Antioxidant and Ultrastructural Alterations in Wheat During Drought-Induced Leaf Senescence

1. Introduction

Wheat is a globally significant crop and a vital source of human nutrition (Cheng et al., 2024). The senescence of wheat leaves is a key physiological process that directly impacts grain yield and quality. Understanding the mechanisms governing leaf senescence is crucial for developing strategies to enhance wheat productivity and resilience (Zhao et al., 2022; Chapman et al., 2021; Hassan et al., 2021).

Leaf senescence is a developmental process characterized by a series of programmed disassembly and degenerative events, such as the accumulation of reactive oxygen species, degradation of macromolecules, breakdown of intracellular organelles, and the transition from nutrient assimilation to remobilization (Li et al., 2024; Woo et al., 2019). Natural leaf senescence is typically associated with age and accelerates as the plant transitions from vegetative to reproductive growth (Aloryi et al., 2023). During physiological leaf senescence, stored nutrients accumulated in the leaves are translocated to actively growing organs, and in cereals, to the grain (Guo et al, 2021). Premature senescence, induced by unfavorable environmental conditions, can reduce crop yields. Various abiotic stressors, including drought, extreme temperatures, salinity, nutrient deficiencies, and light intensity, as well as biotic factors such as pathogen infections and insect infestations, can hasten the leaf senescence process (Tan et al., 2023). Furthermore, genetic factors and hormonal regulation play a pivotal role in controlling the timing and progression of leaf senescence (Cao et al., 2022; Sultana et al., 2021).

In the context of global climate change, water scarcity is expected to have the most significant negative impact on crop yields worldwide. The complex stress caused by drought can trigger premature leaf senescence and lead to early maturation of the entire plant, resulting in reduced productivity (Tan et al., 2023). Drought-induced leaf senescence contributes to plant survival under drought stress in certain species by (i) facilitating the early redirection of resources from vegetative to reproductive development, thereby ensuring the completion of the life cycle in monocarpic species, (ii) enabling the remobilization of nutrients from senescing leaves to younger leaves, which supports the survival of perennial plants, and (iii) reducing water loss at the whole-plant level (Munné-Bosch and Alegre, 2004). It has been observed that under drought conditions, nitrogen availability in the soil decreases, which initiates and accelerates the leaf senescence process (Hajibarat and Saidi, 2022). Research on drought-induced leaf senescence is critical not only for understanding how plants survive under adverse climatic conditions but also for improving agricultural productivity, as crops with delayed leaf senescence can be used to enhance crop yields (Joshi et al., 2019; Gregersen et al., 2013).

The leaf senescence process is characterized by alterations in the expression of thousands of senescence-associated genes (SAGs) (Cao et al., 2022; Camargo Rodriguez, 2021). Studies have shown that numerous transcriptional regulators govern senescence by modulating SAG expression (Cao et al., 2023; Borril et al., 2019). In addition to genetic and environmental factors, epigenetic regulation adds a new layer of complexity to our understanding of the control of leaf senescence. Epigenetic modifications, such as DNA methylation and histone modifications, influence the expression of genes associated with aging, offering additional avenues for research and potential targets for intervention (Woo et al., 2019; Yolcu et al., 2018). Multi-omics approaches have revealed that leaf senescence is governed by multiple layers of regulation, including chromatin remodeling, transcriptional and post-transcriptional processes, as well as translational and post-translational mechanisms (Zhang et al., 2021; Kim et al., 2018).

The antioxidant system plays a crucial role in mitigating the effects of aging and environmental stress (Qi et al., 2023; Nousis et al., 2023). Antioxidants are vital for maintaining cellular homeostasis and protecting plant tissues from oxidative damage during aging. The equilibrium between the production of reactive oxygen species (ROS) and the activity of the antioxidant system is a pivotal factor in determining leaf longevity and overall plant health (Yuan et al., 2023). During leaf senescence, as chlorophyll degrades and photosynthetic activity diminishes, the generation of ROS in chloroplasts increases, leading to oxidative stress (Li et al., 2023; Buet et al., 2019). This prompts the activation of antioxidant enzymes, such as superoxide dismutase, catalase, and peroxidases, to neutralize ROS and protect cellular components from oxidative damage. The dynamic regulation of antioxidant enzyme activity during leaf senescence is a complex process influenced by a range of intracellular and environmental factors (Sachdev et al., 2021; Hönig et al., 2018). Understanding the intricate relationship between these factors and antioxidant enzyme activity is crucial for elucidating the underlying mechanisms of leaf senescence (Chen et al., 2021).

Cellular organelles are known to respond rapidly to environmental stress through various adaptive and avoidance mechanisms. Leaf cell organelles across different plant species exhibit distinct changes, reflecting their survival strategies under stress (Ďúranová et al., 2023). Cell death in senescent leaves is predominantly associated with the deterioration of intracellular organelles (Lim et al., 2007). To date, most research has focused on physiological and biochemical parameters (Tefera et al., 2023) with relatively few studies examining changes at the ultrastructural level. This indicates a gap in the scientific literature regarding the ultrastructural aspects of the aging process (Gan et al., 2022). As plants age, the cell membrane's structure is initially compromised with the subsequent transition of the functional proteins of the membrane through the liquid crystal phase. The accumulation of malondialdehyde, resulting from membrane lipid peroxidation, triggers a series of physiological and biochemical changes. Chloroplasts are reported to undergo degeneration earlier in the aging process compared to mitochondrial and nuclear fragmentation (Tamary et al., 2019).

Our current understanding of leaf senescence is primarily derived from studies using model systems such as Arabidopsis (Yumoto et al., 2024; Xie et al., 2021; Zhao et al., 2023; Kim et al., 2018). Research indicates that the molecular and biochemical pathways involved in leaf senescence are highly conserved across various plant species (Guo et al., 2021; Woo et al., 2019). Investigating these pathways in wheat could yield valuable insights into the genetic and regulatory networks that govern this complex process in cereals. Recently, there has been a growing body of research on the aging process in wheat (Yan et al., 2023; Chapman et al., 2021; Sultana et al., 2021; Borril et al., 2019; Joshi et al., 2019). Transcriptomic analysis has revealed key insights into the genetic regulation underlying leaf senescence in wheat (Sultana et al., 2021; Borril et al., 2019).

Overall, managing senescence in crops such as wheat requires a thorough understanding of complex regulatory networks at various levels, including molecular, biochemical, and physiological processes. In this study, we aim to investigate the mechanisms of drought-induced senescence in contrasting genotypes of durum and bread wheat, explore the role of antioxidant enzymes during flag leaf senescence, and identify ultrastructural changes in leaves during senescence dynamics. Gaining deeper insights into different aspects of leaf senescence may allow us to identify potential targets for manipulating these processes.

2. Materials and Methods

2.1. Cultivation of Plant Material

Seeds of local bread wheat (Triticum aestivum L.) genotypes, including the tolerant Gyrmyzy gul 1 and sensitive Tale 38, as well as durum wheat (Triticum durum Desf.) genotypes, the tolerant Vugar and sensitive Tartar, were obtained from the gene bank of the Research Institute of Crop Husbandry (Baku, Azerbaijan). Five seeds of each genotype were planted in 5 L plastic pots filled with a soil mixture consisting of 40% topsoil and 60% natural sand. Throughout the experiment, the ambient temperature was maintained between 19-29°C, and the relative humidity was between 50% and 65%. The plants were grown in controlled environment chambers with a 16h light/8h dark cycle. The plants were watered 2-3 times weekly to make sure the soil stayed moist. All plants were provided with equal irrigation until the booting stage. Afterward, watering was stopped for the drought-treated plants, while the control plants continued to be watered regularly. Flag leaves were sampled 7, 14, 21, 28, and 35 days after anthesis. The leaf samples were immediately frozen in liquid nitrogen and stored at -80°C until analysis.

2.2. Chlorophyll Content

Total chlorophyll content and chlorophyll a/b ratio were determined spectrophotometrically in the 80% acetone extract according to Porra et al. (1989).

2.3. Cell Membrane Stability

For the membrane stability index (MSI), flag leaf samples (2 cm from the middle sections, with 3 repetitions for each sample) were washed three times in deionized water to remove surface electrolytes. The samples were placed in 10 ml of double-distilled water at 40°C for 30 minutes (Deshmukh et al. 1991). Electrical conductivity (C1) was measured using a conductivity meter. Subsequently, the same samples were placed in a boiling water bath (100°C) for 10 minutes, and their electrical conductivity (C2) was re-recorded. The membrane stability index (MSI) was calculated as MSI = [1 - (C1 / C2)] × 100.

2.4. Lipid Peroxidation Assay

The intensity of lipid peroxidation in plants was assessed by measuring the quantity of malondialdehyde (MDA) in flag leaf samples under both normal watering and drought stress conditions (Wang, 2011). The MDA content was determined spectrophotometrically using the Thermo Scientific Evolution 350 UV/Vis Spectrophotometer (UK, England) through its reaction with 0.5% thiobarbituric acid at wavelengths 532 nm and 600 nm. The formula used for calculation was: A = (D532 - D600) / (E * m). Where, A - represents MDA concentration, D - is the optical density, m - denotes plant wet biomass, and E - is a constant coefficient.

2.5. Total Protein Content

Soluble protein content was determined using the Bradford method (1976) at 595 nm, with bovine serum albumin used to construct the calibration curve.

2.6. Preparation of Enzyme Extract

Flag leaf samples (0.5 g) were crushed in liquid nitrogen and dissolved in a buffer solution containing 100 mM Na-phosphate (pH 7.8), 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride (PMSF), 1% polyvinylpyrrolidone (PVP), and 0.1% Triton X-100. The homogenized samples were then centrifuged at 15,000 g for 20 minutes at 4°C. The resulting supernatant was used to determine the activity of antioxidant enzymes

2.7. Ascorbate Peroxidase (APX, EC 1.11.1.11)

APX activity was measured by monitoring the decrease in optical density at a wavelength of 290 nm using a spectrophotometer (ULTROSPEC 3300 PRO, AMERSHAM, USA), following the method described by Nakano and Asada (1980). The reaction mixture contained 0.1 mM EDTA (pH 8.0), 0.05 mM ascorbate, 0.1 mM H₂O₂, 50 mM sodium phosphate buffer (pH 7.6), and 100 µl of enzyme extract. The molar extinction coefficient used was 2.8 mM⁻¹ cm⁻¹, with enzyme activity expressed in units of µmol ascorbate/ (mg protein·min).

2.8. Guaiacol Peroxidase (GPX, EC 1.11.1.7)

The activity of guaiacol peroxidase (GPX) was determined spectrophotometrically by measuring the change in optical density of the reaction solution at 470 nm over 2 minutes, as described by Zelinova et al. (2010). The reaction medium consisted of 50 mM sodium phosphate buffer (pH 7.0), 25 mM guaiacol, 25 mM H₂O₂, and 20 μl of enzyme extract. The molar extinction coefficient (ε) of 26.6 mM⁻¹ cm⁻¹ was used, with enzyme activity expressed in units of µmol guaiacol/ (mg protein·min).

2.9. Benzidine Peroxidase (BPX, EC 1.11.1.7)

Benzidine peroxidase (BPX) activity was determined spectrophotometrically by monitoring the increase in optical density of the reaction solution at 590 nm for 1 minute, as described by Gechev et al. (2002). The activity of the enzyme was calculated in units of µmol benzidine/ (mg protein·min), using an extinction coefficient (ε) of 39 mM⁻¹ cm⁻¹.

2.10. Catalase (CAT, E.C. 1.11.1.6)

For the determination of catalase (CAT) activity, 1 g of flag leaf tissue was homogenized in 10 ml of 50 mM potassium phosphate buffer (pH 7.0). The homogenate was filtered and centrifuged at 8000 g for 10 minutes to obtain a clear supernatant. For the assay, 2.9 ml of phosphate buffer (pH 7.0) was combined with 25 μl of the enzyme extract. To initiate the reaction, 90 μl of 3% hydrogen peroxide (H₂O₂) was added to the solution. The decrease in optical density at 240 nm was measured per minute using a spectrophotometer. The enzyme activity was calculated in mmol/(g·min) using a molar extinction coefficient (ε) of 39.4 mM⁻¹cm⁻¹ (Kumar and Knowles, 1993).

2.11. Superoxide Dismutase (SOD, EC 1.15.1.1)

To determine the activity of the superoxide dismutase (SOD) enzyme, a specific kit (SOD Assay Kit-WST, Sigma-Aldrich) was employed. In this study, the cytosolic isoform of SOD from plant cells was analyzed. Leaves were homogenized in 50 mM potassium phosphate buffer (pH 7.8), and the homogenate was centrifuged. The supernatant, which contained the cytosolic form of SOD, was used for further analysis. The optical density (OD) of the reaction was measured at a wavelength of 450 nm to assess SOD activity.

2.12. RNA Extraction and cDNA Synthesis

Total RNA was extracted from leaf material using the Monarch Total RNA Miniprep Kit (New England Biolabs, Inc.) following the manufacturer's instructions. Genomic DNA contamination was removed using RNase-free DNase I. The quality and quantity of the extracted RNA were assessed by agarose gel electrophoresis. RNA concentration was measured spectrophotometrically using a NanoDrop Thermo Scientific-2000C (USA). Single-stranded cDNA synthesis was performed from the total RNA using the LunaScript RT SuperMix Kit (New England Biolabs, Inc.) according to the manufacturer’s instructions, in a final volume of 20 µl.

2.13. Quantitative Real-Time PCR

PCR was conducted using a Mic Real-Time PCR system in a total reaction volume of 20 µl. Each reaction mixture contained 10 µl of Luna Universal qPCR Mix (New England Biolabs, Inc.), 1 µl of 1:5 diluted cDNA, 0.5 µl each of forward and reverse primers (10 µM), and 7 µl of nuclease-free water. The PCR protocol included an initial denaturation step at 94°C for 60 seconds, followed by 45 cycles of 95°C for 15 seconds and 60°C for 30 seconds. No-template controls (NTCs) were included for each primer pair. Each reaction was performed in triplicate (technical replicates) for each of the three biological replicates. Elongation factor 1 alpha (Elf1-α) was used as an internal control gene. Primer sequences used for expression analysis are listed in Table 1. Primer efficiency for each pair was determined by the standard curve method using serial dilutions of cDNA, calculated using the formula: Efficiency (%) = (10^(-1/slope) - 1) × 100. Dissociation curves for each amplicon were analyzed to confirm the specificity of the amplification reactions. Fold change in gene expression (stressed versus control) was calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001).

2.14. Analysis of Leaf Mesophyll Cells by Light and Transmission Electron Microscopy (TEM)

Flag leaves were used for ultrastructural examination. The specimens were fixed in a solution containing 2.5% glutaraldehyde, 2% paraformaldehyde, 4% sucrose, and 0.1% picric acid, prepared in 0.1 M phosphate buffer (pH 7.4). The fixed material was then transported to the Electron Microscopy Laboratory at Azerbaijan Medical University (Baku) for further processing. After fixation for one day, the samples were postfixed in 1% osmium tetroxide solution prepared in phosphate buffer (pH 7.4) for two hours. Araldite-Epon blocks were prepared from the material using standard methods in electron microscopy (Kuo, 2014). Semi-thin (1–2 μm) sections were cut from the blocks using a Leica EM UC7 ultramicrotome (Leica, USA), stained with methylene blue, azure II, and basic fuchsin (Morikawa et al., 2018), and viewed under a Primo Star microscope (Zeiss, Germany). Images of the relevant sections were captured with a digital camera EOS D650 (Canon, Japan). Ultrathin sections (50–70 nm) of the blocks, double-stained with uranyl acetate and lead citrate, were examined using a Transmission Electron Microscope JEM-1400 (JEOL, Japan) at a voltage of 80–120 kV. Morphometric analysis of the electron micrographs was performed in TIF format using the TEM Imaging Platform software developed by Olympus Soft Imaging Solutions GmbH (Germany).

2.15. Statistical Analysis

All experiments were conducted in triplicate. Statistical significance was tested using the student's t-test. Differences between mean values were considered significant at p<0.001 (***), p<0.01 (**), and p<0.05 (*).

3. Results

3.1. Chlorophyll Content

The content of photosynthetic pigments—chlorophyll a and chlorophyll b—was measured in flag leaves at five time points (7, 14, 21, 28, and 35 days after anthesis, DAA) during natural (age-induced) and drought-induced senescence in wheat genotypes with contrasting stress tolerance (Table 2). In all plants, the quantities of both chlorophyll a and chlorophyll b progressively decreased as senescence advanced. At 7 DAA, although there was a slight reduction in pigment levels in stress-sensitive genotypes, no significant differences were observed in tolerant plants. In contrast to naturally senescing plants, a sharp decline in pigment levels was observed in the flag leaves of drought-stressed wheat at 21 DAA. This decline was particularly pronounced in sensitive genotypes, with the amount of both chlorophyll a and chlorophyll b being more than twofold lower in drought-exposed tetraploid Tartar and hexaploid Tale 38 compared to 14 DAA. A significant decrease in chlorophyll a content was observed in naturally senescing plants at 35 DAA. Notably, the reduction in chlorophyll b content in stressed plants was detected earlier, at 14 DAA in sensitive genotypes and at 21 DAA in tolerant genotypes.

3.2. Membrane Stability

During the post-anthesis period, due to the aging process, membrane stability in the flag leaves exhibited a declining trend. (Figure 1). At 7 DAA, this parameter was at a similar level across all genotypes. However, in the subsequent periods, membrane stability in stressed plants was lower compared to those under normal conditions. At 21 DAA, the membrane stability index decreased by up to 20% in stress-sensitive genotypes.

3.3. Malondialdehyde Content

An increase in the content of malondialdehyde (MDA), an indicator of lipid peroxidation, was observed in wheat genotypes during natural senescence (Figure 2). The level of MDA was higher during drought-induced senescence. This difference was more pronounced and occurred earlier in stress-sensitive genotypes. At 21 DAA, no significant difference in MDA levels was observed between control and stressed variants of the drought-tolerant durum wheat genotype Vugar, while a 2.3-fold increase was noted in the stress-sensitive bread wheat genotype Tale 38. Towards the end of senescence, the difference in MDA content between control and drought-stressed variants became more pronounced, showing increases of 1.55, 1.73, 2.25, and 2.4-fold in Vugar, Tartar, Gyrmyzy gul 1, and Tale 38, respectively.

3.4. Soluble Protein Content

The total soluble protein content is shown in Figure 3. At 7 DAA, no significant differences were observed between genotypes for this parameter. In naturally senescing plants, the protein content increased until 28 DAA and then sharply decreased at 35 DAA. In stressed plants, the curve reflecting soluble protein content showed changes earlier, at 21 DAA, similar to other parameters. The only exception was the drought-tolerant durum wheat genotype, which, like the control plants, exhibited a decrease in protein content after 28 DAA.

3.5. The Activity of SOD

At 7 DAA, the activity of SOD was similar across all variants (Figure 4). By 14 DAA, enzyme activity increased, with a more significant rise observed in stress-exposed genotypes. In naturally aging wheat, the maximum SOD activity was recorded at 28 DAA. In contrast, during stress-induced senescence, the maximum activity occurred earlier, at 21 DAA. Towards the end of ontogenesis, SOD activity declined. A sharp decrease in enzyme activity was observed in stress-exposed bread wheat compared to durum wheat.

3.6. Gene Expression Analysis of SOD Isoforms

To determine which SOD isoforms are closely involved in the senescence process, gene expression analysis was performed. Transcript levels of CuZnSOD, FeSOD, and MnSOD were examined (Figure 5). The transcript level of CuZnSOD was similar across all variants at 7 DAA. In naturally aging plants, the expression of this isoform increased progressively, reached a peak at 28 DAA, and then decreased at 35 DAA. Under stress conditions, the maximum expression of this gene was observed at 21 DAA, after which the expression decreased, reaching a minimum level at 35 DAA. The expression pattern of FeSOD during flag leaf senescence differed. In all genotypes, the transcription of this isoform significantly increased after flowering, peaked at 21 DAA, and then declined sharply. MnSOD expression was higher towards the end of ontogenesis, with transcript levels reaching a maximum at 35 DAA.

3.7. The Activity of Peroxidases

The activity of peroxidase enzymes, including APX, BPX, and GPX was measured in flag leaves at five stages after anthesis (Figure 6).

In naturally senescing plants, APX activity increased after anthesis, reaching a maximal level at 28 DAA, and then decreased at 35 DAA. Except for the drought-tolerant durum wheat genotype Vugar in stressed plants, APX activity peaked at 21 DAA. In the Vugar genotype, APX activity under drought conditions nearly doubled at 28 DAA, reaching the highest level compared to other genotypes. BPX activity reached a maximum in all genotypes at 28 DAA and sharply decreased at 35 DAA. Under stressed conditions, BPX activity was higher in stressed plants compared to control plants. The activity of GPX in flag leaves of both naturally senescing and drought-stressed wheat genotypes also increased after anthesis, peaking at 28 DAA. Similar to other peroxidases, GPX activity decreased again at 35 DAA, with a significant reduction observed particularly, in bread wheat genotypes.

3.8. The Activity of CAT

In contrast to peroxidases, the CAT enzyme reached its maximum activity earlier (14 DAA). As senescence progressed, CAT activity decreased. In plants subjected to stress, which senesced more rapidly, CAT activity was higher compared to control plants (Figure 7).

3.9. Light Microscopy

Changes occurring in the flag leaves of bread wheat genotypes were examined by light microscopy at 7, 21, and 35 days after anthesis (DAA) (Figure 8 and Figure 9). Figure 8A shows the anatomical structure of the flag leaf of the Gyrmyzy gul 1 genotype under normal watering conditions at 7 DAA.

The anatomy of the flag leaf exhibits distinct features: the epidermis (E), stomata (St), and mesophyll cells (Mc) are visible. The stomata are open, and the mesophyll cells are rich in chloroplasts. At 7 days after anthesis (DAA) under drought stress (Figure 8B), no significant anatomical differences are observed. By 21 DAA, both open and closed stomata are present, and some areas of the epidermis show signs of thickening (Figure 8C). At 35 DAA, all stomata are observed to be closed. In addition to the thickening of various epidermal layers, some mesophyll cell walls exhibit damage (Figure 8D).

In contrast to the tolerant genotype, the Tale 38 genotype exhibits more pronounced changes in anatomical structure during the examination of its flag leaves (Figure 9). At 7 DAA under drought stress, the structural elements of the leaves are similar to those in the control group (Figure 9A). However, at 21 DAA, some stomata are closed, and thickening of the epidermal cell layers and damage to the walls of some mesophyll cells are observed (Figure 9B). By 35 DAA, pathological changes in the flag leaves of the Tale 38 genotype have intensified. Specifically, the epidermal layer shows increased thickening in some areas, with structural integrity compromised, most stomata remain open, and the walls of mesophyll cells are disrupted. Additionally, there is a reduction in the number of chloroplasts in the mesophyll cell cytoplasm (Figure 9C).

3.10. Transmission Electron Microscopy

Changes in the ultrastructure of flag leaves in the Gyrmyzy gul 1 and Tale 38 genotypes during drought-induced leaf senescence were also examined using transmission electron microscopy (TEM) (Figure 10 and Figure 11). In the control leaves of the Gyrmyzy gul 1 genotype, cultivated under normal watering conditions, the ultrastructure clearly shows cell walls (Cw), chloroplasts (Ch), the central nucleus (N), lipid droplets (Ld), mitochondria (M), internal thylakoid membranes of chloroplasts (Th), and plastoglobuli (Pl) (Figure 10A).

Figure 10B shows the ultrastructural appearance of flag leaves at 7 DAA under drought conditions. No significant pathology is observed. At 21 DAA, although the cell walls maintain a normal structure, some organelles with membranous structures, such as mitochondria, exhibit structural damage and transform into myelin-like structures (Ms) (Figure 10C). More severe changes compared to the control are observed at 35 DAA (Figure 10D-F). Specifically, some mesophyll cell walls are damaged (Figure 10D), chloroplast membranes are compromised, and connections between chloroplasts are severed, with alterations in thylakoid membrane structures (Figure 10E). The nuclear envelope of mesophyll cells is thickened and structurally disrupted in some areas (Figure 10F). Figure 10 depicts the changes occurring in the mesophyll cells of the Tale 38 genotype during leaf senescence, as observed using TEM.

Figure 11A illustrates the structural elements of mesophyll cells in flag leaves of plants grown under normal watering conditions (control). At 7 DAA under drought conditions, no significant pathology is observed in the flag leaves (Figure 11B). At 21 DAA, chloroplast membranes are damaged and their connections are severed. The crystals in the mitochondria are not clearly visible, and some mitochondria are severely damaged and completely disintegrated (Figure 11C). At 35 DAA, most mesophyll cell walls are severely damaged and fragmented in several places (Figure 11D). The majority of chloroplast membranes are damaged and fragmented, causing the chloroplasts to disassociate from the cell walls and migrate toward the center of the cytoplasm (Figure 11E). Additionally, most membranous structures in the cytoplasm are also damaged. Ultrastructural analysis of the chloroplasts reveals that the thylakoid membranes are damaged and disintegrated, with vacuoles forming in the intermembrane spaces (Figure 11F).