Photosynthesis and stress response of coal �y ash on stem elongation in wheat

Coal is one of the primary energy sources in China and is widely used for electricity generation. Crops growing in overlapped areas of farmland and coal resources (OAFCR) suffer from coal �y ash stress, especially during stem elongation, which is a key stage that impacts wheat yield and is sensitive to environmental stress. As a primary food crop of China, wheat is essential for food security. However, the characteristics of wheat under the combined stress of �y ash and various heavy metals have not been su�ciently investigated. In this study, we explored the response of stem elongation in wheat to different levels of coal �y ash stress and determined the content of heavy metals (HMs) in wheat leaves. We found that with an increase in �y ash content, the Cu content in the shoots increased, while that in the roots decreased. Coal �y ash exposure reduced the proportions of Pb and Zn in the cytoderm, and the proportion of Cu in the soluble constituents decreased from 58.3–45.7%. Total chlorophyll, chlorophyll a, and chlorophyll b levels decreased signi�cantly, whereas peroxidase (POD) and catalase (CAT) activities generally increased with increasing �y ash dose. Meanwhile, chloroplasts, mitochondria, and their internal structures were damaged, and the cell structures of leaves, such as the internal membrane structure, were damaged.


Introduction
On the eastern plains of China, the overlapped area of farmland and coal resources (OAFCR) covers an area of 1.33 × 10 5 km 2 and accounts for 31.93% of the cultivated land area (Hu et al., 2014).Coal is the primary energy source in China and is widely used for power generation.According to the Statistical Bulletin of 2022 National Economic and Social Development of the People's Republic of China, the installed thermal power capacity was 1332.39 million kW in 2022, an increase of 2.7% from the previous year.To reduce the process of transporting coal, many coal-red power plants have been built near coal resources.The combustion of coal releases particulate matter, including y ash, that can affect the areas surrounding power plants.Furthermore, coal combustion cannot completely destroy inorganic materials such as heavy metals (HMs), which are present in y ash and are an environmental pollutant (Nguyen et al., 2022).The winter heating season has led to a ve-fold increase in PM 2.5 in the air (Xiao et al., 2015) and an increase in HMs attached to PM 2.5 (Deng et al., 2020) compared with normal levels in many parts of China.Particulates and HMs produced by coal combustion can be deposited on the land surrounding coal-red power plants, potentially in uencing the normal growth of crops in OAFCR and threatening food security.
Stem elongation is considered a crucial stage in determining the nal yield of wheat and is sensitive to environmental changes (Kronenberg et al., 2017).Stress can cause an increase in ag leaves and reduce the total chlorophyll content during stem elongation (Fan et al., 2019).Notably, the accumulation of HMs during stem elongation is higher than that in other stages under plant stress (Jin et al., 2010;Su et al., 2019).Moreover, stem elongation is vital for determining the number of fertile orets at anthesis and is directly related to wheat yield (Doghozlou & Emam, 2022).
Similar to the role of plant roots in absorbing HMs, aboveground plant organs, such as leaves, fruits, and owers, also have a strong absorption capacity (Song et al., 2021).In the OAFCR, the HMs carried in y ash are deposited onto plant parts, particularly leaves.Leaves can actively absorb HMs through stomata, surface cracks, aqueous pores, and ectodesmata (Fernandez & Brown, 2013;Kovár et al., 2023).Many factors affect the uptake of HMs on the leaf surface, such as the physical and chemical characteristics of the cuticle, plant physiology, the properties of the metal, and exposure time (Wang et al., 2020).After foliar penetration, HMs can be transported to other plant organs through the phloem vascular system in the same manner as substances are transported during photosynthesis (Shahid et al., 2017).
Subsequently, heavy metals are transported between cells via active transport within the cell symplastic pathway (Carini & Bengtsson, 2001).Cells can mitigate the toxicity of absorbed HMs to organelles by blocking their entry with the cell wall and separating the HMs in vacuoles (Shahid et al., 2014).Foliar absorption of single metals has been widely studied (Cakmak et al., 2000;Gao et al., 2021a).However, y ash contains a wide variety of HMs in complex forms and in different amounts (Altikulac et al., 2022).
The interactions between different HMs are also complicated.Therefore, the mechanisms underlying HM uptake and transport pathways in plants under y ash stress require further in-depth analysis.
Photosynthesis is an essential process for ensuring yield (Han et al., 2016).A high y ash content with attached HMs (Jin et al., 2012;Sagardoy et al., 2010) substantially reduces stomatal conductance, albedo, and photosynthesis (Raja et al., 2014), which inhibits CO 2 and O 2 exchange.Additionally, HMs can change the chlorophyll structure by forming complexes, such as that formed when Cu replaces the Mg atom in the center of the chlorophyll (Petrovic et al., 2006).Many enzymes are involved in the process of photosynthesis, including ribulose-1,5-bisphosphate carboxylase (Rubisco), a key enzyme in CO 2 assimilation that catalyzes the formation of ribulose biphosphate (RuBP), which regulates the Calvin cycle (Han et al., 2016) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-1,6bisphosphate aldolase (FBA), which are involved in glycolysis and the Calvin cycle (Patipong et al., 2019;Yu et al., 2020).Excessive HM content inhibits the activity and function of Rubisco and carbonic anhydrases (Arena et al., 2017), whereas it increases GAPDH activity (Chen et al., 2005).Meanwhile, FBA is sensitive to various stressors, like water shortage or salt stress (Cai et al., 2022) Wheat grown in OAFCR is subjected to y ash and its associated HMs, which causes combined stress with complex effects; however, the mechanisms of the impact of y ash and HMs on wheat require further study.Therefore, in this study, we simulated different levels of y ash stress on both the roots and shoots of wheat during the stem elongation period with the aim of determining: (1) the impact of HM absorption and transport on stem elongation in wheat, (2) the effects of particulate matter stress from coal-burning on wheat photosynthesis, and (3) the changes in the antioxidant mechanisms of wheat under particulate matter stress from coal-burning.

Experimental design
The nutrient matrix (vermiculite:peat soil = 1:1) was purchased from the Xuzhou Moon Bay ower market and y ash was collected from the Huamei Power Plant.The Cu, Zn, and Pb contents of the nutrient matrix and ash are shown in Table 1. ).Subsequently, they were germinated using hydroponics for 72 h (93% germination rate) and then planted in nutrient matrix-lled incubators.Each incubator contained approximately 200 wheat seedlings (Fig. 1).Wheat in the four greenhouses was sprayed with ash through the hole 2-4 times a week.
According to the average daily dust fall ux of 0.4593 g/m 2 in the northern suburbs of Xuzhou (Li Changping et al., 2008) four ash gradients were set (index ×0, ×0.5, ×1, and ×2), which were each applied to one greenhouse during ash spraying: 0 g/d for #1, 0.186 g/day for #2, 0.372 g/day for #3, and 0.744 g/day for #4.When the rst node of the main wheat stem was ~ 1.5-2 cm from the ground, samples were collected for analysis, and three parallel samples were collected from each incubator.

Analysis of heavy metals in wheat
The wheat plants were washed with tap water, rinsed with distilled water, and separated into roots and shoots.Samples were oven-dried at 80°C for 48 h and then ground and passed through a 0.5 mm sieve.
The Pb and Cu contents in the samples were determined through inductively coupled plasma mass spectrometry (ICP-MS) (7900, USA, Agilent), and Zn was determined through ICP optical emission spectroscopy (Optimal 8000, USA, PerkinElmer).Experiments were performed in triplicate for each incubator; thus, nine replicate experiments were conducted for each greenhouse.The reference material GBW10046 was used to analyze the accuracy of the sample analysis procedure.The differences between the measured and standard values did not exceed 15%.

Analysis of subcellular heavy metal distribution
The plant tissue was separated into three subcellular fractions: cytoderm, organelles, and soluble fractions.The following steps were performed at 4 ℃.First, 1 g of fresh wheat leaves was homogenized in a precooled extraction buffer using a mortar and pestle.The extraction buffer contained 0.25 M sucrose, 50 mM Tris-HCl (pH 7.5), and 1 mM dithiothreitol (Lai & Cai, 2016).Next, the homogenate was centrifuged in a high-speed freezing centrifuge at 2000 rpm for 10 min, and the pellet was designated as the cytoderm fraction.Subsequently, the supernatant was centrifuged at 12 000 rpm for 45 min, and the pellet and supernatant solution were identi ed as the organelle and soluble fractions, respectively.Finally, the separated components were oven-dried at 70 ℃ and then digested with HNO 3 -HClO 4 .The HM contents were determined using ICP-MS.

Determination of chlorophyll content
Chlorophyll content was determined using Arnon's method (Arnon, 1949).Fresh wheat leaves were ground in 80% acetone, and the absorbance of the extract was measured at 663 nm and 645 nm.The chlorophyll content of plant leaves was calculated according to the following formulae: where C a and C b are the mean chlorophyll a and b contents (mg/L), respectively; D 663 and D 645 represent the absorbance values of the sample at wavelengths of 663 nm and 645 nm, respectively; and W, V, N, and M are the mean chlorophyll content (mg/g), extract liquid volume (L), dilution ratio, and fresh weight of the sample (g), respectively.

Measurement of photosynthesis and antioxidant enzyme activities
The activities of Rubisco, FBA, GAPDH, and antioxidant enzymes, including SOD, CAT, and POD, were measured using the enzyme-linked immunosorbent assay (ELISA) technique at Beijing Dongge Weiye Technology Co., Ltd.The sample, standard product, and horseradish peroxidase-labeled detection antibody were added sequentially to micro plates (Nunc or Greneir).After incubation and thorough washing, the sample was stained with tetramethyl benzidine, and the absorbance was measured with an ELISA reader (Winooski, VT, USA) at 450 nm to measure the activity of the enzymes.Microscopy was used to observe the ultrastructure of wheat leaves under different y ash doses.
Samples were prepared for observation as follows.Small pieces (1 mm 2 ) of wheat leaves were xed for 72 h at room temperature using 2.5% glutaraldehyde prepared with phosphate buffer solution (PBS) at a pH of 7.4, rinsed four times (20 min each) with PBS, and post-xed in 1% osmium tetroxide prepared with PBS for 2 h at room temperature.The xed samples were dehydrated using a graded ethanol series (30, 40, 50, 60, 70, 80, and 90% for 15 min each and 100% three times for 10 min each).After dehydration, samples were embedded with epoxy propane (Epon 812) and polymerized at 60 ℃ for 2 d.Sections were sliced using a Leica S-type ultramicrotome and stained with 2% uranium acetate for 1 h and lead citrate for 20 min (Chen et al., 2011).The samples were observed and imaged using a G2 T12 transmission electron microscope (TEM) (FEI Company, USA).
For scanning electron microscopy (SEM), a number of pieces (0.5 × 0.5 cm) were cut at the same position on two sides of the midvein.Fixation and dehydration (graded ethanol series) were performed in the same manner as for TEM (Ciorita et al., 2021).The samples were then placed in an isoamyl acetate:ethanol solution (1:3, 1:2, 1:1, and 1:0 for 30 min each) to replace the ethanol.Subsequently, the samples were subjected to critical point drying in carbon dioxide using a Hitachi HCP-2 critical point dryer (Hitachi, Tokyo, Japan).After drying, the samples were placed in a Hitachi E-1010 ion sputtering device (Hitachi, Tokyo, Japan) for gold spraying.The samples were observed and imaged using a HitachiS-3400N SEM (Hitachi, Tokyo, Japan).

Statistical analysis
Nine parallel experimental datasets were used.The data were analyzed using analysis of variance, and signi cance was tested at P = 0.05 using the SPSS statistical package (IBM SPSS Statistics for Windows, version 27.0).

Heavy metals in wheat
As shown in Table 2, the HM content was generally higher in the roots than in the shoots.With an increase in y ash dosage, the Cu content markedly increased in the shoots.

Subcellular distribution
We determined that Pb, Cu, and Zn were mainly distributed in the soluble constituents of the wheat leaf cells (Fig. 2).The content of Pb in soluble constituents accounted for 38.8-67.8% and had the highest proportion at low doses of y ash.The proportion of Cu in the soluble constituents decreased with increasing y ash dose, whereas it increased in the organelles.Meanwhile, Zn had the highest content in the organelles of cells under the low dose of y ash, and the order of Zn content was #2 > #3 > #4 > #1.
Moreover, the proportions of Pb and Zn in the cytoderm were reduced after exposure to y ash, while those in the soluble constituents increased.

Total chlorophyll, chlorophyll a, and chlorophyll b
Generally, as the dose of y ash increased, the total chlorophyll, chlorophyll a, and chlorophyll b contents decreased (Table 3).Compared to that of #1, the total chlorophyll contents of #2, #3, and #4 decreased by 12.90%, 18.36%, and 56.33%, respectively.Furthermore, the chlorophyll a content decreased signi cantly (p < 0.05) from 19.00 mg/L to 10.64 mg/L when y ash dose increased from 0.186 g/d to 0.744 g/d.For chlorophyll b, y ash had the greatest inhibitory effect at the highest dose, with an inhibition rate of 68.63%, and there was a signi cant (p < 0.05) difference in the chlorophyll b contents of #1, #3, and #4.

Activities of enzymes related to photosynthesis
As shown in Fig. 3, GAPDH activity showed substantially differences between #3 and #4, while FBA activity decreased with the content of y ash increasing.There were no considerably differences in GAPDH activity among #1, #2, and #3, and the highest activity occurred under the stress of the highest dose of y ash.As the y ash dose increased, FBA activity was considerably inhibited, and the FBA activity of #1 was 3.6 times that of #4.Meanwhile, at the intermediate dose (#3), Rubisco activity was inhibited by 36.08%, which was the highest inhibitory effect.
Figure 3 The activity of GAPDH, FBA, and Rubisco

Activities of antioxidant enzymes
Fly ash had different effects on the activities of SOD, POD, and CAT (Table 4).The SOD activity declined signi cantly with increasing y ash dose, and it was inhibited by 46.3% in #4 compared with #1.As the y ash dose increased, the POD activity increased from 136.93 U/L in #1 to 213.02 U/L in #4.The present results clearly show a signi cant enhancement of POD activity in #3 and #4 compared to that in #1.After treatment with y ash, a signi cant induction of CAT activity was observed in the experimental groups.Speci cally, CAT activity peaked in #4 and was 1.25 times that in #1.

Ultrastructure of wheat leaves
With increasing y ash dose, the number of particles adsorbed onto the epidermis, trichomes, and stomata increased (Figure S1).Additionally, the adsorption of larger particles and the reduction of trichomes became more evident (Figure S1a).Trichomes in samples from #4 were fractured, and the cuticular wax at the base of the trichomes peeled off (Figure S1b).The number of large particles around the guard cells markedly increased.The cuticular wax of the guard cells peeled off, and a continuous network structure formed, covering nearly half of the stoma in samples from #4 (Figure S1c).Under moderate and high levels of y ash stress, we observed that approximately 50% of the cuticular wax peeled off, and particles attached to the surface of leaves (Figure S1d).
The effects of coal y ash on the ultrastructure of the wheat leaves were also substantially,.The ellipsoidal chloroplasts in the wheat leaves under low y ash doses were closely arranged along the cytoderm with a well-developed thylakoid system (Figure S2a, b).Under medium and high levels of y ash stress, chloroplasts in the leaves swelled to become spherical, and the space between the chloroplasts and cytoderm expanded until the chloroplasts were entirely separated into the cytoplasmic matrix.Chloroplasts in the leaves at high y ash doses showed disorganization of grana stacking and disappearance of the lamellar structure.Plasmolysis was evident in samples from #4 (Figure S2 a).Leaf mitochondria under medium and high levels of y ash stress exhibited vacuolization (Figure S2b).With an increase in y ash dose, the black granular sediment gradually spread from the primary to the secondary cytoderm and entered the interior of the cell.Sediment aggregation occurred in samples from #4 (Figure S2c).

Discussion
There are two main pathways of HM uptake in plants: soil-root transfer and foliar uptake (Uzu et al., 2010).Both methods are involved in the process of HM uptake by wheat.In this study, the Cu content in the aboveground parts substantially increased, whereas the opposite pattern was observed in the roots (Table 2).Under high levels of coal y ash stress, the content of Pb in the roots decreased, but was not signi cant in the aboveground parts, which increased the TF of HMs in wheat (Table 2).Based on our nding that photosynthetic enzyme activities were signi cantly inhibited as the y ash dose increased (Fig. 3), we deduced that weakened photosynthesis in leaves due to y ash stress may lead to less energy generation for root uptake of HMs (Liao et al., 2018).The main organs for the foliar uptake of HMs in plants are the stomata, cuticular wax, and trichomes.Under moderate and high levels of y ash stress, the cuticular wax peeled off (Figure S1d), and the ash particles were deposited around the stomata (Figure S1c).Cuticular wax damage (Shabnam et al., 2021) and stomatal capture of particles (Kozlov et al., 2000;Ma et al., 2021) cause the direct exposure of leaf cells to y ash, which may increase the probability of HMs in y ash entering plant tissue.The sequestration of coal y ash by trichomes protects the other parts of plant from HM toxicity (Gao et al., 2021b).Trichome fracture was observed on the epidermis during high y ash stress (Figure S1b), likely weakening the ability of trichomes to isolate HMs.These injuries to the ultrastructure of the epidermis aggravate the foliar uptake of HMs.In the case of foliar uptake, it has been demonstrated that the transport of HMs from leaves to grains occurs mainly via the phloem through nicotianamine-and phytochelatin-bound forms, which poses a threat to food security (Clemens & Ma, 2016;Li et al., 2020b).
As the rst barrier for wheat cells, the cytoderm can block some HMs, and the cytoderm composition and structure changed (Parrotta et al., 2015).We observed that black granular sediment adhered to the cytoderm (Figure S2c).The proportions of Pb and Zn in the cytoderm decreased with increasing y ash dose (Fig. 2).The cytoderm can bind to HMs at certain concentrations and relieve HM stress (Jia et al., 2020).However, the ability of the cytoderm to bind to HMs decreases when the concentration of HMs is too high (Muschitz et al., 2009).Plants transport HMs into vacuoles to reduce the levels of toxic substances (Hall, 2002).We found that Pb, Cu, and Zn were mainly distributed in the soluble constituents, and their contents changed from 38.8-67.8%under y ash stress.Vacuoles play an integrated role in HM detoxi cation by controlling transporters (Martinoia et al., 2007).The deposition of y ash in vacuoles caused the accumulation of Pb, Cu, and Zn in the soluble constituents.
Photosynthesis is substantially inhibited by particulate matter (Gu et al., 2018), which can damage chloroplasts and affect the activity of photosynthesis-related enzymes.We found that the contents of chlorophyll a, chlorophyll b, and total chlorophyll (Table 2) and the activity of FBA decreased considerably (Fig. 3) with increasing y ash dose.Meanwhile, GAPDH had the highest activity at the highest dose of y ash, whereas Rubisco had the highest activity at a low y ash dose (Fig. 3).The reduction in chlorophyll disrupts the hydrogen transfer reaction chain during photosynthesis (Chen et al., 2022).A decrease in FBA can result in the inhibition of RuBP regeneration (Uematsu et al., 2012), and the change in RuBP content is closely related to the reduction in Rubisco (Schrader et al., 2007).Additionally, Rubisco activity can markedly affect the photosynthetic rate by regulating CO 2 assimilation (Yamori et al., 2012).
Photosynthetic e ciency cannot be in uenced when the RuBP at each Rubisco site is approximately 2 times higher than the maximum CO 2 assimilation rate, regardless of GAPDH activity (Price et al., 1995).
However, when the concentration of CO 2 increases, enhanced GAPDH activity can improve photosynthesis (Suzuki et al., 2021).We found that the y ash particles accumulated around the stoma (Figure S1c) resulted in stoma clogging and closing, which can reduce stomatal conductance (Li et al., 2019).A reduction in stomatal conductance can markedly interfere with the photosynthetic process and decrease the e ciency of photosynthesis (Li et al., 2019).Moreover, chloroplasts are sensitive to stress (Wang et al., 2023).We found that the ellipsoidal chloroplasts swelled to become spherical and separated from the cytoderm under y ash stress.Grana stacking was inhibited and the thylakoid membrane system in the chloroplasts was damaged (Figure S2a).Changes in chloroplast morphology can damage the thylakoid membrane system (Barhoumi et al., 2007), which is the site of photochemical reactions and electron transfer (Li et al., 2020a) and is sensitive to environmental conditions (Kirchhoff, 2019).The reduction in chlorophyll and the activity of enzymes related to photosynthesis both inhibit photosynthesis during stem elongation, which can reduce the yield of wheat.
The accumulation of ROS induced by stressful conditions can lead to nucleic acid injury, enzyme inhibition, and eventually, the death of cells in plants (Imran et al., 2021).The activities of antioxidant enzymes increases during detoxi cation in plants under stressful conditions (Hou et al., 2007;Qadir et al., 2019).Notably, SOD is considered the rst line of defense against ROS, as it decomposes O 2 •− to H 2 O 2 and O 2 .However, SOD activity declined from 278.47 U/mL to 149.56 U/mL in our study, indicating that excess levels of ROS lead to oxidative damage to the SOD enzyme system and inhibit the enzymatic reaction of SOD (Malar et al., 2014).Damage to the SOD enzyme system may lead to the accumulation of O 2 •− (Guo et al., 2007), demonstrating the toxicity of y ash stress in wheat.Oxidative stress induced by excess ROS leads to lipid peroxidation (Hasanuzzaman et al., 2020) and chloroplasts are the leading source of ROS production (Liu et al., 2021).In the present study, we also found that the thylakoid membrane system in chloroplasts was damaged under moderate and high levels of y ash stress (Figure S2a), which may be induced by excessive accumulation of O 2 •− .Meanwhile, POD and CAT scavenge H 2 O 2 and protect plants from oxidative stress (Malar et al., 2014).We found that POD and CAT activities increased as the y ash dose increased and maintained high activities at higher stress levels (Table 3).The enhancement of POD and CAT activity provides better protection against oxidative damage caused by y ash stress (Liu et al., 2010;Tanyolac et al., 2007).In this study, it was evident that a high level of y ash stress disrupted the rst line of defense against ROS induced by y ash exposure, which reduced SOD activity in wheat.However, the activities of POD and CAT were still enhanced to resist the oxidative damage induced by y ash stress.

Conclusions
Under y ash stress, the HM content in wheat roots was higher than that in the leaves, and HMs mainly accumulated in the soluble constituents.The chlorophyll and FBA contents decreased substantially as the y ash dose increased.Meanwhile, POD and CAT activities increased with exposure to y ash.Trichomes and cuticular wax were damaged, chloroplasts were severely deformed, and the internal membrane structure was disorganized.The proportion of heavy metals in the subcellular fractions Stress conditions induced by particulate matter and attached HMs lead to reactive oxygen species (ROS) generation in plants, which causes oxidative stress and damage to essential cell components (Matic et al., 2021).Speci cally, plants increase the activity of superoxide dismutase (SOD) to eliminate O 2•− (Zhang et al., 2021) and then increase the activities of peroxidase (POD) (Gajewska & Sklodowska, 2008) and catalase (CAT) to scavenge H 2 O 2 to maintain homeostasis under oxidative stress induced by environmental stress.

Table 2
The content of Cu in shoots grown in greenhouse #4 was 1.40 times that in shoots grown in #1.Meanwhile, the Cu and Pb contents in #4.The TF of Cu increased by 0.26 from #2 to #4.Compared with that of #2, the TF of Zn increased by 0.1 in #4.The content of Pb, Cu and Zn of different parts of wheat (mg/kg) were markedly reduced in the roots.The content of Pb decreased from 4.88 mg/kg to 1.77 mg/kg, and the lowest content was observed at the highest dose of y ash.The Cu content in the roots decreased by 7.26 mg/kg from #1 to #4.As the y ash dose increased, the translocation factors (TF =[metal]shoot/[metal] root) of Pb, Cu, and Zn showed an upward trend.The TF of Pb increased from 0.17 in #2 to 0.37

Table 3
The content of total chlorophyll, chlorophyll a, chlorophyll b (mg/L)

Table 4
The activity of antioxidant enzymes in elongation stage of wheat (U/mL, U/L)