Differences in instantaneous water use efficiency derived from post-carboxylation fractionation respond to the interaction of CO 2 concentrations and water stress in semi-arid areas

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Introduction
Since the onset of the industrial revolution, the atmospheric CO2 concentration has increased at an annual rate of 0.4%, and is expected to increase further to 700 μmol•mol -1 , together with more frequent periods of low water availability (IPCC, 2014).Increasing atmospheric CO2 concentrations that trigger an ongoing greenhouse effect will not only lead to fluctuations in global patterns of precipitation, but also will amplify drought in arid regions, and lead to more frequent occurrences of extreme drought events in humid regions (Lobell et al., 2014).Accompanying the increasing concentration of CO2, the mean δ 13 C of atmospheric CO2 is depleted by 0.02‰-0.03‰year -1 (data available from the CU-INSTAAR/NOAACMDL network for atmospheric CO2; http://www.esrl.noaa.gov/gmd/).
The carbon isotopic composition determined recently could respond more subtly to environmental changes and their influences on diffusion via plant physiology and metabolic processes (Gessler et al., 2014;Streit et al., 2013).While the depletion of δ 13 C CO 2 has been shown in the atmosphere, variations in CO2 concentration itself also might affect the δ 13 C of plant organs that, in turn, respond physiologically to climatic change (Gessler et al., 2014).The carbon discrimination ( 13 Δ) of leaves could also provide timely feedback about the availability of soil moisture and the atmospheric vapor pressure deficit (Cernusak et al., 2012).Discrimination against 13 C in leaves relies mainly on environmental factors that affect the ratio of intercellular to ambient CO2 concentration (Ci/Ca) and Rubisco activities (Farquhar et al., 1982).As changes in environmental conditions affect photosynthetic discrimination, they are expected to be recorded differentially in the δ 13 C of water-soluble organic matter (δ 13 C WSOM) of the different plant organs.
Meanwhile, several processes during photosynthesis alter the δ 13 C of carbon transported within plants considerably.Carbon-fractionation during photosynthetic CO2 fixation has been described and reviewed well elsewhere (Farquhar et al., 1982;Farquhar and Sharkey, 1982).
Post-photosynthetic fractionation is derived from equilibrium and kinetic isotopic effects, which determines isotopic differences between metabolites and intramolecular reaction positions.Several scholars have defined the carbon isotopic fractionations that occur between the leaf and wood cambium as "post-photosynthetic" or "post-carboxylation" fractionation (Jä ggi et al., 2002;Badeck et al., 2005;Gessler et al., 2008).Post-carboxylation fractionation in plants includes the carbon discriminations that follow carboxylation of ribulose-1, 5-bisphosphate, and internal diffusion (RuBP, 27‰), as well as related transitory starch metabolism (Gessler et al., 2008;Gessler et al., 2014), fractionation in leaves, fractionation-associated phloem transport, the remobilization or storage of soluble carbohydrates, and starch metabolism fractionations in sink tissue (tree rings).In sucrose synthesis, 13 C-depletions of triose phosphates occur during exportation from the cytoplasm, and during production of fructose-1, as does 6-bisphosphate by aldolase in transitory starch synthesis (Rossmann et al., 1991;Gleixner and Schmidt, 1997).Synthesis of two sugars before transportation to the twig is associated with the post-carboxylation fractionation generated in leaves.
It is also important to consider that the carbon cycling time within plants has an absolute influence on the time integration of photosynthetic carbon discrimination.Several studies have indicated that recently-assimilated carbohydrate that is imprinted with environmental signals is mixed with other carbohydrate pools of different ages during transportation along the basipetal tree axis (Brandes et al., 2006;Richardson et al., 2012).It is necessary to avoid confusion of carbon sources, and further, to determine carbon fractionation within leaves following photosynthetic carboxylation.In addition, Discuss., doi:10.5194/bg-2016Discuss., doi:10.5194/bg- -372, 2016 Manuscript under review for journal Biogeosciences Published: 7 September 2016 c Author(s) 2016.CC-BY 3.0 License.whether these fractionations are related to environmental variation has not yet been investigated.

Biogeosciences
The simultaneous isotopic analysis of leaves is a recent refinement in isotope studies that allows us to determine the temporal variation in isotopic fractionation (Rinne et al., 2016), and will help decipher environmental conditions more reliably.Newly assimilated carbohydrates can be extracted, and are defined as the water-soluble compounds (WSCs) in leaves (Brandes et al., 2006;Gessler et al., 2009), which also can be associated with gas exchange properties on a daily basis (Kodama et al., 2008).However, there is dispute whether the post-carboxylation fractionation process may alter the stable signatures of leaf carbon and thence influence instantaneous water use efficiency (iWUE).
In addition, the way in which the iWUE derived from that isotope fractionation responds to different environmental factors, such as elevated [CO2] and/or soil water gradients, has not yet been observed.
Consequently, we investigated δ 13 C of the fast-turnover carbohydrate pool in leaves from saplings of two trees typical in semi-arid areas of China-Platycladus orientalis and Quercus variabilistogether with simultaneous gas exchange measurements in growth chambers .Our goals are to compare the differences in iWUE derived from 13 C-frationation of post-carbonxylation between P. orientalis and Q. variabilis, and to describe how these differences in iWUE respond to the interactive effects of elevated [CO2] and water stress.

Study site and design
Saplings of P. orientalis and Quercus variabilis were selected as experimental material from the Capital Circle forest ecosystem station, a part of the Chinese Forest Ecosystem Research Network (CFERN, 40 º 03'45"N, 116 º 5'45"E) in Beijing, China.This region is populated by warm, temperate, deciduous, broad-leaved trees and mixed tree communities dominated by Quercus spp.and Platycladus orientalis (L.) Franco, respectively.Saplings have similar ground diameters, heights, and growth statuses.The saplings were placed in pots 22 cm in diameter and 22 cm in height.
Undisturbed soil samples were collected from the field in the research region, and the sieved soil (with all particles <10 mm removed) was placed in the pots.A single P. orientalis sapling was transplanted into each pot.The soil bulk density in the pots was maintained at 1.337-1.447g• cm -3 .
After one month of rejuvenation, the potted saplings were placed into chambers for cultivation.
The controlled experimental treatments were conducted in growth chambers (FH-230, Taiwan Hipoint Corporation, Kaohsiung City, Taiwan).To imitate the meteorological factors of the growth seasons in the research region, the daytime temperature in the chambers was set to 25 ± 0.5℃ from 07:00 to 17:00, and the night-time temperature was 18 ± 0.5℃ from 17:00 to 07:00.Relative humidity was maintained at 60% and 80% during the day and night, respectively.The light system was activated in the daytime and shut down at night.The average daytime light intensity was maintained at 200-240 µmol• m -2 • s -1 .CO2 concentration was controlled by the central controlling system of the chambers (FH-230).Two growth chambers (A and B) were used in our study.
We designed a device to water the potted plants automatically to avoid heterogeneity caused by interruptions in the watering process (Fig. 1).It consisted of the water storage tank, holder, controller, soil moisture sensors, and drip irrigation components.Prior to use, the water tank was filled with water, and the soil moisture sensor was inserted to a uniform depth in the soil.After connecting the controller to an AC power supply, specific soil water could be set.The soil volumetric water content (SWC) of the pot soil was monitored by the soil moisture sensors.Through the sensors, the chamber could determine whether to water or stop watering the plants.Two drip irrigation devices were installed in both chambers, respectively.After measuring the average Field Capacity (FC) of the pot soil (30.70%), five levels of SWC were maintained before the orthogonal tests, as follows: 100% FC (or CK) (SWC approximately 27.63%-30.70%),70%-80% of FC (SWC approximately 21.49%-24.56%),60%-70% of FC (SWC approximately 18.42%-21.49%),50%-60% of FC (SWC approximately 15.35%-18.42%),and 35%-45% of FC (SWC approximately 10.74%-13.81%).Each level of soil water was kept within the specific range thereafter by the irrigation device.
After establishing the equilibrium circumstances of elevated CO2 across the soil water gradients, the saplings were ready for investigation.Each orthogonal treatment included three replicates, and each replicate continued for 7 days.Pots were rearranged periodically to minimize non-uniform illumination.

Foliar gas exchange measurement
Fully expanded primary annual leaves of the saplings were measured with a portable infrared gas photosynthesis system (LI-6400, Li-Cor, Lincoln, US) before and after the 7-day cultivation in the chambers.The main photosynthetic parameters, such as net photosynthetic rate (Pn) and transpiration rate (Tr), were measured.Based on the theories proposed by Von Caemmerer and Farquhar (1981), stomatal conductance (gs) and intercellular CO2 concentration (Ci) were calculated by the Li-Cor software.Each leaf was measured three times.Three leaves from each sapling were chosen, and three saplings were measured within one orthogonal treatment.Instantaneous water use efficiency via gas exchange (WUEge) was calculated as the ratio of Pn to E.

Plant material collection and sample preparation
After measuring gas exchange, recently-expanded, sun leaves were removed from the P. orientalis and Quercus variabilis saplings cultivated in the orthogonal treatments, and frozen immediately in liquid nitrogen.A protocol adapted from Gessler et al. (2004) was used to extract the water-soluble compounds (WSCs).All samples were ground to fine powders using mortars and liquid nitrogen.50 mg of ground leaves and 100 mg PVPP (polyvinylpolypyrrolidone) were weighed, mixed evenly, and incubated in 1ml double demineralized water for 60 min at 5℃ in a centrifuge tube.Then, the tubes were heated in 100℃ water for 3 min.After they cooled to room temperature, the supernatant was centrifuged at 12000 xg for 5 min and transferred into tin capsules Biogeosciences Discuss., doi:10.5194/bg-2016-372,2016 Manuscript under review for journal Biogeosciences Published: 7 September 2016 c Author(s) 2016.CC-BY 3.0 License.
to be dried at 70℃.Folded capsules were then ready for δ 13 C analysis of WSOM.
The samples of WSCs from leaves were combusted in an elemental analyzer (EuroEA, HEKAtech GmbH, Wegberg, Germany) and analyzed in the mass spectrometer (DELTA plus XP, ThernoFinnigan).Carbon isotope signatures are expressed in δ-notation in parts per thousand, relative to the international Pee Dee Belemnite (PDB): where δ 13 C is the heavy isotope and   and   refer to the isotope ratio between the particular substance and the corresponding standard, respectively.The precision of the repeated measurements was 0.1 ‰.

Isotopic calculation
Based on the linear model developed by Farquhar and Sharkey (1982), the isotope discrimination factor, Δ, was calculated as: where   13 is the isotope signature of ambient [CO2] in the chamber; C P 13 is the  ∶ 13  12 of the water-soluble compounds extracted from foliage.The Ci : Ca is determined by: where Ci is the intercellular CO2 concentration, and Ca is the ambient CO2 concentration in the chamber; a is the discrimination dependent on a fraction factor (4‰). b is the discrimination during CO2 fixation by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and internal diffusion (27‰).Instantaneous water use efficiency (iWUE) is calculated as: where   is the net carbon assimilation,   is the molar rate of transpiration, and 1.6 is the diffusion ratio of stomatal conductance to water vapor to CO2 in the chamber.∆ is the difference in water vapor pressure between the intracellular in leaves and ambient air, which may be calculated as: where e lf and e atm represent the extra-and intra-cellular water vapor pressure, respectively.T and RH is temperature and relative humidity on leaf surface.
is the ratio between carbohydrates consumed during respiration of the leaves and that of other organs at night (0.3).
When SWC increased, most Pn in P. orientalis peaked at 70%-80% of FC, while that of Q. variabilis reached higher values of 70%-80% of FC and FC.The uptake capacity of carbon was improved significantly with elevated [CO2] at any given soil moisture (p<0.05) for P. orientalis.We observed increased Pn of Q. variabilis after CO2 gradient-fumigation, except under C400.Further, greater magnitudes of increments in Pn of P. orientalis were found at 50%-70% of FC from C400 to C800, which was observed at 35%-45% of FC for Q. variabilis.Instantaneous carbon assimilation capacities of Q. variabilis among all treatments were stronger than were those of P. orientalis (Figs. 2a and 2b, p<0.01).
The gs in P. orientalis coincided with Pn as soil moisture increased, and was highest at 70%-80% of FC in C400, C500, and C800 (Fig. 2c), while it peaked at FC in C600.When SWC increased from 35%-45% to 50%-60% of FC, gs in Q. variabilis moved up sharply and then increased gradually to its maximum in C400, C500, and C800 as soil moisture increased.As the water stress was alleviated (at 70%-80% of FC and FC), the reduction of gs in P. orientalis was more pronounced with elevated [CO2] at a given SWC (p<0.01).Nevertheless, gs of Q. variabilis in C400, C500, and C600 was significantly higher than that in C800 at 50%-80% of FC (p<0.01).gs in Q. variabilis exceeded that in P. orientalis under the same treatments (p<0.01,Figs.2c and 2d).
The Ci in P. orientalis rose gradually as SWC increased, and peaked at FC at any given [CO2].
Under the same conditions of cultivation as P. orientalis, Ci of Q. variabilis reached their maximums at 60%-70% of FC, and declined thereafter with increased SWC.The variation in Ci of the two species was similar and decreased as [CO2] elevated.Ci of Q. variabilis was significantly greater than was that of P. orientalis under the same treatment (p<0.01,Figs.2e and 2f).
The Tr of P. orientalis and Q. variabilis all exhibited single peaks that occurred at 70%-80% of FC in combination with the soil moisture gradient.The Tr of the two saplings in different [CO2] were compared at each SWC (Figs. 2g and 2h).Except for 35%-60% of FC, the Tr of the two saplings in C400 and C500 was significantly higher than that in C600 and C800 (p<0.01).With the same [CO2] and the same SWC, the Tr of Q. variabilis was remarkably larger than was that of P. orientalis (p<0.01).

δ 13 C of water-soluble compounds in leaves
To observe the photosynthetic traits of the two saplings, the same leaf was frozen immediately and the water-soluble compounds (WSCs) were extracted for all orthogonal treatments.δ 13 CWSC (δ 13 C of water-soluble compounds from leaves) of P. orientalis and Q. variabilis saplings cultivated in the four CO2 concentrations all increased as soil moisture improved (Figs. 3a and 3b, p<0.01).

Estimations of WUEge and WUEcp
Instantaneous water use efficiency via gas exchange (WUEge) is calculated as Pn divided by Tr.
Figure 4a shows that increased magnitudes of WUEge of P. orientalis under severe drought (i.e., 35%-45% of FC) were highest at any given [CO2], ranging from 90.70% to 564.65%.As SWC increased, WUEge reduced along a gradient in C400, C500, C600, and C800, while they increased promoted slightly at FC in C600 or C800 as soil water increased (Fig. 4b).The maximum of WUEge in P. orientalis thus occurred at 35%-45% of FC in C800 for all orthogonal treatments, and did so in Q. variabilis as well.Further, elevated CO2 concentrations enhanced the WUEge of Q. variabilis clearly at any SWCs except that at 60%-80% of FC.Based on the comparison between the same [CO2] × SWC treatments, most saplings of P. orientalis had greater WUEge than did Q.variabilis (p<0.05).
The δ 13 C values of water-soluble compounds from leaves of the two saplings were measured, and the instantaneous water use efficiency could be determined from Eq. ( 6) for the δ 13 CWSC of leaves, defined as WUEcp.As illustrated in Fig. 5a, WUEcp of P. orientalis in C600 or C800 climbed up as water stress was reduced beyond 50%-60% of FC, while the water threshold was 60%-70% of FC in C400 or C500.Q. variabilis exhibited no uniform trend of WUEcp with soil wetting.Except for C400, WUEcp of Q. variabilis decreased abruptly at 50%-60% of FC, and rose as soil moisture improved in C500, C600, and C800. Figure 5b shows the effects of elevated CO2 on WUEcp in Q. variabilis.In contrast to the findings for WUEge in the two saplings, the WUEcp of Q. variabilis was more pronounced than was that of P. orientalis for all orthogonal treatments.

Post-carboxylation fractionation generated before photosynthate leaves the leaves
We evaluated the differences between instantaneous water use efficiency via leaf gas exchange and δ 13 C of water-soluble compounds (Table 1), which can retrace 13 C fractionation before carboxylation transport to the twig.When comparing WUEge and WUEcp, the 13 C-depletion of P.
orientalis ranged from 0.0328‰ to 0.0472‰, which was smaller than that of Q. variabilis (0.0384‰ to 0.0466‰).The fractionation effects of P. orientalis were magnified with increased soil moisture, and particularly at 35%-80% of FC from C400 to C800, the magnitudes of the increments reached 21.30%-42.04%.Under C400 and C500 in Q. variabilis, the coefficients were amplified, as SWC increased until 50%-60% of FC, then remained constant.With respect to C600 and C800, the coefficients of Q. variabilis were amplified at 50%-80% of FC, and decreased at FC.The average coefficients of P. orientalis increased gradually as [CO2] rose, while those of Q. variabilis declined sharply from C600 to C800.Coefficients of P. orientalis increased at a faster rate than did those of Q.
variabilis with increased soil moisture.
Stoma are the conduit between the plant and atmosphere.Post-carboxylation fractionation may be correlated with the degree to which the stomata are open and the resistance they exert.Here, we performed linear regressions between gs and the 13 C fractionation coefficient for P. orientalis and Q. variabilis (Fig. 6).It was apparent that the 13 C fractionation coefficient was linearly dependent on the gs (p<0.05), which controls the exchange of CO2 and H2O, and responds to environmental variation.

Photosynthetic traits
The exchange of CO2 and water vapor via stomata is modulated in part by the soil or leaf water potential (Robredo et al., 2010).Saplings of P. orientalis reached their maximums of Pn and gs at 70%-80% of FC under any [CO2].As SWC exceeded the water threshold, elevated CO2 caused a greater reduction in gs, as has been reported for barley and wheat (Wall et al., 2011).Nonetheless, maximal values of gs in Q. variabilis in C400, C500, C600, and C800 were generated successively as soil moisture increased, indicating that drought stimulated the stomata, which are more sensitive to environmental changes.In addition, Ci of Q. variabilis peaked at 60%-70% of FC, following declines as soil moisture increased (Wall et al., 2006;Wall et al., 2011).The gs of P. orientalis and Q. variabilis decreased with elevated [CO2], which was evidenced by FACE and non-FACE experiments (Ainsworth and Rogers, 2007;Wall et al., 2011).This is interpreted as stomata having the tendency to maintain a constant Ci or Ci/Ca when ambient [CO2] increases, which would determine the CO2 used directly in chloroplast (Yu et al., 2010).On the basis of theories proposed by Farquhar and Sharkey (1982) and common experimental technologies (Xu, 1997), this could be explained as a stomatal limitation.However, Ci of P. orientalis was observed to increase considerably when SWC exceeded 70%-80% of FC in all CO2 treatments (Mielke et al., 2000).One factor that can account for this is that plants close their stomata to reduce intensive loss of water during the synthesis of organic matter, simultaneously decreasing the availability of CO2 and generating respiration of organic matter (Robredo et al., 2007).Another explanation is that the limited root volume in potted experiments may not be able to absorb sufficient water to support full growth of shoots (Leakey et al., 2009;Wall et al., 2011).When SWC exceeds the threshold (70%-80% of FC), further elevations in [CO2] may cause nonstomatal limitation, i.e., accumulation of nonstructural carbohydrates in leaf tissue that induces mesophyll-based and/or biochemical-based transient inhibition of photosynthetic capacity (Farquhar and Sharkey, 1982).Xu and Zhou (2011) developed a five-level SWC gradient to examine the effect of water on the physiological and ecological characteristics of perennial Leymus chinensis.They demonstrated that there was an evident threshold in the gradient at which a plant could manage its structure and function to adjust to environmental conditions.Miranda Apodaca et al. (2015) also concluded that, in suitable water conditions, elevated CO2 augmented CO2 assimilation of herbaceous plants.
The Pn of the two saplings increased with increased [CO2] in our study, as was found previously for C3 in woody plants (Kgope et al., 2010).Further, increasing [CO2] appeared to alleviate soil water stress at 35%-45% or 50%-60% of FC, which proves that photosynthetic inhibition produced by water stress (or excess) may be moderated by increased [CO2] (Robredo et al., 2007;Robredo et al., 2010).These results are consistent with a number of studies in which elevated CO2 was expected to ameliorate the adverse effects of drought stress by decreasing plant transpiration (Kirkham, 2016;Kadam et al., 2014;Miranda Apodaca et al., 2015;Tausz Posch et al., 2013).

δ 13 C of water-soluble compounds
Stable isotope ratios of plant tissues have been applied widely to evaluate the ecophysiological processes that interact with environmental variation, especially those that control plant-atmosphere exchanges of mass circulation and energy flow (McCarroll and Loader, 2004;Poussart et al., 2004;Rinne et al., 2010).Based on the relationship between photosynthetic carbon isotope fractionation (Δ 13 C or Δ) and the ratio between internal leaf and ambient CO2 concentration (Ci/Ca: Eq. ( 2) and Eq.(3), Farquhar et al. 1982), the δ 13 C of plant tissue could characterize effects of environmental interaction on internal reactions and processes of photosynthesis (Gessler et al., 2014).Further, the leaf carbon isotope ratios are an excellent surrogate for direct measurement of iWUE (Eq.( 4) shows the determination of iWUE by δ 13 C), which was fractionated over CO2 diffusion into leaf via stomata and carboxylation in chloroplast.The δ 13 C of water-soluble compounds (δ 13 CWSC) extracted from saplings' leaves has been measured to examine the physiological and metabolic responses to current environmental variation (Streit et al., 2013).The authors found that the average δ 13 CWSC of P. orientalis and Q. variabilis were correlated positively with the increment of soil moisture during Biogeosciences Discuss., doi:10.5194/bg-2016-372,2016 Manuscript under review for journal Biogeosciences Published: 7 September 2016 c Author(s) 2016.CC-BY 3.0 License.
35%-80% of FC, which was demonstrated by Adiredjo et al. (2014) as well.Thus, 13 CWSC may respond according to the availability of soil moisture, which was reviewed by Cernusak et al. (2012).
Once it exceeds 70%-80% of FC, the average δ 13 CWSC values of the two saplings with soil moistening were consistent with the trends of gs.Elevated CO2 concentrations affected physiological performance profoundly (Gimeno et al., 2015), especially by increasing the CO2 supply to the chloroplasts and reducing stomatal conductance, which would have influenced δ 13 CWSC indirectly in this study.

Differences between WUEge and WUEcp
The increments of WUEge in P. orientalis and Q. variabilis that resulted from the combination of an increase in Pn and decrease in gs, followed by the reduction of Tr (Figs. 1a,1g,1b and 1h), also were demonstrated by Ainsworth and McGrath (2010).Combining the Pn and Tr of P. orientalis and Q. variabilis in the same treatment, the lower WUEge in Q. variabilis is achieved generally by the plant's physiological and morphological traits, such as larger leaf area, rapid growth, and higher stomatal conductance than that of P. orientalis (Adiredjo et al., 2014).Medlyn et al. (2001) reported that the stomatal conductance of broadleaved species is more sensitive to elevated CO2 concentrations than is that of conifers.Moreover, with respect to the patterns of iWUE, there has been no consensus on soil water states at the leaf level, although some have discussed this topic (Yang et al., 2010).As SWC decreased, the WUEge of P. orientalis and Q. variabilis was enhanced, as presented by Parker and Pallardy (1991), DeLucia andHeckathorn (1989), andReich et al. (1989).Leakey (2009) also concluded that the WUE of stressed plants could be increased substantially, which was shown more clearly with elevated [CO2] in this study.Bögelein et al. (2012) confirmed that WUEcp was more consistent with daily mean WUEge than was WUEphloem.The WUEcp of P. orientalis and Q. variabilis demonstrated similar variations to those of δ 13 CWSC, and water stress was alleviated when combined with elevated [CO2], which differentiated the trends in WUEge.The assumption has been made that δ 13 CWSC is coupled tightly with dynamics in the environment several days before the water-soluble compounds is extracted.
As observed, the WUEcp of P. orientalis and Q. variabilis responded synthetically with increasing SWC across different [CO2] gradients over the course of several days.Consequently, WUEcp and WUEge have different variable curves according to treatments.In addition, there were characteristic species-specific responses of δ 13 CWSC under the same environmental conditions.

Post-carboxylation fractionation generated before photosynthate leaving leaves
Photosynthesis, a biochemical and physiological process (Badeck et al., 2005), is characterized by discrimination against 13 C, which leaves an isotopic signature in the photosynthetic apparatus.
There is already a classic review of the carbon-fractionation in leaves (Farquhar et al., 1989) that covers the significant aspects of photosynthetic carbon isotope discrimination.The transportation route of photosynthate production, from leaf to wood formation, consists of post-assimilation fractionations/processes, referred to as "post-photosynthetic" or "post-carboxylation" fractionation (Jä ggi et al., 2002;Gessler et al., 2008).The post-photosynthetic fractionation associated with the metabolic pathways of non-structural carbohydrates (NSC; defined here as soluble sugars + starch) within leaves, and fractionation during translocation, storage, and remobilization prior to tree ring formation remain unclear (Epron et al., 2012;Gessler et al., 2014;Rinne et al., 2016).
photosynthetic carbon isotope discrimination in terrestrial plants.The 13 C fractionation effects of P. orientalis were enhanced by soil moistening, consistent with that of Q. variabilis, except at FC.The 13 C isotope signature of P. orientalis was dampened by elevated [CO2].Yet, 13 C-depletion was weakened in Q. variabilis in C600 and C800.Linear regression between gs and the 13 C fractionation coefficient indicated that the post-carboxylation fractionation in leaves depended on the variation of gs and stomata aperture correlated with environmental change.5ConclusionsThrough orthogonal treatments of four [CO2] × five SWC, WUEcp calculated by δ 13 C of watersoluble compound and WUEge derived from simultaneous leaf gas exchange for leaves were estimated to differentiate the δ 13 C signal variation before leaf-exported translocation of primary assimilates.In response to the interactive effects of [CO2] and SWC, WUEge of the two species of saplings both decreased with soil moistening, and increased with elevated [CO2] at 35%-80% of FC.We concluded that relative soil drying, coupled with elevated [CO2], could improve WUEge by strengthening photosynthetic capacity and reducing transpiration.WUEge of P. orientalis was significantly greater than was that of Q. variabilis, while the opposite was the case for WUEcp in the two species.Rising [CO2] and/or soil moistening generated increasing disparities between WUEge and WUEcp in P. orientalis; nevertheless, the differences between WUEge and WUEcp in Q. variabilis increased as [CO2] increased and/or water stress was alleviated.The 13 C fractionation also was linearly dependent on gs.With respect to post-photosynthesis fractionation in postcarboxylation and transportation processes, we cannot neglect the fact that the instantaneous water use efficiency derived from the synthesis of sucrose and starch were influenced inevitably by environmental changes.Thus, cautious descriptions of the magnitude and environmental dependence of apparent post-carboxylation fractionation are worth our attention in photosynthetic fractionation.

Figure 1 .
Figure 1.Structural diagram of the device for automatic drip irrigation Arabic numerals indicate the individual parts of the automatic drip irrigation device (No. 1-7).The lower-left corner of this figure presents the detailed schematic for the drip irrigation components (No. 8-12).The lower-right corner of this figure shows the schematic for the drip irrigation component in profile.

Figure 6 .
Figure 6.Regression between stomatal conductance and 13 C fractionation coefficient of P. orientalis and Q. variabilis under four CO2 concentrations × five soil volumetric water contents are established in Figs.6a and 6b.p=0.05, n = 32.