1-FEH activity, the onset of the reduction in soil water potential and in leaf water potential. Increases in 1-FEH and invertase activities led to a high yield of reducing sugars at the 23rd day after water suppression, and together with 1-FFT, 1-FEH also seemed to act in the redistribution of fructan molecules after re-watering. The increase in reducing sugars and in the fructo-oligo:fructo-polysaccharide ratio were associated to the maintenance of rhizophore turgor. Considering that WS plants showed changes in fructan metabolism that favored water retention and absorption after re-watering, the occurrence of osmotic adjustment mechanisms is suggested, reinforcing the hypothesis of fructans as protective agents against abiotic stresses, such as drought.

Low temperature and defoliation affect fructan-metabolizing enzymes in different regions of the rhizophores of Vernonia herbacea.

In addition to the storage function, fructans in Asteraceae from floras with seasonal growth have been associated with drought and freezing tolerance. Vernonia herbacea, native of the Brazilian Cerrado, bears underground reserve organs, rhizophores, accumulating inulin-type fructans. The rhizophore is a cauline branched system with positive geotropic growth, with the apex (distal region) presenting younger tissues; sprouting of new shoots occurs by development of buds located on the opposite end (proximal region). Plants induced to sprouting by excision of the aerial organs present increased 1-fructan exohydrolase (1-FEH) activity in the proximal region, while plants at the vegetative stage present high 1-sucrose:sucrose fructosyltransferase (1-SST) in the distal region. The aim of the present study was to analyze how low temperature (5 degrees C) could affect fructan-metabolizing enzymes and fructan composition in the different regions of the rhizophores of intact and excised plants. 1-SST and 1-fructan:fructan fructosyltransferase (1-FFT) were higher in the distal region decreasing towards the proximal region in intact plants at the vegetative phase, and were drastically diminished when cold and/or excision were imposed. In contrast, 1-FEH increased in the proximal region of treated plants, mainly in excised plants subjected to cold. The ratio fructo-oligo to fructo-polysaccharides was significantly higher in plants exposed to low temperature (1.17 in intact plants and 1.64 in excised plants) than in plants exposed to natural temperature conditions (0.84 in intact vegetative plants and 0.58 in excised plants), suggesting that oligosaccharides are involved in the tolerance of plants to low temperature via 1-FEH, in addition to 1-FFT. Principal component analysis indicated different response mechanisms in fructan metabolism under defoliation and low temperature, which could be interpreted as part of the strategies to undergo unfavorable environmental conditions prevailing in the Cerrado during winter.

Increased expression of fructan 1-exohydrolase in rhizophores of Vernonia herbacea during sprouting and exposure to low temperature

Rhizophores of Vernonia herbacea, an Asteraceae found in the Brazilian Cerrado, store high amounts of fructans that vary in composition over the phenological cycle. Fructan 1-exohydrolase (1-FEH) activity is detectable during the sprouting phase, mainly in the proximal regions of rhizophores, of plants induced to sprout by defoliation and/or cold storage. We found an increase in 1-FEH gene expression during natural and induced sprouting and further enhancement through low-temperature treatment. Furthermore, a comparative analysis of 1-FEH gene expression in different regions of the rhizophores during the transition from dormancy to sprouting is presented. Transcripts were detected mainly in the proximal region, coinciding with high 1-FEH activity and a high concentration of free fructose. Low temperature promoted the accumulation of fructans of a low degree of polymerization (DP) and enhanced 1-FEH activity and gene expression. It is hypothesized that a set of 1-FEH proteins acts in two different ways during fructan mobilization: (1) by hydrolyzing fructo-oligosaccharides and -polysaccharides in sprouting plants (naturally or induced) for carbon supply and (2) by hydrolyzing preferably fructo-polysaccharides under low temperature to maintain the oligosaccharide pool for plant cold acclimation.

Fructan variation in the rhizophores of Vernonia herbacea (Vell.) Rusby, as influenced by temperature

The influence of climatic variations on fructan content in tropical regions is not well known. The present study deals with the effects of temperature on fructan contents in rhizophores of plants of Vernonia herbacea, a native species from the Brazilian cerrado vegetation. Intact plants and fragmented rhizophores were subjected to different temperatures under natural and controlled environmental conditions. Rhizophores of plants in pre-dormant stage (aerial parts showing some yellowish leaves) presented higher fructan content at 5oC than those kept at 25oC, whereas in dormant plants (aerial parts absent) temperature treatments did not affect fructan contents. Fragmented rhizophores obtained from dormant plants presented higher levels of fructo-polysaccharides at the end of the experiment than at the beginning of the treatment, regardless of the temperature they were stored, whereas fragments obtained from vegetative plants showed a decrease in fructan content under the same treatments. It was concluded that variations observed in fructan contents are related to the phenological state of the plants prior to the treatment rather than to extraneous temperatures they are subjected to during this stage.

Cloning, Characterization and Functional Analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5 degrees C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly beta-2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81% in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.

Endogenous hormone concentrations correlate with fructan metabolism throughout the phenological cycle in Chrysolaena obovata

BACKGROUND AND AIMS Chrysolaena obovata, an Asteraceae of the Brazilian Cerrado, presents seasonal growth, marked by senescence of aerial organs in winter and subsequent regrowth at the end of this season. The underground reserve organs, the rhizophores, accumulate inulin-type fructans, which are known to confer tolerance to drought and low temperature. Fructans and fructan-metabolizing enzymes show a characteristic spatial and temporal distribution in the rhizophores during the developmental cycle. Previous studies have shown correlations between abscisic acid (ABA) or indole acetic acid (IAA), fructans, dormancy and tolerance to drought and cold, but the signalling mechanism for the beginning of dormancy and sprouting in this species is still unknown. METHODS Adult plants were sampled from the field across phenological phases including dormancy, sprouting and vegetative growth. Endogenous concentrations of ABA and IAA were determined by GC-MS-SIM (gas chromatography-mass spectrometry-selected ion monitoring), and measurements were made of fructan content and composition, and enzyme activities. The relative expression of corresponding genes during dormancy and sprouting were also determined. KEY RESULTS Plants showed a high fructan 1-exohydrolase (EC 3.2.1.153) activity and expression during sprouting in proximal segments of the rhizophores, indicating mobilization of fructan reserves, when ABA concentrations were relatively low and precipitation and temperature were at their minimum values. Concomitantly, higher IAA concentrations were consistent with the role of this regulator in promoting cell elongation and plant growth. With high rates of precipitation and high temperatures in summer, the fructan-synthesizing enzyme sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) showed higher activity and expression in distal segments of the rhizophores, which decreased over the course of the vegetative stage when ABA concentrations were higher, possibly signalling the entry into dormancy. CONCLUSIONS The results show that fructan metabolism correlates well with endogenous hormone concentrations and environmental changes, suggesting that the co-ordinated action of carbohydrate metabolism and hormone synthesis enables C. obovata to survive unfavourable field conditions. Endogenous hormone concentrations seem to be related to regulation of fructan metabolism and to the transition between phenophases, signalling for energy storage, reserve mobilization and accumulation of oligosaccharides as osmolytes.

Sub-tropical urban environment affecting content and composition of non- structural carbohydrates of Lolium multiflorum ssp. italicum cv. Lema

This study analyzed the relationship between environmental factors, especially air pollution and climatic conditions, and non-structural carbohydrates (NSC) in plants of Lolium multiflorum exposed during 10 consecutive periods of 28 days at a polluted site (Congonhas) and at a reference site in São Paulo city (Brazil). After exposure, NSC composition and leaf concentrations of Al, Fe, Cu, Zn, Pb and Cd were measured. The seasonal pattern of NSC accumulation was quite similar in both sites, but plants at Congonhas showed higher concentrations of these compounds, especially fructans of low and medium degree of polymerization. Regression analysis showed that NSC in plants growing at the polluted site were explained by variations on temperature and leaf concentration of Fe (positive effect), as well as relative humidity and particulate material (negative effect). NSC in the standardized grass culture, in addition to heavy metal accumulation, may indicate stressing conditions in a sub-tropical polluted environment.

Elevated CO2 Atmosphere Minimizes the Effect of Drought on the Cerrado Species Chrysolaena obovata

Chrysolaena obovata stores inulin in the rhizophores, associated with drought tolerance. While crop plants are widely studied concerning the interactive effects of high [CO2] and drought, few studies reported these effects in native species. Here, we evaluated the combined effects of these factors on water status and fructan metabolism in C. obovata, a native Cerrado species. Two lots of plants were kept at 380 and 760 ppm CO2 in open-top chambers. In each, [CO2] plants were divided into four groups and cultivated under different water availability: irrigation with 100 (control), 75 (low), 50 (medium), and 25% (severe drought) of the water evapotranspirated in the last 48 h. In each, water treatment plants were collected at 0, 9, 18, and 27 days. On day 27, all plants were re-watered to field capacity and, after 5 days, a new sampling was made. Water restriction caused a decrease in plant moisture, photosynthesis, and in enzymes of fructan metabolism. These changes were generally more pronounced in 25% plants under ambient [CO2]. In the later, increases in the proportion of hexoses and consequent modification of the fructan chain sizes were more marked than under high [CO2]. The results indicate that under elevated [CO2], the negative effects of water restriction on physiological processes were minimized, including the maintenance of rhizophore water potential, increase in water use efficiency, maintenance of photosynthesis and fructan reserves for a longer period, conditions that shall favor the conservation of this species in the predicted climate change scenarios.