NAA at a high concentration promotes efficient plant regeneration via direct somatic embryogenesis and SE-mediated transformation system in Ranunculus sceleratus.

The novel methods for efficient plant regeneration via direct somatic embryogenesis (SE) and SE-mediated transformation system under high concentration of NAA in Ranunculus sceleratus were established. On MS media containing a high concentration of NAA (10.0 mg/L) in the dark, all inoculated explants (root, stem and leaf) formed somatic embryos at high frequencies, respectively, 66.03, 126.47 and 213.63 embryoids per explant, and 100% of the embryoids developed into plantlets on 1/2 MS rooting media. Morphological and histological analyses revealed that SE in R. sceleratus followed a classical pattern. All inoculated explants can be used as receptors for genetic transformation in R. sceleratus, through direct SE-mediated method after Agrobacterium infection. RcLEC1-B, as a marker gene, changed the number and morphology of flower organs and the development of cuticle in R. sceleratus, which indicated that the efficient transgenic system of R. sceleratus was established. To our knowledge, this is the first observation that both direct SE and transgenic transformation system, via induction of a single plant growth regulator, have been successfully constructed in R. sceleratus.

A little bit of sex prevents mutation accumulation even in apomictic polyploid plants.

BACKGROUND: In the absence of sex and recombination, genomes are expected to accumulate deleterious mutations via an irreversible process known as Muller's ratchet, especially in the case of polyploidy. In contrast, no genome-wide mutation accumulation was detected in a transcriptome of facultative apomictic, hexaploid plants of the Ranunculus auricomus complex. We hypothesize that mutations cannot accumulate in flowering plants with facultative sexuality because sexual and asexual development concurrently occurs within the same generation. We assume a strong effect of purging selection on reduced gametophytes in the sexual developmental pathway because previously masked recessive deleterious mutations would be exposed to selection. RESULTS: We test this hypothesis by modeling mutation elimination using apomictic hexaploid plants of the R. auricomus complex. To estimate mean recombination rates, the mean number of recombinants per generation was calculated by genotyping three F1 progeny arrays with six microsatellite markers and character incompatibility analyses. We estimated the strength of purging selection in gametophytes by calculating abortion rates of sexual versus apomictic development at the female gametophyte, seed and offspring stage. Accordingly, we applied three selection coefficients by considering effects of purging selection against mutations on (1) male and female gametophytes in the sexual pathway (additive, s = 1.000), (2) female gametophytes only (s = 0.520), and (3) on adult plants only (sporophytes, s = 0.212). We implemented recombination rates into a mathematical model considering the three different selection coefficients, and a genomic mutation rate calculated from genome size of our plants and plant-specific mutation rates. We revealed a mean of 6.05% recombinants per generation. This recombination rate eliminates mutations after 138, 204 or 246 generations, depending on the respective selection coefficients (s = 1.000, 0.520, and 0.212). CONCLUSIONS: Our results confirm that the empirically observed frequencies of facultative recombination suffice to prevent accumulation of deleterious mutations via Muller's ratchet even in a polyploid genome. The efficiency of selection is in flowering plants strongly increased by acting on the haplontic (reduced) gametophyte stage.

A molecular basis behind heterophylly in an amphibious plant, Ranunculus trichophyllus.

Ranunculus trichophyllus is an amphibious plant that produces thin and cylindrical leaves if grown under water but thick and broad leaves if grown on land. We found that such heterophylly is widely controlled by two plant hormones, abscisic acid (ABA) and ethylene, which control terrestrial and aquatic leaf development respectively. Aquatic leaves produced higher levels of ethylene but lower levels of ABA than terrestrial leaves. In aquatic leaves, their distinct traits with narrow shape, lack of stomata, and reduced vessel development were caused by EIN3-mediated overactivation of abaxial genes, RtKANADIs, and accompanying with reductions of STOMAGEN and VASCULAR-RELATED NAC-DOMAIN7 (VDN7). In contrast, in terrestrial leaves, ABI3-mediated activation of the adaxial genes, RtHD-ZIPIIIs, and STOMAGEN and VDN7 established leaf polarity, and stomata and vessel developments. Heterophylly of R.trichophyllus could be also induced by external cues such as cold and hypoxia, which is accompanied with the changes in the expression of leaf polarity genes similar to aquatic response. A closely-related land plant R. sceleratus did not show such heterophyllic responses, suggesting that the changes in the ABA/ethylene signaling and leaf polarity are one of key evolutionary steps for aquatic adaptation.

Submerged freshwater plant communities do not show species complementarity effect in wetland mesocosms.

It is a generally accepted theory that ecological functions are enhanced with increased diversity in plant communities due to species complementarity effects. We tested this theory in a mesocosm study using freshwater submerged plant beds to determine if increasing species number caused overyielding and species complementarity. We applied a maximum of four species in the plant beds corresponding to the typical species number in natural freshwater plant beds. We found no clear effects of species number (1-4) on biomass production and thus no conclusive overyielding and complementarity effect. This may be explained by low species differentiation among the four species in plant traits relevant for resource acquisition in freshwater, or that other species interactions, e.g. allelopathy, were inhibiting overyielding. The existing knowledge on species complementarity in aquatic plant communities is sparse and inconclusive and calls for more research.

Two distinct plant respiratory physiotypes might exist which correspond to fast-growing and slow-growing species.

The origin of the carbon atoms in CO2 respired by leaves in the dark of several plant species has been studied using 13C/12C stable isotopes. This study was conducted using an open gas exchange system for isotope labeling that was coupled to an elemental analyzer and further linked to an isotope ratio mass spectrometer (EA-IRMS) or coupled to a gas chromatography-combustion-isotope ratio mass spectrometer (GC-C-IRMS). We demonstrate here that the carbon, which is recently assimilated during photosynthesis, accounts for nearly ca. 50% of the carbon in the CO2 lost through dark respiration (Rd) after illumination in fast-growing and cultivated plants and trees and, accounts for only ca. 10% in slow-growing plants. Moreover, our study shows that fast-growing plants, which had the largest percentages of newly fixed carbon of leaf-respired CO2, were also those with the largest shoot/root ratios, whereas slow-growing plants showed the lowest shoot/root values.

Plant population differentiation and climate change: responses of grassland species along an elevational gradient.

Mountain ecosystems are particularly susceptible to climate change. Characterizing intraspecific variation of alpine plants along elevational gradients is crucial for estimating their vulnerability to predicted changes. Environmental conditions vary with elevation, which might influence plastic responses and affect selection pressures that lead to local adaptation. Thus, local adaptation and phenotypic plasticity among low and high elevation plant populations in response to climate, soil and other factors associated with elevational gradients might underlie different responses of these populations to climate warming. Using a transplant experiment along an elevational gradient, we investigated reproductive phenology, growth and reproduction of the nutrient-poor grassland species Ranunculus bulbosus, Trifolium montanum and Briza media. Seeds were collected from low and high elevation source populations across the Swiss Alps and grown in nine common gardens at three different elevations with two different soil depths. Despite genetic differentiation in some traits, the results revealed no indication of local adaptation to the elevation of population origin. Reproductive phenology was advanced at lower elevation in low and high elevation populations of all three species. Growth and reproduction of T. montanum and B. media were hardly affected by garden elevation and soil depth. In R. bulbosus, however, growth decreased and reproductive investment increased at higher elevation. Furthermore, soil depth influenced growth and reproduction of low elevation R. bulbosus populations. We found no evidence for local adaptation to elevation of origin and hardly any differences in the responses of low and high elevation populations. However, the consistent advanced reproductive phenology observed in all three species shows that they have the potential to plastically respond to environmental variation. We conclude that populations might not be forced to migrate to higher elevations as a consequence of climate warming, as plasticity will buffer the detrimental effects of climate change in the three investigated nutrient-poor grassland species.

Effect of temperature on the progamic phase in high-mountain plants.

Progamic processes are particularly temperature-sensitive and, in lowland plants, are usually drastically reduced below 10 °C and above 30 °C. Little is known about how effectively sexual processes of mountain plants function under the large temperature fluctuations at higher altitudes. The present study examines duration and thermal thresholds for progamic processes in six common plant species (Cerastium uniflorum, Gentianella germanica, Ranunculus alpestris, R. glacialis, Saxifraga bryoides, S. caesia) from different altitudinal zones in the European Alps. Whole plants were collected from natural sites shortly before anthesis and kept in a climate chamber until further processing. Flowers with receptive stigmas were hand-pollinated with allopollen and exposed to controlled temperatures between -2 and 40 °C. Pollen performance (adhesion to the stigma, germination, tube growth, fertilisation) was quantitatively analysed, using the aniline blue fluorescence method. Pollen adhesion was possible from -2 to 40 °C. Pollen germination and tube growth occurred from around 0 to 35 °C in most species. Fertilisation was observed from 5 to 30-32 °C (0-35 °C in G. germanica). The progamic phase was shortest in G. germanica (2 h at 30 °C, 12 h at 5 °C, 24 h at 0 °C), followed by R. glacialis (first fertilisation after 2 h at 30 °C, 18 h at 5 °C). In the remaining species, first fertilisation usually occurred after 4-6 h at 30 °C and after 24-30 h at 5 °C. Thus, mountain plants show remarkably flexible pollen performance over a wide temperature range and a short progamic phase, which may be essential for successful reproduction in the stochastic high-mountain climate.

Effects of ozone on species composition in an upland grassland.

Northern hemispheric background concentrations of ozone are increasing, but few studies have assessed the ecological significance of these changes for grasslands of high conservation value under field conditions. We carried out a 3-year field experiment in which ozone was released at a controlled rate over three experimental transects to produce concentration gradients over the field site, an upland mesotrophic grassland located in the UK. We measured individual species biomass in an annual hay cut in plots receiving ambient ozone, and ambient ozone elevated by mean concentrations of approximately 4 ppb and 10 ppb in the growing seasons of 2008 and 2009. There was a significant negative effect of ozone exposure on herb biomass, but not total grass or legume biomass, in 2008 and 2009. Within the herb fraction, ozone exposure significantly decreased the biomass of Ranunculus species and that of the hemi-parasitic species Rhinanthus minor. Multivariate analysis of species composition, taking into account spatial variation in soil conditions and ozone exposure, showed no significant ozone effect on the grass component. In contrast, by 2009, ozone had become the dominant factor influencing species composition within the combined herb and legume component. Our results suggest that elevated ozone concentrations may be a significant barrier to achieving increased species diversity in managed grasslands.

Apomixis is not prevalent in subnival to nival plants of the European Alps.

BACKGROUND AND AIMS: High alpine environments are characterized by short growing seasons, stochastic climatic conditions and fluctuating pollinator visits. These conditions are rather unfavourable for sexual reproduction of flowering plants. Apomixis, asexual reproduction via seed, provides reproductive assurance without the need of pollinators and potentially accelerates seed development. Therefore, apomixis is expected to provide selective advantages in high-alpine biota. Indeed, apomictic species occur frequently in the subalpine to alpine grassland zone of the European Alps, but the mode of reproduction of the subnival to nival flora was largely unknown. METHODS: The mode of reproduction in 14 species belonging to seven families was investigated via flow cytometric seed screen. The sampling comprised 12 species typical for nival to subnival plant communities of the European Alps without any previous information on apomixis (Achillea atrata, Androsace alpina, Arabis caerulea, Erigeron uniflorus, Gnaphalium hoppeanum, Leucanthemopsis alpina, Oxyria digyna, Potentilla frigida, Ranunculus alpestris, R. glacialis, R. pygmaeus and Saxifraga bryoides), and two high-alpine species with apomixis reported from other geographical areas (Leontopodium alpinum and Potentilla crantzii). KEY RESULTS: Flow cytometric data were clearly interpretable for all 46 population samples, confirming the utility of the method for broad screenings on non-model organisms. Formation of endosperm in all species of Asteraceae was documented. Ratios of endosperm : embryo showed pseudogamous apomixis for Potentilla crantzii (ratio approx. 3), but sexual reproduction for all other species (ratios approx. 1·5). CONCLUSIONS: The occurrence of apomixis is not correlated to high altitudes, and cannot be readily explained by selective forces due to environmental conditions. The investigated species have probably other adaptations to high altitudes to maintain reproductive assurance via sexuality. We hypothesize that shifts to apomixis are rather connected to frequencies of polyploidization than to ecological conditions.

In vitro propagation through axillary shoot culture of Ranunculus asiaticus L.

Ranunculus asiaticus is an important ornamental species mainly cultivated in the countries surrounding the Mediterranean sea. So far, the multiplication of this plant has been mainly carried out by seed and tuberous root division; however, these systems present many drawbacks. Tissue culture is an attractive alternative for accelerated propagation of selected and indexed genotypes. In this chapter, we present a flow chart for the commercial production of Ranunculus clones by using in vitro axillary budding. Although the price of micropropagated plants is higher compared to traditional material (seedlings and tuberous roots from seed populations), we need to consider that micropropagation helps to supply growers with more performant and healthy genotypes, and a better production schedule can be envisaged for cultivation and packaging.

Life form dependent impacts of macrophyte vegetation on the ratio of resuspended nutrients.

The effects of floating-leaved and submerged macrophytes on sediment resuspension and on the ratio of resuspended nitrogen and phosphorus were studied by sediment traps in the Kirkkojärvi basin in southern Finland. The effect of submerged macrophytes on preventing sediment resuspension was stronger than the effect of floating-leaved plants. On average, among submerged plants the resuspension rate of suspended solids was 43%, and among floating-leaved plants 87% of that in the open water. The floating-leaved Nuphar lutea had a reductive effect on P resuspension but no significant effect on N resuspension. The impact on P resuspension was strong, because root uptake by Nuphar lutea reduced the P content of the sediment. N:P ratio in resuspended nutrients was 6.7 among the plants and 4.1 in the open water. Among suzbmerged plants, sediment N content was strongly increased but P content was not affected due to the pleustophytic life form of the dominant plants (Ceratophyllum demersum, Ranunculus circinatus). The effect of pleustophytes on sediment nutrients was weak, because their nutrient uptake is mostly foliar. The N:P ratio of resuspended nutrients was 7.9 among the submerged plants and 7.0 in the open water. The results suggested that depending on the life form, macrophytes can modify the flux of N and/or P to the water column through their effects on nutrient resuspension and possibly modify phytoplankton communities via their effects on the N:P ratio. If the overall nutrient level is the most important factor for the dominance of cyanobacteria, submerged macrophytes can have stronger effects on phytoplankton community structure than floating-leaved species. If N:P ratio is of importance, the effects of floating-leaved species may be more pronounced.

No genetic diversity at molecular markers and strong phenotypic plasticity in populations of Ranunculus nodiflorus, an endangered plant species in France.

BACKGROUND AND AIMS: Although conservation biology has long focused on population dynamics and genetics, phenotypic plasticity is likely to play a significant role in population viability. Here, an investigation is made into the relative contribution of genetic diversity and phenotypic plasticity to the phenotypic variation in natural populations of Ranunculus nodiflorus, a rare annual plant inhabiting temporary puddles in the Fontainebleau forest (Paris region, France) and exhibiting metapopulation dynamics. METHODS: The genetic diversity and phenotypic plasticity of quantitative traits (morphological and fitness components) were measured in five populations, using a combination of field measurements, common garden experiments and genotyping at microsatellite loci. KEY RESULTS: It is shown that populations exhibit almost undetectable genetic diversity at molecular markers, and that the variation in quantitative traits observed among populations is due to a high level of phenotypic plasticity. Despite the lack of genetic diversity, the natural population of R. nodiflorus exhibits large population sizes and does not appear threatened by extinction; this may be attributable to large phenotypic plasticity, enabling the production of numerous seeds under a wide range of environmental conditions. CONCLUSIONS: Efficient conservation of the populations can only be based on habitat management, to favour the maintenance of microenvironmental variation and the resulting strong phenotypic plasticity. In contrast, classical actions aiming to improve genetic diversity are useless in the present case.

Connectivity, habitat heterogeneity, and population persistence in Ranunculus nodiflorus, an endangered species in France.

Here, we explore the role of habitat spatial structure in the maintenance of metapopulations of Ranunculus nodiflorus. This rare species grows in puddles that can be connected occasionally by flooded corridors. We monitored five locations in the Fontainebleau forest, France, since 2002 and recorded the presence of corridors among puddles and evaluated their impact on puddle demography and plant fitness. We showed that connections increased population size, by increasing both the number of puddles occupied by the species and the density of individuals within puddles, but seemed to have no direct influence on plant fitness. We found no evidence of a large persistent soil seed bank. Natural corridors are likely to decrease the extinction probability of the populations, most probably by allowing recolonization of empty puddles after extinctions. Therefore, the preservation of corridors appears crucial for the conservation of R. nodiflorus in its natural habitat.

Ecological factors influencing tetraploid speciation in snow buttercups (Ranunculus Adoneus): niche differentiation and tetraploid establishment.

Chromosome doubling plays an important role in generating new species of flowering plants. However, reproductive incompatibilities between newly formed tetraploid plants and their diploid progenitors are expected to create a significant barrier to the persistence and establishment of neopolyploid populations. Ecological differentiation can reduce this barrier via prezygotic isolation arising from spatial separation. Alternatively, superior viability or fecundity of neotetraploid plants might compensate for the reproductive cost of incompatible pollen from diploid neighbors. The performance of plants of both cytotypes can be assessed in their respective habitats through reciprocal transplants, although such experiments have not been used previously in the study of tetraploid speciation. We used a series of seed and seedling transplant experiments to assess ecological differentiation and competitive ability during early establishment phases for tetraploid and diploid forms of the snow buttercup (Ranunculus adoneus). At two sites, seeds from diploids and tetraploids had similar germination probabilities. Tetraploid snow buttercup seedlings had a significant growth advantage in a controlled environment chamber experiment. However, in the field diploid and tetraploid buttercup seedlings did not differ consistently in survival or growth, nor did the two cytotypes show reciprocal advantages in performance, as expected if ecological differentiation has occurred. At the seed and seedling stages, neither niche differentiation nor tetraploid competitive superiority appears sufficient to explain neotetraploid success in the presence of their diploid progenitors.

Does competition for phosphate supply explain the invasion pattern of Elodea species?

Two invasive aquatic plants, Elodea canadensis and Elodea nuttallii, occurred in north-eastern France. In this study, we examine the influence of phosphorus availability in soft water streams to explain the invasion pattern of exotic species (E. nuttallii and E. canadensis) compared to native plants (Callitriche platycarpa, Ranunculus peltatus). Total phosphorus was measured in these four aquatic macrophytes. Sediment total phosphorus and water-soluble reactive phosphorus were also analysed each season in 2001. Phosphorus content in the two invasive species and in R. peltatus was higher than in C. platycarpa. Elodea species are adapted to the seasonal phosphorus fluctuations as well as R. peltatus and exhibited high phosphorus storage ability. The high fluctuation availability of resources in space or/and time favoured the spread of the invasive plants and confirms the theory of invasibility of Davis et al. [2000. Fluctuating resources in plant communities: a general theory of invasibility. J. Ecol. 88, 528-534]. The eutrophication process increases the invasibility of E. nuttallii's, while inducing competition between E. nuttallii and native macrophyte species.

Genomic compatibility occurs over a wide range of parental genetic similarity in an outcrossing plant.

The theory of inbreeding and outbreeding suggests that there is a hump-shaped relationship between the genetic similarity of sexually reproducing parents and the performance of their offspring. Inbreeding depression occurs when genetic similarity is high, whereas hybrid breakdown is expected when genetic similarity is low. Between these extremes, the effect of genetic similarity on fitness is unclear. We studied the shape of this relationship by crossing 65 target genotypes of the clonal, self-incompatible Ranunculus reptans with partner genotypes spanning a broad scale of genetic similarity, ranging from crosses within populations to between-population crosses and hybridisation with a closely related species. Offspring were raised in outdoor tubs. Results revealed a quadratic relationship between parental genetic distance and offspring performance, with the clonal component of fitness more strongly hump-shaped than the sexual component. Optimal genetic similarity encompassed a broad range of within-population and between-population crosses. This pattern of genomic compatibility has important implications for the evolution of mating systems and mate choice.

To succeed globally, disperse locally: effects of local pollen and seed dispersal on tetraploid establishment.

Newly formed tetraploid plants in sympatry with their diploid progenitors should face significant obstacles to persistence and population establishment because of low-fitness triploids formed by cross-ploidy pollinations. Prior models have found restrictive conditions for a minority tetraploid subpopulation to persist. A stochastic spatial model, parameterized using snow buttercups (Ranunculus adoneus), was used to examine the influence of limited seed and pollen dispersal distances on the success of minority tetraploids and the interaction of these factors with different rates of self-pollination and tetraploid advantage. Higher rates of self-pollination and increased tetraploid advantage increase the probability of tetraploid persistence. Limiting the dispersal of seeds and pollen further increases the positive impact of any given level of self-pollination and tetraploid advantage. Taxa with short-distance seed and pollen dispersal should face much less stringent barriers to sympatric polyploid speciation than taxa with long-distance dispersal patterns. With short-distance seed and pollen dispersal, polyploid speciation should be possible in the absence of ecological differentiation or recurrent polyploid formation through unreduced gametes.

Experimental life-history evolution: selection on growth form and its plasticity in a clonal plant.

The growth form along the continuum from compact phalanx plants to more loosely packed guerilla plants is an important life-history trait in clonal plants. Prerequisite for its evolution is heritable genetic variation. Starting with 102 genotypes of the stoloniferous herb Ranunculus reptans, we performed one selection experiment on spatial spread per rosette as measure of guerillaness (broad-sense heritability 0.198) and another on plasticity in this trait in response to competition (broad-sense heritability 0.067). After two generations, spatial spread was 36.9% higher in the high line than in the low line (realized heritability +/- SE 0.149 +/- 0.039). Moreover, compared with the low line genotypes of the high line had fewer rosettes, a lower proportion of flowering rosettes, a higher proportion of rooted rosettes, more branches per rosette, longer internodes and longer leaves. In the second experiment, we found no significant direct response to selection for high and low plasticity in spatial spread (realized heritability +/- SE -0.029 +/- 0.063), despite a significant correlated response in plasticity in the length of the first three stolon internodes. Our study indicates a high potential for further evolution of the clonal growth form in R. reptans, but not for its plasticity, and it demonstrates that the clonal growth form does not evolve independently of other clonal life-history characteristics.

Effects of submerged macrophytes on sediment resuspension and internal phosphorus loading in Lake Hiidenvesi (southern Finland).

The effects of submerged macrophytes on sediment resuspension and internal phosphorus loading in the shallow Kirkkojärvi basin of Lake Hiidenvesi were studied by sedimentation traps and sediment and water samples. During the 83 d study period, 793 g DW m(-2) of sediment was resuspended within the stand formed by Ranunculus circinatus, Ceratophyllum demersum and Potamogeton obtusifolius. Outside the stand, 1701g DW m(-2) sediment resuspension was measured during the same period. Water turbidity and concentration of suspended solids (SS) were significantly lower within the plant bed compared with the surrounding water area. Despite the higher concentration of inorganic suspendoids, the concentration of chlorophyll a was higher in the open water than within the submerged plant bed, owing to the enhanced nutrient recycling rate in the absence of submerged plants. With the resuspended sediment, 11.8 mg Pm(-2)d(-1) was brought into the water column within the stand and 24.5 mg Pm(-2)d(-1) outside the stand. Within the macrophyte stand, resuspended particles absorbed phosphorus from the water (indicated by the inverse relationship between SS and soluble reactive phosphorus), which was probably connected to the lowered phosphorus concentration of surface sediment due to uptake by macrophytes.

Survival of Ranunculus repens L. (creeping buttercup) in an amphibious habitat.

The turlough form of Ranunculus repens is subjected to several months' complete inundation with hard groundwater. Experimental flooding to the level of the soil surface had no effect on turlough or ruderal populations relative to drained controls. Experimental submergence resulted in direct tissue death of the ruderal population but did not affect the turlough population relative to drained controls. There was no detectable difference in the proportion of aerenchyma in drained, flooded and submerged roots of plants from either population. The proportion of aerenchyma increased with root age in the ruderal population. Up to twice the proportion of aerenchyma occurred in the lower third of the root in the turlough population relative to the middle and upper thirds. Submergence in artificially hardened tap water increased the amount of tissue death in the ruderal population, whereas it appeared to enhance the growth of plants from the turlough population relative to that of plants submerged in tap water. Only the ruderal population demonstrated a depth accommodation response in submerged conditions. Root concentrations of ethanol-soluble carbohydrates were up to three times higher in a field- collected turlough population during winter and autumn months than those in a ruderal population. Low levels of ethanol-insoluble carbohydrates were present in the turlough population but were absent from the ruderal population. Starch concentrations fluctuated greatly in the turlough population and were generally higher than those in the ruderal population. These results, together with those from previous investigations, suggest that the turlough population survives prolonged submergence by maintaining low levels of submerged photosynthesis, which may circulate oxygen within the plant tissues, and by utilizing storage carbohydrates for maintenance respiration.