Salt stress inhibits photosynthesis and destroys chloroplast structure by downregulating chloroplast development–related genes in Robinia pseudoacacia seedlings

doi:10.21203/rs.3.rs-2431422/v1

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Salt stress inhibits photosynthesis and destroys chloroplast structure by downregulating chloroplast development–related genes in Robinia pseudoacacia seedlings

https://doi.org/10.21203/rs.3.rs-2431422/v1

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Soil salinization is an important factor limiting food security and ecological stability. As a commonly used greening tree species, Robinia pseudoacacia often suffers from salt stress that can manifest as leaf yellowing, decreased photosynthesis, disintegrated chloroplasts, growth stagnation, and even death. To elucidate how salt stress decreases photosynthesis and damages photosynthetic structures, we treated R. pseudoacacia seedlings with different concentrations of NaCl (0, 50, 100, 150, and 200 mM) for 4 weeks and then measured their biomass, ion content, organic soluble substance content, reactive oxygen species (ROS) content, antioxidant enzyme activity, photosynthetic parameters, chloroplast ultrastructure, and chloroplast development-related gene expression. NaCl treatment significantly decreased biomass and photosynthetic parameters, but increased ion content, organic soluble substances, and ROS content. High NaCl concentrations (100-200 mM) also led to distorted chloroplasts, scattered and deformed grana lamellae, disintegrated thylakoid structure, irregularly swollen starch granules, and larger, more numerous lipid spheres. Compared to control (0 mM NaCl), the 50 mM NaCl treatment significantly increased antioxidant enzyme activity while upregulating expression of the ion transport-related genes HhNHX1 and HhSOS1 and the chloroplast development-related genes psaA, psbA, psaB, psbD, psaC, psbC, ndhH, ndhE, rps7, and ropA. Additionally, high concentrations of NaCl (100-200 mM) decreased antioxidant enzyme activity and downregulated the expression of ion transport- and chloroplast development-related genes. These results showed that although R. pseudoacacia can tolerate low concentrations of NaCl, high concentrations (100-200 mM) can damage chloroplast structure and disturb metabolic processes by downregulating gene expression.

Robinia pseudoacacia seedlings

photosynthesis

chloroplast

salt stress

Salt stress inhibits photosynthesis and destroys chloroplast structure.

Soil salinization is already a global problem that climate change, inappropriate irrigation methods, and natural disasters such as hurricanes and tsunamis threaten to exacerbate (Hasegawa, 2013). It is estimated that about 70% of the world's agricultural drylands (5.2 billion hectares) are affected by erosion, soil degradation, and salinization (Riadh et al. 2010). Saline environments account for nearly 10% of the world's total land area (950 Mha), and 50% of irrigated land (230 Mha), which seriously impedes agricultural development (Ruan et al. 2010). It is therefore urgent that we develop and utilize saline areas to solve the problem of land shortages and ensure food security (Shahid et al. 2018; Egamberdieva et al. 2019).

High concentrations of salt ions in soil cause osmotic stress, ion toxicity, and nutrient imbalance in plants, which can seriously disrupt normal growth (Ghars et al. 2008; Abbasi et al. 2016). Salt ions in plants can also damage organelle structure; affect cellular processes including photosynthesis; alter mRNA and protein synthesis; and disrupt energy metabolism, amino acid biosynthesis, and lipid metabolism (Tseng et al. 2007; Koussevitzky et al. 2007; Abdelhamid et al. 2020). In addition, salt stress causes reactive oxygen species (ROS) accumulation, causing oxidative stress to plants. Some halophytes and salt-tolerant plants can eliminate ROS by increasing the content and activity of antioxidant enzymes under salt stress, thus reducing damage to the plant. However, most crop plants cannot remove ROS effectively, leading to metabolic disorders and inhibited plant growth (Lu et al. 2016; Liu et al. 2017; Siddiqui et al. 2017).

Photosynthesis, as the source of energy for plant growth and development, is the most basic and critical physiological process in green plants (Xu et al. 2015; Jin et al. 2020; Zhang et al. 2020). Salt stress seriously affects photosynthesis in plants (Chaves et al. 2009). Large amounts of Na⁺ and Cl⁻ in the leaves leads to water loss from guard cells and therefore to changes in guard cell and stomata morphology, activity, and/or density numbers, which can reduce or prevent CO₂ entry and limit normal photosynthesis (Parida et al. 2003; Debez et al. 2004; Sui et al. 2015; Zhao et al. 2018). Moreover, salt stress leads to ion toxicity and oxidative stress, particularly in chloroplasts, the main sites of ROS production. Salt stress affects the electron transport chain (ETC) from the photosynthetic complex photosystem II (PSII) to PSI, resulting in copious production of ROS that causes oxidative damage to nucleic acids, proteins, lipids, membranes, and some photosynthetic enzymes, reducing CO₂ oxidation and crop yield (Gulzar et al. 2021; Hameed et al. 2021). Salt stress can also damage the structural integrity of chloroplasts, which is essential to the photosynthetic light reactions and carbon assimilation processes (Bose et al. 2017). Under salt stress, salt-tolerant plants can maintain the normal morphology of photosynthetic structures to sustain their functions. However, when salt stress intensity exceeds the tolerance capacity of the plants, various organelles are damaged accompanied with changed ultrastructure (Gu et al. 2017). Studies showed that under salt stress, the chloroplast membrane system was damaged, with the shape of the chloroplasts changing from ovoid to spherical, the inner lamellae becoming slightly swollen, the number of basal lamellae decreasing, and the number of starch grains, lipid droplets and osmiophilic globules in the chloroplasts increasing (Yin et al. 2011; Qin et al. 2013; Lu et al. 2020). Many genes involved in the development and maintenance of the normal chloroplast structure that is integral to photosynthesis are known, and salt stress has been shown to affect the expression of such gene, leading to aberrant chloroplast structure. Huang et al. found that the expression of Ndhf genes related to light response, Rbcl genes related to dark response, and Matk genes related to chloroplastic intron splicing were downregulated under 150 mM NaCl in Eucalyptus robusta Smith chloroplasts, which led to decreased photosynthesis and deformed chloroplasts (Huang et al. 2019).

Robinia pseudoacacia L. also known as black locust or false acacia, is native to the United States and belongs to the Faboideae subfamily of the Fabaceae. R. pseudoacacia is often used as an afforestation species in saline areas because it reproduces easily and grows rapidly, while also having a high tolerance for salinity, drought, soot, and dust. Therefore, it plays an important role in improving saline environments, preventing soil erosion and regulating hydrology (Kleinbauer et al. 2010). However, Lu et al. found severe degradation of black locust forests in the Yellow River Delta, manifested by yellowing leaves and dead tree tips (Lu et al. 2021). This suggests that salt stress can affect photosynthesis in black locusts, but there is no report on how salt stress affects chloroplast structure or how chloroplasts adapt to changes during salt stress. In this study, we treated R. pseudoacacia seedlings with different concentrations of NaCl to elucidate these mechanisms by measuring photosynthetic indicators, chloroplast structure, and changes in gene expression.

Plant material and seed germination

The R. pseudoacacia seeds were Luci 103 (provided by Daqingshan forest farm in Feixian County, Shandong Province). Seeds were sterilized in 75% ethanol for 15 min, then in NaClO (3%) solution for 15 min, and washed with sterile water 3 times. Sterilized, washed seeds were placed in a culture bottle with sterile water, which was put into an 85°C water bath for 15 min followed by a 45°C water bath for 12 h.

Seeds were evenly sown in nutrient soil in a greenhouse (the growth conditions were consistent with those of Li et al. 2020), irrigated with water, and germinated for 3–5 days, and NaCl treatment was started when the seedlings had grown to 13–15 cm in length. Five NaCl concentrations of 0, 50, 100, 150, and 200 mM were used, with five replicates per treatment. Plants were watered regularly with 200 mL per pot at 8:30 am daily. To avoid salt shock, salt treatments were increased gradually (increasing by 50 mM per day) until the final desired concentration was reached (the fourth day). Measurements were taken after 2 weeks at that concentration.

Measurement Of Plant Height, Root Length, Dry Weight, Fresh Weight And Water Content

Plant height and root length of R. pseudoacacia seedlings were measured to the nearest millimeter. Fresh weights of the shoots and the roots (FW1 and FW2, respectively) were taken, and then the samples were heated to 105°C for 20 min. Samples were then kept in an 80°C oven until weights remained constant (about two days), and then their dry weights (DW1, DW2) were measured.

Water content of shoot = (FW1 – DW1) / FW1 × 100%

Water content of root = (FW2 – DW2) / FW2 × 100%

Extraction And Quantification Of Ion Contents

Leaf samples (0.3 g) were weighed, boiled in water for 3.5 h, and then filtered to a constant volume of 25 mL. Na⁺ and K⁺ contents were measured using a flame spectrophotometer (Model 2655-00 Digital Flame Analyzer; Cole-Parmer Instrument Co. Vernon Hills, Illinois, USA). Cl⁻ content was measured using an ICS-1100 ion chromatography system (Dionex Corporation, Sunnyvale, CA, USA).

Determination Of Soluble Organic Matter

A 0.3 g subsample of fresh leaves was ground together with 2.4 mL of prepared phosphate-buffered saline (PBS) solution (183:17 (v/v) 0.2 mM Na₂HPO₄ solution:0.2 mM NaH₂PO₄ solution in 1000 mL ddH₂O, pH 7.8) and then centrifuged at 10,000 g for 10 min at 4°C.

The concentration of total soluble sugars was measured using the anthracene-sulfur colorimetric method (Zhang, 1985; Lin et al. 2018). Soluble protein content was determined using Coomassie Brilliant Blue G250 and BSA (Bradford, 1976). Proline content was determined using ninhydrin (Tamás, et al. 2008). Fresh leaves from the same plants were also ground with 5 mL dH₂O, heated in a water bath at 85 ℃ for 30 min, allowed to cool, and filtered, and the solution volumes were standardized to 0.1 L. Six drops of phenolphthalein solution (1%) were added to 0.05 L of the extract and titrated with NaOH solution (10 mM) until it turned pink. The volume of NaOH required was recorded and used to calculate the content of organic acids.

Determination of MDA, O₂^-, H₂O₂

The fully expanded leaves of third branches of Tamarix seedlings subjected to different NaCl treatments were used to measure H₂O₂, O₂^–, and malondialdehyde (MDA). Detailed experimental procedures were according to Li et al. (2022).

Leaves of 0.3 g FW were cut into pieces and ground with 2 mL 1% trichloroacetic acid solution and then was placed in a test tube containing 0.5% thiobarbituric acid solution. The test tube was boiled for 10 min and then the solution was filtered. OD values of the filtrate were measured at 532 nm and 600 nm to determine malondialdehyde content.

Leaves of 0.5 g FW were cut into pieces and ground with 5 mL 50 mM PBS (pH 7.8) at 4 ℃. The solution was filtered and the filtrate was placed in a test tube containing 5 mL reaction solution (17 mM aminobenzenesulfonic acid: 7 mM α-naphthylamine, 1:2 (V/V)). Let stand for 30 min after mixing and then was measured OD value at 530 nm to determine malondialdehyde O₂⁻ content.

Leaves of 0.5 g FW were cut into pieces and ground with 5 mL acetone at 4 ℃. The solution was centrifugated at 5000 g for 8 min. 2 mL supernatant was placed in a test tube containing 0.1 mL 20% TiCl₄ and 0.4 mL strong ammonia solution and then mixed and centrifugated at 8000 g for 7 min. After being washed 3–5 times with acetone, the precipitate was dissolved with 5 ml 2 M H₂SO₄ and to be measure the OD value 415 nm to determine the H₂O₂ content.

Determination Of Electrical Conductance

Leaves were cleaned and punched into leaf discs. The leaf discs were soaked in dH₂O for 2 hours, which called solution 1. The conductance of solution 1 was measured with a conductivity measuring instrument (DDG-5205A, Leici, Shanghai, China) and the conductance value is represented by L1. The solution 1 then was in a boiling water bath for 15–20 min, and then called solution 2. The solution 2 was cooled to room temperature and the leaf discs were removed. The conductance of solution 2 was represented by L2. Electrical conductance was calculated using the following formula:

Electrical conductance = (L1/L2)*100%

Antioxidant Activity Assays

Fresh leaves (0.3 g) were ground in PBS solution (2.4 mL) and then centrifuged at 10000 rpm for 10 min at 4°C; the supernatant was then used as the enzyme solution for testing.

The SUPEROXIDE DISMUTASE (SOD) activity of the enzyme solution was measured by the nitro blue tetrazolium (NBT) photochemical reduction method and PEROXIDASE (POD) activity was measured using guaiacol. CATALASE (CAT) activity was measured in 0.2 mL enzyme solution by adding 6 ml of reaction solution, which consisted of 200 ml PBS with 0.3092 ml of 30% H₂O₂. ASCORBATE PEROXIDASE (APX) activity was determined using 0.1 mL enzyme solution by adding 1.7 mL of PBS containing EDTA-Na₂ (0.1 mM), followed by 5 mM ascorbate (ASA) (0.1 ml), adding 20 mM H₂O₂ (0.1 mL), and immediately (within 40 s) measuring the light absorption at suitable wavelengths.

Measurement Of Gas Exchange Parameters

After exposure to different NaCl concentrations for 15 days, photosynthetic parameters were measured from 14:00 h to 17:00 h using a Li-6000 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). Five replicates were used for each treatment.

Preparation of temporary mount on the leaf surface of R. pseudoacacia

The leaves of R. pseudoacacia were put into Carnot fixative fluid (3:1 (v/v) ethanol:glacial acetic acid) for 1 h, and the temporary mount was prepared by adding Hoyers solution (40 ml lactic acid with chloral hydrate until saturation) for more than 30 min, which was observed under a differential interference (DIC) microscope (ECLIPSE80i Differential Interference Fluorescence Microscope, Nikon, Japan).

Ultrastructure of chloroplasts in seedlings of R. pseudoacacia

1 cm² of leaf tissue was placed in 2.5% glutaraldehyde (pH = 6.8) fixative fluid. The samples were immersed in the fixative by evacuating the air and left for 2 h at 4 ℃. The fixed samples were removed and rinsed with 0.1 mol/L phosphoric acid buffer (pH = 6.8) at 2 h intervals (4 ℃). This was followed by sequential dehydration with 50%, 70%, 95%, and 100% ethanol. Dehydrated samples were transferred to 100% acetone for displacement, then epoxy-impregnated, encapsulated, and polymerized. Sections were made with an ultrathin sectioning machine (Leica EM UC7, Germany). The sections were then double stained with uranyl acetate dioxide-lemon lead was used and examined under a transmission electron microscope (Hitachi HT7800, Japan).

Qrt-pcr Analysis

The chloroplast development-related gene sequences (RppsbA, RppsbD, RppsaA, RppsaB, RpndhE, RpndhH, RppsaC, RppsbC, Rprps7, and RpropA) of R. pseudoacacia seedlings were obtained from the NCBI website, and primers were designed using primer5 (Table S1). Total RNA was extracted from the leaves of R. pseudoacacia seedlings using the Rapid Universal Plant RNA Extraction Kit 3.0 (Hua Yueyang Biotechnology Co. Ltd.). cDNA synthesis was performed using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (Code No. AG11728). Real-time quantitative reverse-transcription PCR (qRT-PCR) was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Changsha, China). The relative expression of each gene was calculated using the 2^−△△Ct method to calculate the relative expression of each gene (Gao et al. 2018). The housekeeping gene LbTUBULIN was used as a control.

Statistical analysis

Statistical analyses were made using SPSS software (version 19.0; IBM, Armonk, New York, USA). Significance (P < 0.05) was determined using a Duncan's multiple range test.

Effect of NaCl stress on the growth, ions content and organic soluble substances contents of R. pseudoacacia seedlings

NaCl stress significantly inhibited the growth of R. pseudoacacia seedlings. Although we did not detect significant differences from the 0 mM NaCl control at 50 mM NaCl, at higher NaCl concentrations (100, 150, and 200 mM), plant height, root length, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight all significantly decreased with increasing NaCl (Fig. 1).

Compared with those in the control, the Na⁺ content, Cl⁻ content, and Na⁺/K⁺ ratio significantly rose with increasing NaCl concentrations, while the K⁺ content fell significantly (Fig. 2A,B,C,D). At 50 mM NaCl, the relative expression of HhNHX1 and HhSOS1 was significantly upregulated compared to the control, whereas it was significantly downregulated at higher NaCl concentrations (100 mM, 150 mM, and 200 mM) (Fig. 2E,F).

Compared with the control values, soluble protein, soluble sugar, and proline contents significantly increased with increasing NaCl concentration (Fig. 3). Organic acid content showed no significant difference at 50 mM NaCl compared to the control, but rose at higher salt concentrations (100 mM, 150 mM, 200 mM). The increase in proline content was significantly greater than the increases for other osmotic regulators. At 50 mM, 100 mM, 150 mM, and 200 mM treatments, the proline contents were 1.31 times, 2.17 times, 3.36 times, and 3.68 times those of the control, respectively.

Effect of NaCl stress on MDA, H ₂ O ₂, and O⁻ ₂ contents and electrical conductance and antioxidant enzyme activities of R. pseudoacacia seedlings

Compared with those in the control, the MDA, H₂O₂, and O⁻ ₂ contents and the electrical conductivity significantly increased along with increasing salt concentrations (Fig. 4), indicating that a large amount of ROS was produced and suggesting the plasma membrane may have suffered damage.

With increasing salt concentrations, the activities of the antioxidant enzymes CAT, APX, SOD, and POD first increased and then decreased compared with the control (Fig. 5). All four enzymes showed their highest activities at 50 mM NaCl. The activities of CAT and APX were also elevated at 100 mM NaCl, but reduced under higher-salt conditions (150 mM NaCl and 200 mM NaCl), while the activity of SOD increased at 100 and 150 mM NaCl and then decreased at 200 mM NaCl. Notably, the activity of POD showed the sharpest drop-off under high-salt conditions, being decreased at 100, 150, and 200 mM NaCl.

Effect of NaCl stress on photosynthesis, chlorophyll content and leaf grease content and chloroplast structure of R. pseudoacacia seedlings

Compared with those in the control, the net photosynthetic rate, transpiration rate, and stomatal conductance significantly decreased with increasing NaCl concentrations, while intercellular CO₂ concentration significantly increased, suggesting that NaCl stress inhibited the reduction of CO₂ (Fig. 6).

With increasing salt concentrations, total chlorophyll, chlorophyll a, and chlorophyll b contents significantly decreased compared to the control (Fig. 7). At 200 mM NaCl, the total chlorophyll, chlorophyll a, and chlorophyll b contents were 55.2%, 47.1%, and 48.7% of the control group abundances, respectively.

With increasing salt concentrations, both the grease content and the degree of chloroplast structural damage increased compared to the control (Fig. 8A,B). At 100 mM NaCl, the chloroplasts began to distort, the grana lamellae scattered and deformed, the structure of the thylakoids began to disintegrate, the starch granules started to become larger, swollen, and irregular, and the number and volume of lipid spheres increased. At 200 mM NaCl, the chloroplast structure further swelled and deformed, the thylakoids continued to expand and became disorderly, distorted, and deformed, filamentous lamellae appeared, starch granules continued to become larger and more irregular, and the number and volume of lipid spheres increased further.

Effect of NaCl stress on the expression of key genes in chloroplast development of R. pseudoacacia seedlings

The relative expression of key genes (psbA, psbD, psaA, psaB, ndhE, ndhH, psaC, psbC, rps7, and ropA) during the chloroplast development of R. pseudoacacia seedlings were significantly upregulated under 50 mM NaCl treatment for 24, 48 and 72 h, but was significantly downregulated at higher NaCl levels (100 mM, 150 mM, 200 mM) (Fig. 9).

Salt stress is an important environmental factor that restricts plant growth and development and reduces crop yield (Wang et al. 2015). Under salt stress, plants exhibit slow growth and development, metabolic inhibition, and, in severe cases, wilting and even death. Here, we showed that NaCl treatment significantly inhibited the growth of R. pseudoacacia seedlings (Fig. 1). This is consistent with the results of Gan et al. and Ren et al. (Ren et al. 2014; Gan et al. 2020).

Salt stress has a broad range of effects on plant metabolism, including the contents of other ions and organic soluble substances, various aspects of cell metabolism, and the expression of corresponding genes (Liu et al. 2018). For example, in this experiment, with increased NaCl concentrations, Na⁺ and Cl⁻ accumulated in large quantities in plants, while K⁺ decreased, resulting in a high Na⁺/K⁺ ratio (Fig. 2A,B,C,D). Under salt stress, plants tend to upregulate Na⁺/H⁺ antiporter genes to maintain Na⁺/K⁺ homeostasis and avoid Na⁺ accumulation in the cytoplasm. One of Na⁺/H⁺ antiporter located on the plasma membrane, SALT OVERLY SENSITIVE 1 (SOS1), can be activated by phosphorylation to transport Na⁺ to the extracellular matrix. Similarly, Na⁺/H⁺ EXCHANGER 1 (NHX1) is a Na⁺/H⁺ antiporter located on the tonoplast that can be activated by phosphorylation to pump excess intracellular Na⁺ into the vacuole (Qiu et al. 2002; Miranda et al. 2017; Zhang et al. 2017). An increasing number of experiments have demonstrated the role of Na⁺/H⁺ antiporters in salt resistance: for example, heterologous expression of AoNHX1 (from Avicennia officinalis L.) increases salt tolerance in rice and Arabidopsis (Krishnamurthy et al. 2019), RtNHX1 (from Reaumuria trigyna) enhances salt tolerance in transgenic Arabidopsis plants by sequestering Na⁺ into the vacuole and decreasing the Na⁺/K⁺ ratio in the cytoplasm (Li et al. 2017). In this experiment, the relative expression of RpNHXl and RpSOS1 was higher than in the control under 50 mM NaCl treatment, but lower at higher NaCl concentrations (100 mM, 150 mM, and 200 mM) (Figs. 2E,F). This indicates that R. pseudoacacia seedlings under 50 mM NaCl treatment can effectively maintain ion homeostasis in the cytoplasm by either compartmentalizing Na⁺ into the vacuole or transporting Na⁺ to the extracellular matrix, thereby reducing damage caused by salt stress, but this mechanism becomes insufficient at higher salt concentrations. This is consistent with past findings (Li et al. 2017; Krishnamurthy et al. 2019).

Salt ions in soil can cause osmotic stress in plants, which prompts them to accumulate various organic substances in their roots to reduce osmotic potential, improve cellular water retention capacity, and mitigate the damage caused by osmotic stress. These substances are either small molecules such as proline and betaine, or structural substances such as sucrose and starch (Yang and Xu, 2003). Organic osmoregulatory substances play an important role in plant tolerance to salt stress. Under salt stress, the P5CS1 (DELTA1-PYRROLINE-5-CARBOXYLATE SYNTHASE1) gene in the Arabidopsis myc2 mutant is upregulated to synthesize proline, thereby enhancing salt tolerance (Verma et al. 2020). The enrichment of soluble sugars in rice can also effectively relieve osmoregulatory effects and enhance salt tolerance (Boriboonkaset et al. 2013). In this experiment, concentrations of organic substances increased under salt stress in R. pseudoacacia seedlings (Figs. 3). The most significant increase was in the proline content, suggesting that proline plays a major role in osmotic adjustment (Verma et al. 2020; Boriboonkaset et al. 2013).

Besides ion and osmotic stress, salt stress also produces another, secondary stress in plants, namely oxidative stress (Yang et al. 2018). In R. pseudoacacia seedlings subjected to salt stress, the O⁻ ₂, H₂O₂, and MDA contents increased, as did electrical conductivity (Fig. 4), which are signs of oxidative stress (Gao et al. 2019; Siddiqui et al. 2019). However, salt-tolerant plants tend to adapt to salt stress by increasing ROS scavenging capacity using enzymatic antioxidants (SOD, POD, APX, CAT, etc.) and non-enzymatic antioxidants (AsA, GSH, etc.) (Mittler et al. 2002). Among them, SOD is the key enzyme for O⁻ ₂ scavenging, and CAT, APX, and POD are the key enzymes for H₂O₂ scavenging (Liang et al. 2018). Under salt stress, cucumber responded by adjusting CAT and APX antioxidant enzyme activities (Naliwajski and Skłodowska, 2021), whereas the CAT and SOD activities in sweet sorghum increased and then decreased under NaCl stress (Wang et al. 2022). In our experiments, the activities of SOD, POD, CAT, and APX increased at low concentrations of NaCl, but decreased at high concentrations of NaCl (Fig. 5). This indicates that R. pseudoacacia seedlings can tolerate low concentrations of NaCl and effectively scavenge ROS by increasing antioxidant enzyme activity. However, at high concentrations of NaCl, the ROS scavenging ability of seedlings decreased, resulting in ROS accumulation and subsequent damage to the plants’ growth and development (Naliwajski and Skłodowska, 2021; Wang et al. 2022).

Photosynthesis is sensitive to salt stress (Yang et al. 2020). Changes in photosynthetic parameters, including net photosynthetic rate, transpiration rate, and stomatal conductance, are direct reflections of photosynthetic function (Poór, et al. 2019). In this experiment, Pn, Tr, and Gs of R. pseudoacacia seedlings decreased under NaCl treatment, indicating that photosynthesis was inhibited (Fig. 6). In this experiment, chlorophyll content was significantly reduced under NaCl treatment (Fig. 7), which likely contributed to reduced photosynthesis. Because they serve as the main site of photosynthesis, the structural and functional integrity of chloroplasts is another prerequisite for photosynthesis (Kirchhoff, 2019). Chloroplasts are also the organelles most sensitive to salt stress, which often damages the chloroplast membrane system and deforms chloroplast structure. In Cornus hongkongensis subsp. elegans, Vitis amurensis Rupr, and Trifolium repens L. salt stress led to disorganized cystoid, basidiome, and stroma lamellae structures, which seriously damaged the integrity of the chloroplast ultrastructure (Yin et al. 2011; Qin et al. 2013; Lu et al. 2020). The results of our experiments showed that the chloroplast ultrastructure in R. pseudoacacia seedlings was clearly damaged by salt stress, with the chloroplasts being distorted and deformed, and the starch granules swollen and irregular (Fig. 8).

The photosynthetic complex PSII is an important site for the absorption, transmission, and conversion of light energy in the light reactions, and the core proteins D1, V2, and D2 are encoded by the chloroplast genes psbA, psbC, and psbD. In this experiment, the expression of psbA, psbC, and psbD was significantly upregulated under 50 mM NaCl treatment (Fig. 9B,D,F), which is consistent with previous studies (Yu et al. 1995), indicating that R. pseudoacacia seedlings tolerated low concentration NaCl stress. However, these genes were downregulated at elevated salt concentrations, suggesting that these core PSI proteins are important factors affecting photosynthetic efficiency during salt stress. psaA has a light-induced regulatory function during the transition from proplastid to chloroplast in C4 plants (such as sorghum) (She et al. 2007; Gong et al. 1992). In the present study, the expression of psaA, psaB, and psaC was also significantly upregulated under low-concentration NaCl treatment but was significantly downregulated under high-concentration NaCl treatment (Fig. 9A,C,E). We speculate that upregulation of these genes at lower NaCl concentrations can compensate for the damage to chloroplasts caused by salt stress, but this compensatory mechanism becomes insufficient at higher NaCl concentrations.

We also found that the expression of rps7, which encodes a chloroplast ribosomal protein that plays an important role in maintaining chloroplast function, was significantly upregulated under low-concentration NaCl treatment but significantly downregulated under high-concentration NaCl treatment (Fig. 9I), indicating that excess NaCl severely affected the synthesis of chloroplast ribosomal proteins. The NADH complex encoded by the chloroplast genome contributes to the adaptation of chloroplasts to environmental stresses and plays an important role in photosynthetic efficiency and response to stress. The expression of ndhE and ndhH, encoding an NADH subunit and the PSⅠ psaC subunit, respectively (Yukawa et al. 2005), was significantly downregulated under high concentrations of NaCl (Fig. 9G,H). We presume that these high NaCl concentrations severely disrupted signaling processes regulated by ndhE and ndhH, preventing the synthesis of the NADH complex. Several studies have demonstrated a strong relationship between ropA expression and abiotic stresses. In Arabidopsis, low levels of oxidative stress activate ropA, leading to the production of H₂O₂ and ethanol dehydrogenase to resist hypoxic stress (Baxter-Burrell et al. 2002). In the present experiment, the expression of ropA was upregulated at low concentrations of NaCl, indicating R. pseudoacacia seedlings are tolerant of low-concentration NaCl treatment (Fig. 9J), which is consistent with the antioxidant results. However, at high concentrations of NaCl (100 mM, 150 mM, and 200 mM), the above genes regulating chloroplast development were significantly downregulated, affecting the development and structure of chloroplasts and leading to structural abnormalities and functional damage.

In all, our results showed that R. pseudoacacia seedlings can tolerate low levels of NaCl. Although biomass decreased, photosynthetic structures were not damaged and the plants continued to grow in the presence of low concentrations of NaCl. However, the high-concentration NaCl treatment downregulated ion transport- and chloroplast development-related gene expression, leading to ion accumulation and damage to photosynthetic structures, and thus resulting in growth arrest and sometimes death. These results will provide theoretical guidance for planting R. pseudoacacia on saline-alkali land.

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-Availability of data and material

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-Competing interests

The authors declare that they have no conflict of interest.

-Funding

This work was supported by the NSFC (National Natural Science Research Foundation of China, project nos. 32170281; 31600488), Shandong Provincial Agricultural Variety Improvement Project of Shandong Province (2022LZGC018).

-Authors' contributions

MC and HF designed the research, LL, XL and FM performed the experiments. LL and XL wrote the paper with contributions from the other authors. AJ and CL analyzed the data. MC revised the paper. All authors read and approved the final manuscript.

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Salt stress inhibits photosynthesis and destroys chloroplast structure by downregulating chloroplast development–related genes in Robinia pseudoacacia seedlings

Status:

Version 1

Abstract

Figures

Key Message

Introduction

Materials And Methods

Plant material and seed germination

Measurement Of Plant Height, Root Length, Dry Weight, Fresh Weight And Water Content

Extraction And Quantification Of Ion Contents

Determination Of Soluble Organic Matter

Determination of MDA, O₂^-, H₂O₂

Determination Of Electrical Conductance

Antioxidant Activity Assays

Measurement Of Gas Exchange Parameters

Qrt-pcr Analysis

Statistical analysis

Results

Discussion

Declarations

References

Supplementary Table S1

Status:

Version 1

Salt stress inhibits photosynthesis and destroys chloroplast structure by downregulating chloroplast development–related genes in Robinia pseudoacacia seedlings

Status:

Version 1

Abstract

Figures

Key Message

Introduction

Materials And Methods

Plant material and seed germination

Measurement Of Plant Height, Root Length, Dry Weight, Fresh Weight And Water Content

Extraction And Quantification Of Ion Contents

Determination Of Soluble Organic Matter

Determination of MDA, O2-, H2O2

Determination Of Electrical Conductance

Antioxidant Activity Assays

Measurement Of Gas Exchange Parameters

Qrt-pcr Analysis

Statistical analysis

Results

Discussion

Declarations

References

Supplementary Table S1

Status:

Version 1

Determination of MDA, O₂^-, H₂O₂