• The annual developmental cycle of tuberous roots of Ranunculus asiaticus was studied with respect to structure and content of their cells, to understand how these roots are adapted to desiccation, high temperature and rehydration.
• Light microscopy, histochemical analysis, and protein analyses by SDS-PAGE were employed at eight stages of annual root development.
• During growth and maturation of the roots, cortical cells increased in size and their cell walls accumulated pectin materials in a distinct layer to the inside of the primary walls, with pits between adjoining cells. The number of starch granules and protein bodies also increased within the cells. Several discrete proteins accumulated. Following quiescence and rehydration of the roots there was a loss of starch and proteins from the cells, and cell walls decreased in thickness.
• The resurrection geophyte R. asiaticus possesses desiccation-tolerant annual roots. They store carbon and nitrogen reserves within their cells, and pectin within the walls to support growth of the plant following summer quiescence and rehydration.
Ranunculus asiaticus (Turban Buttercup, Persian Crowfoot) is a perennial geophyte with sparingly branched stems up to 30 cm high, bearing bright flowers varying in colour from white and yellow to pink and red. One of the most attractive native geophytes of the Mediterranean region, Ranunculus was introduced to horticulture many years ago and is now produced as an ornamental crop in southern California, France, Israel, and South Africa (De Hertogh, 1996). This species originated in southwestern Asia and the Mediterranean region, and in natural habitats is found on rocky or grassy hillsides, pastures, and foothills. In the wild, plants flower from Feb to May, and then enter a summer quiescence period for 5–6 months; active growth resumes with the onset of cooler temperatures and rain (Meynet, 1993). Underground storage organs of R. asiaticus are annual crowns with several renewal buds and tuberous roots, which form during vegetative growth of the parent plant in winter and spring. Following senescence and shriveling of the above-ground parts of the parent plant, these newly formed storage organs remain underground during summer and serve as a source of storage material during resumption of growth and initial formation of leaves in autumn.
Evolution of geophytes in climatic areas with marked seasonal changes has led to their adaptation to periods of high and low temperatures and/or drought. In order to survive extreme environments, geophytes have undergone adaptations that may include increased capacity for water binding, tolerance of and/or resistance to desiccation and drought and development of subterranean organs that contain specialized storage compounds (De Hertogh & Le Nard, 1993; Kamenetsky, 1997; Kamenetsky et al., 2003).
A major characteristic of geophytes is the presence of large quantities of one or more storage (reserve) compounds in their underground storage organs. A variety of carbohydrates, including starch, soluble sugars, glucomannans, and fructan are variously present in geophytes (De Hertogh & Le Nard, 1993; Miller et al., 1997). In addition, storage proteins may serve as a reserve of nitrogen in roots of several perennial species, particularly in members of the Compositae, Euphorbiaceae, and Leguminosae (Bewley, 2002). In some species, the amount of root proteins fluctuates seasonally. In spite of the fact that storage proteins are usually only a relatively small component of the total nitrogen pool within the root, their accumulation is associated with overwintering. There is a possibility that in herbaceous perennials, some proteins act as a temporary store for nitrogen, and are reutilized to support plant growth (Cyr & Bewley, 1989, 1990a,b).
The underground organs of R. asiaticus are adapted to survive summer drought and high temperatures in a state of quiescence, when the above-ground vegetative tissues desiccate and die. Contrary to many geophytes (e.g. bulbs), in which the process of organogenesis occurs in underground buds during the quiescence period (Hartsema, 1961; De Hertogh & Le Nard, 1993; Kamenetsky & Rabinowitch, 2002), the shoot apical meristem of R. asiaticus remains inactive during the 5–6 months of summer but is able to renew itself during the short winter period when rains occur and temperatures are mild (R. Kamenetsky, unpublished).
Only a small group of higher plants can tolerate such long periods of extreme heat and dehydration. This unique trait is usually restricted to specialized tissues of seeds and pollen but, in some species of monocots and dicots, vegetative tissues also show high drought tolerance (Bewley & Krochko, 1982; Proctor & Pence, 2002; Scott, 2000; Bernacchia & Furini, 2004). These species are able to dehydrate, remain quiescent during long periods of drought, and then resurrect upon rehydration.
Although cellular desiccation tolerance has been studied in seeds, seedlings and leaves of several plant species adapted to extreme drought (Walters et al., 2002), desiccation tolerance of underground storage organs, especially roots, is hardly known. As noted by Proctor & Pence (2002), desiccation tolerance is known in bulbils of Oxalis species and in underground storage organs of Anemone coronaria and Ranunculus asiaticus (Antipov & Romanyak, 1983), but ‘there seems to have been little systematic work to determine which of the more persistent of these are truly desiccation tolerant’. R. asiaticus represents a special type of resurrection geophyte, which survives unfavorable environmental conditions in the form of underground storage organs, and is ecologically adapted to an annual cycle of desiccation and resurrection; it may provide a useful model plant for investigating mechanisms of plant adaptations to long periods of heat and drought.
Here we provide evidence that changes occur to the structure and contents of the cells within tuberous roots of R. asiaticus during their seasonal cycle of desiccation and re-hydration. Light microscopy, histochemical and protein analyses were employed at eight stages of annual root development, including new root formation, desiccation, quiescence, rehydration and ultimately, root deterioration.
Materials and Methods
Plant material and growth conditions
Tuberous roots of Ranunculus asiaticus L., cv. Aviv, were produced from seeds during the 2002–03 growth season under field conditions in Israel (Asa Flower Bulbs, Bizaron, Israel). The mature underground crowns with tuberous roots were harvested from one isolated plot in April 2003, subsequently sorted for uniformity, cleaned, dried and stored at ambient conditions in Israel for 6 months. In October 2003, the dry crowns were transferred to Guelph, Canada. Before planting, they were imbibed in tap water for 24 h and then planted in a moist medium (Sunshine #4 Aggregate Plus substrate, SunGro Horticulture, Westerville, OH) in 2-l containers. The containers were initially stored at 4°C for 30 d. During root hydration and storage at low temperatures, roots were sampled at three time points in order to study the dynamics of structural changes (Table 1). On December 12, 2003, plants were placed in the glasshouse at a temperature of 14–16°C under natural photoperiod and were grown for 4 months until full flowering. To simulate natural environmental conditions, in April 15, 2004 growth temperatures were increased to 22–25°C, and irrigation was stopped. Fully formed underground storage organs were harvested in May 12, 2004.
Table 1. Sampling of tuberous roots of Ranunculus asiaticus at different stages of development
Dry tuberous roots were received from a commercial source in Israel. They were imbibed in moist mixture at 4°C for 30 d, and planted in the glasshouse at 14–16°C on December 12, 2003. During their growth, roots were collected from plants in a glasshouse at the University of Guelph. Root samples were used for histological observations, as well as for biochemical analysis for proteins.
Newly formed young white tuberous roots, 0.5–1 cm long, from plants in their growth stage. New plants were produced from the old tuberous roots planted in November 2003
Newly formed young white tuberous roots, 1–2 cm long, from plants in their growth stage
Newly formed young roots 3–5 cm long from plants in their growth stage, before flowering
Newly formed young roots from plants at the end of flowering; beginning of foliage senescence
Newly formed tuberous roots from plants with completely dry above-ground organs
Newly formed tuberous roots, dried for 1 month after harvest and stored at ambient temperatures
Old tuberous roots, after 1–3 h of hydration
Old tuberous roots, after 24 h of hydration and exposure to 5°C for 5 d
Old tuberous roots, after 24 h of hydration and exposure to 5°C for 30 d
Depleted tuberous roots from growing plants
Phenological observations were made throughout the plant growth cycle. Samples of tuberous roots for histological and biochemical studies were collected at different stages of plant development (Table 1).
Roots of four to six plants were sampled for water content at each stage of development. Water content was determined by drying the dissected roots in a forced oven at 80°C until constant weight was attained.
Roots were sampled at the times indicated in Table 1 and prepared for light microscopy, either by cutting hand sections with a two-sided razor blade or fixing root material in 2.5% glutaraldehyde in 0.07 m Sorensen's phosphate buffer, pH 6.8, dehydrating tissue in a graded ethanol series and embedding in LR White resin (CANEMCO, Lachine, Quebec, Canada). Sections of embedded material were cut with glass knives and heat-fixed to microscope slides before staining. For general observations of root anatomy and developmental changes, fresh and embedded tissues were stained either with 0.05% Toluidine Blue 0 (TBO) in citrate buffer, pH 4.5 (O’Brien & McCully, 1981) or with 1% methylene blue in 1% borax, counterstained with 1% aqueous basic fuchsin (Ruzin, 1999). Although roots at all stages of development were examined for anatomical characteristics, we concentrated mainly on the desiccation/rehydration stages for histochemical analyses of storage reserves and cell walls.
Histochemical analysis of cell walls
Fresh and embedded tissues were stained with 0.05% TBO in citrate buffer, pH 4.5 (O’Brien & McCully, 1981), 0.01% aqueous Calcofluor White M2R (Ruzin, 1999) and with 0.005% aqueous ruthenium red (Ruzin, 1999). As controls for pectin staining, fresh sections were treated at room temperature for 2 h with Pectolyase Y-23 (5 µg ml−1 in 0.1 m HEPES buffer, pH 4.7) obtained from Seishen Pharmaceutical, Tokyo, before staining with ruthenium red. Fresh sections were also treated with Aspergillus endo-β-mannanase (Megazyme, Cork, Eire) for 24 h to test for the presence of mannans in the thickened cell walls of tuberous roots.
Histochemical analysis of storage compounds
Fresh sections were stained with a 1% aqueous solution of I2KI for starch (Ruzin, 1999), 0.07% Sudan IV in 70% ethanol for lipids (Ruzin, 1999), and 1% aniline blue black in 7.0% acetic acid for proteins (O’Brien & McCully, 1981). Sections of embedded tissues were also stained for proteins using the latter method. Controls for protein involved treating sections of embedded tissue with a saturated solution of Proteinase K (Fermentas) in 0.1 m HEPES buffer, pH 4.7 for several hours at room temperature before staining with aniline blue black.
Roots for chemical analysis were rinsed in water, then rapidly immersed in liquid nitrogen, and subsequently stored at −80°C. For protein extraction, root samples (c. 130–140 mg) were rapidly ground in liquid nitrogen with a mortar and pestle with washed sea sand. Soluble proteins were extracted in 1.5 ml 0.1 m HEPES-KOH buffer, pH 8.0, and quantified before loading on the gel by the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA), using bovine serum albumin standard, diluted in 0.1 m HEPES-KOH buffer. The microassay procedure used the Microtiter Plate protocol (Bio-Rad, according to the manufacturer's instructions), with color development read on a spectrophotometer set to 595 nm.
Protein separation was performed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), as described by Cyr & Bewley (1990a). The separations were performed using 12% (w/v) acrylamide minigels. Seven µl of sample, containing c. 20 µg of protein were loaded in each lane.
Annual development cycle
Under our experimental conditions, Ranunculus asiaticus produced new leaves from reactivated shoot meristems in December and January. The first floral stems were visible in February, and flowering occurred in March. During the initial stages of plant growth, storage reserves within the tuberous roots were depleted causing them to shrivel. A new adventitious root system was formed from underground shoot tissues during the development of foliage leaves. Initially, the young root system consisted of enlarged basal parts, and thinner, slightly branched apical parts (Fig. 1, stages 1–3). Following flowering and senescence of the above-ground organs, the basal parts of the roots enlarged considerably, and a brown outer layer developed (Fig. 1, stage 4). At the end of the growing season, in April and May, the above-ground parts withered completely, new-formed tuberous roots became desiccated and remained underground, and plants entered a summer quiescence period (Fig. 1, stages 5–6). At this stage, the tuberous roots were harvested (Fig. 1, stage 5). In the wild, these roots remain underground during summer and serve as a source of storage material for growth resumption and the initial formation of foliage leaves during the subsequent autumn period. Under our experimental conditions, storage organs were dried and stored at ambient conditions.
Following the period of quiescence, re-hydration of the tuberous roots led to their swelling and to activity of the apical meristems (Fig. 1, stage 7). After spouting and leaf formation, the annual roots became depleted (Fig. 1, stage 8) and eventually disintegrated.
Structural changes during root development and histochemistry of cell walls Young adventitious roots (Fig. 1, stages 1–3) consisted of a vascular cylinder of alternating xylem and phloem with parenchymatous pith, and a cortex of thin-walled parenchyma cells with some intercellular space development (Fig. 2a,d,e). With continued development, roots increased in diameter (Fig. 1, stage 4), largely through an increase in the number of cortical cells, but also some cell enlargement in the cortex (Fig. 2b). At the beginning of April, following flowering (Fig. 1, stage 4), large vacuoles and accumulation of starch grains were visible in cortical parenchyma cells (Fig. 2b,f). During the senescence stage of the above-ground parts and at the end of the growing season, roots became mature (Fig. 1, stage 5). At this stage, cortical cell walls had thickened considerably and obvious pits were present in secondary cell walls (Fig. 2c,g).
In the initial stages of root development (Fig. 1, stages 1–4), cortical cells had only primary walls that stained positively for cellulose/hemi-cellulose (Fig. 3a). With root maturation, the secondary cell walls that were deposited showed very little birefringence because of the relatively low cellulose content relative to pectin compared with the primary cell walls when sections were viewed with a polarizing microscope with the polarizing filters in the crossed polars position. Secondary cell walls stained red with ruthenium red, indicating a high concentration of pectin (Jensen, 1962). Treatment of fresh sections of hydrated roots with endo-β-mannanase for 24 h did not diminish the amount of cell wall material (data not shown), showing that the thickened walls did not contain appreciable amounts of mannans.
Following quiescence and before planting, the dehydrated tuberous roots (Fig. 1, stage 7) were subjected to hydration and storage in moist medium at low temperatures. During this treatment, the cellular structure of tuberous roots was studied at three time points: after 1–3 h of hydration, after 24 h of hydration and 5 d of storage at low temperature, and after 24 h of hydration and 30 d of storage at low temperatures (Table 1). Dry roots contained c. 10% of water, and consequently, parenchyma cells were highly dehydrated. Hydration of the dry tuberous roots caused marked changes in their appearance (Fig. 1, stage 7) that was accompanied by rapid changes in the structure of the cortical cells.
Dehydrated roots that were allowed to rehydrate during fixation in 2.5% glutaraldehyde showed immediate swelling of secondary walls in the cortical cells (Fig. 3c). However, although the cell walls expanded very quickly (almost immediately after rehydration), the cytoplasm remained shrunken for a longer period of time (Fig. 3c). Roots fixed in glutaraldhyde and embedded in resin, showed swollen secondary cell walls with pits, the sites of plasmodesmatal connections between cortical cells (Fig. 3d).
In hand sections made from intact roots after 5 d of rehydration, which were then allowed to imbibe in TBO, marked changes in cell wall expansion were visible in cortical cells (Fig. 3e). Imbibition and storage for 30 d caused further enlargement of the secondary cell walls (Fig. 3f).
At the last stage of root development, during storage compound breakdown and root deterioration, the cortical cell walls became thinner, as the secondary layer gradually degraded (Fig. 3g).
The expanded secondary cell walls after 30 d of hydration stained positively with ruthenium red for pectins (Fig. 4a); controls treated with pectolyase before staining with ruthenium red (Fig. 4b) confirmed that these walls contained a high concentration of pectin.
Histochemical identification of storage compounds
Starch: Starch grains were present in cortical cells of roots during their development (Fig. 4c). The first appearance of these grains was visible in newly formed roots (Fig. 1, stages 3–4; Fig. 2f). A massive presence of starch was also characteristic of roots at maturation stage (Fig. 1, stage 5; Fig. 2g).
Lipids: Few lipid bodies were evident in roots at later stages of development, including in the rehydrated tuberous roots (Fig. 1, stage 7; Fig. 4d).
Proteins: Young roots lacked protein bodies and although some protein bodies were present in developing tuberous roots, they were most numerous during later stages of development. Protein bodies were especially evident in tuberous roots rehydrated for 1–3 h (Fig. 1, stage 7; Fig. 4e), as well as for roots rehydrated for 24 h and stored at low temperatures for 5 and 30 d (data not shown). Most of the protein bodies were removed by treatment with proteinase K (Fig. 4f).
Changes in water content during the annual cycle
Measurements of the water content in the roots revealed substantial changes during the annual cycle (Fig. 5). Young roots at stages 1–3 contained 80–92% water; desiccation at stages 5 and 6 resulted in quiescent roots containing only 8–9% water. Re-exposure of the roots to water resulted in a large increase in water content, and in depleted roots (stage 8) it was as high as 80%.
Changes in root proteins during the annual cycle
SDS-PAGE analysis of proteins extracted at stages 1–8 showed that they were prominent only from stages 4–7, there being a marked initial increase at the time of foliage senescence; their decline began during rehydration and exposure to cool temperatures, and they were depleted as the old roots degenerated (Fig. 6).
Traces of c. 44, 23, 14 and 12 kDa proteins were evident at stages 2 and 3, but there was a substantial increase in these and at least five other proteins between stages 3 and 4, with a peak in some at stage 5 (e.g. 44, 35, 23, and 14 kDa) and an c. 13 kDa protein at the next stage. By this stage the 44 kDa protein had declined substantially, to increase transiently at stage 7, along with an c. 19 kDa protein. The major changes occurred before the roots were subjected to desiccation and storage. The most prominent protein of 14 kDa remained at reduced amounts even in the depleted root (Fig. 1, stage 8).
While members of the Ranunculaceae are widely distributed in temperate and subtropical regions, R. asiaticus is common in the eastern Mediterranean and eastwards to southwest Asia. In its natural habitat, this perennial plant with annually replaced roots and monocarpic shoots is adapted to the extreme seasonal changes. Every year, tuberous roots form de novo during flowering and senescence of the plant in the spring, then desiccate and remain in the soil to survive summer drought and high temperatures for 5–6 months. New shoots develop in the autumn, when temperatures decrease and rains occur (Fig. 1; Gutterman, 2002).
R. asiaticus might be regarded as a type of resurrection geophyte, which is able to survive severe desiccation in the vegetative state of its underground organs. In general, the vast majority of desiccation-tolerant plants belong to the lower groups of the plant kingdom, but some members of angiosperms are also known for their desiccation tolerance (Gaff, 1977; Bewley & Krochko, 1982; Oliver & Bewley, 1997; Proctor & Pence, 2002). Most resurrection plant species are native to arid climates in the world such as southern Africa, southern America and Western Australia (Gaff, 1977; 1987). The growth and reproduction of these plants occur in wet seasons, but upon drying the plants can remain quiescent for a considerable time. Mechanisms that protect plants from water stress are also frequently effective against large fluctuations in temperature (Hartung et al., 1998) and other environmental stresses (Gaff & Wood, 1988; Gaff, 1989).
It was postulated that, in order to tolerate desiccation, resurrection plants have to overcome a number of stresses and to have evolved three main adaptations: first they are able to protect their tissues from excessive damage during drought stress; second they can maintain their tissues’ physiological integrity during dehydration; and third upon rehydration, they are able to repair any damage that has been caused by desiccation (Bewley, 1979; Bewley & Oliver, 1992; Swayze, 2004). Although these characteristics were proposed for the above-ground vegetative tissues, they are also relevant to the storage tuberous roots of R. asiaticus.
In the natural habitats of R. asiaticus, the beginning of the growing season in the autumn is unpredictable, but the beginning of the dry and hot season is more expected, and coincides with long photoperiods and gradual increases in temperature (Gutterman, 2002). Hence, adaptations of the plants to summer quiescence can be viewed as a mechanism to protect them from desiccation-induced cellular damage. As the roots of R. asiaticus grow in the spring, their cortical tissues undergo marked changes in structure: relatively small cells with primary cell walls, large vacuoles and negligible amounts of storage compounds (Figs 2a,d,e, 3a, 5 and 6) develop into larger cells with secondary walls and appreciable amounts of starch and protein bodies (Figs 2c,f,g, 3b, 5 and 6). Similar to other geophytes (De Hertogh & Le Nard, 1993), starch accumulation begins in the roots following flowering and senescence of underground parts (Fig. 1, stage 4; Fig. 2f), which coincides with root tuberization.
Root desiccation is accompanied by modulation of the mechanical properties of the cell wall during dehydration. A similar process was shown in the leaf tissue of the model resurrection species Craterostigma wilmsi: an extensive shrinkage during drying and dehydration induced an alteration of polysaccharide content and structure in the cell wall (Vicréet al., 2004). In Ranunculus, this transformation is accompanied by rapid water loss in root tissues. The extent of desiccation of the vegetative tissues of R. asiaticus is very high (8–10% of water), compared with other geophytes which contain fairly high moisture levels (c. 70% of water), but have limited desiccation ability (De Hertogh & Le Nard, 1993). Such low water content, known only for seeds and several resurrection plant species, provides evidence of high ability to maintain physiological integrity of root tissues during their desiccation period.
It was not possible to study the root cellular structure in their dried state during the quiescence period, because all employed microscopic techniques utilized liquid components for tissue fixation or staining, and thus resulted in cell rehydration. Hence, the condition of cell walls and cytoplasm contents in desiccated roots remains undetermined.
During rehydration of the roots, secondary cell walls expand rapidly upon water influx, presumably due to the presence of pectin in the secondary cell walls; in some cells walls appeared to expand even faster than the cytoplasm (Fig. 3c). In other desiccation-tolerant vegetative tissues, rehydration causes immediate cellular damage manifested as cellular disruption and leakage of cellular ions (Bewley & Krochko, 1982; Oliver & Bewley, 1997). However, in Ranunculus roots, the initial binding of water by pectin in the cell walls might serve as a protective mechanism of allowing water to enter the cytoplasm more slowly, thus affording defense against rehydration-induced damage due to rapid water influx to the cells.
Prolonged root rehydration at low temperatures results in additional enlargement of the cell walls (Fig. 3f). Cell walls become degraded as the above-ground organs grow and become established, and there is a depletion of the starch and protein storage reserves. These presumably provide an initial supply of carbon and nitrogen for growth of the aerial organs, and they are depleted by the end of the root life cycle (Fig. 3g).
The pattern of proteins observed by separation using SDS-PAGE (Fig. 6) coincides with the appearance and decline in protein bodies within the cells, as revealed by microscopy. Deposition, remobilization and reutilization of proteins occurs in the perennial roots of a number of species, including several members of the Compositae, Leguminosae and Euphorbiaceae (Bewley, 2002). Protease digestion shows that protein bodies are present within cortical cells (Fig. 4e,f); it is likely that at least some of the predominant ones are sequestered and can be regarded as storage proteins. The most evident proteins in the mature roots have a molecular mass of less than 14 kDa, which is lower than that of predominant proteins in the roots of other species, for example alfalfa (Avice et al., 1996) and other legumes (Corre et al., 1996), chicory and dandelion (Cyr & Bewley, 1990a,b; Xu et al., 2000). In both of the latter, the major protein is of c. 18 kDa (Xu et al., 2000; Richard-Molard et al., 2004), and has similar properties to allergen and pathogenesis-related proteins.
The pattern of the other major proteins in R. asiaticus is also considerably different with respect to the other studied species, of which none has so many prominent bands when separated by SDS-PAGE. Many of the species appear to possess root proteins between 29 and 36 kDa, but it is not known if these proteins have homologies with each other, or to the ones noted here. It is possible that there are species- or family-specific proteins.
Many plants synthesize low molecular weight heat shock proteins (small HSPs) in response to high temperature stress (Waters et al., 1996). Since the roots of R. asiaticus and other geophytes are subjected to high temperature in their quiescent state, they might synthesize such proteins in anticipation of a high-temperature perturbation. However, small HSPs are generally 17–26 kDa in mass (Waters et al., 1996); hence, the major low molecular mass proteins in the roots of R. asiaticus are unlikely to be of this type. Also, the majority of HSPs is located within the cytosol, or within organelles, and is not abundant in protein bodies (Waters, 1995). An HSP of 12 kDa has been identified in yeast, which has desiccation-protection properties of a late embryogenesis abundant protein (LEA) (Sales et al., 2000); it can only remain as speculation as to whether similar proteins occur in plant roots.
In conclusion, the roots of R. asiaticus undergo profound changes in their cellular structure and contents during their annual life cycle, incorporating phases of growth, cell wall and protein deposition, desiccation, and degradation of the wall and cellular contents. The accumulation of proteins presumably serves as a store for nitrogen to support early re-establishment of the shoots, and some proteins may have a protective function under high-temperature and/or desiccation conditions. In addition, binding of water by pectin in the cell walls could serve as a protection mechanism during desiccation and rehydration to limit stress-induced damage to the cells, as well as serving as a potential source of carbon for the growing plant when mobilized.
This study was performed in the framework of the sabbatical leave of RK, and supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grants (A2210 to JDB and 5470–2002 to RLP); CFM is supported by Coordenacão de Aperfeiçoamento de Pessoal de Nìvel Superior, Brazil.