Whole-Canopy Photosynthetic Characterization of Apple Tree and the Effects Induced by Grafting on Rootstocks with Different Vigor

Abstract

Leaf photosynthesis is pivotal for the synthesis of carbohydrates; however, the growth and development of horticultural crops are more closely related to canopy photosynthetic capacity. Measurements of canopy photosynthesis allow a better evaluation of the fruit tree performance at the canopy scale. Therefore, an open chamber system to determine instantaneous canopy apparent photosynthesis (CAP_i) was investigated. CAP_i slightly overestimated the biomass accumulation by 5.9%, which revealed that the CAP_i method is valuable for quantifying canopy photosynthesis. Further, many woody horticultural plants are usually grafted for propagation, such as apples, whereas the rootstocks always influence the canopy size and photosynthesis. In this study, the effect of dwarfing, semi-dwarfing, and vigorous apple rootstocks on canopy photosynthesis was studied. Compared to vigorous rootstock, dwarfing rootstock significantly reduced the leaf net photosynthetic rate and total leaf area by 20.8% and 53.1%, respectively, and resulted in a 59.7% reduction in CAP_i. Throughout the study, CAP_i was an effective method that should be considered for canopy photosynthesis measurement of horticultural crops in the future.

1. Introduction

Canopy photosynthesis highly depends on the photosynthetic capacity of leaves, which is affected by the interaction among various internal and external elements. A leaf serves as a specialized organ for photosynthesis [1], and the loosely stacked leaves compose the principal canopy volume [2]. The spatial distribution of leaves in the canopy has noticeable impacts on leaf age [3], physiology, and anatomical traits [4]. The structure of the canopy modifies its microenvironment conditions [5], such as photosynthetic photon flux density (PPFD) [6], temperature [7] and humidity [8]. Furthermore, in some woody plants, fruits [9], stems [10], and flowers [11] perform photosynthesis or respiration as well. Thus, the whole plant’s photosynthetic capacity is a comprehensive characteristic including the performance of various organs in different orientations [12].

Dwarfing and vigorous rootstocks have crucial effects on apple tree performance, as indicated by the modified canopy structure and photosynthesis. The application of dwarfing rootstock has a significant constraint on tree vigor and canopy size [13]. The growth of grafted trees is primarily dependent on photosynthesis, which may vary with the combinations of different scions and rootstocks [14]. It has been reported that apple scions which have been grafted on dwarfing rootstocks had lower leaf photosynthesis than those on vigorous rootstocks [15,16]. Physiological processes driven by leaf fluxes have been demonstrated to scale to the canopy [17]. Hence, the corresponding leaf-related patterns may be observed on the canopy level. Whole-canopy photosynthetic assimilations of apple trees grafted on various rootstocks were modeled before. Zhou et al. [18] elucidated that photosynthesis at the canopy and leaf level have similar variation tendencies among different grafted combinations. Given the significance of canopy photosynthesis and the limited research on rootstocks, accurate measurement based on gas exchange data appears to be particularly important.

Efforts aimed at measuring whole-canopy photosynthesis have been initiated, and improvements in measurement accuracy are underway. In earlier studies, closed chamber systems were used to calculate photosynthesis [19], whereas continuous photosynthesis may have generated a decrease in CO₂, which deviated from the chamber background concentration and caused a bias error [20]. Subsequently, semi-closed chamber systems were proposed to be an alternative solution. CO₂ was delivered or removed to maintain a relatively constant CO₂ atmosphere within the semi-closed chamber. However, it also failed to amend the insensitivity of transient and short-term fluctuation, as existed in closed chamber systems [21]. Open chamber systems relatively coupled the external environment and the plants in chambers. Canopy photosynthesis was assessed by sampling the airflow rate through a chamber, and CO₂ concentration differences between the inlet and outlet of the chamber. An open chamber system was applied to monitor canopy photosynthesis on annual wheat [22], and the subsequent calculation of canopy photosynthesis was based on the covered area of the growth chamber. This method is more suitable for the determination of small crops, and imposes restrictions on planting density. The gas exchange characteristics of a single plant would put forward a promising solution for the determination of canopy photosynthesis in perennial fruit trees. A gas exchange system has been gradually applied to explore the influence of plant and external factors on apple tree canopy carbon assimilation. The canopy net carbon exchange rate of ‘Empire’/M.9 has been explored in polyester plastic chambers [23]. Untiedt and Blanke measured canopy photosynthesis by putting the apple tree in transparent polyethylene film sacks [24]. Validation tests based on growth data are required to ensure the accuracy of canopy photosynthesis measurement systems, but there is a lack of apple tree whole-canopy measurements that were previously reported. Furthermore, in apple cultivation, rootstocks greatly affect plant growth, which is potentially induced by altered canopy level photosynthesis, but few reports mentioned this aspect. Hence, the objectives of the present study were to verify the accuracy of the open chamber system in the determination of canopy photosynthesis of apple plants; and then, to explore the influence of apple rootstocks of different vigor on apple-canopy-level photosynthesis.

2. Materials and Methods

2.1. Plant Materials and Culture Conditions

One-year-old M.9 rootstocks (

Table 1. Basic information of apple rootstocks in this study.

Rootstock	Latin Name	Dwarfing Class	Origin	Parentage
M.9	Malus pumila Mill.	Dwarfing	East Malling Research Station, Kent, United Kingdom	Natural seedlings
M.26	Malus pumila Mill.	Semi-dwarfing	East Malling Research Station, Kent, United Kingdom	M.16 × M.9
Chistock-1	Malus xiaojinensis	Semi-dwarfing	China Agricultural University, Beijing, China	Natural seedlings
Baleng	Malus robusta Rehd.	Vigorous	Huailai, Hebei, China	Natural seedlings

) were grown in pots filled with a mixed substrate of peat and vermiculite (1:1, v/v), and were cultured in a greenhouse at China Agricultural University, Beijing, China (40°0′37″ N, 116°21′56″ E, elevation 44 m). A photoperiod of 14 h/10 h (day/night) was set with the fixed artificial PPFD of 1000 µmol photon m⁻² s⁻¹ at the canopy top during the day. The artificial radiation was supplied by light-emitting diode (LED) lights. The equipped fan-pad evaporative cooling system of the greenhouse was operated to simulate the mean air temperature (24–31 °C) and relative humidity (about 84%) throughout the day in the field. To fully accommodate the environment mentioned above for photosynthetic accumulation, the M.9 clones were prepared in May 2021 and were cultured in a controlled environment until an experiment that lasted for 30 days was conducted on 1 July 2021. Growth and photosynthetic parameters were measured on 16 well-grown apple clones with a completely randomized design.

Three-year-old apple trees (Malus domestica Borkh. cv. Red Fuji) grafted onto vigorous rootstock, Baleng; semi-dwarfing rootstock, Chistock-1 and M.26; and dwarfing rootstock, M.9 (Table 1) were transplanted into pots filled with a mixed substrate of vermiculite and peat soil (1:1, v/v), and were cultured under the same greenhouse management conditions mentioned above. The mean air temperature was maintained at 18–27 °C, and the relative humidity was about 66% throughout the day. Experimental parameters were determined from 17 May 2021 to 13 June 2021. A randomized block design was performed with four plots and at least two replicates for each grafted combination per plot. Hereafter, each scion–rootstock combination will be referred to by the rootstock names.

2.2. Growth Parameters

M.9 clones were grown in the controlled environment for 30 days, and the growth parameters were measured on Day 0 and Day 30 with three replications per measurement. M.9 clones were harvested and separated into leaves, stems, and roots, and, subsequently, were washed thoroughly with distilled water. The fresh mass of samples was separately weighed using an analytical balance (FA2204E, Xingyun, Changzhou, Jiangsu, China) after blotting excess moisture. The dry mass of samples was separately determined after drying at 80 °C to constant mass. The whole plant water content (%) was calculated as plant water content = (fresh mass − dry mass)/fresh mass, where the fresh mass and dry mass represent the weight (g) of the fresh and dry tissue sample, respectively. The images of leaves on a whole plant were separately scanned by a camera, and were subsequently analyzed by ImageJ software (1.51J8, Bethesda, MD, USA) to evaluate the leaf area [25]. The total leaf area was the sum of individual leaf areas.

Morphological parameters and the soil–plant analysis development (SPAD) value of each scion-rootstock combination were measured. The total leaf area was obtained as described above. The plant height and canopy width along east–west and north–south directions were determined using a tape measure. The average canopy width was the average canopy width measured along east–west and north–south directions. The SPAD values of scion leaves were determined by the SPAD chlorophyll meter (502, Minolta, Osaka, Japan).

2.3. Leaf Photosynthetic Characterizations

The photosynthetic characteristics of functional leaves on three M.9 clones were monitored throughout the day. The leaf net photosynthetic rate (P_N) and dark respiration rate (R_D) were measured and recorded by the LI-6400XT portable photosynthesis system (Li-Cor, Lincoln, NE, USA), interfaced with a 2 cm × 3 cm 6400-02 B leaf chamber which provided a 1000-µmol photon m⁻² s⁻¹ artificial red/blue mixed LED light source. During the measurements, the leaf chamber conditions were controlled at constant: the sample chamber CO₂ level of 400 μmol m⁻² s⁻¹ supplied by the CO₂ mixer, the leaf-to-air vapor pressure deficit was around 1.3 kPa, the block temperature was controlled between 19.8 °C and 28.2 °C, and the flow rate was set at 500 μmol s⁻¹.

The P_N, intracellular CO₂ concentration (C_i), transpiration rate (E), and stomatal conductance (g_s) of the fully expanded scion leaf of at least eight trees in each scion-rootstock combination were measured from 08:00 to 17:00. The water-use efficiency (WUE) and leaf carboxylation efficiency (CE) of four grafted combinations were expressed as WUE = P_N/E and CE = P_N/C_i [16]. The stomatal threshold (LS) was calculated following LS = 1 − C_i/C_a [26], where C_a is the ambient CO₂ concentration. The photosynthetic light response curves of four grafted combinations were measured on the same leaves after the gas exchange parameters had been determined. The measurements were conducted by an automatic program in LI-6400XT with a PPFD gradient of 2000, 1500, 1200, 1000; 500, 200, 100, 50, 20, and 0 μmol photon m⁻² s⁻¹. The parameters of the photosynthetic light response curves were generated using a ‘Photosyn Assistant’ program (Dundee Scientific, Dundee, UK). The non-rectangular hyperbola and its general equation described by Thornley [27] in Formula (1) were fitted via IBM SPSS Statistics (Version 25, SPSS, Chicago, IL, USA):

$$P_N=\frac{{\mathsf{\phi }\times \mathrm{PPFD}+P}_{\mathrm{Nmax}}-\sqrt{{{(\mathsf{\phi }\times \mathrm{PPFD}+P}_{\mathrm{Nmax}})}^2-{4\times \mathsf{\theta }\times \mathsf{\phi }\times \mathrm{PPFD}\times P}_{\mathrm{Nmax}}}}{2\times \mathsf{\theta }}-{\mathrm{R}}_{\mathrm{D}}$$

(1)

where P_N is the leaf net photosynthetic rate (μmol CO₂ m⁻² s⁻¹), PPFD is the photosynthetic photon flux density (µmol photon m⁻² s⁻¹), φ is the apparent quantum yield, P_Nmax is the light-saturated net photosynthetic rate (μmol CO₂ m⁻² s⁻¹), θ is the curvature of the curve, and R_D is the respiration rate (μmol CO₂ m⁻² s⁻¹).

2.4. Canopy Photosynthetic Characterization

The instantaneous canopy apparent photosynthesis (CAP_i) of M.9 clones was measured throughout the day in cylinder-shaped chambers (15.7 L) (Figure 1a). The ambient air was sucked into a gas-mixing device, and was fully blended by two fans installed reversely. The mixed air was shunted into transparent glass growth chambers. Adjustable fans were installed to provide appropriate wind speed around the canopy to eliminate boundary layer effects [26,28] and mix the gas sufficiently [22]. The water-sealed chamber bases ensured a gas tightness of chambers [29], and avoided potential CO₂ contamination or leakage [30]. Artificial LED lighting sources were installed above chambers to provide 1000 µmol photon m⁻² s⁻¹ PPFD at the top of the canopy to avoid temperature rises in the chambers caused by the light source. One of the chambers was equipped with a HOBO data logger to monitor the photon flux density at the top of the canopy, and the other three chambers each contained a plant for measurement. The inlet flow rate of each chamber was controlled and recorded by a combination of a gas rotometer (LZB-3WB, Shuanghuan, Shiyan, Hubei, China) and electronic gas flowmeter (MF5706-25, Siargo, Santa Clara, CA, USA) to guarantee a decreased CO₂ concentration of around 400 µmol mol⁻¹ in each chamber. Parameters in formulas were recorded, and the values were then used to calculate CAP_i. Based on the previous studies conducted on the open gas system [21,22], Formulas (3) and (4) were modified as follows:

PV = nRT

(2)

where P is the barometric pressure (Pa), V is the volume of gas (m³), n is the amount of substance of gas (mol), R is the universal gas constant (m³ Pa mol⁻¹ K^−l), and T is the absolute (K).

$${\mathrm{AIR}}_{\mathrm{flux}}=\frac{\mathrm{n}\times {\mathrm{AIR}}_{\mathrm{flow}}}{60\mathrm{V}\times {10}^3}$$

(3)

where AIR_flow is the flow rate (L min⁻¹) measured by the flowmeter, and AIR_flux is the flow rate (mol s⁻¹) converted from AIR_flow.

CAP_i = (C₀ − C₁) × AIR_flux

(4)

where CAP_i is instantaneous canopy apparent photosynthesis (μmol s⁻¹), C₀ is the CO₂ concentration (μmol mol⁻¹) of air flow at the inlet of a chamber, and C₁ is the CO₂ concentration (μmol mol⁻¹) of air flow at the outlet of the same chamber.

The daily accumulation of CAP_i (μmol day⁻¹) in M.9 clones is the integral of CAP_i and time in a day. The daily accumulation of accumulated leaf photosynthesis (μmol CO₂ day⁻¹) was the integral of P_N, total leaf area, and time in a day. The amount of substance of CO₂ was converted to the amount of substance of C₆H₁₂O₆, and then the corresponding mass was calculated.

For the scion-rootstock combinations, CAP_i was measured as described above, in a chamber (1 × 1 × 2 m) (Figure 1b) from 08:00 to 17:00.

2.5. Statistical Analysis

Statistical analyses were performed using IBM SPSS Statistics (Version 25, SPSS, Chicago, IL, USA) and Origin software (2022, OriginLab, Northampton, MA, USA). The differences in growth parameters of M.9 on Day 0 and Day 30 were tested by the independent-samples t-test, and the ‘*’, ‘**’, and ‘ns’ indicated significance levels with p < 0.05, p < 0.01, and p ≥ 0.05, respectively. Photosynthesis variations among various time points of M.9 clones and the parameter differences among four apple scion-rootstock combinations were carried out using a one-way variance (ANOVA), and the significant difference was determined by the least significant difference (LSD) test with p < 0.05. Different letters after mean values ± standard error in the same column in Tables or above individual columns in Figures indicate the significant difference determined by the least significant difference (LSD) test with p < 0.05. The light response curves were analyzed via a non-rectangular hyperbola in IBM SPSS Statistics. The sssociations among the parameters of apple-scion–rootstock combinations were additionally assessed using Pearson’s two-tailed correlation. Principal component analysis (PCA) of grafted apple tree was conducted using Origin software. Figures were generated by Origin and Microsoft Excel (version 2020, Microsoft, Redmond, WA, USA).

3. Results

3.1. Environmental Condition

The air temperature and relative humidity were maintained at relatively stable levels during the measurements on scion-rootstock combinations and M.9 clones. The mean daily air temperatures were 22.5 °C and 26.9 °C. The mean daily air relative humidity during the measurement periods averaged 66.4% and 84.1% (Figure 2).

3.2. Photosynthate Accumulation of M.9 Clones Based on Leaf and Canopy Photosynthesis

• The dry mass and total leaf area of M.9 clones increased significantly within the 30 days, whereas the plant water content showed no evident change (Figure 3). The CAP_i fluctuated over time throughout the day, with a variation of 201.8%, and peaked in the forenoon, with a value of 0.27 μmol CO₂ s⁻¹. During the night, the CAP_i was maintained at around −0.14 μmol CO₂ s⁻¹ (Figure 4a). The P_N of functional leaves varied 13.62 % throughout the day, and the respiration rates were about −1.47 μmol CO₂ m⁻² s⁻¹ during the night (Figure 4b). Compared to the gained dry mass of 2.85 g per plant, the simulated photosynthate based on accumulated leaf photosynthesis was 20.39 g per plant, 231.7 times higher than that simulated by CAP_i (3.02 g).

3.3. Leaf and Canopy Photosynthetic Parameters of Scions Influenced by Rootstocks

The cv. Red Fuji was grafted on Baleng rootstock, with a superior height and larger canopy size, which were 30.3% and 59.7% higher than that of M.9, respectively. The total leaf area and SPAD induced by Baleng were significantly higher than M.26 and M.9, but no significant difference was found between Baleng and Chistock-1 (

Table 2. Growth parameters and soil-plant analysis development (SPAD) values of scion-rootstock combinations.

Rootstock	Plant Height (cm)		Canopy Width (cm)		Total Leaf Area (cm2)		SPAD
M.9	137.4 ± 4.3	c	58.4 ± 10.2	b	1796.8 ± 72.1	c	45.7 ± 2.4	c
M.26	148.2 ± 7.6	bc	76.4 ± 10.4	ab	2790.0 ± 281.6	b	47.6 ± 0.6	bc
Chistock-1	157.3 ± 3.3	b	83.3 ± 4.6	ab	3580.0 ± 235.9	ab	51.5 ± 0.9	ab
Baleng	179.1 ± 2.5	a	93.3 ± 8.7	a	3829.9 ± 425.3	a	56.7 ± 0.5	a

SPAD was measured on the same functional leaves. Data in the table are mean values (n = 3–4) ± standard error (SE). Different letters after mean values ± standard error in the same column indicate the significant difference determined by the least significant difference (LSD) test with p < 0.05.

). The application of dwarfing rootstock showed parallel decreases in CAP_i and P_N both in the morning and afternoon (Figure 5). In the morning, the CAPi on dwarfing rootstock, M.9, and semi-dwarfing rootstock, M.26, were significantly lower than those on vigorous rootstock Baleng, whereas no significant difference was observed on Baleng and Chistock-1. There was no significant difference in CAP_i between Baleng and Chistock-1 both in the morning and the afternoon. In the afternoon, the P_N (6.8–14.8%) and CAP_i (30.7–44.5%) of four rootstocks were significantly reduced. In the afternoon, scion leaves on M.9 had significantly higher leaf WUE and LS than those on semi-dwarfing and vigorous rootstocks (Figure 6d,f). An analysis of photosynthetic light response curves, as indicated in Figure 7 and

Table 3. Parameters of photosynthetic light response curves on grafted apple combination, including light-saturated net photosynthetic rate (P_Nmax), dark respiration rate (R_D), light compensation point (LCP), light saturation point (LSP), and apparent quantum yield (φ).

Rootstock	PNMAX (μmol CO2 m−2 s−1)		RD (μmol CO2 m−2 s−1)		LCP (μmol m−2 s−1)		LSP (μmol m−2 s−1)		φ
M.9	15.2 ± 0.5	b	0.62 ± 0.12	c	17.3 ± 2.7	b	1137.3 ± 59.6	a	0.037 ± 0.004	b
M.26	15.2 ± 0.2	b	0.95 ± 0.15	bc	25.3 ± 5.8	ab	1136.0 ± 56.0	a	0.037 ± 0.003	b
Chistock-1	16.8 ± 0.8	ab	1.49 ± 0.05	a	30.7 ± 1.3	a	1088.0 ± 64.2	a	0.049 ± 0.003	a
Baleng	18.5 ± 0.9	a	1.11 ± 0.13	ab	25.3 ± 1.3	ab	1250.7 ± 97.8	a	0.043 ± 0.002	ab

Parameters were measured on the same functional leaves. Data in the table are mean values (n = 3) ± standard error (SE). Different letters after mean values ± standard error in the same column indicate the significant difference determined by the least significant difference (LSD) test with p < 0.05.

, illustrated that among scion–rootstock combinations, higher maximum net CO₂ assimilation rate (P_Nmax) were recorded on Baleng than M.26 and M.9 with a value of 18.5 μmol photon m⁻² s⁻¹, whereas the difference of LSP was not significant. The results presented in Figure 6 indicated that scion leaves grafted on vigorous rootstock, Baleng, had higher g_s, E, and CE than M.9. Additionally, higher WUE and LS were found in leaves grafted on the dwarfing rootstock M.9 in the afternoon.

• Figure 8a visualized the correlation matrix of plant growth and photosynthetic parameters via a correlation heat map. CAP_i was positively correlated with g_s (r = 0.965, p = 0.044, two-tailed), P_N (r = 0.988, p = 0.012, two-tailed), total leaf area (r = 0.972, p = 0.028, two-tailed), and canopy width (r = 0.999, p = 0.001, two-tailed). P_N was positively correlated with g_s (r = 0.962, p = 0.038, two-tailed), E (r = 0.960, p = 0.040, two-tailed), CE (r = 0.935, p = 0.047, two-tailed), CAP_i (r = 0.988, p = 0.012, two-tailed), total leaf area (r = 0.966, p = 0.034, two-tailed), and canopy width (r = 0.986, p = 0.033, two-tailed). According to PCA in Figure 8b, two main comprehensive evaluation factors accounting for 85.3% variation were extracted, including P_N, g_s, E, total leaf area, and canopy width in PC1, which explained 69% of the total variation, and CAP_i, CE, LS, C_i, WUE and plant height in PC2, which explained the 16.3% of the total variation.

4. Discussion

The accumulated photosynthate calculated by CAPi largely reflected the actual biomass accumulation. A significant increase in dry mass and total leaf area, as well as the stable leaf water content (Figure 3), indicated the steady growth of M.9 clones in this experiment. Plant photosynthetic capacity varies with daily fluctuation of environmental factors such as temperature, air vapor pressure deficit (VPD), and PPFD throughout the day [31,32,33]. It was measured that the diurnal fluctuation of CAP_i (201.8%) was much wider than that of P_N (13.62%) (Figure 3). In the case of the same total leaf area and uniform illumination, the difference may be explained by the different photosynthesis in leaves of various positions or leaf ages [34]. In actively growing plants, photosynthetic apparatus harvest solar energy incorporates CO₂ into carbon, ultimately increasing the biomass production of plants [35]. To explore the authenticity of accumulated CAP_i for simulated biomass gain, corresponding comparisons were processed. A growth chamber system for the measurements of gas exchange at the canopy scale was applied to soybeans in 2017, and overestimated total carbon uptake by 7.6% [30], whereas CAP_i measured in the open chamber system well simulated canopy photosynthesis with only 5.9% overestimation. The above results emphasized the significance of canopy level photosynthesis in performing photosynthetic assessments. As the application of rootstocks altered the production and allocation of carbohydrates, more accurate verification based on carbon content is worth further exploration. Although the precision of CAP_i is expected to be perfected by methods such as taking the photosynthesized carbon released into the soil via root and mycorrhizal fungi into account [36], it can still be regarded as a low-cost and promising modeling method.

The less developed canopy and limited leaf photosynthesis jointly accounted for the constrained canopy photosynthesis on dwarfing rootstock. As CAP_i was significantly correlated with total leaf area (r = 0.972, p = 0.028, two-tailed) (Figure 8a), compared to scions on vigorous rootstocks, the limited leaf area of scions on dwarfing apple trees may produce less photoassimilate [37]. Hence, the plant height and canopy width of apple trees grafted on Baleng were 30.3% and 59.7% greater than that on M.9 (Table 2). The greater canopy on Baleng potentially induced a much lower temperature and light interception on shaded leaves, subsequently affecting the related biochemical processes and assimilate distribution [34]. Previous studies showed that scion leaf photosynthesis was affected by rootstocks [38]. As indicated by PCA (Figure 8b), the PC1, with an explained variation of 69%, mainly characterized the capacity for photosynthesis and vigorous growth. The sequence of M.9, M.26, Chistock-1, and Baleng, along the PC1 direction, also showed the increased photosynthetic capacity with the vigor of rootstocks. The SPAD values of Baleng and Chistock-1 were 1.13 and 1.24 times greater than M.9, indicating greater capacities to absorb, transmit, and utilize solar energy [18]. Our results were in agreement with the previous findings that scion leaves grafted on apple dwarfing rootstocks inhibited photosynthesis [15]. The reduced C_i caused by lower g_s may explain the inhibited P_N and elevated WUE in M.9 [32]. The lower CE relevant to Rubisco activity [39] potentially resulted from the lower chlorophyll content [40], and repressed P_N in M.9 leaves. Compared with dwarfing trees, higher g_s of scion leaves induced by grafting on Baleng promoted CO₂ diffusion inside the leaf, and significantly increased C_i. Moreover, a more efficient leaf carboxylation rate of Baleng further up-regulated P_N. The results above seemed to reveal the difference in P_N induced by rootstocks, which may be explained by the integrative effects of light capture, gas exchange, and photosynthetic enzyme activity at the leaf level. Canopy photosynthesis constituted by the photosynthetic rate of single leaves furtherly affects productivity [41]. Grafting Baleng on apple trees increased total leaf area while enhancing P_N, which, in turn, could collectively contribute to higher CAP_i. Higher photosynthesis on Baleng is beneficial for the synthesis of carbohydrates and their subsequent allocation to roots. The promoted roots system development thereafter facilitated nutrient and water acquisition, which plays a key role for apple plants to face environmental stress. The smaller effective photosynthetic area potentially constrained by total area for light harvest and the lower photosynthetic capacity per unit leaf area (Figure 6b) may give an integrated interpretation of the reduced CAP_i in dwarfing rootstock.

The canopy complexity and heterogeneity triggered by apple rootstocks gave priority to quantifying photosynthetic variation more accurately by CAP_i rather than P_N. Differing from cereal crops such as rice and corn, the apple tree has a more complex canopy, including leaves, branches, and fruits [42]. Compared to the morning, CAP_i declines more severely than P_N among four grafted combinations in the afternoon, which may be explained by the greater leaf physiological status switch of old leaves [43]. Compared to vigorous rootstock, dwarfing rootstock significantly reduced CAP_i and P_N by 59.7% and 20.8%. There were multiple aspects that induced these phenomena. On the one hand, the P_N was determined only by functional leaves, which resulted in a portion of photosynthesis that cannot be reflected by P_N, whereas CAPi compensated for this deficiency greatly. On the other hand, leaf photosynthetic characteristics were affected by leaf age [34], spatial position within the canopy [44], and environmental factors [12]. Canopy structure and spatial position of leaves influenced the leaf microenvironment and further induced variations in leaf physiological properties [44,45]. There also exists an underlying possibility of CO₂ exchange in the non-leaf organs of some plants. The branches of some woody plants were evidenced to have photosynthetic capacities [10]. Additionally, several fruits, such as apple [46], peach [47], and citrus [9], could perform photosynthesis and respiration. A very limited study of CAP_i was performed on rootstock-influenced apple trees, which emphasized the necessity of evaluating photosynthetic characterization at the canopy scale. Moreover, under abiotic or biological stresses, plants may exhibit symptoms of puckered, malformed, and even cracked leaves [48], which are impossible to perform leaf gas exchange measurements and photosynthetic evaluation from. Consequently, CAP_i could serve as an effective tool, coupling with phenotypic analysis, for research on perennial fruit trees under various stresses.

5. Conclusions

Instantaneous canopy apparent photosynthesis (CAP_i) was proven as a reliable methodology to determine photosynthesis at the canopy scale validation with biomass. In contrast to vigorous and semi-dwarfing rootstocks, the application of dwarfing rootstock generated a remarkable reduction in CAP_i, which mainly results from the smaller canopy size and lower leaf photosynthesis capacity. Light capture, gas exchange, and photosynthetic enzyme activity resulted in constrained leaf photosynthesis comprehensively. The rootstock-induced canopy microenvironment and leaf physiological variation gave CAP_i superiority over P_N in the photosynthetic evaluation, especially for apple trees grafted on vigorous rootstock. CAP_i proved to be promising in future research on whole-canopy photosynthetic phenotyping.