Stem Photosynthesis in ‘Hybrid Poplar 275’ Remains Stable Following Defoliation Induced by Severe Drought

Abstract

Drought is a major stressor affecting tree physiology and is expected to intensify under climate extremes. Stems, partly due to their photosynthetic capacity, tend to be more drought-resilient than leaves. This study aimed to assess stem photosynthetic and its impact on carbon balance in leafless stems under drought conditions. Severe drought caused a marked decline in stem and root water potential (Ψ) and reduced stem water vapor conductance (g_tw) by about 40%. Despite this, stems retained the capacity for active gas exchange: though with reduced stem CO₂ efflux (ECO₂) and enhanced CO₂ refixation, which increased from about 40% under control conditions to ~55%–60% after drought, accompanied by a twofold increase in intrinsic water use efficiency (iWUE). Chlorophyll a fluorescence and pigment analyses indicated that the integrity of photosystem II (PSII) was preserved under drought, supporting sustained corticular photosynthesis. Concentrations of chloride, malate, and citrate in the xylem sap did not change significantly under drought, indicating a high capacity of stems to maintain homeostasis. Stable isotope analyses revealed drought-induced shifts in δ¹³C, consistent with altered carbon allocation following leaf abscission. These results confirm that stem photosynthesis and CO₂ reassimilation contribute significantly to stem metabolic resilience, mitigating drought-induced carbon losses and helping to preserve plant survival.

1. Introduction

Drought is among the most significant environmental stresses affecting tree physiology, growth, and survival, and its frequency and intensity are expected to increase under the ongoing climate change scenarios [1]. While the responses of leaves to drought have been extensively studied, particularly the role of stomatal closure in balancing carbon uptake and water loss [2,3,4], less is known about how stems contribute to whole plant acclimation during prolonged water deficits [5,6]. Apart from its conductive role, the stem is also metabolically active and, capable of respiration [7], photosynthesis [8], and internal CO₂ recycling [9]. Understanding how these processes are modulated during drought is essential for evaluating tree resilience and predicting forest responses to climatic extremes [10].

Stem photosynthesis is a particularly intriguing process, occurring from the earliest developmental stages, when stems are still fully green across their cross-section [11], and persisting even in mature trees for up to 100 years, as reported for species such as beech [12]. However, the conditions for corticular photosynthesis seem to become increasingly challenging as the stem develops. Light availability is usually limited by the thickening periderm or cork layers [13], which often leads to reduced chlorophyll levels in the central stem tissues, thereby restricting it mainly to the cortex. Under such conditions, stem photosynthesis serves several important functions: it provides oxygen for respiration, reduces internal CO₂ concentrations, and generates energy to sustain the living cortical tissues, particularly conducting cells [14]. At the same time, stems are rich in CO₂, with concentrations considerably higher than in the atmosphere [15,16], which can enhance Rubisco activity by mitigating photorespiration [17]. Moreover, the produced sugars represent not only an additional metabolic gain but also play a crucial role under water stress by helping to prevent cavitation and subsequent xylem embolism—air blockages that disrupt water transport and may result in irreversible hydraulic failure [18,19]. Due to lower water conductance compared with leaves, stems also exhibit higher water-use efficiency (WUE), highlighting their potential role in maintaining plant performance during drought [20].

Depending on climatic conditions, two main types of stem photosynthesis can be distinguished [21,22]. The first type—stem net photosynthesis (SNP), found in desert and tropical environments—has high conductance and is equipped with stomata, allowing for significant CO₂ uptake from the atmosphere under favorable conditions. The second type -stem recycling photosynthesis (SRP), originating from temperate climates, possesses unregulated lenticels, where net photosynthesis is rarely observed. Instead, CO₂ reassimilation occurs, and only a reduction in CO₂ efflux (ECO₂) under illumination is observed.

Stem gas exchange, particularly stem ECO₂, has recently been recognized as a complex indicator of metabolic activity, consisting of respiration, corticular photosynthesis, CO₂ transport in the xylem sap, and anaplerotic CO₂ fixation by phosphoenolpyruvate carboxylase (PEPC) [23]. Several studies have shown that SRP can substantially reduce apparent respiratory losses by reassimilating internally produced CO₂, with reported contributions ranging from 10% to over 100% of ECO₂ depending on species and environmental conditions [24,25]. The extent to which these processes buffer stem metabolism under drought conditions has also been the focus of recent investigations [6,22,23,26,27,28].

Up to now, drought-stressed tree stems performing SRP have typically responded to water deficit with a reduction in ECO₂, while maintaining photosynthetic activity. In contrast, stems with SNPs, such as in Parkinsonia florida, showed a strikingly different response: plants subjected to drought without light access to the stem exhibited 100% mortality, whereas in the group where stems had light access, mortality did not exceed 17%. In the case of plants performing SRP, however, the examined drought conditions were relatively mild [26,27,29,30]. This limited drought effect prompted us to test whether, under conditions of prolonged severe drought leading to defoliation, stem photosynthesis would also remain largely stable, thereby contributing to tree survival, and it was the first objective of this work. Moreover, we aimed to investigate how the mentioned conditions affect stem physiology, to better understand the mechanisms sustaining proper carbon management in trees when foliar photosynthesis is inhibited. This knowledge may also support the selection and management of woody plants better adapted to stresses induced by climate change.

2. Materials and Methods

2.1. Plant Material

Two-year-old Populus ‘Hybrid 275’ (NE 42; Populus maximowiczii × P. trichocarpa) clones were used in the experiment. Twenty-two branch cuttings were planted in March 2022 into 20 L containers filled with standard horticultural substrate (N:P:K = 9:5:10; pH 6.0–6.5), periodically fertilized with Substral Osmocote (Bayer, Leverkusen, Germany), and watered daily if necessary. The plants were grown under open-field conditions in Kraków, Poland (N 50°4′7.36″, E 19°50′29.66″), exposed to natural sunlight, ambient precipitation and temperature fluctuations. After two growing seasons, the plants reached a height of 1.3 to 1.5 m.

The drought experiment commenced on 1 September 2023 by withholding irrigation from a subset of 11 plants for 14 days to induce drought stress. Throughout the experiment, both drought-stressed and well-watered control plants were shielded from natural precipitation using a transparent foil roof. During the 14-day period, daytime temperatures ranged from 19 °C to 32 °C, nighttime temperatures from 12 °C to 18 °C, and relative humidity fluctuated between 40% and 70%. On 14 September 2023, drought-exposed plants showed visibly desiccated foliage, which subsequently abscised.

At the end of the stress period, physiological measurements were performed, including chlorophyll a fluorescence, leaf water potential, and gas exchange parameters. Xylem sap and cortex tissues were sampled for subsequent biochemical and isotopic analyses. All collected tissues were immediately flash-frozen in liquid nitrogen and stored at −80 °C until further analyses.

2.2. Water Potential

Water potential (Ψ) in different plant organs was measured using a Scholander-type pressure chamber (Model 600, PMS Instruments, Corvallis, OR, USA). Measurements were carried out between 11:00 a.m. and 1:00 p.m. on fully expanded, mature leaves collected from the uppermost parts of the stems, but only from well-watered plants. One leaf was collected from each individual plant, with each plant representing one replicate. Stem water potential was measured on excised stem segments taken approximately 70 cm above the soil surface, while root water potential was assessed using the thickest roots from the same plant. One measurement per plant constituted one replicate, and five replicates were performed for both drought-stressed and well-watered treatments. The same stem segments used for water potential measurements were also used for xylem sap extraction, which was subsequently collected and stored for biochemical analysis.

2.3. Gas Exchange

Photosynthetic parameters were measured using a LI-6400XT Portable Photosynthesis System (LI-COR Inc., Lincoln, NE, USA) equipped with a 6 × 4 cm transparent conifer chamber (6400-04) and an LED light source SL 3500 providing white light (Photon Systems Instruments, Brno, Czech Republic). Measurements were carried out on stem segments located approximately 70 cm above the base of the plant. One measurement per plant was considered a single replicate, and four replicates were performed per treatment. Measurements were performed under controlled conditions: a constant block temperature of 30 °C, a CO₂ concentration of 400 µmol mol⁻¹, and relative humidity maintained at 35%–40%. After a 30 min acclimation period in darkness within the cuvette, photosynthetic light response curves were recorded. Light response measurements were conducted at ascending irradiance levels of 25, 50, 100, 500, 1000, 1500, and 2000 µmol PAR m⁻² s⁻¹. Following light acclimation, stems were exposed to an irradiance of 1500 µmol PAR m⁻² s⁻¹, and CO₂ response curves were generated at ascending concentrations of 50, 200, 400, 700, 1500, and 2000 µmol mol⁻¹. At each irradiance or CO₂ concentration level, gas exchange was monitored until a steady-state plateau was reached prior to data logging.

The values of water vapor conductance (g_tw) were calculated according to von Caemmerer and Farquhar [31]:

$${\mathrm{g}}_{\mathrm{t}}\mathrm{w}={\displaystyle \frac{\mathrm{E}\left(1000-\frac{\mathrm{W}\mathrm{c}+\mathrm{W}\mathrm{s}}{2}\right)}{\mathrm{W}\mathrm{c}-\mathrm{W}\mathrm{s}}}$$

(1)

where E is transpiration, and Wc and Ws are the molar concentrations of water vapor within the stem cortex and in the air sample, respectively. Wc was calculated according to formula:

$$\mathrm{W}\mathrm{c}={\displaystyle \frac{\mathrm{e}\left(\mathrm{T}\mathrm{c}\right)}{\mathrm{P}}}\ast 1000$$

(2)

where e(Tc) is saturation vapor pressure at temperature of cortex (Tc) and P is a total atmospheric pressure [31]. The efflux of CO₂ (ECO₂) was calculated according to von Caemmerer and Farquhar [31]:

$${\mathrm{E}\mathrm{C}\mathrm{O}}_2={\displaystyle \frac{\mathrm{F}\left(\mathrm{C}\mathrm{r}-\mathrm{C}\mathrm{s}\left(\frac{1000-\mathrm{W}\mathrm{r}}{1000-\mathrm{W}\mathrm{s}}\right)\right)}{100}}\ast \left(-1\right)$$

(3)

where F is the air flow rate, Cr and Cs are the CO₂ concentrations in the reference and sample air streams, respectively, and Wr is the molar concentration of water vapor in the reference air. The values of intrinsic water-use efficiency (iWUE) were calculated according to Ávila-Lovera [22] with own modifications:

$$\mathrm{i}\mathrm{W}\mathrm{U}\mathrm{E}={\displaystyle \frac{\mathrm{d}{\mathrm{E}\mathrm{C}\mathrm{O}}_2-\mathrm{i}{\mathrm{E}\mathrm{C}\mathrm{O}}_2}{{\mathrm{g}}_{\mathrm{t}}\mathrm{w}}}$$

(4)

where iECO₂ represents the efflux of CO₂ under illumination (PAR > 0), dECO₂ represents the efflux of CO₂ in darkness (PAR = 0) and g_tw is the water vapor conductance. The percentage of CO₂ refixation was calculated according to Cernusak and Marshall [32]

$$\% \mathrm{r}\mathrm{e}\mathrm{f}\mathrm{i}\mathrm{x}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}={\displaystyle \frac{\mathrm{d}{\mathrm{E}\mathrm{C}\mathrm{O}}_2-\mathrm{i}{\mathrm{E}\mathrm{C}\mathrm{O}}_2}{\mathrm{d}{\mathrm{E}\mathrm{C}\mathrm{O}}_2}}\ast 100$$

(5)

where iECO₂ represents the efflux of CO₂ under illumination (PAR > 0), and dECO₂ represents the efflux of CO₂ in darkness (PAR = 0). According to Wittman et al. [17], the corticular CO₂ concentration (Ci) in the intercellular system of the stem cortex was calculated as:

$$\mathrm{C}\mathrm{i}=\mathrm{C}\mathrm{a}-{\displaystyle \frac{{\mathrm{E}\mathrm{C}\mathrm{O}}_2}{{\mathrm{g}}_{\mathrm{t}}\mathrm{c}}}$$

(6)

where Ca is the concentration of CO₂ in the atmosphere, g_tc is the total conductance of the stem surface to CO₂, calculated according to the formula [17]:

$${\mathrm{g}}_{\mathrm{t}}\mathrm{c}={\displaystyle \frac{{\mathrm{g}}_{\mathrm{t}}\mathrm{w}}{1.56}}$$

(7)

2.4. Fluorescence Measurement

The fluorescence of chlorophyll a was measured using the PSI FluorCam 700MF Chlorophyll Fluorescence Imaging System (PSI, Brno, Czech Republic). Stem segments from well-watered and drought-treated plants were analyzed in the laboratory. Seven segments, each from an individual plant, were measured per treatment. Fluorescence yield was assessed following 30 min of dark adaptation. Saturating pulse of white light (2000 μmol m⁻² s⁻¹) was applied to determine minimal fluorescence (Fo), maximal fluorescence (Fm), and variable fluorescence (Fv). The maximum quantum efficiency of photosystem II (Fv/Fm) was calculated according to Genty et al. [33]. Subsequently, the material was exposed to actinic light at an intensity of 250 μmol m⁻² s⁻¹, followed by 10 saturation pulses, allowing the determination of minimal fluorescence in the light-adapted state (Fo′), maximal fluorescence in the light-adapted state (Fm′), and steady-state fluorescence (Fs). The effective quantum yield of PSII photochemistry (ΦPSII) was calculated as [33]:

$$\mathsf{\Phi }\mathrm{P}\mathrm{S}\mathrm{I}\mathrm{I}={\displaystyle \frac{{\mathrm{F}\mathrm{m}}^{\prime }-\mathrm{F}\mathrm{s}}{{\mathrm{F}\mathrm{m}}^{\prime }}}$$

(8)

Non-photochemical quenching (NPQ), representing thermal dissipation of excess excitation energy, was calculated as [34]:

$$\mathrm{N}\mathrm{P}\mathrm{Q}={\displaystyle \frac{\mathrm{F}\mathrm{m}-{\mathrm{F}\mathrm{m}}^{\prime }}{{\mathrm{F}\mathrm{m}}^{\prime }}}$$

(9)

The photochemical quenching coefficient (qP), which reflects the proportion of open PSII reaction centers, was calculated using the equation [34]:

$$\mathrm{q}\mathrm{P}={\displaystyle \frac{{\mathrm{F}\mathrm{m}}^{\prime }-\mathrm{F}\mathrm{s}}{{\mathrm{F}\mathrm{m}}^{\prime }-\mathrm{F}\mathrm{o}}}$$

(10)

2.5. Photosynthetic Pigments Content

Tissue samples for pigment analysis were collected from stem segments located approximately 70 cm above the ground. One sample was taken from each individual plant, and five samples were analyzed per treatment. Photosynthetic pigments were extracted from the frozen stem cortex tissue using a mortar and 80% (v/v) acetone. Chlorophyll a (Chl a), chlorophyll b (Chl b), and total carotenoid concentrations were determined spectrophotometrically with a UV-VIS Helios Gamma spectrophotometer (Thermo Spectronic, Waltham, MA, USA). An aliquot (1 mL) of the uppermost supernatant was used for absorbance measurements at 470, 649, and 665 nm, following the method of Wellburn [35]. Pigment contents were calculated and expressed as mg m⁻².

2.6. Xylem Sap Composition

Analyses of chloride, citrate, and malate were conducted on previously frozen xylem sap samples, each collected from an individual plant during Ψ measurements. Eleven samples were analyzed per treatment. The analyses were performed using a Hewlett-Packard HPCE 3D capillary electrophoresis system, following a modified protocol based on Geiser et al. [36]. Instrument control and data processing were performed using ChemStation A10.02 software (Agilent Technologies, Santa Clara, CA, USA). Separations were carried out in a fused silica capillary (50 µm I.D., 32.5 cm total length) using a separation buffer composed of 15 mmol dm⁻³ Tris-HCl (pH 8.3), 15 mmol dm⁻³ pyromellitic acid, and 0.2 mmol dm⁻³ tetradecyltrimethylammonium bromide (TTAB). The buffer was filtered through a 0.22 µm syringe filter prior to use. Electrophoretic separation was performed at 45 °C under reversed polarity (anode at the detector end), with an applied voltage of 4 kV. The capillary was preconditioned with the separation buffer for 3 min before each injection. Samples were pressure-injected at 50 mbar for 2 s and analyzed using UV detection at 350 nm with a reference wavelength of 220 nm. Each sample was mixed with an internal standard (sodium sulfate), filtered through 0.22 µm centrifuge filters, and subjected to analysis. Quantification was based on 7-point external calibration curves (0–10 mg mL⁻¹) using sodium chloride, citric acid, and malic acid as standards.

2.7. Stable Isotope Composition

Samples for isotope composition analysis were taken from stem segments located approximately 70 cm above the ground. One stem cortex sample was collected from each individual plant, and five samples were analyzed per treatment. Frozen samples were oven-dried for 24 h at 105 °C and subsequently ground to a fine powder for stable isotope analysis. About 3 mg of well-dried (24 h in an oven at 105 °C) leaf samples have been placed in tin capsules (Elemental Microanalysis Ltd., Okehampton, UK). The ¹³C and ¹⁵N isotopes analyses have been conducted by the Stable Isotope Facility (SIF), University of California (Davis, CA, USA) using Carlo Erba NC2500 elemental analyser (Thermo Fisher Scientific, Waltham, MA, USA) interfaced to a Sercon 20–22 isotope ratio mass spectrometer (IRMS) (Sercon Ltd., Cheshire, UK). Tissue samples were combusted at 1000 °C in a reactor with chromium and silver copper oxide. To ensure complete combustion, the oxygen was dosed with sample introduction. After combustion, residual oxygen and nitrogen oxides were removed using reduced copper. Carbon dioxide and nitrogen were separated by a gas chromatography method. After separation, the analyte gases were transferred to the IRMS for measurement. The calculation was based on internal standards (caffeine, glutamic acid, glutathione and nylon powder) application. Final results of carbon isotope composition (δ¹³C) was calculated as:

$${\delta }^{13}\mathrm{C}=\left({\displaystyle \frac{\mathrm{R}}{\mathrm{R}\mathrm{s}\mathrm{t}\mathrm{n}\mathrm{d}}}-1\right)\ast 1000$$

(11)

where R is the isotope ratio ¹³C/¹²C in the sample, and Rstnd is the isotope ratio ¹³C/¹²C in international standard PDB (Pee Dee Belemnite) [37]. Nitrogen isotope composition (δ¹⁵N) was calculated as:

$${\delta }^{15}\mathrm{N}=\left({\displaystyle \frac{\mathrm{S}}{\mathrm{S}\mathrm{s}\mathrm{t}\mathrm{n}\mathrm{d}}}-1\right)\ast 1000$$

(12)

where S is the isotope ratio ¹⁵C/¹⁴C in the sample, and Sstnd is the isotope ratio ¹⁵N/¹⁴N in the air [37].

2.8. Statistics

Statistical analyses were performed using Statistica 12.0 (StatSoft, Tulsa, OK, USA). The obtained parameters were evaluated using Student’s t-test. Detailed information on the statistical tests applied and the number of replicates is provided in the descriptions of the tables and figures.

3. Results

Drought stress markedly affected several plant physiological parameters, particularly those related to water content. In well-watered poplar plants, water potential (Ψ) followed the typical gradient that facilitates water transport through the plant (Figure 1), with a systematic decrease from roots (−2.5 MPa), through stems (−4.8 MPa), to leaves (−6.4 MPa). Under severe drought stress, which ultimately led to leaf abscission, Ψ values dropped significantly, averaging approximately −9 MPa in roots and falling to −12.5 MPa in stems, indicating a pronounced water deficit in these tissues as well.

Alterations in plant water potential were reflected in the efflux of H₂O, presented as a light response curve dependent on photosynthetically active radiation (PAR). The total conductance to water vapor (g_tw) remained relatively constant with increasing light intensities in both treatments, but values were consistently lower under drought stress (Figure 2a). While well-watered stems reached maximum conductance values of approximately 2.5 mmol H₂O m⁻² s⁻¹, drought-stressed stems remained around 1.5 mmol H₂O m⁻² s⁻¹. This indicates a reduction in g_tw by approximately 40% compared to the control. The efflux of CO₂ (ECO₂) (Figure 2b), which was light-dependent, showed a decreasing trend with increasing photosynthetically active radiation (PAR). The shape of the response curves was similar in both treatments, indicating the same pattern of decline. In well-watered plants, ECO₂ decreased from approximately 2.0 to 1.4 µmol CO₂ m⁻² s⁻¹ over the measurement range. Under drought conditions, the decline followed a similar shape, with values falling from about 1.5 to 0.9 µmol CO₂ m⁻² s⁻¹, corresponding to a reduction of roughly 25%–35% compared to the control.

Based on ECO₂ measurements, CO₂ refixation was calculated as a percentage (Figure 2c), where 100% corresponds to ECO₂ measured at 0 µmol PAR. Under drought stress, CO₂ refixation increased significantly, reaching 55%–60% at high PAR levels. In contrast, refixation in well-watered poplar stems reached only about 40%. This indicates a greater significance of refixation under drought conditions in comparison with well-watered plants. Further evidence of more efficient use of stem-fixed CO₂ relative to water loss is provided by the pattern of intrinsic water use efficiency (iWUE) curves (Figure 2d). iWUE exhibited the strongest differences above 500 µmol PAR, reaching 0.25–0.32 µmol CO₂ mmol⁻¹ H₂O in controls and 0.5–0.55 under drought, i.e., a 2-fold enhancement. The relationship between CO₂ efflux and internal CO₂ concentration (Ci) further confirmed a heterogeneity in metabolic activity (Figure 3).

In both well-watered and drought-stressed stems, ECO₂ showed a negative correlation with Ci. In control plants, ECO₂ ranged from 0.8 to 1.7 µmol CO₂ m⁻² s⁻¹ at low Ci, decreasing to 0.2–0.6 µmol CO₂ m⁻² s⁻¹ at high Ci. Under drought conditions, ECO₂ was consistently lower, remaining within 0.6–0.9 µmol CO₂ m⁻² s⁻¹ across the Ci range. At 1450 µmol CO₂ mol⁻¹ Ci, CO₂ efflux dropped to zero, and at even higher Ci levels, a small net influx appeared, reaching 0.2 µmol CO₂ m⁻² s⁻¹. This suggests that the mechanism for CO₂ refixation in stems remains active under elevated CO₂ concentrations, even during drought stress.

The analysis of chlorophyll fluorescence parameters (Figure 4) revealed that drought stress had minimal impact on the primary photochemical efficiency of photosystem II (PSII). The maximum quantum efficiency (Fv/Fm) remained stable under drought conditions compared to the control, with values around 0.83 in both treatments, indicating that the structural integrity of PSII was maintained. Similarly, the photochemical quenching coefficient (qP) showed no significant change, suggesting that the capacity of PSII to utilize absorbed light in photochemical processes was unaffected by water deficit.

Similarly, no significant differences was observed in non-photochemical quenching (NPQ) under drought stress. This indicates a stabile capacity for dissipating excess excitation energy as heat, which could potentially protect the photosynthetic apparatus to photodamage. Also, the effective quantum yield of PSII (ΦPSII) remained largely unchanged, reflecting stable photosynthetic performance even under limited water availability.

Results obtained from chlorophyll a fluorescence are consistent with the pigment content in the stem cortex of poplar (

Table 1. Photosynthetic pigment content and anions xylem abundance in control and drought-stressed two-year-old stems of poplar. Photosynthetic pigments were determined from the cortex of stems (n = 5) and anions were analyzed in the xylem sap (n = 11). No significant differences were found between the corresponding groups according to Student’s t-test at p ≤ 0.05 have been noted.

	Treatment
	Control	Drought
Parameter
Chl a [mg m−2]	128.50 ± 28.94	118.49 ± 17.43
Chl b [mg m−2]	63.60 ± 17.81	63.07 ± 13.98
Chl a/b	2.10 ± 0.53	1.93 ± 0.37
Chl a + b [mg m−2]	192.10 ± 43.31	181.55 ± 26.58
Carotenoids [mg m−2]	31.41 ± 20.14	30.20 ± 16.63
Cl− [mg ml−1]	0.287 ± 0.328	0.123 ± 0.073
Malate [mg ml−1]	0.370 ± 0.167	0.513 ± 0.400
Citrate [mg ml−1]	0.017 ± 0.063	0.003 ± 0.009

). Pigment contents during drought remained stable: the concentration of chlorophyll a ranged between 118 and 129 mg m⁻², chlorophyll b remained unchanged (~63 mg m⁻²), and carotenoids also showed no consistent trend (31.4 to 30.2 mg m⁻²). In a similar manner, the chlorophyll a/b ratio ranged from 1.93 to 2.10.

Table 1 also presents the concentrations of selected anions measured in the expressed xylem sap. The values were widely dispersed. Statistical analysis showed that the mean concentrations of Cl⁻, citrate, and malate did not differ significantly between treatments. Consequently, it was not possible to demonstrate that the applied drought induced changes in xylem anion levels. In the case of citrate, concentrations were trace and close to the detection limit of the measuring equipment.

Stable isotope analyses of cortex tissues are presented in Figure 5. The carbon isotope composition (δ¹³C) decreased from −31.84‰ in the control group to −32.64‰ under drought conditions, which is associated with changes in carbon metabolism. The nitrogen isotope composition (δ¹⁵N) ranged from −2.24‰ in controls to −0.76‰ under drought. However, this difference was not statistically significant and does not suggest altered nitrogen assimilation and/or transport during water deficit.

4. Discussion

Leaves represent the most drought-sensitive organs and rapid stomatal closure plays a crucial role in limiting water loss during drought while simultaneously helping to maintain the stem water potential. In our case, the drought stress was severe, resulting in significant water potential reduction (Figure 1) that was accompanied by visible leaf desiccation and abscission. Leaf shedding is recognized as a protective strategy under acute drought, minimizing transpiration demand and thus preserving stem integrity [38,39,40]. Although embolism is likely under such conditions, the absence of transpiring leaves may promote passive refilling of xylem conduits through capillary-driven flow, which occurs independently of transpiration and is driven by water potential gradients [41,42].

Leaves represent the most drought-sensitive organs, and rapid stomatal closure plays a crucial role (within minutes) in limiting water loss during drought [2], while simultaneously helping to maintain the water potential of the whole plant, including the stem. In many cases, this process is reversible; however, when drought stress is very strong, such adaptation becomes irreversible [1]. Nevertheless, in plants—especially trees—growing in regions with moderate climatic conditions, this response enables the development of new leaves in the following season, thereby ensuring plant survival.

In our study, drought resulted in a significant decline in leaf water potential (Figure 1), but had no measurable effect on xylem sap anion concentration (Table 1). In citrus trees, xylem Cl⁻ levels have been shown to respond rapidly to environmental changes, serving as a sensitive indicator of chloride uptake [43]. Similarly, in Laurus nobilis, drought triggered an increase in xylem ion concentrations, interpreted as a compensatory response to sustain water transport under reduced hydraulic conductivity [44]. In our case, although severe drought stress caused visible leaf desiccation and abscission, it did not lead to detectable changes in xylem sap anion concentration. Leaf shedding is recognized as a protective strategy under acute drought, minimizing transpiration demand and thus preserving stem integrity [38,39,40]. Although embolism is likely under such conditions, the absence of transpiring leaves may facilitate passive refilling of xylem conduits through capillary-driven flow, which occurs independently of transpiration and is driven by water potential gradients [41,42]. The question arises as to whether photosynthetic and photochemical activity is disturbed under such physiological circumstances.

In our research, decline in stem conductance confirmed stem-level dehydration (Figure 2a). Specifically, stem water vapor conductance (g_tw) decreased from ~2.5 mmol m⁻² s⁻¹ to ~1.5 mmol m⁻² s⁻¹. The values obtained in this study fall within the range of 1.0–27.3 mmol m⁻² s⁻¹ reported for deciduous trees originating from temperate climate zones [17,20,45], e.g., ca. 1.0 mmol m⁻² s⁻¹ in four-year-old stems of Pinus monticola [32] or 1.1 mmol m⁻² s⁻¹ in young stems of Betula pendula Roth [17]. Significantly lower conductance was recorded in the stems of plants exhibiting exceptional drought resistance about 0.15 to 0.20 mmol m⁻² s⁻¹ in eight to ten year-old stems of Clusia multiflora and Clusia rosea [14,16]. The results of conductance values indicate that even under drought conditions, poplar stems retained the capacity for intensive gas exchange, including the efflux of CO₂ (Figure 2b).

In recent years, CO₂ efflux from plant stems has become an important metric for estimating stem metabolism, not only in the context of respiration, photosynthesis, and radial and axial CO₂ transport, but also in plants under drought conditions. To quantify stem metabolic activity and monitor its temporal dynamics, measurements of stem CO₂ efflux (ECO₂) are widely employed. ECO₂ in our light-dependent measurements ranged between 0.5 and 2.0 µmol m⁻² s⁻¹ and was similar to the values reported by De Roo et al. [26] but significantly lower compared to other papers concerning Populus, e.g., Salomon et al. [46] recorded as much as 3–4 µmol m⁻² s⁻¹ in 3-year-old P. canadensis. Under drought conditions, a decrease in respiration is expected due to reduced cell turgor [47]. However, according to other researchers drought may enhance respiration intensity in stems [48], such response was also observed in roots and leaves [49,50]. Another factor related to the decrease in stem turgor is the reduction in resistance for gases including CO₂ moving outside the stem, which should affect the increase in ECO₂ [51,52]. The rate of ECO₂, however, is not solely determined by “in situ” respiratory activity but is also modulated by the transport and redistribution of CO₂ within the stem, particularly via the xylem sap [15,16]. In well-watered stems, CO₂ dissolved in xylem accounts for 16%–27% of ECO₂ [6,26,46]. In our case, following leaf abscission—in the case of drought—ECO₂ is expected to decline to negligible values due to reduced transpiration-driven fluxes.

An additional mechanism that could influence ECO₂ is anaplerotic CO₂ fixation that has been widely postulated [16,53,54]. Nevertheless, a similar level of malate concentrations in the xylem sap (Table 1) suggests that phosphoenolpyruvate carboxylase (PEPC) activity was not changed under drought conditions. Consequently, the observed difference in ECO₂ (measured in darkness) between drought and irrigated treatments, approximately 0.6 µmol m⁻² s⁻¹, can be attributed solely to the reduced amount of CO₂ transported upwards with the transpiration stream as well as depression in respiration. Moreover, light-response curves under both control and drought conditions demonstrated a standard response to illumination [20]. The difference between dark and light was of similar magnitude, about 0.8 µmol m⁻² s⁻¹, for drought and control. In our case, a decrease in efflux was observed under both well-watered and drought conditions. These patterns correspond well with previously reported findings [5,27,55] and indicate a strong role of photosynthetic CO₂ fixation by the stem photosynthetic apparatus. Based on the reduction of ECO₂ this also contributes to higher intrinsic water use efficiency (iWUE) during drought (Figure 2d). WUE is a key physiological factor [1,22] that demonstrates efficient carbon utilization in poplar stems under drought conditions.

Numerous studies have analyzed the distribution of chlorophyll in stems, which is typically concentrated beneath the periderm. Chlorophyll concentration is connected with photosystem II (PSII) presence and performance parameters [8,11,56] and more recently photosystem I (PSI) investigated by Natale et al. [57]. In our experiments, chlorophyll a fluorescence parameters measured directly beneath the cork (Figure 4) suggest that stem photosynthetic activity did not decline significantly under drought. Likewise, photosynthetic pigment concentrations remained unchanged under water deficit, in contrast to previously studied stems. In Vitis (grapevine), drought was reported to increase chlorophyll a and b contents in stems, suggesting that pigment accumulation in the cortex (and other stem tissues) may represent an adaptive mechanism [58]. In contrast, in avocado (Persea americana), drought stress (with a ~60% reduction in stem photosynthesis) and light exclusion did not significantly alter bark chlorophyll concentration, indicating that changes in photosynthetic capacity were not directly linked to pigment content [59].

Putting all these facts together, these results confirm that stem photosynthesis actively contributes to CO₂ reassimilation (Figure 2c). Based on ECO₂ measurements, reassimilation accounted for approximately 35% under irrigated conditions and up to 50% under drought. Other studies have reported a wide range of stem CO₂ reassimilation values, from 10% to 127% of efflux, depending on species and environmental conditions [6,21,24,25]. Kharouk et al. [60] also reported 30%–50% reassimilation of respiratory CO₂ by the bark of Populus tremuloides, which contributes 10%–15% to CO₂ acquisition during the summer months. Studies of Dukat et al. [25] demonstrated that stem CO₂ efflux comprises stem photosynthesis (up to 13%), varying seasonally. Importantly, De Roo et al. [6] found that woody tissue photosynthesis reduced CO₂ efflux by up to 50%, and this effect remained unchanged under drought stress. Cernusak and Hutley [61] estimated that 11% of wood in the branch of Eucalyptus miniata originates from corticular photosynthate. The above results (Figure 2) refer to ECO₂ under atmospheric conditions, i.e., a CO₂ flux to the atmosphere at 0.04% CO₂. Measurements under elevated CO₂ (Figure 3) showed that, despite drought-induced reductions in stem CO₂, the capacity for assimilation remained largely maintained.

Leaf senescence, aging, and eventual abscission are processes typically associated with the remobilization of assimilates and nitrogen compounds. Inter-organ fluxes can be traced using isotopic techniques, notably δ¹³C and δ¹⁵N (Figure 5). In our study, δ¹³C decreased under drought, whereas δ¹⁵N increased, although more as a trend than as a statistically significant change. In stems of woody plants, effluxed CO₂ is ¹³C enriched [62], and the remaining organic matter is ¹³C depleted [61,63]. Moreover, the intensity of stem photosynthesis is visible in the δ¹³C value due to CO₂ released from stems: Cernusak et al. [64] revealed strong stem photosynthesis modulation in δ¹³C of CO₂ released from stems of two woody plants. Similarly to Saveyn et al. [63] and Cernusak and Hutley [61] as result of darkening, an increased δ¹³C in organic matter of stems was observed. However, in our case, the decrease in δ¹³C under drought conditions is most likely associated with the allocation of carbon withdrawn from the leaves [62,65].

5. Conclusions

Our findings demonstrate that, despite severe reductions in water potential, desiccation, and abscission, poplar stems maintained functional photosynthetic activity. Under water limitation, chlorophyll a fluorescence parameters and photosynthetic pigment contents remained unaffected. Stems sustained light-dependent reductions in ECO₂, indicating a substantial contribution of corticular photosynthesis. Reassimilation of CO₂ accounted for approximately 40% of ECO₂ under well irrigated conditions and up to 60% under drought, a pattern that also corresponded with increased intrinsic water use efficiency under water stress. Stable isotope analyses further supported these adjustments, revealing drought-induced shifts in δ¹³C that are consistent with altered carbon allocation following leaf abscission. Future studies could focus on the rehydration phase, which would enable assessment of actual plant survival. In summary, these findings confirm that stem photosynthesis and CO₂ reassimilation play a significant role in enhancing stem metabolic resilience, mitigating drought-induced carbon losses, and supporting overall plant stress tolerance.