Growth duration is a better predictor of stem increment than carbon supply in a Mediterranean oak forest: implications for assessing forest productivity under climate change.

Understanding whether tree growth is limited by carbon gain (source limitation) or by the direct effect of environmental factors such as water deficit or temperature (sink limitation) is crucial for improving projections of the effects of climate change on forest productivity. We studied the relationships between tree basal area (BA) variations, eddy covariance carbon fluxes, predawn water potential (Ψpd ) and temperature at different timescales using an 8-yr dataset and a rainfall exclusion experiment in a Quercus ilex Mediterranean coppice. At the daily timescale, during periods of low temperature (< 5°C) and high water deficit (< -1.1 MPa), gross primary productivity and net ecosystem productivity remained positive whereas the stem increment was nil. Thus, stem increment appeared limited by drought and temperature rather than by carbon input. Annual growth was accurately predicted by the duration of BA increment during spring (Δtt0-t1 ). The onset of growth (t0 ) was related to winter temperatures and the summer interruption of growth (t1 ) to a threshold Ψpd value of -1.1 MPa. We suggest that using environmental drivers (i.e. drought and temperature) to predict stem growth phenology can contribute to an improvement in vegetation models and may change the current projections of Mediterranean forest productivity under climate change scenarios.

In situ assessment of the velocity of carbon transfer by tracing 13 C in trunk CO2 efflux after pulse labelling: variations among tree species and seasons.

Phloem is the main pathway for transferring photosynthates belowground. In situ(13) C pulse labelling of trees 8-10 m tall was conducted in the field on 10 beech (Fagus sylvatica) trees, six sessile oak (Quercus petraea) trees and 10 maritime pine (Pinus pinaster) trees throughout the growing season. Respired (13) CO2 from trunks was tracked at different heights using tunable diode laser absorption spectrometry to determine time lags and the velocity of carbon transfer (V). The isotope composition of phloem extracts was measured on several occasions after labelling and used to estimate the rate constant of phloem sap outflux (kP ). Pulse labelling together with high-frequency measurement of the isotope composition of trunk CO2 efflux is a promising tool for studying phloem transport in the field. Seasonal variability in V was predicted in pine and oak by bivariate linear regressions with air temperature and soil water content. V differed among the three species consistently with known differences in phloem anatomy between broadleaf and coniferous trees. V increased with tree diameter in oak and beech, reflecting a nonlinear increase in volumetric flow with increasing bark cross-sectional area, which suggests changes in allocation pattern with tree diameter in broadleaf species. Discrepancies between V and kP indicate vertical changes in functional phloem properties.

Comparison of seasonal variations in water-use efficiency calculated from the carbon isotope composition of tree rings and flux data in a temperate forest.

Tree-ring δ(13) C is often interpreted in terms of intrinsic water-use efficiency (WUE) using a carbon isotope discrimination model established at the leaf level. We examined whether intra-ring δ(13) C could be used to assess variations in intrinsic WUE (W(g), the ratio of carbon assimilation and stomatal conductance to water) and variations in ecosystem WUE (W(t) , the ratio of C assimilation and transpiration) at a seasonal scale. Intra-ring δ(13) C was measured in 30- to 60-µm-thick slices in eight oak trees (Quercus petraea). Canopy W(g) was simulated using a physiologically process-based model. High between-tree variability was observed in the seasonal variations of intra-ring δ(13) C. Six trees showed significant positive correlations between W(g) calculated from intra-ring δ(13) C and canopy W(g) averaged over several days during latewood formation. These results suggest that latewood is a seasonal recorder of W(g) trends, with a temporal lag corresponding to the mixing time of sugars in the phloem. These six trees also showed significant negative correlations between photosynthetic discrimination Δ calculated from intra-ring δ(13) C, and ecosystem W(t), during latewood formation. Despite the observed between-tree variability, these results indicate that intra-ring δ(13) C can be used to access seasonal variations in past W(t).

Impact of carbohydrate supply on stem growth, wood and respired CO2 delta13C: assessment by experimental girdling.

The present study examines the impact of the C source (reserves vs current assimilates) on tree C isotope signals and stem growth, using experimental girdling to stop the supply of C from leaves to stem. Two-year-old sessile oaks (Quercus petraea) were girdled at three different phenological periods during the leafy period: during early wood growth (Girdling Period 1), during late wood growth (Girdling Period 2) and just after growth cessation (Girdling Period 3). The measured variables included stem respiration rates, stem radial increment, delta(13)C of respired CO(2) and contents of starch and water-soluble fraction in stems (below the girdle) and leaves. Girdling stopped growth, even early in the growing season, leading to a decrease in stem CO(2) efflux (CO(2R)). Shift in substrate use from recently fixed carbohydrate to reserves (i.e., starch) induced (13)C enrichment of CO(2) respired by stem. However, change in substrate type was insufficient to explain alone all the observed CO(2R) delta(13)C variations, especially at the period corresponding to large growth rate of control trees. The below-girdle mass balance suggested that, during girdling periods, stem C was invested in metabolic pathways other than respiration and stem growth. After Girdling Period 1, the girdle healed and the effects of girdling on stem respiration were reversed. Stem growth restarted and total radial increment was similar to the control one, indicating that growth can be delayed when a stress event occurs early in the growth period. Concerning tree ring, seasonal shift in substrate use from reserves (i.e., starch) to recently fixed carbohydrate is sufficient to explain the observed (13)C depletion of tree ring during the early wood growth. However, the inter-tree intra-ring delta(13)C variability needs to be resolved in order to improve the interpretation of intra-seasonal ring signals in terms of climatic or ecophysiological information. This study highlighted, via carbohydrate availability effects, the importance of the characterization of stem metabolic pathways for a complete understanding of the delta(13)C signals.

Is there any 12C/13C fractionation during starch remobilisation and sucrose export in potato tubers?

The delta(13)C (carbon isotope composition) variations in respired CO(2), total organic matter, proteins, sucrose and starch have been measured during tuber sprouting of potato (Solanum tuberosum) in darkness. Measurements were carried out both on tubers and on their growing sprouts for 23 days after the start of sprout development. Sucrose was slightly (13)C-depleted compared with starch in tubers, suggesting that starch breakdown was associated with a small isotope fractionation. In sprouts, all biochemical fractions including sucrose were (13)C-enriched compared with source tuber-sucrose, suggesting that sucrose translocation from tuber to sprouts fractionated against (12)C. However, both apparent fractionations were explained by the consumption of (13)C-depleted carbon for respiration or growth that enriched in the (13)C sucrose molecules left behind. In addition, whole tuber sucrose is constantly composed of recent sucrose from starch breakdown and old sucrose associated with an inherited, slightly (13)C-depleted pool. We therefore conclude that any fractionation at either the starch breakdown or the sucrose translocation level is unlikely under our conditions.

Leaf and twig delta13C during growth in relation to biochemical composition and respired CO2.

In deciduous trees, the delta(13)C values of leaves are known to diverge during growth from those of woody organs. The main purpose of this study is to determine whether the divergence in delta(13)C between leaves and current-year twigs of Fagus sylvatica (L.) is influenced by changes (i) in the relative contents of organic matter fractions and (ii) in the delta(13)C of respired CO(2). The delta(13)C values of bulk matter, extractive-free matter, lignin, holocellulose, starch, soluble sugars, water-soluble fraction and respired CO(2), as well as their relative contents in bulk matter were determined. The delta(13)C values of biochemical fractions and respired CO(2) showed very similar temporal variations for both leaves and twigs. Variations in bulk matter delta(13)C during growth were, therefore, poorly explained by changes in biochemical composition or in respiratory fractionation and were attributed to the transition from (13)C-enriched reserves (mainly starch) to (13)C-depleted new photoassimilates. The divergence between leaves and twigs was related to higher values of soluble sugar delta(13)C in twigs. However, the difference between lignin and holocellulose delta(13)C varied during growth. This phenomenon was attributed to the delay between holocellulose and lignin deposition. These results may have implications for analysis of organic matter delta(13)C in trees and forest ecosystems.

Evidence of a seasonal trade-off between growth and starch storage in declining beeches: assessment through stem radial increment, non-structural carbohydrates and intra-ring δ13C.

Forest decline is reported in recent decades all over the world. However, developing a clear vision of the associated tree dysfunctioning is still a challenge for plant physiologists. In this study, our aim was to examine the seasonal carbon adjustments of beech trees in the case of a long-term drought-induced decline. We compared healthy and declining trees in terms of stem radial growth, phloem sugar content and δ13C, together with xylem carbohydrates and intra-ring δ13C patterns. The radial growth of declining trees was clearly reduced by lower growth rates and shorter growing season length (44 days compared with healthy trees). The soluble sugar content was higher in the xylem of declining trees compared with the healthy ones, but similar in the phloem except at the end of their growth. Declining trees increased their levels of xylem starch content from budburst until the date of maximal growth rate. These reserve dynamics revealed an early trade-off between radial growth and starch storage that might be the result of an active or passive process. For declining trees, the slight decrease of intra-ring cellulose δ13C pattern during the early growing season was attributed to the synthesis of 13C enriched starch. For healthy trees, δ13C patterns were characterized by a progressive 13C increase along the ring, attributed to increased water-use efficiency (WUE) in response to decreased water availability. Individual variations of the crown area were negatively correlated to the intra-ring δ13C amplitude, which was ascribed to variations in canopy WUE and resource competition for healthy trees and partly to variations in the amount of reserves accumulated during spring for declining ones. Our study highlights the carbon physiological adjustment of declining trees towards reducing spring growth while storing starch, which can be reflected in the individual intra-ring cellulose δ13C patterns.

Carbon-use strategies in stem radial growth of two oak species, one Temperate deciduous and one Mediterranean evergreen: what can be inferred from seasonal variations in the δ13C of the current year ring?

Tree ring synthesis is a key process in wood production; however, little is known of the origin and fate of the carbon involved. We used natural 13C abundance to investigate the carbon-use process for the ring development in a temperate deciduous (Quercus petraea (Matt.) Liebl.) and a Mediterranean evergreen (Quercus ilex L.) oak. The sapwood carbon reserves, phloem sucrose contents, stem respired CO2 efflux and their respective carbon isotope compositions (δ13C) were recorded over 1 year, in the native area of each species. The seasonal δ13C variation of the current year ring was determined in the total ring throughout the seasons, as well as in slices from the fully mature ring after the growth season (intra-ring pattern). Although the budburst dates of the two oaks were similar, the growth of Quercus ilex began 50 days later. Both species exhibited growth cessation during the hot and dry summer but only Q. ilex resumed in the autumn. In the deciduous oak, xylem starch storage showed clear variations during the radial growth. The intra-ring δ13C variations of the two species exhibited similar ranges, but contrasting patterns, with an early increase for Q. petraea. Comparison between δ13C of starch and total ring suggested that Q. petraea (but not Q. ilex) builds its rings using reserves during the first month of growth. Shifts in ring and soluble sugars δ13C suggested an interspecific difference in either the phloem unloading or the use of fresh assimilate inside the ring. A decrease in ring δ13C for both oaks between the end of the radial growth and the winter is attributed to a lignification of ring cell walls after stem increment. This study highlighted the differences in carbon-use during ring growth for evergreen and deciduous oaks, as well as the benefits of exploring the process using natural 13C abundance.

Biochemical composition is not the main factor influencing variability in carbon isotope composition of tree rings.

From June to December, we determined the effects of variations in biochemical composition on delta(13)C of tree rings of 2-year-old oaks (Quercus petraea (Matt.) Liebl.) growing under semi-natural conditions, and the dependence of these effects of water stress during the growth season. Percent abundance, carbon concentration and delta(13)C were measured in holocellulose, lignin, extractive-free wood, starch and a water-soluble fraction. Relative concentrations of lignin and holocellulose in the extractive-free wood varied little during the season or in response to water stress, indicating that these compounds could not quantitatively explain the variations in whole-wood delta(13)C. Among all sampled tree rings, the relative concentration of each structural compound (holocellulose and lignin) accounted for less than 5% of the delta(13)C variability in whole wood. Variations in holocellulose and extractive-free wood delta(13)C between tree rings were almost identical (r > 0.95), whereas variations in lignin delta(13)C were less well correlated to these compounds. Whole-wood delta(13)C had a slightly altered isotopic signal compared with that of the structural compounds because of the presence of the extractive component. These results showed that variations in lignin delta(13)C and lignin concentration have little influence on extractive-free wood delta(13)C and whole-wood delta(13)C. Rather, holocellulose influences delta(13)C the most. Thus, we confirmed that, for climatic reconstruction from tree rings, removal of extractives by soxhlet is generally sufficient and sometimes unnecessary. Our findings also indicate that, in the case of rapid and severe water stress, the structural component did not accurately record the associated increase in delta(13)C because of dilution with previously formed organic matter and cessation of trunk growth. The effect of drought on carbon isotope ratios was more pronounced in the extractive compounds, making them good water stress indicators but only on a scale of days to months.

Interspecific variability of stem photosynthesis among tree species.

The photosynthetic characteristics of current-year stems of six deciduous tree species, two evergreen tree species and ginkgo (Ginkgo biloba L.) were compared. Gas exchange, chlorophyll concentration, nitrogen concentration and maximum quantum yield of PSII were measured in stems in summer and winter. A light-induced decrease in stem CO(2) efflux was observed in all species. The apparent gross photosynthetic rate in saturating light ranged from 0.72 micromol m(-2 )s(-1) (ginkgo, in winter) to 3.73 micromol m(-2) s(-1) (Alnus glutinosa (L.) Gaertn., in summer). Despite this variability, a unique correlation (slope = 0.75), based on our results and those reported in the literature, was found between gross photosynthetic rate and dark respiration rate. Mass-based gross photosynthetic rate decreased with stem mass per area and correlated to chlorophyll concentration and nitrogen concentration, both in summer and winter. The radial distribution of stem chlorophyll differed among species, but all species except ginkgo had chlorophyll as deep as the pith. In summer, the maximum quantum yield of stem PSII (estimated from the ratio of variable to maximal fluorescence; F (v)/F (m)) of all species was near the optimal value found for leaves. By contrast, the values were highly variable in winter, suggesting large differences in sensitivity to low-temperature photoinhibition. The winter values of Fv/Fm were only 31-60% of summer values for the deciduous species, whereas the evergreen conifer species maintained high F (v)/F (m) in winter. The results highlight the interspecific variability of gross photosynthesis in the stem and its correlation with structural traits like those found for leaves. The structural correlations suggest that the selection of photosynthetic traits has operated under similar constraints in stems and leaves.

Comparing the intra-annual wood formation of three European species (Fagus sylvatica, Quercus petraea and Pinus sylvestris) as related to leaf phenology and non-structural carbohydrate dynamics.

Monitoring cambial phenology and intra-annual growth dynamics is a useful approach for characterizing the tree growth response to climate change. However, there have been few reports concerning intra-annual wood formation in lowland temperate forests with high time resolution, especially for the comparison between deciduous and coniferous species. The main objective of this study was to determine how the timing, duration and rate of radial growth change between species as related to leaf phenology and the dynamics of non-structural carbohydrates (NSC) under the same climatic conditions. We studied two deciduous species, Fagus sylvatica L. and Quercus petraea (Matt.) Liebl., and an evergreen conifer, Pinus sylvestris L. During the 2009 growing season, we weekly monitored (i) the stem radial increment using dendrometers, (ii) the xylem growth using microcoring and (iii) the leaf phenology from direct observations of the tree crowns. The NSC content was also measured in the eight last rings of the stem cores in April, June and August 2009. The leaf phenology, NSC storage and intra-annual growth were clearly different between species, highlighting their contrasting carbon allocation. Beech growth began just after budburst, with a maximal growth rate when the leaves were mature and variations in the NSC content were low. Thus, beech radial growth seemed highly dependent on leaf photosynthesis. For oak, earlywood quickly developed before budburst, which probably led to the starch decrease quantified in the stem from April to June. For pine, growth began before the needles unfolding and the lack of NSC decrease during the growing season suggested that the substrates for radial growth were new assimilates of the needles from the previous year. Only for oak, the pattern determined from the intra-annual growth measured using microcoring differed from the pattern determined from dendrometer data. For all species, the ring width was significantly influenced by growth duration and not by growth rate, which differs from previous studies. The observed between-species difference at the intra-annual scale is key information for anticipating suitability of future species in temperate forests.

Tree stem phosphoenolpyruvate carboxylase (PEPC): lack of biochemical and localization evidence for a C4-like photosynthesis system.

Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (Km) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mM and a IC50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway).

Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest.

The stable C isotope composition (delta13C) of CO2 respired by trunks was examined in a mature temperate deciduous oak forest (Quercus petraea). Month-to-month, day-to-day and diurnal, measurements were made to determine the range of variations at different temporal scales. Trunk growth and respiration rates were assessed. Phloem tissue was sampled and was analysed for total organic matter and soluble sugar 13C composition. The CO2 respired by trunk was always enriched in 13C relative to the total organic matter, sometimes by as much as 5 per thousand. The delta13C of respired CO2 exhibited a large seasonal variation (3.3 per thousand), with a relative maximum at the beginning of the growth period. The lowest values occurred in summer when the respiration rates were maximal. After the cessation of radial trunk growth, the respired CO2 delta13C values showed a progressive increase, which was linked to a parallel increase in soluble sugar content in the phloem tissue (R=0.95; P<0.01). At the same time, the respiration rates declined. This limited use of the substrate pool might allow the discrimination during respiration to be more strongly expressed. The late-season increase in CO2 delta13C might also be linked to a shift from recently assimilated C to reserves. At the seasonal scale, CO2 delta13C was negatively correlated with air temperature (R=-0.80; P<0.01). The diurnal variation sometimes reached 3 per thousand, but the range and the pattern depended on the period within the growing season. Contrary to expectations, diurnal variations were maximal in winter and spring when the leaves were missing or not totally functional. By contrast to the seasonal scale, these diurnal variations were not related to air temperature or sugar content. Our study shows that seasonal and diurnal variations of respired 13C exhibited a similar large range but were probably explained by different mechanisms.

(13)C/(12)C isotope labelling to study leaf carbon respiration and allocation in twigs of field-grown beech trees.

In situ (13)C/(12)C isotopic labelling was conducted in field-grown beech (Fagus sylvatica) twigs to study carbon respiration and allocation. This was achieved with a portable gas-exchange open system coupled to an external chamber. This method allowed us to subject leafy twigs to CO(2) with a constant carbon isotope composition (delta(13)C of -51.2 per thousand) in an open system in the field. The labelling was done during the whole light period at two different dates (in June 2002 and October 2003). The delta(13)C values of respiratory metabolites and CO(2) that is subsequently respired during the night were measured. It was found that night-respired CO(2) is not completely labelled (only ca. 58% and 27% of new carbon is found in respired CO(2) immediately after the labelling in June 2002 and October 2003, respectively) and the labelling level progressively disappeared during the next day. It is concluded that the carbon respired by beech leaves after illumination was supplied by a mixture of carbon sources in which current carbohydrates were not the only contributors. In addition, as has been found in herbaceous plants, isotopic data before labelling showed that carbon isotope discrimination favoring the (13)C isotope occurred during the night respiration of beech leaves.

Mixotrophy in orchids: insights from a comparative study of green individuals and nonphotosynthetic individuals of Cephalanthera damasonium.

Some green orchids obtain carbon (C) from their mycorrhizal fungi and photosynthesis. This mixotrophy may represent an evolutionary step towards mycoheterotrophic plants fully feeding on fungal C. Here, we report on nonphotosynthetic individuals (albinos) of the green Cephalanthera damasonium that likely represent another evolutionary step. Albino and green individuals from a French population were compared for morphology and fertility, photosynthetic abilities, fungal partners (using microscopy and molecular tools), and nutrient sources (as characterized by 15N and 13C abundances). Albinos did not differ significantly from green individuals in morphology and fertility, but tended to be smaller. They harboured similar fungi, with Thelephoraceae and Cortinariaceae as mycorrhizal partners and few rhizoctonias. Albinos were nonphotosynthetic, fully mycoheterotrophic. Green individuals carried out photosynthesis at compensation point and received almost 50% of their C from fungi. Orchid fungi also colonized surrounding tree roots, likely to be the ultimate C source. Transition to mycoheterotrophy may require several simultaneous adaptations; albinos, by lacking some of them, may have reduced ecological success. This may limit the appearance of cheaters in mycorrhizal networks.