Nitrate reduction and nitrate content in ash trees (Fraxinus excelsior L.): distribution between compartments,site comparison and seasonal variation

Summary Nitrate reductase activity (NRA), nitrate content and biomass components of leaflets, leaf stalks, old stem, current-year stem and roots of ash trees (Fraxinus excelsior L.) growing in their natural habitats were investigated. In addition, NRA, total nitrogen and nitrate concentration were analyzed in the leaves and roots of ash trees from four different field sites. The highest NRA per gram biomass and also per total compartment biomass was found in the leaflets, even though root biomass was much higher than total leaflet biomass. The highest nitrate concentrations were found in the leaf stalks. Correlations between nitrate availability in the soil and NRA in leaves were not significant due to high variability of the actual soil nitrate concentrations. The seasonal variation in foliar NRA, nitrate concentration and total nitrogen concentration is much smaller in F. excelsior than reported for herbaceous species and is mainly caused by changes in the actual soil nitrate availability and by senescence of the leaves.     

Seasonal changes in nitrate use by three woody species: the importance of the leaf-expansion period

Seasonal changes in plant NO₃ ⁻-N use were investigated by measuring leaf nitrate reductase activity (NRA), leaf N concentration, and leaf expansion in one evergreen woody species (Quercus glauca Thunb.) and two deciduous woody species [Acer palmatum Thunb. and Zelkova serrata (Thunb.) Makino]. Leaf N concentration was highest at the beginning of leaf expansion and decreased during the expansion process to a steady state at the point of full leaf expansion in all species. The leaf NRA of all species was very low at the beginning of leaf expansion, followed by a rapid increase and subsequent decrease. The highest leaf NRA was observed in the middle of the leaf-expansion period, and the lowest leaf NRA occurred in summer for all species. Significant positive correlations were detected between leaf NRA and leaf expansion rates, while leaf N concentrations were negatively correlated with leaf area. In the evergreen Q. glauca, the N concentration in current buds increased before leaves opened; concurrently, the N concentration in 1-year-old leaves decreased by 25%. Our results show that the leaf-expansion period is the most important period for NO₃ ⁻-N assimilation by broadleaf tree species, and that decreases in leaf N concentration through the leaf-expansion period are at least partly compensated for by newly assimilated NO₃ ⁻-N in current leaves.     

Uptake,demand and internal cycling of nitrogen in saplings of Mediterranean<Emphasis Type="Italic"> Quercus</Emphasis> species

Nitrogen uptake, nitrogen demand and internal nitrogen cycling were studied to address the question of the importance of nutrient storage in Quercus species with contrasting leaf longevities. We carried out this study at the whole-plant level with young trees (2-4 years old) of three Mediterranean Quercus species: the evergreen Q. ilex, the marcescent/evergreen Q. faginea, and the deciduous Q. pyrenaica. Seasonal dynamics of nitrogen in all compartments of the plant were followed for 3 years. Nitrogen losses were measured through litter production, herbivory and fine root shedding. Nitrogen uptake was estimated using increments of nitrogen plant content plus accumulative nitrogen losses. Nitrogen uptake was limited to a few months during late winter and spring. Before budbreak, acquired nitrogen was stored in old-leaf cohorts of evergreen and woody compartments. After budbreak, Quercus species relied first on soil uptake and second on nitrogen retranslocation to supply new growth requirements. However, in most cases we found a high asynchrony between nitrogen demand by growing tissues and soil supply, which determined a strong nitrogen retranslocation up to 88.4% of the nitrogen demand throughout leaf expansion. Except for the first year after planting, the above- and underground woody fractions provided more nitrogen to the new tissues than the old leaf cohorts. Differences in the benefit of nitrogen withdrawn from senescent and old leaves were not found between species. We conclude that sink/source interaction strength was determined by differences between nitrogen demand and uptake, regulating internal nutrient cycling at the whole plant level.     

Nitrate reductase activity (NRA) in Asphodelus aestivus Brot. (Liliaceae): distribution among organs,seasonal variation and differences among populations

Nitrate reductase activity (NRA) in different compartments (leaves, inflorescence stalks, flowers and tuberous roots) of Asphodelus aestivus Brot. (Liliaceae) and actual mineral nitrogen (NO₃⁻-N and NH₄⁺-N) in soil surrounding the roots were investigated over one year. Although the highest NRA was found in the leaves, the other plant compartments, such as flowers and tuberous roots, also have nitrate assimilation capacity. High nitrate assimilation capacity under suitable conditions is considered to be a good strategy for development and dominance of this species in Mediterranean environments. There was a seasonal variation in nitrate assimilation in leaves and actual NO₃⁻-N content of soils. Depending on actual nitrate content of soils, nitrate assimilation increased in winter.     

Relationships between phenology and the remobilization of nitrogen, phosphorus and potassium in branches of eight Mediterranean evergreens

Few studies have examined the effects of plant growth on nutrient remobilization in phenologically contrasting species. Here we evaluated the consequences of above-ground seasonality of growth and leaf shedding on the remobilization of nutrients from branches in eight evergreen Mediterranean phanaerophytes that differ widely in phenology. Vegetative growth, flower bud formation, flowering, fruiting, leaf shedding, and the variations in nitrogen (N), phosphorus (P) and potassium (K) pools in branches throughout the year were monitored in each species. Nitrogen and P remobilization occurred in summer, after vegetative growth and synchronously with leaf shedding. Despite the time-lag between growth and remobilization, the branches that invested more nutrients in vegetative growth also remobilized more nutrients from their old organs. Potassium remobilization peaked in the climatically harshest periods, and appears to be related to osmotic requirements. We conclude that N and P remobilization occurs mainly associated with leaf senescence, which might be triggered by factors such as the replenishment of nutrient reserves in woody organs, the hormonal relations between new and old leaves, or the constraints that summer drought poses on the amount of leaf area per branch in summer.     

Seasonal Differences in Photochemical Efficiency and Chlorophyll and Carotenoid Contents in Six Mediterranean Shrub Species under Field Conditions

The effects of summer and winter stress on the chlorophyll and carotenoid contents and photosystem 2 efficiency were examined in six Mediterranean scrub species. These six species belong to two different plant functional types: drought semi-deciduous (Halimium halimifolium L., Rosmarinus officinalis L., Erica scoparia L.) and evergreen sclerophylls (Juniperus phoenicea L., Pistacia lentiscus L., Myrtus communis L.). Two sites with different water availability were chosen. In the xerophytic site, despite they belong to two different functional types, R. officinalis and J. phoenicea showed a similar response. These were the most affected species in summer. H. halimifolium showed optimal values of F_v/F_m and non-significant seasonal changes in xanthophyll content. In the mesic site, E. scoparia and M. communis were apparently the most affected species by winter climatic conditions. P. lentiscus presented a pattern similar to H. halimifolium, except for elevated F₀ values. In all the studied species, lutein plus zeaxanthin content was negatively correlated with F_v/F_m in summer and with leaf water potential, thus indicating that the thermal dissipation of energy was a general pattern for all species. Under stress, plant response is more species-specific than dependent on its functional type.     

Seasonal Patterns of Photosystem 2 Photochemical Efficiency in Evergreen Sclerophylls and Drought Semi-Deciduous Shrubs under Mediterranean Field Conditions

Photochemical efficiency of photosystem 2 (PS2), assessed from in situ chlorophyll (Chl) fluorescence measurements, was seasonally monitored in five evergreen sclerophyll and five malacophyllous drought semi-deciduous species, co-occurring in the same Mediterranean field site. In evergreen sclerophylls, a considerable drop in the variable (F_v) to maximum (F_m) Chl fluorescence ratio coincided with the lowest winter temperatures, indicating low PS2 efficiency during this period. Summer drought caused a comparatively slight decrease in F_v/F_m and only in three of the five evergreen sclerophyll species tested. In drought semi-deciduous shrubs, the winter drop in F_v/F_m was much less conspicuous. During the summer, and in spite of the severe and prolonged desiccation of their malacophyllous leaves, F_v/F_m was maintained high and only in one species the PS2 efficiency was transiently suppressed, when the leaf relative water content became lower than 30 %. Thus evergreen sclerophylls are more prone to photoinhibition by low winter temperatures, while the sensitivity of drought semi-deciduals depends on the extent and duration of summer drought. This revised version was published online in September 2006 with corrections to the Cover Date.     

Nitrate concentration and nitrate reductase activity in the leaves of three legumes and three cereals*

Nitrate concentration and nitrate reductase activity (NRA) were studied in the leaves of soybean (Glycine max), groundnut (Arachis hypogaea and cowpea (Vigna unguiculata) and sorghum (Sorghum bicolor), pearl millet (Pennisetum americanum) and maize (Zea mays) at three nitrogen fertiliser levels in two field experiments. Higher nitrate concentrations were detected in the leaves of groundnut, cowpea and pearl millet than in sorghum and maize. Nitrate content in the leaves and leaf NRA were not related across crop species, nor was a generalised pattern of leaf NRA and leaf nitrate observed within legumes or within cereals. Nitrogen application resulted in higher nitrate availability in the leaves, with varied leaf NRA.     

Fluctuations in nitrate reductase activity,and nitrate and organic nitrogen concentrations of succulent plants under different nitrogen and water regimes

The CAM (Crassulacean acid metabolism) succulent species Kalanchoe daigremontiana, K. tubiflora and Crassula argentea, and the succulent C₃ species Peperomia obtusifolia, were cultivated in pure culture in open-air conditions under two different regimes of nitrogen and water supply. At specified intervals during the course of vegetative growth, biomass, nitrate reductase activity (NRA), nitrate concentration, and organic nitrogen concentration of whole plants were measured. After 100 days of cultivation the leaf conductance of Crassula and Peperomia was measured at intervals for the duration of a day. Behaviour of all four species was strongly influenced by the cultivation regime. This was apparent in terms of productivity and variable flucturations in NRA, nitrate concentration, and organic nitrogen concentration during the vegetative period. Increase in biomass was mostly connected with a decrease in all other investigated parameters, especially under conditions of water and/or nitrogen deficiency. The typical reaction of the CAM species Crassula to limited netrogen but adequate soil water was to reduce leaf conductance during light, whereas the C₃ plant Peperomia increased conductance in comparison with plants having a nitrogen suppy. The NRA of all plant species was reduced by both soil nitrate deficiency and drought. The succulent plant species, which are specially adapted to drought, neither took up nor used nitrate when water was limited. This was particularly the case for the CAM species, but less so for the C₃ Peperomia, which showed very high concentrations of nitrate and organic nitrogen, but low NRA and biomass gain. A formula was derived to express the nitrogen use efficiency (NUE) of the species, i.e. the ability of a plant to use nitrogen over a specific period of growth. NUE was shown to increase with age for the crassulacean species but to decrease for the C₃ Peperomia. Furthermore, NUE varied with the different nutrient levels in a species-specific manner, with high values for NUE not necessarily coupled to high productivity, and with NUE of the C₃ species generally higher than that of CAM species.     

Nitrate assimilation in coexisting vascular plants in mire and swamp forest habitats in Central Sweden

Summary In order to monitor the nitrate assimilation capability of mire plants, in vivo current and maximally induced nitrate reductase activity (NRA) were investigated in 14 species of vascular plants from four different sites in a central Swedish mire. One of the sites was a swamp forest. The plants studied included species with both wide and restricted ecological ranges, and the mire sites selected covered a wide range of plant productivity. At the most productive site, current NRA differed among coexisting species. This differentiation in the use of nitrate as a source of nitrogen suggested the possibility of resource partitioning with regard to nitrogen acquisition. Maximally induced NRA, measured 3 days after an addition of nitrate, was highest at the most productive sites and differed among coexisting species. Plant species characteristic of rich fens had the highest maximally induced NRA. In all species, there was a positive correlation between the ability to assimilate peaks of available nitrate and total leaf nitrogen concentration.     

Mediterranean sea cliff plants: morphological and physiological responses to environmental conditions

Aims The plants of Mediterranean sea cliff ecosystems are resistant to several environmental challenges. In this study, six species typical of the coastal rocky cliffs have been analyzed in order to evaluate their diverse morphological and physiological responses to their environment across the seasons, and to examine the strategy of the ecological group to which each species belongs. Since these species are widespread across the Mediterranean region, our aim was also to highlight their ecophysiological features in habitats where the direct influence of the sea is stronger.Methods The selected species are characteristic of the sea cliffs of Elba island (Tyrrhenian sea, Italy): the halophyte Crithmum maritimum, the semideciduous Helichrysum italicum and Lavandula stoechas and the sclerophylls Myrtus communis, Quercus ilex and Rhamnus alaternus. Four morphological traits—canopy height, leaf area, specific leaf area and leaf dry matter content—and two physiological traits—leaf water potential (LWP) and photosynthetic efficiency (PE), measured before the dawn and at midday—were analyzed. Water potential was measured by a pressure chamber and photosynthetic efficiency was determined by the analysis of chlorophyll fluorescence. Plant performance was also evaluated by calculating chronic (PI chr) and dynamic photoinhibition (PI dyn).Important findings Crithmum maritimum showed high resistance to the recurrent dry periods, because of the high water storage capacity of its leaves and its PE declined markedly only in July, under the harshest climatic conditions. Semideciduous taxa utilize primarily an avoidance strategy, which aims at reducing the overall leaf surface, while sclerophylls mostly show a tolerance strategy towards the prevailing stressors, as demonstrated by LWP and PE, that are lower in the sclerophylls than in the semideciduous taxa during summer, due to osmoregulation and photoinhibition, respectively. Furthermore, variability of physiological parameters was higher in the sclerophylls than in the semideciduous taxa, because the former had to withstand wider oscillations of their LWP and PE. The sclerophyllous taxa underwent a slight loss of PE also in winter, likely owing to the combined action of low temperature and high irradiance. In Mediterranean sea cliff ecosystems, the stressful combination of high irradiance, high temperatures and low rainfall typical of the summer season may have been intensified by the shallow soil which displays a poor water storage capacity. On the other hand, winter stress, caused by high solar radiation and low temperatures, does not seem to seriously affect the performance of the studied species.     

A study on nitrate reductase activity (NRA) of geophytes from Mediterranean environment

Nitrate reductase activity (NRA) in different compartments of 14 Mediterranean geophytes (bulbous, tuberous and rhizomatous) and actual mineral nitrogen (NO₃⁻ and NH₄⁺) in their soils were investigated. The nitrate reduction capacities of each species were determined as NRA per total plant material. Differences among compartments for NRA were significant in all species. The highest NRA was found in leaves of tuberous species (Anemone coronaria, Cyclamen coum) and of most bulbous species (Allium flavum, Allium guttatum, Bellevelia sarmatica, Galanthus plicatus, Leucojum aestivum, Ornithogalum nutans, Tulipa sylvestris). Therefore, in this group of species the contribution of the leaves to total plant NRA was the highest. The other bulbous species (Allium scorodoprasum, Crocus chrysanthus, Fritillaria bithynica, Muscari neglectum) and one rhizomatous taxon (Iris suaveolens) have a different NRA distribution within the plants. In these species the highest values of NRA were found in different organs. For example, in Allium scorodoprasum the highest NRA was in tunics, and in flowers in M. neglectum. Although leaves are the main compartments reducing nitrate in most of the studied geophytes, other compartments also contribute to total plant nitrate reduction.Our results show that the nitrate reduction capacity is different among geophyte species. Even if it roughly reflects the nitrogen supply in a habitat, differences in nitrate reduction capacities of different species collected from same sites indicate that the nitrate reducing capacity is species-specific.     

Site fertility and leaf nutrients of sympatric evergreen and deciduous species of Quercus in central coastal California

Leaf and soil nutrient levels interact with and may each influence the other. We hypothesize that to the extent soil fertility influences the nutritional state of trees, soil fertility should correlate with summer leaf nutrient levels, whereas to the extent that trees influence soil nutrient levels, the quality of leaf litterfall should correlate with soil fertility. We examined these correlations for five sympatric oak species (genus Quercus) in central coastal California. Soil fertility, including both nitrogen and especially phosphorus, correlated significantly with summer leaf nutrient levels. In contrast, phosphorus, but not nitrogen, in the leaf litterfall correlated positively with soil nutrients. These results suggest that soil nitrogen and phosphorus influence tree nutrient levels and that leaf phosphorus, but not leaf nitrogen, influence soil fertility under the trees. Feedback between the soil and the tree for phosphorus, but not nitrogen, is apparently significant and caused by species-specific differences in leaf quality and not by litterfall quality differences within a species. We also compared functional differences between the evergreen and deciduous oak species at our study site. There were no differences in soil nitrogen and only small differences for soil phosphorus between the phenological types. Differences in leaf nutrient concentration were much more pronounced, with the evergreen species having substantially lower levels of both nitrogen and phosphorus. Evergreen species conserved more phosphorus, but not more nitrogen, than the deciduous species, but there was no consistent relationship between retranslocation and either soil nitrogen or phosphorus. These results do not support the hypothesis that evergreenness is an adaptation to low soil fertility in this system.     

Nitrogen use strategies of neotropical rainforest trees in threatened Atlantic Forest

The characteristics of nitrogen acquisition, transport and assimilation were investigated in species of an Atlantic Forest succession over calcareous soil in south‐eastern Brazil. Differences in behaviour were observed within the regeneration guilds. Pioneer species showed high leaf nitrogen contents, a high capacity to respond to increased soil nitrogen availability, a high capacity for leaf nitrate assimilation and were characterized by the transport of nitrate + asparagine. At the other end of the succession, late secondary species had low leaf nitrogen contents, little capacity to respond to increased soil nitrogen availability, low leaf nitrate assimilation and were active in the transport of asparagine + arginine. The characteristics of nitrogen nutrition in some early secondary species showed similarities to those of pioneer species whereas others more closely resembled late secondary species. Average leaf δ¹⁵N values increased along the successional gradient. The results indicate that the nitrogen metabolism characteristics of species may be an additional ecophysiological tool in classifying tropical forest tree species into ecological guilds, and may have implications for regeneration programmes in degraded areas.     

Seasonal fluctuations in the concentration of UV-absorbing compounds in the leaves of some Mediterranean plants under field conditions

Leaves of 14 representative Mediterranean plant species were collected on a monthly basis and assayed for UV-absorbing compounds concentration, either on an area or a dry mass basis, from 1995 to 1997. Strong seasonal fluctuations were observed in eight species (all evergreens, two phrygana, one deciduous, one summer perennial and one winter perennial). Two different patterns of changing concentrations of UV-absorbing compounds were observed. In the first, concentration of these compounds was higher in young developing leaves and concentration declined during maturation, whereas in other plants, the opposite trend was observed. These differences could be attributed to the particular leaf surface morphology of each plant. The observed seasonal fluctuations of UV-absorbing compounds seem to be more correlated to developmental processes, than to seasonal fluctuations of the naturally occurring UV-B radiation. Most of the winter perennials did not show strong fluctuations during the period of development. The concentration of these compounds varied not only on a seasonal basis among the examined plants, but between different life forms as well: during winter, examination of the leaves of 13 species showed that evergreen sclerophylls and phrygana had at least two-fold higher concentration of UV-B-absorbing compounds on a leaf area basis than winter perennials. In addition, during the same season and irrespective of life form and species, the absorbance at 300 nm per unit of mature leaf area followed an asymptotic exponential decrease when specific leaf area increased. The UV-B radiation screening capacity of the leaves of these plants is discussed in relation to each adaptive strategy.     

Nitrate reductase activity in vegetation below an arctic bird cliff,Svalbard, Norway

Abstract. Vegetated sites below bird-nesting cliffs are uniquely nutrient-rich habitats in the otherwise nutrient-poor arctic environment. Plants from six distinct vegetation zones below such a cliff at 79° N, Svalbard, Norway, were collected for analysis under greenhouse conditions. Leaf nitrate reductase activity (NRA) was analysed in 42 species representing 25 % of the Svalbard vascular flora. The species mean NRA values ranged from 0.37 to 8.34 μmols of nitrite ions formed per gram of plant fresh weight per hour. Species in the vegetated zone growing closest to recent guano deposits had the highest NRA values, (mean = 4.47) whereas plants growing farther below the cliff had significantly lower values (mean = 0.55). A similar pattern was detected in a duplicate set of plants induced with 15 mM KNO₃; vegetation zone means for NRA ranged from 5.08 to 0.98 μmols of nitrite ions formed per gram of plant fresh weight per hour. Maximally induced species NRA values were highest in the first zones below the cliff and decreased downslope. This gradient paralleled the steep soil nitrate gradient, which decreased from 13.84 mg/l at the cliffbase to 1.03 mg/l downslope. Correspondingly, soil ammonium ions in the vegetation zones ranged between 1.96 mg/l at the cliff-base to 0.03 mg/l downslope. Correlations between NRA and soil nitrate provide a systematic basis for assigning scalar ‘nitrogen figures’ as indicators of habitat preference, here for the first time applied to arctic species.     

Nitrate reductase activity in high-mountain plants: a test across species,growth form and habitat type

Aims For many terrestrial plants, nitrate is the most important form of available soil nitrogen for growth. However, many plant species, which grow on acidic, ammonium-dominated soils, exhibit a constantly low level of nitrate reductase activity (NRA). Little is known about NRA in high-mountain vascular plants in similar conditions. We tested the hypothesis that high-mountain vascular plants in acidic and ammonium-dominated habitats have low levels of NRA.     

The potential of NO3--N utilization by a woody shrub species Lindera triloba: a cultivation test to estimate the saturation point of soil NO3--N for plants

Responses of seedlings of a shrub species, Lindera triloba, grown in perlite culture medium, to nitrate (NO3--N) supply were investigated to estimate the saturating point of available NO3--N for plant utilization. NO3--N concentration and nitrate reductase activity (NRA) in leaves and roots were used as indicators of NO3--N uptake and assimilation by L. triloba. Root NRA increased with NO3--N supply when concentrations were low and reached a plateau at high NO3--N concentrations. On the other hand, root NO3--N concentration increased linearly with NO3--N supply; therefore, it is suggested that NO3--N uptake did not limit NO3--N assimilation by L. triloba. In contrast, leaf NRA and leaf NO3--N concentration were low and were not influenced by NO3--N supply. This may be caused by the lack of transport of NO3--N from roots to leaves. The NO3--N retained in perlite was compared with NO3--N pool sizes in soils from a forest where L. triloba occurs naturally to estimate the level of NO3--N availability to plants in the forest soil. The maximum NO3--N pool size in the forest soil was comparable to concentrations at which root NRA reached a plateau in perlite cultures. These results indicate that soil NO3--N availability is below the saturation point for NO3--N uptake by L. triloba, and it is the limiting factor of NO3--N utilization by L. triloba under field conditions in which this species naturally occurs.     

Nitrate reduction in ramets of a clonal plant Eichhornia crassipes responding to nitrate availability during clonal growth stage

Effect of nitrate availability on nitrate reduction was examined in inter-connected ramets of invasive clonal plant Eichhornia crassipes grown with two nitrate supply regimes during different clonal growth stage. Increase of nitrate availability accelerated nitrate reductase activity (NRA) in parent and offspring ramets of E. crassipes, and there was greatly different pattern in inter-connected ramets during clonal growth stage. Leaf NRA was lower in offspring than that in parent ramets in phase 1, while significantly higher leaf NRA in offspring ramets was detected during phase 2. The results indicated NRA in inter-connected ramets of E. crassipes was highly dependent on nitrate availability and growth stage.     

Relative growth rate of leaf biomass and leaf nitrogen content in several mediterranean woody species

In many plant species, herbivory is a major determinant of leaf mortality and it can cause a strong reduction in productive potential. Most predation occurs on young, expanding leaves. Thus, a rapid growth of the leaves can reduce the impact of predation. Furthermore, in cold Mediterranean climates the length of the growing season is constrained to a short period in spring and early summer owing both to low winter temperatures and drought stress in early summer. Therefore, a rapid deployment of leaf area and a high photosynthetic capacity during the spring and early summer might have important positive effects on the final carbon balance of the leaf population. Relative growth rates (RGR) of leaf biomass were measured in 19 woody species typical of Central Western Spain with deciduous and evergreen habits. Highly significant differences were detected in the leaf growth rate of the different species. The differences between species, however, did not correlate either with the mean leaf life-span of each of the species or with other leaf traits such as photosynthetic capacity, specific leaf area or nitrogen content. Leaf growth rate was positively correlated with time elapsed between leaf initiation and fruit maturation, so that species with fruit dispersal in spring and early summer in general had lower leaf growth rates than species with autumn fruit shedding. This relationship shows the effects of the concurrence between vegetative and reproductive organs for nutrients and other resources. Nitrogen concentration in the leaves was very high at the time of bud break, and declined during leaf expansion owing to the dilution associated with the increase in structural components. The rate of nitrogen dilution was, thus, positively related to the leaf growth rate. Relative growth rates calculated for nitrogen mass in leaves were very low compared to the growth in total mass. This suggests that most leaf nitrogen is translocated from the plant stores to the leaf biomass before the start of leaf expansion and that the contribution of root uptake during leaf expansion is comparatively low.