Changes in growth, ammonium assimilation and amino acid levels in leaves of Arabidopsis thaliana under cadmium treatment

Cadmium is the most harmful soil pollutant due to its long biological half-life. In the present study, the effect of Cd on ammonium assimilation in Arabidopsis thaliana wild-type Col0 was investigated. Thirty-day-old Arabidopsis seedlings were exposed to 20 µM CdCl2 during different exposure times (0, 1, 2, 3, and 7 days). Seedling growth decreased under Cd stress mainly after 7 days of Cd exposure. Cd stress caused a gradual decrease in soluble leaf protein and induced an increase in leaf ammonium and the content of free amino acids such as glutamine (Gln), glutamate (Glu), asparagine (Asn), and proline (Pro), which may be related to the increase in protease activity. The results showed that the activities of Glutamine Synthetase (GS) and glutamate dehydrogenase (GDH) were inversely related. Cd stress led to an increase in GDH activity, whereas GS activity decreased. GDH activity on polyacrylamide gels showed that Cd induced both β- and α-enriched isoforms. Therefore, this study confirms that the ammonium assimilation process plays an important role for plants in adapting to Cd stress.


Introduction
A member of the mustard family, Arabidopsis thaliana is a little flowering plant [1].Its generation time is 4-5 weeks and it is studied as plant model in plant physiology.Cadmium contamination of agricultural lands is largely due to the application of Cd-containing fertilizers and sewage sludge [2].Cd is considered one of the most highly toxic pollutants to all living organisms [3,4].Several studies have demonstrated its cytotoxicity on plant growth and physiology [3,5,6].Cd stress in plants leads to various stress symptoms such as chlorosis, disturbances in mineral nutrition, nitrogen and carbohydrate metabolism and a reduction in biomass production [6,7].The photosynthetic apparatus is particularly vulnerable to Cd, and a reduction in photosynthesis is a common response in plants exposed to Cd [8].
Cd decreased plant growth, photosynthetic rate and nitrogen metabolism [9][10][11].Cd can also alter plant mineral uptake through its effects on the availability of minerals from the soil or by reducing the population of soil microbes [12].Several hyperaccumulating plant species have been used for soil Cd decontamination as tobacco plants [9,13].
A growing number of studies address the effects of Cd stress on photosynthetic CO 2 fixation [14,15].Although photosynthetic CO 2 fixation plays an important role in plant yield, ammonium assimilation contributes greatly to plant production.However, the effects of Cd stress on ammonium assimilation have been poorly studied.

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Therefore, in the present study, we attempted to explain the Cd-induced temporal changes in ammonium assimilation in Arabidopsis thaliana.Ammonium assimilation by metabolism of ammonium into amino acids and amides is part of the process of ammonium detoxification in plants [16].Glutamate dehydrogenase (GDH) and glutamine synthetase (GS) are among the enzymes involved in ammonium assimilation.GDH activity is thought to occur in mitochondria, whereas GS activity occurs in chloroplasts [16].Therefore, the present study investigated the Cd-induced temporal changes in the activity of these two key enzymes involved in ammonium assimilation, GDH and GS.

Plant material and growth conditions
Arabidopsis thaliana wild-type Col0, used for the experiments, were obtained from research lab of "Adaptation des Plantes à leur Environnement", INRA France.Seeds were sterilized and stratified at 4 °C for 4 days.Plants were grown under hydroponic culture in a growth chamber with an 8-h-light/16-h-dark cycle and 80% relative humidity.The plants were supplied with the basic nutrient medium containing 8 mM KNO 3 , 1 mM MgSO 4 , 1 mM KH 2 PO 4 , 2 mM CaNO 3 , 5 µM MnSO 4 , 30 µM H 3 BO 3 , 1 µM ZnSO 4 , 1 µM CuSO 4 , and 30 µM K-iron-EDTA.Plants were grown for 4 weeks and were then divided into two groups.The first was maintained on the same media (control).The second was transferred to basic nutrient medium containing 20 μM of CdCl 2 solution.The treatments were applied for different times (1 day, 2 days, 3 days and 7 days).

Ammonium content
Ammonium was extracted from the leaves at 4 °C with 0.3 mM H 2 SO 4 and 0.5% (w/v) polyclar AT.The ammonium content was quantified according to the reaction of Berthelot modified by Weatherburn [17].

Protein content
Soluble protein contents in both lines and all treatments were quantified using Coomassie Brilliant Blue with bovine serum albumin as a protein standard [18].

Protease activity
Protease activity was measured using the method of Weckenmann and Martin [19], using azocasein as substrate.Absorbance of the released-azo-dye was measured at 340 nm and one unit of activity was defined as the activity producing an increase of 0.01 unit of absorbance during the 1 h incubation.

Metabolite's measurement
Total amino acid content was assayed by the method of Rosen [20] using glutamine as a reference.Individual amino acid composition was determined using ion exchange chromatography [21].

Glutamine synthetase
The enzyme essay was carried out on each line and each treatment.Frozen samples were homogenized in a cold mortar and pestle with grinding medium containing 25 mM Tris-HCl buffer (pH 7.6), 1 mM MgCl 2 , 1 mM EDTA, 14 mM 2-mercaptoethanol and 1% (w/v) polyvinylpyrrolidone (PVP).The homogenate was centrifuged at 25,000g for 30 min at 4 °C (centrifuge refrigerator Eppendorf 5810r).GS activity was determined using hydroxylamine as a substrate, and the formation of γ-glutamylhydroxamate (γ-GHM) was quantified with acidified ferric chloride [22].

Glutamate dehydrogenase
The enzyme essay was carried on each line and on each treatment.GDH extractions were performed according to the method described by Magalhaes and Huber [23].Frozen samples were homogenized in a cold mortar and pestle with 100 mM Tris-HCl (pH 7.5), 14 mM 2-mercaptoethanol, and 1% (w/v) PVP.The extract was centrifuged at 12,000g for 15 min at 4 °C.GDH activity was determined by following the absorbance changes at 340 nm.

Detection of GDH activity on the gel
Leaf soluble proteins were extracted from frozen material in cold extraction buffer containing 100 mM Tricine, 1 mM EDTA, 40 mM CaCl 2 , 0.5% (w/v) PVP, 0.1% (v/v) 2-mercaptoethanol, and 1 mM AEBSF (4-(2-aminoethyl)-benzenesulfonyl fluoride).The protein separation was carried out in a 1 mm-thick non-denaturing gel, as described previously.Equal amounts of protein (60 µg) were loaded into each gel track.Native PAGE of the partially purified GDH extracts was performed by the method of Davis [24] on 5% running gel with a 4% stacking gel.The buffer system was 100 mM Tris-Glycine adjusted to pH 8 with HCl.Running was at 4 °C, 120 V, for about 2 h, and bands containing GDH activity were visualized with the tetrazolium system.The staining solution contained 150 mM Tris-HCl (pH 8.8), 50 mM glutamate, 0.5 mM NAD + , 0.5 mM NBT (Nitro Blue Tetrazolium chloride), and phenazine.

Statistical analysis
The data presented in the figures is the average of at least six replicates per exposure treatment and means ± confidence limits at alpha = 0.05 level.Two-way analysis of variance (ANOVA) and Tukey's HSD tests was used to determine significant differences between means treatments at probability level ≤ 0.05, using the SPSS software 20.1 (Version IBM.20.0.2004).

Plant growth
Cd treatment decreased seedling growth in Arabidopsis thaliana, mainly after 7 days of the treatment (Fig. 1).The growth response of Arabidopsis thaliana plants to 20 µM CdCl 2 during different exposure time (0, 1, 2, 3, and 7 days) is shown in Fig. 2. Since the first day of Cd exposure, seedling fresh weight (FW) decreased by about 35% compared to control (Fig. 2).The FW reduction became less important after 2 and 3 days, and it didn't exceed 13% compared to the control.The effect of Cd treatment was more pronounced at the 7 day of Cd exposure, and FW decreased more to reach more than 49%.

Soluble protein content
The results showed that the leaf soluble protein (SP) content decreased (about 15%) since the first day of Cd treatment compared to the control.After 7 days of Cd treatment the leaf soluble protein decreased more than 30% compared to the control (Fig. 3).

Ammonium content
The presence of Cd in the growth media induced an endogenous ammonium accumulation in leaves of about 25% since the first day of treatment, compared with the control and a gradual increase was associated with the increasing time of exposure.This increase was more pronounced in the 7 day of Cd addition, the endogenous ammonium content increased by 50%, in comparison with the control (Fig. 4).

Protease activity
The Cd treatment induced an increase of protease activity since the first day of treatment.A remarkable increase in the protease activity was noticed After 2 days of Cd treatment and the percentage of the increase was about 40% compared to control.After 7 days of treatment, the protease activity increased by only 30% refers to control (Fig. 5).

Glutamine synthetase activity
Results showed that after only 1 day of Cd exposure, GS activity decreased.The GS activity decreased progressively with time of Cd exposure.After 3 days, the decrease of GS activity remained important (20%) referring to the control.
While, the Cd treatment for 7 days induced a small increase of GS activity by about 4% refer to control (Fig. 6).

Glutamate dehydrogenase activity and GDH subunit patterns
As shown in Fig. 7A, the Cd presence in growth media caused a significant stimulation of GDH dehydrogenase activity.This increase happened especially after 7 days of exposure.The GDH activity rise of about 13%, 20% and 50% respectively after 2, 3 and 7 days of treatment.GDH activity on a polyacrylamide gel revealed the presence of seven GDH isoform patterns formed by the binding of six of the two α and β subunits (Fig. 7B).
The upper GDH band was formed by six β subunits and the base band by six α subunits.
Detection of GDH activity on gel and quantification of proteins showed higher staining intensity, especially in his fourth band.Results showed that Cd induced both α and β subunits.GDH activity increased gradually until his 7 days of Cd exposure, and the band density of the fourth isoform was more than twice as dense compared to controls.GDH activity on polyacrylamide gels was consistent with GDH measured in Arabidopsis leaves after different Cd exposure times under Cd treatment.Also in this experiment, the overall increase in NADH-GDH activity paralleled the decrease in GS activity.

Free amnio acid level
During the experimental period, total amino acid levels remained unchanged in the leaves after 1 day of Cd exposure (Fig. 8A).In the stressed plants, amino acid levels increased visibly after 2 days of Cd treatment by about 17% referring to control (Fig. 8A).After 2 days of Cd exposure, a slight increase in Glu (Glutamate) and Asn (Asparagine) levels was detected in leaves (Fig. 8B).A small Cd inhibitory effect was revealed on Gln level (Fig. 8B).In contrast, Proline (Pro) level was increased by about 23% referring to control in treated leaves.

Discussion
The incorporation of nitrogen into the formation of various cellular components, such as proteins and nucleic acids, constitutes a significant mechanism through which nitrogen metabolism facilitates plant growth.It can be inferred that any circumstance, including both biotic and abiotic stresses, which cause a decrease in the availability of nitrogen, particularly through ammonium absorption, will have an adverse impact on the overall growth and productivity of plants.Several studies explain the close relationship between nitrogen supply and cadmium.In 2020, Yang et al. demonstrated that nitrogen supply increases Cd uptake and accumulation in plants [25].On the other hand, Vazquez et al. explained that ammonium, as the only source of nitrogen, decreases Cd accumulation and allows Cd retention in roots [26].
In fact, it has been found that the presence of Cd in plants has a negative impact on their productivity and has a tendency to accumulate in various plant organs, which can have a detrimental effect on vital physiological processes [27].The exposure of plants to cadmium is recognized to result in a decrease in measurable growth parameters such as height, root length, fresh weight (FW), and dry weight (DW).The magnitude of deleterious effects exhibited significant variation in relation to the particular plant species, genotype, and plant tissue.Several sensitive growth parameters have been utilized as indicators of phytotoxicity induced by Cd [28,29].
The toxicity of Cd in plants is known to vary based on several factors such as growing conditions, exposure time, exposure length, and plant age, as documented by previous literature [30].Several plant species, comprising tobacco, Pisum sativum, and chickpea, have been associated with cadmium toxicity [9,[29][30][31].A plausible explanation for the observed decline in protein concentration [32] could be a deceleration in protein synthesis or acceleration in protein degradation.
The ammonium ions hold significant importance in the nitrogen metabolism of plants, as cited in several studies [16].The formation of ammonium arises as a consequence of nitrogen assimilation through nitrate reduction, deamination of amino acids, and photorespiration, as reported by a previous study [16].Previous studies have reported analogous elevations in the accumulation of endogenous ammonium with Cd exposure in seedlings of tomato and rice [33,34].The stimulation of protease activity was observed under cadmium-induced stress conditions in various plant species, such as tobacco [9], tomato seedlings [34], and barley [35].The elevation observed in the concentration of ammonium in foliage can plausibly be attributed to heightened protease activity (refer to Fig. 4).GS enzyme serves as the principal catalyst accountable for the process of ammonium assimilation in plants [16].The introduction of Cd resulted in a significant reduction in the overall activity of GS, which had a prominent involvement in the process of ammonium assimilation [36,37].It is evident that the accretion of ammonium in leaves is consequent upon the attenuation in the functionality of the enzyme glutamine synthetase.Although the function of GDH in higher plants remains controversial, the current findings indicate a negative correlation between the enzymatic activities of GS and GDH.The concurrent decline in GS activity and augmentation in GDH activity may plausibly be accounted for by this phenomenon.Therefore, the elevation in GDH activity may facilitate the assimilation of accumulated ammonia subsequent to the decline in GS activity.Henceforth, the observed rise in GDH activity may potentially serve as a compensatory mechanism to counterbalance the reduction in GS activity.This finding is in accordance with prior studies conducted on tomato seedlings that have undergone treatment with Cd [34].
The aminating activity of GDH is believed to be linked to the process of ammonium detoxification under Cd stress [34].The present investigation's results provide corroboration for the postulation that the glutamine synthetase/ glutamate synthase (GS/GOGAT) pathway, responsible for ammonium assimilation, may undergo a change towards the GDH pathway when subjected to Cd treatment [35].
Exposure to Cd led to a decrease in Gln level could be due to a decrease in GS activity.
On the other hand, as depicted in Fig. 8B, Cd induced an increase in glutamate (Glu) and asparagine (Asn) levels in leaves, the increase in Glu and Asn content was reported by Zhu, et al., in C. crepidioides plants [38].The greater accumulation of free amino acids, especially Gln and Asn, might be related to Cd tolerance [38].
Proline acts as a heavy metal chelator, thereby alleviating heavy metal stress [39,40].Cd treatment can lead to an increase in proline content in many plant species and appears to correspond with Cd-induced changes in water balance.Pandey and Sharma [41] demonstrated that Cd induces an increase in Proline concentration in cabbage leaves and suggested a relationship with the altered water status of treated plants.Free Proline chelates Cd ions and forms a non-toxic Cd-proline complex [42].Proline is also involved in antioxidant protection.Matysik et al. investigated the role of proline in plant protection against reactive oxygen species damage [43].Proline as osmoprotectant, biochar [44], Acetate [45] and nitrogen salts [46] were different solutions proposed to reduce Cd toxicity in plants.

Conclusion
The plant growth and ammonium assimilation appear to be affected by Cd.In the current study, we investigated the link between the exposure period in Arabidopsis thaliana seedlings under Cd stress and the two essential enzymes involved in ammonium assimilation, GS and GDH.

Fig. 3 Fig. 4 Fig. 5 Fig. 6
Fig. 3 Cadmium effect on leaf soluble protein (SP: mg/g FW) in Arabidopsis thaliana after different Cd exposure time (0, 1, 2, 3, and 7 days).The data presented are the means values of six replicates, ± standard error.Lowercase letters (a, b) compare control and treated samples at the same Cd exposure time.Bars denoted by same letters do not significantly differ by the Tukey test at 5% probability

Fig. 7 Fig. 8
Fig. 7 Cadmium effect on A glutamate dehydrogenase activity (µmol NADH oxidized /mg SP/h) in leaves of Arabidopsis thaliana after different Cd exposure time (0, 1, 2, 3, and 7 days).Data are means of six replicates ± CL at 0.05 levels.Lowercase letters (a, b) compare control and treated samples at the same Cd exposure time.Bars denoted by same letters do not significantly differ by the Tukey test at 5% probability.B The amount of each GDH protein was expressed as % relative to the corresponding protein amount prior to Cd treatment