Figure shows variations of δ13
C of bulk needle material and sucrose (water soluble organic fraction consisted of ≥97% sucrose) synthesized in both light and dark conditions from 14 June 2006 to 28 July 2006. This period includes (i) the phase of active growth (the first half of the vegetation period) in the twigs and new needles until the beginning of July and (ii) the phase of final growth (the second half of the vegetation period). The growing season in this region is less than 60
Figure 2 δ13C values of sucrose* (a) and bulk needle material (b) of 2-year-old pine branches, placed in light (thin lines) and dark (thick lines) chambers during the vegetation period in summer 2006. Stars mark the sharp peaks on the δ13C curves. (more ...)
The measured δ13
C of both sucrose and biomass of needles fluctuates in the light and dark chambers throughout the observed period. Both components display different temporal shifts of the observed peaks in the dark and light conditions (Figure ). Sucrose is the main form of the transported substrates and a carbon resource for the metabolite synthesis [e.g. [32
]. Therefore, the δ13
C course of the biomass generally follows the δ13
C level of sucrose but is 13
C-enriched compared to sucrose. Considering sucrose as the main source of carbon for biomass synthesis, which is accompanied by isotope effects of the pyruvate decarboxylation, we can conclude that the carbon isotope composition of the biomass should differ from that of sucrose to some degree due to respiration.
The CMOM suggests three isotopically different carbohydrate pools formed by photosynthesizing cells in needles with various pathways and relevant for different time intervals according to the respective metabolic needs. As a result, the complete mixing of carbon flows does not occur in the process of the metabolism and the initial differences of δ13C values of carbohydrates from the individual pools remain.
Analyzing the isotopic changes in CO2
respired by the tree leaves in the light and in the dark Tcherkez et al. [9
] supposed that the reasons for the changes might be the difference in metabolic pathways which may change with time, but not the pools having different carbon isotope ratios as follows from CMOM.
De Wit et al. [34
] suggested that a carbohydrate pool develops during photosynthesis, which is used by needles for respiration and for tree growth throughout the vegetation period. Its dynamics are regulated by the concentration of labile carbohydrates such as glucose, sucrose and other assimilates (raffinose, galactose, etc.) in various organs of a tree [36
]. We link this putative pool of carbohydrates to carbohydrates formed in the gluconeogenesis phase via the glycolytic oscillations. This CMOM assumption explains relatively “light” (similar to lipids) isotopic signatures of fresh bulk needle material and sucrose in the initial phase of active growth. It is likely the RCP is developed during the final phase of the preceding vegetation period, once the growth is reduced or terminated and the tree is transitioning to winter dormancy and the onset of the next vegetation period.
The sharp peaks on the δ13
C curves of sucrose (Figure ) can be explained by a change in response of isotopically different carbohydrate flows over time. At night, when the Calvin cycle is not activated, the “heavy” photorespiratory carbohydrate flow from old needle cells ceases, whereas the 13
C-depleted “light” flow from the reserve photosynthetic carbohydrate pool (RCP) continues. The existence of different carbohydrate pools as well as the pool of recent photosynthates was assumed in [10
], but it was admitted that the contribution of these pools to growth and maintenance remains unknown. Furthermore, it was suggested that these C stores are formed at different stages of the growth period.
Figure and show distinct 13C depletion events for the needle sucrose on July 5th and 10th in the light and July 12th in the dark chambers. The 13C depletion in bulk needle material was documented on July 3rd in the dark chamber and July 21st in the light chamber. These events tend to take place around the middle of the growth period and probably indicate the transition from the first active phase of the vegetation period to the second one. The reason for the splitting of 13C depletion of sucrose into two peaks in the dark chamber (Figure ) is not yet clear.
The nature of the 13
C depletion in both needle sucrose and biomass (as growing parts of the trees) cannot be explained by the influence of environmental factors alone. It is unlikely that 13
C depletion signatures are highly correlated with observations of light intensity, precipitation, soil moisture and temperature. For example, a 3-day rainstorm (9 July 07 – 11 July 07) did not affect the carbon isotopic signatures. All together, the effect of 13
C-depleted RCP and the variable carbohydrate flow probably mask the influence of immediate variations in temperature, light etc. The “mask” effect was noticed in [7
], but the authors related it to phloem loading and transport processes.
Clearly, the variations in the use of the RCP cause the fluctuations in carbon isotope signatures of biomass components of the studied pines and mask the short-term relationship between the δ13C values and environmental conditions. We assume that the 13C depletion probably works like an ontogenetic clock. When the newly formed needle cells start their own photosynthetic assimilation, the assimilate flow associated with the Calvin cycle is insufficient to feed the glycolytic chain with substrates, and the RCP compensates this deficit. The flow of “light” substrates originating from the RCP leads to a further 13C depletion while passing through the pyruvate dehydrogenase complex, and develops extremely “light” carbohydrates that may cause the abrupt depletion events (Figure ). Therefore, these phenomena can be linked to the transition from the phase of active growth to the phase of assimilate accumulation. These effects were registered under various environmental conditions in all our experimental data of bulk needle material and its sucrose from the dark and the light treatments (Figures and ), and δ13C of twig cellulose in the current and previous vegetation seasons (Figure ). This supports the tentative hypothesis of ontogenetic origin of the 13C-depleted carbohydrates, which needs to be additionally verified.
Figure 3 Courses of δ13C values of sucrose* (a) and bulk needle material (b) in needles of first (continuous black lines) and second (dotted black lines) year for the vegetation period 2006. Dotted vertical lines mark the halves of growing period for the (more ...)
Furthermore, the common pattern of the δ13C signatures among all studied (Figure and ) plots includes: (i) sawtooth-like variations of δ13C components and (ii) abrupt decrease in δ13C values during the transition from the first half of vegetation period to the second half. The pattern also includes various offsets of negative sucrose δ13C peaks and the time intervals between corresponding peaks of bulk needle material. The peaks in sucrose appear much closer to each other than those in bulk needle material, which is hard to explain by means of the CMOM.
Figure shows δ13
C signatures of sucrose and biomass of the year 2006 and year 2005 in needles sampled from the light chambers. The sucrose 13
C of the first-year needles is substantially (
= 1.97; Φ
0.05) depleted in comparison to sucrose 13
C of the second-year needles. Student’s t-test (
= 0.58; Φ
21) shows insignificant differences in the mean of the needle biomass δ13
C of the first year and the second year (Figure ).
Metabolism coupled with the process of respiration results in gradual scavenging of 12
C isotopes in needles, since the RCP does not contribute significantly to the needle formation in the following year. Similar processes trigger the isotopic imbalance in needle and biomass carbohydrates as well. However, the 12
C-scavenging effect in the case of biomass is weaker. The result agrees with conclusions of other studies e.g. [37
], which suggest the remobilization of carbohydrate reserves stored in the prior year occurs predominantly during the process of wood formation at the beginning of the vegetation period.
Newly formed RCPs may impact the carbon isotope composition of tree-ring cellulose components for several years. Boettger and Friedrich [39
] found a one-year memory effect in δ13
C-time series of Pinus sylvestris
L. growing at the northern tree line of the Khibiny Mountains (NW Russia) and firs (Abies alba
Mill.) growing in Franconia (central Germany). Moreover, individual trees show even longer memory effects lasting for two and four years in pine and fir, respectively [39
]. The deferral pattern in the RCP depletion was explained by: i) limitations in the availability of the reserve substances in the trees growing at their distribution limit in contrast to the trees growing in favorable conditions, and ii) differences in storage of the carbohydrates by different tree species [40
The results shown in Figures and indicate that during the first half of the vegetation period the bulk needle material is slightly enriched with 13C, in contrast with needle sucrose. The enrichment occurs during the period of heterotrophic growth due to the isotope effect of pyruvate decarboxylation in the glycolytic chain due to respiration releasing 13C-depleted CO2 (Figure ). In the second half of the vegetation period, 13C enrichment of sucrose increases relative to 13C biomass because the newly formed cells begin photosynthesis and the influx of carbohydrates from the CCP increases significantly due to photorespiration.
Figure 4 Differences (Δ) between δ13C values of sucrose* and δ13C of biomass from needles measured in the light and in the dark during the vegetation period 2006. *Water soluble organic fraction consisted of ≥97% sucrose (more ...)
This effect is less pronounced in the dark chambers as compared to the light chambers. The slopes of differences between δ13C values of sucrose and biomass of needles sampled in the dark and in the light conditions (Δ13C) are 0.03 and 0.10, respectively (Figure ). The gradient in the dark chamber values is small because the Calvin cycle does not operate in the darkness, and the “heavy” substrates formed in the oxygenase phase probably do not contribute significantly to the biomass formation. Thus, the δ13C variations in sucrose and biomass of the first and second year needles confirm once again the fluctuations of isotopic signatures of sucrose and biomass over time.
Figure shows clear evidence of 13C depletion for sucrose and bulk needle material in the middle of the vegetation period. The correlation of δ13C trends indicates a close relationship between all studied components, which confirms the regular replacement of labile carbohydrates from the RCP and the CCP in the first half and the second half of the vegetation period, respectively.
Figure 5 a) δ13C course of 1-year old bulk needle material (thin line), cellulose of 1-year-old (dotted black line) and 2–year-old (thick black line) twigs and phloem sucrose* (thick gray line) in the light during the vegetation period 2006; b) (more ...)
C trends of sucrose and proteins isolated from the biomass of needles grown in the light chamber (Figure ) show significant 13
C enrichment in proteins. This is a result of carbon isotope fractionation in pyruvate decarboxylation corresponding to the Rayleigh effect and isotopic balance. Sucrose is the initial substrate feeding the glycolytic chain, whereas proteins are the product of their next transformation including those occurring in the pyruvate dehydrogenase complex (Figure ). The role of pyruvate in C allocation between respiratory pathways, which may be different in growth, maintenance and post-photosynthetic metabolism, was underlined in [8
]. This is in line with the CMOM’s assertion of a different role of the reaction in carbon isotope distribution at different functional states of the organism.
The carbon isotopic composition of sucrose and proteins depends on various factors. The sucrose δ13C depends mainly on the isotope effects during CO2 assimilation and photorespiration, while the protein δ13C varies depending on the isotope effects of the pyruvate decarboxylation and the lipid-carbohydrate and protein-carbohydrate metabolism. Likewise, oxygen resulting from water reduction during CO2 assimilation is another important player in the processes of photosynthesis and photorespiration. In order to demonstrate the interactive relationship between oxygen and carbon composition, fluctuations in the oxygen and carbon isotope ratios from pine biomass components were compared.
Figure clearly shows a negative correlation between the δ13
C and δ18
O values of all corresponding variables. The correlation coefficients between δ13
C and δ18
O values of biomass and proteins from the light chamber and δ13
C and δ18
O values of biomass from the dark chamber are significant and equal to −0.70 (n
14) at p
0.01, -0.50 (n
18) at p
0.05 and −0.71 (n
9) at p
0.05, respectively. Interestingly, the corresponding isotope ratios of carbon and oxygen in the stem cellulose are significantly correlated (r
−0.86 at p
11) as well (not shown in Figure ). However, the correlation between carbon and oxygen isotope signatures in the sucrose is insignificant (Figure and ) for both the light and dark chambers, and proteins synthesized in the dark (Figure ).
Figure 6 The courses of δ13C (black lines) and δ18O (dotted gray lines) of sucrose* (a,d), biomass (b,e) and proteins (c,f) from the light (upper panel) and dark (lower panel) chambers during the growth season 2006. *Water soluble organic fraction (more ...)
Clearly, the isotopic characteristics of CO2
are strongly correlated since these gases participate at the same stages of the photosynthesis process. It is well known that the isotope composition of oxygen in photosynthesis corresponds to the rate of oxygen consumption in photorespiration e.g. [41
]. The fractionation of oxygen isotopes results in the preferred involvement of 16
O in the process of photorespiration and, partially, in the process of biosynthesis, whereas the “heavy” isotope 18
O is accumulated in O2
] and released by the cells into the atmosphere [44
The negative relationship between δ13C and δ18O values (Figure ) is possibly caused by the Rayleigh effect on the CO2-assimilated carbon isotope and the oxygen isotope absorbed during photorespiration due to photosynthetic oscillations. The rate of 18O-enrichment in molecular oxygen depends on the degree of O2 involvement in photorespiration, which differs from the amount of O2 generated through the photosynthetic reduction of CO2. The amount of produced O2 is determined by the proportion of the amount of reduction of CO2 diffused into the cell and CO2 available for assimilation. This means that as more CO2 is involved in assimilation and as the assimilated carbon (biomass) becomes more 13C-enriched, more oxygen will be produced.
Furthermore, the greater the proportion of produced oxygen, the more “lighter” oxygen (16O-enriched) is included in the process of photorespiration and biomass synthesis (Figure and ).
The δ13C and δ18O values of bulk needle material, needle sucrose and needle protein are negatively correlated (Figure , and ). However, the relationships between the δ13C and δ18O values of biomass components are not strong, since most of the oxygen in the biomass oxygen originates from CO2. The results suggest a reciprocal interaction between the Rayleigh effects during CO2 assimilation and photorespiration.