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Article

Morpho-Physiological Responses During Dark-Induced Leaf Senescence in Cunninghamia lanceolata Seedlings

by
Zhijun Huang
1,2,
Qingqing Liu
1,*,
Xianhua Zou
1,2,
Liqin Zhu
1,2,
Xiangqing Ma
3,* and
Rongzhen Huang
1,2
1
School of Soil and Water Conservation, Jiangxi University of Water Resources and Electric Power, Nanchang 330099, China
2
Jiangxi Key Laboratory for Intelligent Monitoring and Integrated Restoration of Watershed Ecosystem, Nanchang 330099, China
3
College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
Forests 2025, 16(9), 1372; https://doi.org/10.3390/f16091372
Submission received: 14 July 2025 / Revised: 9 August 2025 / Accepted: 25 August 2025 / Published: 26 August 2025
(This article belongs to the Special Issue Ecophysiological Responses of Forest Tree Growth to Environmental Change)
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Abstract

Low inner leaves in the thick canopy of dense Chinese fir plantations frequently show premature senescence and dieback regardless of age. To elucidate the underlying mechanisms, a 28-day growth chamber experiment was conducted under dark conditions to induce leaf senescence. Changes in leaf area, photosynthetic performance, and the responses of carbon metabolism and the antioxidant defense system were analyzed. Leaf area decreased significantly with time in darkness. The photosystem II reaction center was damaged, and fluorescence parameters and chlorophyll contents decreased, resulting in reduced light energy capture and conversion efficiencies. Photosynthetic rate, apparent quantum yield, stomatal conductance, transpiration rate, and light use efficiency all decreased, while the light compensation point and intercellular CO2 concentration increased. Antioxidant enzyme activities initially increased but eventually collapsed as the stress continued and H2O2 and malondialdehyde accumulated, causing membrane conductivity, i.e., membrane permeability, to increase by 122%. Meanwhile, reduced non-structural carbohydrates, especially total non-structural carbohydrates content, decreased by 45.32%, triggering sugar starvation and accelerating aging. Our study provided new physiological evidence for light-stress response mechanisms in Chinese fir. Specifically, it revealed that dark-induced leaf senescence was mainly caused by irreversible damage to the photosynthetic apparatus and oxidative stress, which together led to carbon starvation and ultimately death.

1. Introduction

Plant senescence is a highly coordinated process initiated and driven by a combination of age-dependent developmental and environmental biotic and abiotic factors. Additionally, several environmental stressors, such as shading, extreme temperatures, drought, and nutritional deficiencies, among others, can trigger undue senescence of affected plant organs, a phenomenon known as artificially induced senescence [1,2,3,4,5].
Senescence exerts an important impact on plant growth and development, as it directly affects whole-plant photosynthesis, carbon assimilation, biomass accumulation, and final productivity. By and large, leaves account for overall plant photosynthesis, and, most importantly, they are the most sensitive tissues to senescence. Moreover, targeted leaf senescence can be initiated if the functional capacity of the leaf is severely affected by developmental or external factors. Indeed, leaf senescence has been proposed as a plant strategy to dispose of inefficient and highly consumptive, aging photosynthetic organs [6]. Considering the central role of leaves in plant photosynthesis, light deprivation or dark treatment can give rise to senescent leaf phenotypes, a process known as dark-induced leaf senescence [5,7]. Therefore, it has come to be widely used experimentally to initiate a process of synchronized senescence under controlled conditions by subjecting young seedlings to prolonged darkness, which is equivalent to extreme shading.
One of the first signs of leaf senescence is the degradation of chloroplasts, followed by a decline in photosynthetic efficiency. Such degradation is a feature shared by both developmental and dark-induced senescence [8]. Further, numerous studies have found that dark treatment effectively induces leaf senescence, and the senescence pattern is essentially the same as that observed in the natural process [9,10,11]. Therefore, artificially dark-induced senescence is widely used for simulating natural leaf senescence under controlled conditions to study the process and the underlying physiological mechanisms [9,12,13].
Owing to high-quality timber, fast growth, and a long cultivation history of over one thousand years, Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) has become an important plantation-tree species in subtropical regions. Based on our previous field-localization study of the leaf wilting process in C. lanceolata plantations, we found that, when the natural leaf senescence phenomenon dependent on age factors is excluded, leaves of the same age at different positions on C. lanceolata saplings showed differences in morphological and physiological characteristics [14].
The single most significant difference among different leaf positions in a tree canopy is the amount and quality of incident light in each case. Therefore, we speculated that light is the key environmental variable causing premature leaf aging and death at the bottom and inside the canopy of high-density C. lanceolata plantations. Premature leaf aging reduces plantation timber yield by limiting sapling growth. However, owing to the difficulty in controlling environmental factors, including light, water, and soil fertility in an open field, our study could not provide an in-depth understanding of the morphological and physiological mechanisms involved in the wilting process of C. lanceolata leaves. Additionally, we previously conducted research on the effects of reduced light intensity and changes in light quality on the morphological and physiological characteristics of C. lanceolata leaves [15,16,17]. However, these studies focused primarily on photosynthetic acclimation mechanisms under conditions of suboptimal light-energy supply. Therefore, here, we used dark-induced senescence as an experimental model to trace any differences in leaf morphological characteristics, photosynthetic performance, chlorophyll fluorescence, chlorophyll contents, non-structural carbohydrate contents, internal homeostasis, and other indexes in C. lanceolata seedlings under an increasing duration of the dark treatments. Our study aimed to exclude the influence of multiple environmental factors on the C. lanceolata leaf wilting process that unavoidably occur in the field, while simultaneously aiming to achieve artificially induced leaf senescence in a relatively short period of time to gain a deeper understanding of the morpho-physiological response mechanisms underlying the process of leaf senescence and wilting in C. lanceolata seedlings grown in the dark.
Sudden light deprivation represents an extreme scenario that accelerates the leaf senescence process over a relatively short period of exposure, thereby offering the opportunity to enrich our understanding of the basic biological processes involved. Moreover, it will allow us to focus on revealing the survival thresholds of C. lanceolata under extreme shading conditions (such as those prevailing in high-density plantations), while simultaneously providing important guidance for light regulation during seedling cultivation and for the improvement of Chinese fir plantation timber yield and quality.

2. Materials and Methods

2.1. Plant Material and Treatments

The experimental plant material consisted of one-year-old clonal C. lanceolata seedlings (Yang-061), cultivated in the Nanping Yangkou State-owned Forest Farm, in Fujian, China. These seedlings were transplanted into pots (inner diameter 28 cm and 30 cm height; one seedling per pot), containing a 2:1 (by volume) mixture of peat soil and vermiculite, and acclimated for one month in a greenhouse prior to the initiation of the experiments. At the end of the acclimation period, well-developed C. lanceolata seedlings of uniform size (mean height 48.13 ± 7.32 cm, mean root-collar diameter 6.40 ± 0.41 mm; n = 15) were selected and transferred to an artificial growth chamber for dark treatment under controlled conditions (25 °C, 75% humidity) to induce leaf senescence. The initial sampling and index measurements conducted before the dark treatment were recorded as the original values and served as the experimental control (0 d). After that, dark-induced senescence was initiated, and leaves were selected for the determination of morpho-physiological indices on days 7, 14, 21, and 28 (hereafter denoted by 7 d, 14 d, 21 d, and 28 d). Considering each pot as a relatively independent individual, each pot was treated as an experimental replicate. Three seedlings randomly selected for measurement at each sampling timepoint were labeled to avoid repeated use.

2.2. Methods

2.2.1. Leaf Morphology Measurements

Fifteen mature and healthy upper leaves located at comparable heights on each test seedling were randomly sampled for measurements. Refer to the methods described previously [14,15] for leaf scanning and analysis (including leaf length, width, and area). For immediate enzyme inactivation after scanning, each sampled leaf was placed separately in an envelope, numbered, and oven-dried at 105 °C for 30 min, following which the temperature was adjusted to 80 °C to complete drying to constant mass. Subsequently, leaves were weighed individually to record their biomass, and specific leaf area (SLA, cm2·g−1) was calculated as the ratio of leaf area to leaf dry mass.

2.2.2. Measurement of Leaf Photosynthesis and Gas Exchange

Leaf photosynthetic photon-flux density (PPFD) response curves were obtained using an LI-6400XT portable photosynthesis system (LI-COR Biosciences Inc., Lincoln, NE, USA) on intact seedlings. Before measurement, all light sources in the laboratory were turned off and the windows covered with blackout curtains to ensure total darkness in the surroundings. To avoid overlapping, test leaves were spread in an integrated fluorescence-chamber head and exposed in a stepwise manner to a gradient from high to low PPFD levels (2000, 1500, 1000, 800, 500, 200, 150, 100, 80, 50, 20, and 0 µmol·m−2s−1). Gas exchange parameters, including stomatal conductance (gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr), were measured with the same apparatus and recorded when a steady-state net photosynthetic rate (Pn) was attained. The data acquisition time was set to 2 min at each PPFD level using the automated measurement program of the photosynthesis assay system, which was automatically calibrated after each count. Environmental conditions in the leaf chamber were set as follows: CO2 concentration 400 µL·L−1, block temperature 25 ± 5 °C, chamber flow rate 300–500 μmol·s−1, relative humidity 50 ± 10% [14]. Additionally, leaves were photographed after measurements for photosynthetic leaf-area estimation and photosynthetic data correction.
A rectangular hyperbola model [18] was used to fit the leaf PPFD response curves. This fitting allowed accurate calculation of photosynthetic parameters, including maximum net photosynthetic rate (Pmax), dark respiration rate (Rd), apparent quantum yield (AQY), light compensation point (LCP) and light saturation point (LSP) [19,20].

2.2.3. Measurement of Chlorophyll Fluorescence

Chlorophyll fluorescence parameters including potential photochemical efficiency ratio of photosystem II (PSII) (Fv/Fo), maximum quantum yield of PSII (Fv/Fm), non-photochemical quenching (NPQ), photochemical quenching (qP), effective quantum yield of PSII (ΦPSII), and fluorescence decline ratio (Rfd, i.e., the potential photosynthetic quantum-conversion efficiency of leaves), were measured using a FluorPen FP 100 (Photon Systems Instrument, Drásov, Czech Republic) [21]. These fluorescence parameters were measured in the same seedlings and leaves as those sampled for the determination of light response curves, and averaged over six replicate measurements per seedling.

2.2.4. Measurement of Leaf Chlorophyll Contents

After the measurements described above, the sampled leaves were immediately harvested to determine photosynthetic pigment concentrations through extraction using an 80% acetone solution. After that, chlorophyll a (Chl a), b (Chl b), and total chlorophyll [Chl (a + b)] concentrations were converted into contents as described by Qiu et al. [22] and were expressed as g·kg−1 of fresh weight.

2.2.5. Determination of Leaf Antioxidant Enzyme Activities

Leaf samples were rinsed and wiped dry with blotting paper, and grinded into a powder for the preparation of a leaf antioxidant-enzyme solution using phosphate buffer. Superoxide dismutase (SOD) activity was determined by the nitrogen blue tetrazolium (NBT) method [23]; in turn, peroxidase (POD) activity was determined by the guaiacol method [24], while catalase (CAT) activity was determined after Yang et al. [25].

2.2.6. Determination of Leaf Membrane Stability and Extent of Lipid Peroxidation

The enzyme solution prepared above was also used to determine malondialdehyde (MDA) content by the thiobarbituric assay as a proxy of lipid peroxidation in C. lanceolata leaves [26]. In turn, leaf H2O2 content was determined by observing the formation of a yellow titanium peroxide complex between H2O2 and titanium sulfate after [27]. Leaves from each sampling timepoint were washed, wiped, and cut into pieces. Then, 0.1 g fresh leaf tissue samples with veins removed were added to 1 mL of phosphate buffer for ice-bath homogenization, transferred to conical tubes, and made up to the mark with phosphate buffer before centrifuging at 8000× g at 4 °C for 10 min. The supernatants were mixed with 0.325 mL of 0.1% titanium chloride (20% (v/v) H2SO4) and centrifuged at 8000× g 4 °C for 10 min. Subsequently, samples were incubated in the greenhouse for 5 min. Then, 200 μL of each sample mixture was transferred to a 96-well plate to measure absorbance at 415 nm. Finally, H2O2 content was calculated according to an H2O2 standard curve.
Relative conductivity was used as a proxy for the relative permeability of leaf cell membranes. Briefly, leaves were collected from the same height and the same part of each seedling at each sampling timepoint, rinsed twice with deionized water, and blotted dry with filter paper; then, the leaves were cut into 0.5–1.0 cm segments. Subsequently, leaf samples (0.5 g fresh weight with veins removed) were placed in an Erlenmeyer flask and soaked with 30 mL distilled water as an experimental group. A control group was set up, which consisted of 30 mL of deionized water only, without any leaf sample included. All flasks were subjected to vacuum extraction for 15 min to make the leaf samples all immersed in water for 1 h at room temperature to measure initial conductivity (R1) of the experimental group using a DDS-II conductivity meter (Leici, Shanghai, China), then boiled for 20 min. The value of final conductivity (R2) and the conductivity of the deionized water (R0) were determined after samples reached room temperature. Leaf relative conductivity was quantified as (R1 − R0)/(R2 − R0) × 100% [28].

2.2.7. Measurement of Leaf Non-Structural Carbohydrates Content

Non-structural carbohydrates, NSCs, mainly comprise soluble sugars and starch. Soluble sugar and starch contents were determined using the anthrone colorimetric method, and total non-structural carbohydrates content was calculated as the sum of soluble sugar and starch contents [16].

2.3. Experimental Design and Statistical Analysis

Differences in the measured parameters were analyzed using one-way ANOVA followed by the least significant difference (LSD) test, if necessary. Means were considered as significantly different at p < 0.05. All figures were elaborated using Origin 2018 software. Data shown in the figures and tables are means ± standard deviation values.

3. Results

3.1. Leaf Morphology Responses to Dark Treatment for Induction of Senescence

Leaf length decreased significantly with increasing duration of dark stress (Figure 1A). In turn, leaf width initially increased and then decreased, with the lowest value being observed at 28 d after initiation of dark stress (Figure 1B); meanwhile, leaf area was significantly reduced, showing the smallest value after 28 d of dark stress, at which time mean leaf area was 20.15% smaller than that at 0 d (Figure 1C). Lastly, specific leaf area gradually increased, although differences among treatments were not significant (p = 0.124) except for that between 0 and 28 d (Figure 1D).

3.2. Photosynthetic Response to Dark Treatment for Senescence Induction

Net photosynthetic rate (Pn) decreased with increasing duration of the dark treatment (Figure 2A). Especially after 28 d of darkness, Pn became negative across PPFD levels, indicating that the photosynthetic capacity of C. lanceolata leaves was severely affected and respiratory carbon loss became greater than photosynthetic carbon assimilation. Consistently, both gs and Tr gradually decreased with increasing duration of dark treatment, with the highest and lowest values recorded at 0 and 28 d after dark treatment initiation, respectively (Figure 2B,C). Concomitantly, Ci increased with increasing duration of darkness (Figure 2D), likely owing to the enhancement of CO2 release via dark respiration. Conversely, Ci decreased with increasing PPFD across sampling timepoints during dark treatment. Furthermore, under the same PPFD level, light use efficiency (LUE) decreased with increasing duration of dark stress (Figure 2E); thus, the longer the dark stress duration, the greater the PPFD required for leaf LUE to peak, and the lower the peak value became.
Maximum net photosynthetic rate (Pmax) in leaves of C. lanceolata seedlings decreased significantly with increasing duration of dark treatment, such that the mean value was negative at 28 d after initiation of dark treatment and decreased by 8.08 μmol·m−2·s−1 compared with that at the beginning of the dark treatment (Table 1). In turn, AQY decreased significantly and then stabilized with increasing stress duration, with the mean value at 0 d being significantly higher than that of any other sampling timepoint. Indeed, AQY was 10.17 times higher at 0 than at 28 d of dark induction. Similarly, LSP and Rd were significantly reduced, whereas LCP increased significantly, such that it was 1.21, 5.69 and 15.23 times higher than that of 0 d at 7, 14, and 21 d of dark induction, respectively, while the value could not be measured at 28 d of dark induction.

3.3. Chlorophyll Fluorescence Response to Dark Treatment for Senescence Induction

All leaf-fluorescence parameters measured, namely, Fv/Fo, Fv/Fm, and ΦPSII decreased significantly as the duration of dark stress increased; in contrast, Rfd and NPQ increased initially and then decreased, with both peaking after 7 d under dark induction and then decreasing significantly. Meanwhile, qP values generally showed a significant decrease, with the smallest values observed at 14, 21, and 28 d after treatment initiation, and did not significantly differ among the three sampling timepoints (Table 2).

3.4. Chlorophyll Contents Response to Dark Treatment for Senescence Induction

All Chl a, Chl b, and Chl (a + b) contents in C. lanceolata leaves decreased significantly with increasing duration of the dark treatment (Figure 3A,B,D). Further, the trend followed by the three parameters showed that they all decreased slowly at the beginning of the dark stress, then decreased significantly towards the end and reached the lowest level at 28 d. The ratio of Chl a to Chl b increased significantly with duration of the dark treatment and peaked at 28 d, when it was approximately 2.10 times greater than the value observed at 0 d (Figure 3C).

3.5. Antioxidant Enzyme Activities Response to Dark Treatment for Senescence Induction

Superoxide dismutase (SOD) activity in C. lanceolata leaves was 236.09 U·g−1 at 28 d after dark treatment initiation, at which point it was 3.11, 2.80, 1.78, and 1.58 times higher than that at 0, 7, 14, and 21 d, respectively (Figure 4A). In turn, POD activity was progressively enhanced with increasing duration of dark treatment, and the increase in POD activity range within each adjacent 7 d interval was 31.66–35.69 U·g−1, although no significant differences were observed (Figure 4B). Lastly, CAT activity was also significantly enhanced with increasing duration of dark treatment, and although a slight decrease occurred at 28 d, it remained significantly higher compared with that at 0 d (Figure 4C).

3.6. Leaf Membrane Permeability Response to Dark Treatment for Senescence Induction

The H2O2 content in C. lanceolata leaves increased significantly with increasing duration of darkness and peaked at 28 d, when it was 1.71-fold greater than that observed at 0 d (Figure 5A). Similarly, leaf MDA content increased significantly with the duration of dark exposure (Figure 5B). In particular, MDA showed a slight early increase (0–7 d), followed by a sharp rise after 7 d, then a slower increase from 14 to 21 d, peaking at 28 d with a 71.25% increase compared to 0 d. Consistently, membrane conductivity increased significantly with increasing duration of dark treatment (Figure 5C). The highest conductivity values were observed at 28 d and were significantly higher than those at any other sampling timepoint and 2.22 times higher than that at 0 d.

3.7. Leaf Non-Structural Carbohydrate Contents Response to Dark Treatment for Senescence Induction

Soluble sugar content of C. lanceolata leaves decreased significantly with increasing duration of dark treatment. The largest decrease (51.67%) was recorded at 28 d (Figure 6A). Both starch and total non-structural carbohydrates content (i.e., the sum of soluble sugar content and starch content) showed significant decreasing trends with increasing treatment duration (Figure 6B,C). In particular, leaf total non-structural carbohydrates content at 28 d was only 54.68% of that recorded on day 0 of dark induction treatment. Finally, the ratio of soluble sugar to starch content was highest at 0 d and decreased significantly at 7 d, after which the ratio remained relatively stable with increasing duration of darkness (Figure 6D).

4. Discussion

Leaf morphology generally shows great plasticity, such that plants effectively adjust it in an effort to adapt to environmental changes. Consistent with findings by Liu et al. [17] on C. lanceolata, and similar to those by Dai et al. [29] on Tetrastigma hemsleyanum Diels et Gilg and those by Liu [30] on Malus sieversii (Led.) Roem, in this study, we found that, in C. lanceolata, leaf area decreased and specific leaf area increased with increasing dark treatment duration. Furthermore, we speculate that this is a plant strategy in response to a low-light environment and may be applicable to fine-leaf and broadleaf plants alike, whereby a conservative strategy for the use of resources is adopted such that investment of carbon in the leaves is relocated towards the sustainment of essential life activities. Indeed, by the time changes in plant leaf phenotype become evident, the physiology of the leaf has previously changed. Namely, the structural breakdown and functional degradation of chloroplasts and the associated decrease in photosynthetic capacity are the initial and main internal physiological responses induced by darkness in plant leaves [13,31].
Most catabolic processes in plants during leaf senescence occur in the chloroplasts and are mainly characterized by a decrease in photosynthesis-related activities [13]. Consistently, we found that, concomitant with a decrease in gs, Tr, and LUE, and an increase in Ci, Pn gradually decreased with increasing duration of dark treatment and was negative at 28 d after its initiation. This phenomenon may be attributed to the loss of photosynthetic capacity as a result of severe damage to the structure of photosynthetic tissue, which in turn resulted in a decrease of CO2 fixation efficiency, the enhancement of mitochondrial respiration, and a large amount of CO2 being released, which altogether led to the increase of Ci in the leaves of the C. lanceolata seedlings kept in darkness for an extended period of time. These findings partially account for the effect of dark induction on the photosynthetic performance of C. lanceolata leaves and allows us to infer that the dark-induced reduction of Pn is mainly explained by non-stomatal limitations [31].
Changes in AQY reflect the changes in the ability of leaves to absorb, convert, and use light energy in the dark. Our data showed that AQY decreased significantly as early as 7 d after initiation of darkness induction, although the differences in AQY values among 14, 21, and 28 d of dark treatment initiation were not significant. This finding indicated that most of the light energy-utilization activity in the leaves had been lost as early as 7 d after darkness induction and that leaves initiated an irreversible senescence and death process with the extension of the dark treatment. At the same time, Pmax, LSP, and Rd values significantly declined, while LCP significantly increased, likely owing to the continuous degradation of photosynthetic proteins under continuous low light or dark conditions, thereby reducing the ability to use light and the tolerance to high light intensity; at this time, the leaves would be prone to photoinhibition if subjected to high light intensities.
Current technology for the determination of chlorophyll fluorescence is a fast, sensitive, non-invasive, and reliable method to assess changes in plant physiological status and the relationship between such status and the environment [31,32]. Therefore, chlorophyll fluorescence parameters are often used as important indicators capable of characterizing plant responses to stress. Among chlorophyll fluorescence parameters, Rfd was determined as the earliest parameter that correlated strongly with the cessation of photosynthesis [13], as it can reflect the activity of PSII and carbon assimilation potential. Specifically, the larger the Rfd value, the greater the photosynthetic vigor. Thus, a decline in Rfd indicates an impaired photosynthetic apparatus or a reduced efficiency of energy conversion. In this study, Fv/Fo gradually decreased, and Rfd first increased and then decreased as darkness was prolonged, consistent with the results of Paluch-Lubawa et al. [33] on Hordeum vulgare L. Presumably, once the plant perceives darkness, it will adapt to the low-light environment through stress acclimation strategies in order to enhance primary photochemical efficiency at the early stage of darkness induction. However, if continued, darkness will induce severe damage to chlorophyll, followed by an irreversible reduction in energy conversion efficiency.
Photosynthetic activity is often measured using the Fv/Fm, which indicates the extent to which the existing photoreactive centers are actually available for photosynthesis [34]. Under field conditions, Fv/Fm values of plant leaves growing normally remain within the range from 0.75 to 0.85 [35]. In this study, Fv/Fm values in C. lanceolata leaves were significantly reduced upon dark induction, particularly after 14 d of darkness, indicating that PSII had experienced stress injury by that sampling timepoint. Furthermore, the decrease in Fv/Fm was concomitant with a significant decrease in ΦPSII, as was also reported by Špundová et al. [36]. These findings indicate that PSII was damaged with a severity that was dependent on the duration of the dark treatment.
Chlorophyll is involved in absorbing and transferring light energy and causing primary photochemical reactions during the early phases of plant photosynthesis [37]. Hence, it is not surprising that any changes in chlorophyll content affect the absorption and utilization of light energy by plants. Indeed, usually, photosynthetic pigment contents are significantly and positively correlated with the exuberance of plant life and negatively correlated with leaf senescence. Moreover, the degradation of chlorophyll is one of the most obvious features of leaf senescence [13,38]. Accordingly, chlorophyll content of C. lanceolata leaves decreased with increasing dark-induction time. This result can also explain the occurrence of reduced photosynthetic rate in dark-induced senescence. In summary, dark stress hindered photosynthetic efficiency in C. lanceolata leaves through the inhibition of PSII activity and a reduction in chlorophyll content. Additionally, whereas leaf photosynthetic and fluorescence parameters responded rapidly to darkness induction, chlorophyll degradation and content reduction occurred at a much slower pace.
In addition to the blockage of energy capture, the metabolism of C. lanceolata leaves changed in response to dark-induced senescence. Generally, when plants are subjected to stress, the dynamic equilibrium between the production and scavenging of reactive oxygen species (ROS) in the plant body is reportedly disrupted, while macromolecule degradation further contributes to the generation of ROS, whose excessive accumulation ultimately disrupts intracellular homeostasis [9,39,40]. In turn, the accumulation of ROS triggers an increase in the activity of SOD, which is the first line of defense of the plant antioxidant-enzyme system [41]. SOD catalyzes the dismutation of ROS into H2O2 and O2 and is followed by POD and CAT activities, which synergistically convert H2O2 to water [40,42]. Our data confirmed the induction of these enzyme activities upon dark treatment of C. lanceolata leaves. However, as dark stress was prolonged, the rate of ROS accumulation surpassed the capacity of the leaf detoxifying machinery, causing oxidative damage to key cellular constituents, as has been previously reported [43], leading to peroxidation of the cell membrane lipids.
Malondialdehyde is the end product of membrane lipid peroxidation, and changes in MDA content reflect the degree of cell-membrane damage [40]. In this study, we found that, between 0 and 7 d of dark induction, C. lanceolata leaves showed relatively stable MDA and H2O2 contents, i.e., the antioxidant enzyme system was able to function in a timely manner under short-term dark induction to maintain a relatively stable state within the leaf cells. However, a prolonged exposure of the leaves to darkness caused MDA and H2O2 contents to significantly increase, in which case, the resulting increase in SOD, POD, and CAT activities failed to cope with the excessive accumulation of MDA and H2O2, ultimately leading to severe and irreversible cell-membrane damage. The data showed that C. lanceolata leaves entered the dark-induced senescence stage, indicating that these antioxidant enzymes did not contribute to the observed MDA burst. Concomitantly, a large accumulation of H2O2 led to an increase in membrane lipid peroxidation in the chloroplasts, and severe cell-membrane damage was signaled by the corresponding increase in membrane permeability, as evidenced by a significant increase in membrane conductivity. In fact, it has been proposed that the role of POD in plants is dual. In the early stage of senescence, POD plays a positive effect by scavenging H2O2; then, in the late stage of natural senescence or under prolonged dark stress, POD appears to play a negative role, mainly involved in ROS production and chlorophyll degradation, thereby triggering membrane lipid peroxidation and ultimately realizing cellular oxidative damage [44].
Several abiotic stress factors, including darkness, high temperature, drought, and N-deficiency, inhibit leaf photosynthesis and CO2 assimilation [45,46,47], sucrose synthesis, and the conversion of starch to soluble sugars [48]. In particular, NSCs mainly composed of soluble sugars and starch are the products of plant photosynthesis. They are an important source of energy for plant growth and metabolism, and a metabolically active carbon pool for the plant. Typically, senescent-associated sugar starvation occurs only if photosynthetic capacity is severely compromised [49,50]. The application of a dark treatment is a stable and controllable method to achieve a carbon starvation-induced senescence system, which essentially translates as an energy-deficit stress on energy metabolism in plants [51]. The results of this study showed that photosynthetic efficiency declined significantly as soluble sugars, starch, and total non-structural carbohydrates content decreased in C. lanceolata leaves with increasing duration of the dark stress. This result suggests that in senescent C. lanceolata leaves, biochemical changes occurred concomitant with a decline in assimilation and depletion of assimilates. Specifically, C. lanceolata leaves exposed to prolonged light deprivation or low light intensities (below LCP) experienced an inability to conduct sufficient carbon assimilation, which in turn disrupted energy supply, metabolic biosynthesis, and carbon balance. Furthermore, the conversion of starch to soluble sugars was blocked, making it difficult to sustain the energy supply required for survival in the later part of the experimental dark period used herein. Under these conditions, plants must and can only rely on stored reserves to sustain growth, which leads to a rapid decline in NSC levels in leaf tissues, carbon starvation, and, ultimately, increased mortality rates [9,52]. This sequence of events partially accounts for a common phenomenon observed in the production of C. lanceolata plantation forests, namely, the extensive senescence dieback of lower branches and leaves in high-density or high-canopy-closure stands. Early senescence and dropping of C. lanceolata leaves may be a strategy to dispose of inefficient and highly consumptive photosynthetic organs to instead store energy for normal growth of upper leaves, such as to ensure seedling survival. Consistently, Wiley and Helliker (2012) showed that trees store NSCs at the expense of growth in order to ensure survival during periods of negative carbon equilibrium [53]. In contrast, leaf NSC concentration in Quercus mongolica Fisch. ex Ledeb. seedlings decreased rapidly, and all seedlings died after 12 weeks of darkness [54]. Meanwhile, Weber et al. [55] observed that Picea abies (L.) H. Kars. and Pinus sylvestris L. seedlings sacrificed young needles and leaves, and broadleaf species like Acer pseudoplatanus L. and Quercus petraea L. developed new etiolated shoots. Altogether, these findings suggest different strategies at play to cope with dark stress in broadleaf trees versus conifer tree species.
Leaf-soluble sugars are regarded as plant sensors of external stimuli, thus contributing to the regulation of plant growth processes, including, photosynthesis, carbon allocation, seed germination, and leaf senescence [49,50,56,57,58], and plant responses to various abiotic stress factors. Significantly reduced concentrations of total soluble sugars in leaf tissues are commonly found in plants exposed to severe abiotic stressors and during age-dependent leaf senescence. Further, several studies have suggested that an insufficient supply of carbon nutrition resulting from reduced sugar levels in leaves causes oxidative stress, which in turn may activate sugar signaling to induce leaf senescence [59]. As proposed by Buchanan-Wollaston et al. [9], in dark-induced senescence the main signal for the process may be a rapid reduction in NSCs levels, resulting in a greater reliance on lipid degradation for energy. In fact, our findings lend support to this idea. Specifically, we observed that dark-induced senescent C. lanceolata leaves showed significantly lower NSC and higher MDA and H2O2 contents. Eventually, C. lanceolata leaves gradually senesced and irreversibly entered the apoptotic metabolic pathway due to carbon starvation and the collapse of the antioxidant enzyme systems. These are the reasons why senescence and eventual death of C. lanceolata leaves occurred under long-term dark treatment.
A word of caution seems necessary. In scientific studies, the use of darkness as a stress treatment may lead to dramatically different results due to differences in specific experimental methods. Whole C. lanceolata seedlings placed in darkness were used for the experiments summarized herein. Whether leaves show different metabolic strategies in response to darkness if a few of them on a seedling are darkened while the rest remain exposed to light, or if detached leaves are cultured in darkness, are questions that warrant further research. Moreover, the detailed molecular basis of the mechanisms responsible for plant responses to dark-induced senescence remains unclear. Therefore, we recommend that these aspects be the focus of future research in pursuit of true advancement of our understanding of dark-induced senescence to help provide new study models or change current ones, creating, ultimately, scientifically based strategies for stand management to maximize productivity.

5. Conclusions

This study provides the first evidence that dark-induced senescence in C. lanceolata involves multifaceted effects of photosynthetic apparatus, oxidative damage, and carbon starvation. Darkness induced strong inhibition of chlorophyll, causing an irreversible reduction in energy conversion efficiency. The observed increase in the light compensation point, concomitant with a reduction in the light saturation point, maximum net photosynthetic rate, and dark respiration rate can be explained by the degradation of photosynthetic proteins under continuous darkness, thus reducing the ability to use light. Furthermore, PSII was inhibited with a severity that was dependent on the duration of the dark treatment. Thus, overall, dark treatment hindered photosynthetic efficiency in C. lanceolata leaves through the inhibition of PSII activity and reduction in chlorophyll content. Additionally, leaf photosynthetic and fluorescence parameters responded rapidly to darkness induction; chlorophyll degradation and content reduction occurred at a much slower pace. Consistently, the rate of accumulation of reactive oxygen species surpassed the capacity of the leaf detoxifying machinery, causing oxidative damage to key cellular constituents, leading to peroxidation of cell membrane lipids. Carbon metabolism analysis showed that NSCs were subject to active decomposition and consumption upon dark stress initiation, leading to a disruption of overall carbon balance.
Altogether, our data unequivocally indicate that C. lanceolata leaves exposed to prolonged light deprivation experienced an inability to conduct sufficient carbon assimilation, which in turn disrupted photoassimilate supply, metabolic biosynthesis, and carbon balance. Eventually, C. lanceolata leaves gradually senesced and irreversibly entered the apoptotic metabolic pathway due to the collapse of the energy capture and conversion pathways and the antioxidant enzyme systems and the ensuing carbon starvation.

Author Contributions

Conceptualization, Q.L. and X.M.; data curation, Z.H., Q.L., L.Z., X.M., and R.H.; formal analysis, Z.H., X.Z., and L.Z.; funding acquisition, Q.L., Z.H., and X.Z.; investigation, Z.H. and Q.L.; methodology, X.M. and R.H.; project administration, Q.L., X.M., and R.H.; writing—original draft, Z.H.; writing—review and editing, Q.L., X.Z., X.M., and R.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Research Start-up Funds of Jiangxi University of Water Resources and Electric Power [grant numbers 2022kyqd009 and 2022kyqd010], and Jiangxi Provincial Program for Academic and Technical Leaders Training of Major Disciplines of Science and Technology Department of Jiangxi Province [grant number 20232BCJ23043].

Data Availability Statement

Data used in this study will be made available upon reasonable request to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, nor in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Leaf morphological characteristics in leaves of Cunninghamia lanceolata seedlings growth in the dark for senescence induction: (A) leaf length; (B) leaf width; (C) leaf area; (D) specific leaf area. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
Figure 1. Leaf morphological characteristics in leaves of Cunninghamia lanceolata seedlings growth in the dark for senescence induction: (A) leaf length; (B) leaf width; (C) leaf area; (D) specific leaf area. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
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Figure 2. Light-response curves for leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) net photosynthetic rate; (B) stomatal conductance; (C) transpiration rate; (D) intercellular CO2 concentration; (E) light use efficiency. Pn, net photosynthetic rate; gs, stomatal conductance; Tr, transpiration rate; Ci, intercellular CO2 concentration; LUE, light use efficiency.
Figure 2. Light-response curves for leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) net photosynthetic rate; (B) stomatal conductance; (C) transpiration rate; (D) intercellular CO2 concentration; (E) light use efficiency. Pn, net photosynthetic rate; gs, stomatal conductance; Tr, transpiration rate; Ci, intercellular CO2 concentration; LUE, light use efficiency.
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Figure 3. Leaf chlorophyll contents in leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) Chl a content; (B) Chl b content; (C) Chl a to Chl b ratio; (D) Chl (a + b) content. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
Figure 3. Leaf chlorophyll contents in leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) Chl a content; (B) Chl b content; (C) Chl a to Chl b ratio; (D) Chl (a + b) content. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
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Figure 4. Antioxidant enzymes activity in leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) SOD activity; (B) POD activity; (C) CAT activity. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
Figure 4. Antioxidant enzymes activity in leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) SOD activity; (B) POD activity; (C) CAT activity. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
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Figure 5. Biochemical and physiological markers of lipid peroxidation and membrane permeability in leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) H2O2 content; (B) MDA content; (C) membrane conductivity. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
Figure 5. Biochemical and physiological markers of lipid peroxidation and membrane permeability in leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) H2O2 content; (B) MDA content; (C) membrane conductivity. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
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Figure 6. Leaf non-structural carbohydrate contents in Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) soluble sugar content; (B) starch content; (C) total non-structural carbohydrates content; (D) soluble sugar to starch ratio. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
Figure 6. Leaf non-structural carbohydrate contents in Cunninghamia lanceolata seedlings grown in the dark for senescence induction: (A) soluble sugar content; (B) starch content; (C) total non-structural carbohydrates content; (D) soluble sugar to starch ratio. Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints.
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Table 1. Leaf photosynthetic performance in leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction.
Table 1. Leaf photosynthetic performance in leaves of Cunninghamia lanceolata seedlings grown in the dark for senescence induction.
Dark Stress DurationPmax
(μmol·m−2·s−1)		AQY
(mol·mol−1)		LCP
(μmol·m−2·s−1)		LSP
(μmol·m−2·s−1)		Rd
(μmol·m−2·s−1)
0 d7.96 ± 1.02 a0.061 ± 0.004 a7.61 ± 1.18 c1199.23 ± 56.72 a0.444 ± 0.060 a
7 d4.10 ± 1.18 b0.018 ± 0.008 b9.21 ± 1.06 c1127.68 ± 20.22 ab0.421 ± 0.059 a
14 d1.36 ± 0.52 c0.010 ± 0.004 bc43.33 ± 13.66 b1051.05 ± 127.96 b0.325 ± 0.091 a
21 d0.10 ± 0.00 cd0.008 ± 0.003 c115.88 ± 25.06 a654.69 ± 10.89 c0.317 ± 0.107 a
28 d−0.12 ± 0.02 d0.006 ± 0.005 c——485.59 ± 27.52 d0.159 ± 0.057 b
Note: Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints. “——” in the table indicates that the instrument cannot measure the value. Pmax, maximum net photosynthetic rate; AQY, apparent quantum yield; LCP, light compensation point; LSP, light saturation point; Rd, dark respiration rate.
Table 2. Leaf chlorophyll fluorescence parameters measured in Cunninghamia lanceolata seedlings grown in the dark for senescence induction.
Table 2. Leaf chlorophyll fluorescence parameters measured in Cunninghamia lanceolata seedlings grown in the dark for senescence induction.
Dark Stress DurationFv/FoFv/FmΦPSIIRfdNPQqP
0 d4.66 ± 0.22 a0.82 ± 0.01 a0.71 ± 0.01 a1.85 ± 0.22 b1.90 ± 0.22 b0.92 ± 0.01 a
7 d3.54 ± 0.52 b0.78 ± 0.03 b0.41 ± 0.03 b2.60 ± 0.22 a2.23 ± 0.31 a0.75 ± 0.04 b
14 d1.74 ± 0.26 c0.63 ± 0.04 c0.31 ± 0.05 c1.60 ± 0.24 c1.16 ± 0.15 c0.63 ± 0.07 c
21 d1.00 ± 0.17 d0.50 ± 0.04 d0.30 ± 0.08 c0.59 ± 0.19 d0.42 ± 0.11 d0.65 ± 0.13 c
28 d0.86 ± 0.15 d0.46 ± 0.04 e0.25 ± 0.07 d0.50 ± 0.16 d0.39 ± 0.10 d0.61 ± 0.10 c
Note: Data are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among sampling timepoints. Fv/Fo, potential photochemical efficiency ratio of PSII; Fv/Fm, maximum quantum yield of PSII; ΦPSII, effective quantum yield of PSII; Rfd, fluorescence decline ratio; NPQ, non-photochemical quenching; qP, photochemical quenching.
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Huang, Z.; Liu, Q.; Zou, X.; Zhu, L.; Ma, X.; Huang, R. Morpho-Physiological Responses During Dark-Induced Leaf Senescence in Cunninghamia lanceolata Seedlings. Forests 2025, 16, 1372. https://doi.org/10.3390/f16091372

AMA Style

Huang Z, Liu Q, Zou X, Zhu L, Ma X, Huang R. Morpho-Physiological Responses During Dark-Induced Leaf Senescence in Cunninghamia lanceolata Seedlings. Forests. 2025; 16(9):1372. https://doi.org/10.3390/f16091372

Chicago/Turabian Style

Huang, Zhijun, Qingqing Liu, Xianhua Zou, Liqin Zhu, Xiangqing Ma, and Rongzhen Huang. 2025. "Morpho-Physiological Responses During Dark-Induced Leaf Senescence in Cunninghamia lanceolata Seedlings" Forests 16, no. 9: 1372. https://doi.org/10.3390/f16091372

APA Style

Huang, Z., Liu, Q., Zou, X., Zhu, L., Ma, X., & Huang, R. (2025). Morpho-Physiological Responses During Dark-Induced Leaf Senescence in Cunninghamia lanceolata Seedlings. Forests, 16(9), 1372. https://doi.org/10.3390/f16091372

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