Photosynthetic parameters and stomatal conductance in attached and detached balsam fir foliage

Abstract Leaf level gas‐exchange measurements can be made on detached foliage to address the challenge of access to the crown of tall trees. However, detachment may impact leaf gas exchange. This necessitates the study of gas‐exchange characteristics of foliage on detached branches to assess the feasibility of using detached branches for gas‐exchange analysis. We compared photosynthetic parameters and stomatal conductance in foliage of attached and detached branches of balsam fir [Abies balsamea (L.) Mill.] during the growing season. Data were analyzed using a linear mixed‐effect model, with fixed and random effects (branch status and measurement month, and tree number, respectively). Branch detachment had no significant effects on: (i) photosynthesis at the current ambient CO2 concentration (400 µmol mol−1, A 400); (ii) maximum rates of Ribulose‐1,5‐bisphosphate (RuBP) carboxylation (V cmax) and regeneration (J max); (iii) the ratio of J max to V cmax (i.e., J max:V cmax), and (iv) stomatal conductance (g s) during the study period (p = 0.120–0.335). There was a strong seasonal effect on all gas‐exchange variables (p ≤ 0.001–0.015). Gas‐exchange measurements made on detached foliage during the warm summer months should be performed with care. Reliable gas‐exchange measurements can be obtained using balsam fir foliage on detached branches 50–80 cm in length, in cooler growing‐season months, up to 30 min after detachment.

The process of detaching foliage from trees, that is, branch cutting, the interval between branch cutting and gas-exchange measurements, or both, can affect the variables being assessed (Richardson & Berlyn, 2002). The photosynthetic capacity of cut foliage may be affected by resultant changes in cell turgor or stomatal aperture (Clark, 1954;Lundegardh, 1931), and drawdown of moisture which may occur because of continued transpiration (Meng & Arp, 1993).
However, detachment is still a viable method for measuring gas exchange (see researchers listed in Table 1), but few studies have reported on its effects on gas-exchange measurements (Clark, 1954;Gauthier & Jacobs, 2018;Koike & Sakagami, 1984;Meng & Arp, 1993). Clark (1954), Koike and Sakagami (1984), Meng and Arp (1993), and Gauthier and Jacobs (2018)  and found that the period within which gas-exchange rates remained unchanged ranged from 3 to 20 min in foliage of differing sizes.
Gas-exchange studies available in the literature have generally assessed the impacts of detachment on the rate of photosynthesis (A) in tree foliage, and even fewer, on stomatal conductance (g s ).
This study reports on the impacts of detachment on photosynthetic parameters, including maximum rate of Ribulose-1,5-bisphosphate (RuBP) carboxylation (V cmax ; μmol m −2 s −1 ) and the maximum rate of RuBP regeneration (J max ; μmol m −2 s −1 ), derived from measurements of A (μmol m −2 s −1 ) relative to intercellular carbon dioxide (C i ; μmol mol −1 ), in response to changing levels of carbon dioxide (alias CO 2 response or A-C i curves). This is important because the Farquhar et al. (1980) model is the most widely used in the analysis of CO 2 -photosynthesis response in trees. Two key parameters in the model, that is, V cmax and J max , have been reported on, in several in situ gas-exchange studies (e.gDiaz-Espejo et al., 2006;Fujita et al., 2012;Wilson et al., 2000;Xu & Baldocchi, 2003). Several researchers have reported on these parameters derived from measurements directly obtained from detached foliage (Table 1). Studies by Wang et al. (2008) and Whitehead et al. (2011) only reported V cmax . Warren et al. (2003), Ethier et al. (2006), Goodine et al. (2008), Merilo et al. (2009), Woodruff et al. (2009), Drake et al. (2010, and Katahata et al. (2014) referred to preliminary measurements conducted in attached and detached foliage to compare gas-exchange rates prior to conducting measurements on which their studies were based. The study by Drake et al. (2010) is the only one we are aware of that has reported on the effects of foliage detachment on V cmax and J max . To our knowledge, this is the first study that assesses V cmax and J max of attached and detached foliage in balsam fir, and the response of these parameters in such foliage to seasonal variation. The objective of this study was to report field observations of the parameters of photosynthesis and stomatal conductance before and after branch detachment during early summer-to-fall conditions.

| Study site
The study was conducted at the University of New Brunswick Woodlot (45° 56ʹ N, 66° 40ʹ W), New Brunswick, Canada, which lies within the Maritime Lowlands Ecoregion of the Atlantic Maritime Ecozone (Ecological Stratification Working Group, 1996). Humoferric podzols and gray luvisols are the dominant soil types in the Ecoregion, with significant areas of gleysols, fibrisols, and mesisols (Ecological Stratification Working Group, 1996). The soils are welldrained, sandy-clay loams, from the soil surface to a depth of 30 cm with <20% coarse fragments. The depth to the compacted layer ranges from <30 to 30-65 cm (Ecological Stratification Working Group, 1996; Canadian Soil Information Service, 2019).

| Gas-exchange measurements
Nine balsam fir trees, ranging in age from 15 to 20 years, with diameter at breast heights (DBH) between 9 and 19 cm, were selected.
Photosynthesis measurements were made in July, August, October, with photosynthetically active radiation (PAR) ranging from 1190 to 1200 μmol m −2 s −1 supplied by a tungsten halogen light unit (PP Systems). Reference CO 2 concentration (C ref ) was changed in descending steps of 400, 200, 100, and 50, and ascending steps of 400, 800, 1,000, 1,400, and 1,800 μmol mol −1 . Flow rate was set at 400 ml min −1 , chamber temperature at 18℃, and relative humidity at 70%. Chamber temperature was set at 18℃ because conifers are known to undergo optimal photosynthetic activity at temperatures lower than 25℃ (Lin et al., 2012;Wieser et al., 2010), the reference temperature on which the Farquhar et al., (1980)

| Data analysis
The Farquhar et al., (1980)  where k c and k o are the turnover rates for RuBP carboxylase and RuBP oxygenase (2.5s −1 and 0.55 s −1 or 0.22k c ; after Farquhar et al., 1980).
The key parameters V cmax and J max in eqn.'s 1 and 2 were estimated from regression analysis of A-C i curves (Wullschleger, 1993), which necessitates the designation a priori of a C i -threshold, at which there is a switch between RuBP-saturated and limited portions of the curve (Manter & Kerrigan, 2004). Generally, it is assumed that The parameters V cmax and J max were estimated using the lower section of the response curve, when C i was approximately ≤300 µmol mol −1 , and the entire curve, respectively (Farquhar et al., 1980;Wullschleger, 1993;Xu & Baldocchi, 2003), that is, after Farquhar et al., 1980 andvon Caemmerer, 2000), where CE and V c in eqn.'s 4 and 5 are the carboxylation efficiency, representing the initial slope of the CO 2 response curve and the rate of carboxylation, respectively. The equation constants, that is, K c , K o , and Г * , having been derived at an ambient temperature of 25℃ were converted to values at the reference temperature (i.e., 18℃) with the following equations: Lambers et al., (1998), where T is the reference temperature at which the CO 2 response curve was developed.
The linear mixed-effects model option in SPSS Statistical software (ver. 24.0, IBM Corp.) was used to analyze the maximum rate of photosynthesis at the current ambient CO 2 concentration (i.e., photosynthesis at 400 µmol mol −1 , A 400 ), V cmax , J max , ratio of J max to V cmax (i.e., J max :V cmax ), and mean g s . Branch status (attached or detached foliage) and measurement month (July, August, October, and November) were the fixed factors, and tree number was the random factor. This analysis was done as a result of the non-independence of measurements made on both foliage types from each tree, and repeated sampling during the study period.
The model equations used are as follows, In eqn.'s 10 and 11, Y is the gas-exchange parameter of interest, β 0 , β 1 , β 2 , β 3 , and b 1 are regression coefficients to be estimated, and ε is the regression error term.

F I G U R E 3
Box plots of (a) the maximum rate of ribulose-1,5-bisphosphate carboxylation (V cmax ; µmol m −2 s −1 ), (b) the maximum rate of ribulose-1,5-bisphosphate regeneration (J max ; µmol m −2 s −1 ), and (c) the ratio of the maximum rates of ribulose-1,5-bisphosphate regeneration (J max ;µmol m −2 s −1 ) to carboxylation (V cmax ;µmol m −2 s −1 ) of attached and detached balsam fir foliage assessed in July through to November 2019. See Figure 2 for box-plot descriptions. Similar lowercase letters above box plots indicate no significant difference regarding branch status (p > 0.05). Differences in uppercase letters above box plots indicate significant differences regarding measurement month (p < 0.05). Comparisons were made in relation to reference values for branch status and measurement month . . a A 400 = rate of photosynthesis at 400 µmol mol −1 ; V cmax = maximum rate of ribulose-1,5bisphosphate carboxylation; J max = maximum rate of ribulose-1,5-bisphosphate regeneration; J max :V cmax = ratio of the maximum rates of ribulose-1,5-bisphosphate regeneration to carboxylation; g s = stomatal conductance; df = degrees of freedom. b Reference set to zero.

TA B L E 2
Output of mixed-effect model analysis (fixed effects; branch status and month) of A 400 , V cmax , J max , J max :V cmax , and g s in attached and detached balsam fir foliage assessed in July through to November 2019 The opening and closing of stomata embedded in the epidermis simultaneously control plants' water loss during transpiration and their uptake of CO 2 . Water loss through plant leaves during transpiration, which results in stomatal closure, occurs from an imbalance between water effluxes and influxes. During transpiration, water-conducting elements contain water columns, which are under tension. Consequently, a water-potential gradient between mesophyll tissue and xylem elements occurs, ensuring the movement of water against flow resistances (Buckley, 2005;Heber et al., 1986).
Stomatal response to foliage detachment is a consequence of the positive relationship between stomatal aperture and turgor pressure of the guard cells, which form the pore, but it has a negative relationship with turgor pressure of adjacent epidermal cells, the more effective, of these two opposing pressures, in regulating aperture (Buckley, 2005).
The rate at which CO 2 enters the leaf is regulated by resistances caused by the opening and closing of the stomata, and the internal pathways within the leaf mesophyll. The reduction in plant photosynthesis arising from moisture stress is generally explained by an increase in the resistance to the movement of CO 2 through the stomata and mesophyll pathways to the site of fixation in the chloroplast. These resistances are substantially increased under such conditions because of low leaf water potentials (Beadle et al., 1973;Brix, 1962;Crafts, 1968;Puritch, 1973;Slatyer, 1967).
Generally, an increase in water stress results in a two-phase photosynthetic response, with a xylem water potential threshold, above which little or no change in photosynthesis occurs, and below which it rapidly decreases. This two-phase response varies among species (Melzack et al., 1985) and has been observed in conifers in studies of the effects of water stress in Pinus taeda L., (Brix, 1962)  Lasiocarpa (Hook.) Nutt., and A. Grandis (Doug) Lindl. ;Puritch, 1973], and Picea sitchensis (Bong.) Carr. (Watts & Neilson, 1978). Brix (1962) found that photosynthesis in P. taeda, under water stress was stable until it increased to 405 kPa, following which there was a decline. Puritch (1973) reported similar stability of photosynthesis under water stress in the four species of the genus Abies studied, up until stresses between 900 and 1,100 kPa followed by a decline. Watts and Neilson (1978) observed stable photosynthesis in P. sitchensis under water stress up until 1500 kPa, after which there was a decline. The results of this study may, therefore, be an indication that the period during which a CO 2 response curve was assessed for detached foliage, occurred at a xylem water potential that allowed photosynthesis to remain at rates that were largely like those in attached foliage samples.
The values of A 400 , V cmax , J max , and J max :V cmax for attached and detached foliage all showed strong seasonal variation (p ≤ 0.001-0.013). The studies by Xu and Baldocchi (2003) and Wilson et al., (2000) showed seasonal variation in the gas-exchange parameters measured, with peak values recorded for the gas-exchange parameters measured in spring and summer, respectively, with declines in subsequent seasons. In this study, however, A 400 , V cmax , and J max increased from summer to fall. This is attributable to the fact that Xu and Baldocchi (2003) (Lin et al., 2012), such as obtained during fall.
The pattern of seasonal variation in J max :V cmax , in attached and detached samples showed that the highest value in both foliage types occurred in July. Though the pattern of seasonal variation in J max :V cmax is a departure from those seen in A 400 , V cmax , and J max , the values ranged between 1 and 3, the range within which J max :V cmax is reported to fluctuate, and reflects the balance between RuBP carboxylation and regeneration (Kattge & Knorr, 2007;Onoda et al., 2005;Robakowski et al., 2002;Walcroft et al., 1997;Wullschleger, 1993). Mean g s across C ref -levels showed a seasonal trend and peaked in October. A similar trend was reported by Beadle et al., (1985) in their study of Pinus sylvestris L.
(another conifer). The seasonal effect on detached foliage also varied in relation to prevailing atmospheric conditions with a general trend of significantly lower gas-exchange parameters in August compared to those in October and November. This is because of higher temperatures and accompanying greater vapor pressure deficit between foliage and the air in the summer, than in the fall (Berry & Bjorkman, 1980).

| CON CLUS IONS
A comparison was made of photosynthetic parameters and stomatal conductance in foliage of attached and detached branches of balsam fir at different times during the growing season, using a linear mixed-effect model. Branch detachment did not have a significant effect on gas-exchange parameters studied, in particular A 400 , V cmax , J max , J max :V cmax , and g s . There was, however, a strong seasonal effect on the parameters. The trend of the data from this study indicates that the photosynthetic parameters and rate of photosynthesis were observed to be highest in October and November, an indication that TA B L E 3 Output of mixed-effect model analysis (random effect; tree number) of A 400 , V cmax , J max , J max :V cmax , and g s in attached and detached balsam fir foliage samples assessed in July through to November 2019 the optimum temperature of balsam fir is low. The impact of detachment on g s during July and August indicated that caution is required when gas-exchange measurements are made over long durations, during warm summer months. We can conclude that detachment had a negligible impact on gas-exchange measurements in balsam fir foliage when carried out on branches 50-to 80-cm long, for up to 30 min. The results from balsam fir are more reliable when gasexchange measurements are done during cooler months.

ACK N OWLED G M ENT
This study was supported with funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) in the form of a Discovery Grant to CPAB.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N
MEA, FRM, and CPAB conceived the ideas; MEA collected the data, designed the methods used, did the data analysis, and led the writing of the manuscript; FRM and CPAB reviewed the drafts, made amendments to the manuscript, and gave final approval for its publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in figshare at https://doi.org/10.6084/m9.figsh are.14791716.