Journal of Applied Botany and Food Quality 95, 23 - 30 (2022), DOI:10.5073/JABFQ.2022.095.004 1Laboratory of Legumes and Sustainable Agrosystems, Centre of Biotechnology of Borj-Cedria, Hammam-Lif, Tunisia 2Faculty of Sciences of Gabès, University of Gabes, Gabes, Tunisia 3Laboratory of Extremophiles Plants, Centre of Biotechnology of Borj-Cedria, Hammam-Lif, Tunisia Effect of increasing zinc levels on Trigonella foenum-graecum growth and photosynthesis activity Fadwa Melki1, 2, Ons Talbi Zribi3, Sabrine Jeder1, 2, Faten Louati1, Issam Nouairi1, Haythem Mhadhbi1, Kais Zribi1* (Submitted: September 21, 2021; Accepted: January 17, 2022) * Corresponding author Summary Zinc is an indispensable element for the plant growth and the cellu- lar metabolism. However, this mineral element becomes harmful at high quantities. The effects of high zinc supply on different physio- logical parameters were investigated in fenugreek. Seedlings were grown in plastic pots filled with inert sand under five ZnSO4 treat- ments (C: control :1.5 μM Zn; 1 mM, 2 mM, 3 mM and 4 mM ZnSO4). Results showed a decrease of 56% to 75% in shoot dry weight and a decrease of 65% to 90% in roots dry weight, relatively to the control. In addition we showed a significant reduction in photosynthetic para- meters, with the highest value of CO2 assimilation under 1 mM Zn (3.3 μmol CO2, m-2·s-1) and a lower value under 4 mM Zn (0.5 μmol CO2, m-2·s-1). The concentration of zinc in plant shoot was around two folds the control under 1, 2 and 3 mM Zn and about four folds under the maximal concentration, 4 mM Zn. In roots, we showed a progressive increase of zinc content. Increasing zinc concentration induced a significant decrease of phosphorus concentration in shoot. Fenugreek was mainly affected by zinc excess greater than 1 mM ZnSO4, however at the highest concentration, fenugreek plants ex- hibited different adaptation strategies. Key words: Trigonella foenum-graecum, heavy metals toxicity, soil contamination, mineral nutrition, plant physiology Introduction Zinc (Zn) is an essential micronutrient that is needed by plants for growth and varied biochemical and physiological pathways (Dinesh et al., 2018). It plays a vital role as a constituent of metalloenzyme and a cofactor for some enzymes such as dehydrogenases, oxidases and peroxidases (Broadley et al., 2007). It is involved also in the syn- thesis of chlorophyll and carotenoids, auxin, nucleic acids and pro- teins. Furthermore, it plays an important role in photosynthesis and in the regulation of cytoplasmic concentration of nutrients, structural stability of cell membranes and proteins, as well as protection of bio- membranes against oxidative damage (Cakmak, 2000; Paunov et al., 2018; Dos Santos et al., 2019). Nevertheless, high levels of this ele- ment can induce toxic effects to sensitive plants. Indeed, frequent Zn contamination of surface soils originating from prolonged use of Zn fertilizers, input from industrial pollution, and mining activities may lead to Zn toxicity in plants (Martens and Smolders, 2013). Broadley et al., (2007) pointed out that high levels of Zn could in- hibit plant growth and evolution by inducing a perturbation in the absorption and the translocation of nutrients or by interfering with metabolic processes and antioxidant defense system. Indeed, excess Zn induce generally functional disorders in plants such as plasma membrane permeability damage and photosynthesis inhibition, as well as interference with phosphorous, magnesium, and manganese uptake (Sagardoy et al., 2009; Paunov et al., 2018; Khan et al., 2019; Szopiński et al., 2019; Dobrikova et al., 2021). Zn toxicity symptoms include stunted growth, leaf chlorosis, necro- tic spots, root system damage and disturbance in water balance (Sagardoy et al., 2010). Plants differ in their ability to tolerate ele- vated concentrations of Zn in the soil (Rascio and Navari-Izzo, 2011; Andresson et al., 2018). Similarly, to other countries, Tunisia faces the problem of Zn toxicity, notably in soils around open-cast mining. The culture of legumes in marginal and moderately contaminated soils could be beneficial in double ways. In fact, legumes with their specific characteristic to fix symbiotically the nitrogen in associa- tion with rhizobia can contribute to the rehabilitation and the de- contamination of these soils. In the other way, moderately contami- nated soils could be additional areas for the culture of some heavy metals-tolerated legumes. To verify this hypothesis, the selection of legumes species according to their Zn tolerance and metal accumula- tion potential was a subject of several researches. Zribi et al. (2015) showed that Medicago sativa under nitrogen fixing symbiosis could tolerate 2 mM Zn and accumulate Zn in their roots and promoting the phytostabilisation process. A study carried out on the behaviour of Trifolium repens growing in a field polluted with Cd, Pb and Zn showed that the metals were preferentially accumulated in the roots than in the aerial part and exposed the plants to the oxidative stress (Bidar et al., 2007). Fenugreek (Trigonella foenum-graecum L.) belongs to the family of Fabaceae and it is cultivated in many countries such as India, Pakistan, Egypt, France, Yemen, Spain, Turkey, Morocco, China and Tunisia. India is the largest producer in the world (Zandi et al., 2015). Fenugreek can be grown easily in low-input, marginal environment. It is a precious aromatic and medicinal plant used for its healthy and nutritious benefits, as it contains a considerable number of vitamins, proteins and minerals (Zandi et al., 2015; Ahmad et al., 2016). By contrast, these virtues of fenugreek cannot deny that this plant is somehow known by its considerable ability to stock enormous amounts of particular heavy metals (Pattnaik and Reddy, 2011). The aim of this study was to investigate the behavior of fenugreek under Zn excess in term of growth, photosynthesis activity and nutri- tion and to verify if this legumes species is appropriate to be culti- vated in Zn contaminated soils. Materials and methods Plant material and germination conditions Seeds of fenugreek plant (Trigonella foenum-graecum L.) were dis- infected and sterilized with ethanol (70%) for 1 minute, then they are rinsed several times with distilled water. The seeds were soaked in distilled water for 3 hours and germinated in darkness at 25 °C in in Petri dishes on filter paper moistened with distilled water. Three- day-old seedlings were then transferred for 52 days into plastic pots (0.5 L) filled with sterile sand (three seeds were replicated in every pot and before the start of treatments only the performant seedling was retained). The experiment was carried out in controlled green- house (25 °C/19 °C, 16 h photoperiod, and 60% relative humidity). 24 F. Melki, O.T. Zribi, S. Jeder, F. Louati, I. Nouair, H. Mhadhbi, K. Zribi Plants were irrigated with nutrient solutions (Vadez et al., 1996) added by different Zn concentrations. The nutrient solution con- tained the following concentrations of macro and micro-elements: KH2PO4 (0.36 mM); K2SO4 (0.7 mM); MgSO4, 7H2O (1 mM); CaCl2 (1.65 mM); HBO3 (4μM); MnSO4, H2O (6.6 μM); ZnSO4, 7H2O (1.55 μM); CuSO4, 5H2O (1.56 μM); Na2MoO4, 7H2O (0.12 μM); CoSO4, 7H2O (0.12 μM) and Fe (1.26 mg·l-1). The pH of the nutrient solution was adjusted to 7. A concentration of 1.55 μM ZnSO4 was used as control (C), and the excess Zn treatments were 1 mM (Zn1), 2 mM (Zn2), 3 mM (Zn3) and 4 mM ZnSO4 (Zn4). The experiment was set up in a completely randomized design with five replications (5 pots per treatment) and the irrigation was performed homoge- neously with 40 ml of the nutritive solution in each pot (estimated after field capacity calculation), generally two times per week. Growth parameters Fifty-two days old plants were harvested and separated into shoots and roots. Roots were rinsed three times with cold distilled water and blotted with filter paper. The fresh weight (FW) was immediately determined, while the dry weight (DW) was determined after drying in an oven at 60 °C until constant weight. The length of the primary root was also measured at the final harvest with a ruler. Tolerance index (TI) was calculated as the ratio between whole plant dry weights of plants cultivated in presence of Zn and the whole plant dry weight of control plants (Ullah et al., 2020). Water relations Tissue water content (TWC) was determined using the following equation: TW (ml·g-1 DW) = (FW-DW)/DW Where FW is fresh weight determined 2 h after harvest, and DW is dry weight obtained after drying at 60 °C to constant weight. Pigment content and gas exchange Leaf chlorophyll and carotenoid concentrations (mg·g-1 FW) were determined spectrophotometrically according to Arnon (1949) and Mc Kinney (1941). Five ml of acetone 80% were added to fresh leaf samples. Chlorophyll (a, b) and carotenoid concentrations were mea- sured at 645, 663 and 460 nm respectively according to the equations reported by Mc Kinney (1941). CO2 assimilation rate (A), stomatal conductance (gs), transpiration rate (E) and water use efficiency [WUE = A/E] were determined on fenugreek leaves just before harvest by using a portable infrared CO2/H2O gas exchange system (LCPro+, UK). Measurements were carried out between 10:00 and 13:00 h on the youngest fully emerged leaf (n = 3 leaf samples taken from three different plants per treat- ment). Data were automatically collected every minute after the photosynthesis rate had stabilised. Ions content Desiccated shoot and root samples were ground to a fine powder us- ing porcelain mortar and pestle, and then ions digestion was achieved in 4/1 (v/v) HNO3/HClO4 mixture (Somer and Unlu, 2006). Zn, magnesium (Mg) and iron (Fe) concentrations were determined by atomic absorption spectroscopy (AAS) (PERKIN ELMER Ana- lyst 300) and phosphorus was assayed using the vanado-molybdate method (Fleury and Leclerc, 1943). Translocation factor (TF) was calculated as the ratio of the metal concentration in shoots to metal concentration in roots (Zhou et al., 2013). Proline content Proline content was determined according to Bates et al. (1973). Samples were homogenized in 3% (w/v) sulfosalicylic acid and cen- trifuged for 15 min at 14000 g. The supernatants added with ninhy- drin and glacial acetic acid were then incubated for 1 h in boiling water. After cooling in an ice bath, toluene was added and proline was assayed with a spectrophotometer at 520 nm. Proline content was calculated against standard proline. Statistical analyses Data were analysed using the statistical software XL Stat (ANOVA I). The number of repetitions was three for ions content and gas exchange parameters and five for other parameters. Significant differences be- tween means were separated using the Duncan test (P = 0.05). Results Zinc effects on plant growth and water content A significant reduction in shoot DW was observed in fenugreek plants grown in sand culture supplemented with 1, 2, 3 and 4 mM ZnSO4 (56%, 68%, 73% and 75% relatives to control, respectively) (Fig. 1A). Root DW was more sensitive to Zn toxicity than shoot DW. Indeed, root biomass decreased by 65, 73, 77 and 90% in presence of 1, 2, 3 and 4 mM ZnSO4 respectively as compared to control plants (Fig. 1B). Shoot and root lengths are similarly affected by Zn supply (Fig. 1C and 1D). Indeed, these parameters significantly decreased by 29, 39, 45 and 53% in presence of 1, 2, 3 and 4 mM ZnSO4 respectively as compared to control plants. Thus, it appears that biomass accumula- tion is more susceptible to Zn toxicity than length. Root/shoot (R/S) DW ratio decreased by 17, 20, 32 and 68% in plants grown with 1, 2, 3 and 4 mM ZnSO4 in the culture medium respec- tively when compared to controls. Interestingly, Zn supply has no significant effect on both shoot and root water content of fenugreek plants as compared to control plants (Fig. 2). Leaf gas exchange and pigment concentration Variations in main gas exchange parameters were similar to those observed for plant biomass production. Control plants displayed the highest values of net CO2 assimilation rate (A) (Fig. 3A), stomatal Fig. 1: Shoot (A) and root dry weight (B) and shoot (C) and root length (D) of fenugreek plants grown with different ZnSO4 concentrations for 52 days. Values on the error bars of A and B correspond to the root:shoot DW ratio, Values (means ± SE of five replicates) fol- lowed by the same letter are not significantly different (Duncan test, P = 0.05). Zinc effect on physiology of fenugreek 25 conductance (gs), (Fig. 3B) and leaf transpiration rate: E (Fig. 3C). With the increase in soil Zn content, A, gs, and E in leaves de- creased significantly. CO2 assimilation rate and transpiration rate decreased by approximately 34%, 52%, 69% and 74% in presence of 1, 2, 3 and 4 mM ZnSO4 respectively as compared to control plants. Nevertheless, stomatal conductance decreased by 50, 72, 84 and 85% in Zn1, Zn2, Zn3 and Zn4 treatments respectively comparing to treated plants. It is worth mentioning that Zn supply has no signifi- cant effect on WUE in fenugreek plants (Fig. 3D). The changes in pigments contents showed similar trends, decreas- ing with the increase of soil Zn content compared to control plants (Tab. 1). Chl a, b and total content decreased significantly by ap- proximately 43, 75, 79 and 84% in presence of 1, 2, 3 and 4 mM ZnSO4 respectively. Total carotenoid content decreased also by 57, 78, 85 and 89% in Zn1, Zn2, Zn3 and Zn4 plants respectively as com- pared to control ones. The Chl a/b ratio decreased by almost 18% in all treated plants. However, the Caro/Chl ratio decreased by 32% in presence of 1, 3 and 4 mM ZnSO4 as compared to control plants. Nutrition status Root and shoot Zn concentrations of fenugreek plants increased generally in response to increasing Zn concentrations in the growth medium. Root Zn concentrations increased by 1.9; 2.2; 2.9 and 3.3- fold in presence of 1, 2, 3 and 4 mM ZnSO4 respectively. However, shoot Zn concentrations increased by 2 -fold in Zn1 and Zn2 treat- ments and by 2.3 and 3.7-fold in Zn3 and Zn4 treatments respectively comparing to treated plants. It is worthy to indicate that under control conditions as well as in presence of 1, 2, and 4 mM ZnSO4 the ac- cumulation of Zn in fenugreek plants was approximately the same between roots and shoots (Fig. 4). The rate of Zn translocation from roots to shoots was about 0.90, 0.95 and 1.0 in control and Zn1 and Zn4 treatments. It is worth mention- ing that there is no significant difference in this parameter between control and Zn- treated plants. Phosphorus (P) concentrations decreased significantly by 65% in shoots of fenugreek plants cultivated in presence of 3 and 4 mM ZnSO4 but did not change significantly in roots (Fig. 5). C 1 2 3 4 ZnSO4 (mM) a a a a a 0 2 4 6 8 10 12 Sh oo t w at er c on te nt (m l. g- 1 D W ) a a a a a R oo tw at er c on te nt (m l. g- 1 D W ) C 1 2 3 4 ZnSO4 (mM) 0 2 4 6 8 10 12 C 1 2 3 4 ZnSO4 (mM) a a a a a 0 2 4 6 8 10 12 Sh oo t w at er c on te nt (m l. -1 D W ) a a a a a R oo tw at er c on te nt (m l. g- 1 D W ) C 1 2 3 4 ZnSO4 (mM) 0 2 4 6 8 10 12 24 a b bc c c 0 2 4 6 8 A ( m ol C O 2. m -2 s- 1 ) cc bc b a 0 0.1 0.2 0.3 0.4 gs (m m ol H 2O . m -2 s- 1 ) dd c b a 0 0.5 1.0 1.5 2.0 E ( m ol . m -2 s- 1 ) 0 a a a a b 2 4 6 W U E (A /E ) C 1 2 3 4 ZnSO4 (mM) C 1 2 3 4 ZnSO4 (mM) a b bc c c 0 2 4 6 8 A ( m ol C O 2. m -2 s- 1 ) cc bc b a 0 0.1 0.2 0.3 0.4 gs (m m ol H 2O . m -2 s- 1 ) dd c b a 0 0.5 1.0 1.5 2.0 E ( m ol . m -2 s- 1 ) 0 a a a a b 2 4 6 W U E (A /E ) C 1 2 3 4 ZnSO4 (mM) C 1 2 3 4 ZnSO4 (mM) c bc b a a R oo t Z n co nc en tr at io n (m g. g -1 D W ) a b cbc d 0 0.5 1.0 1.5 2.0 2.5 Sh oo t Z n co nc en tr at io n (m g. g -1 D W ) 0 0.5 1.0 1.5 2.0 2.5 ab ab bc c a 0 0.3 0.6 0.9 1.2 1.5 Z n tr an sl oc at io n fr om ro ot s to s ho ot s C 1 2 3 4 ZnSO4 (mM) (A) (B) (C) c bc b a a R oo t Z n co nc en tr at io n (m g. g -1 D W ) a b cbc d 0 0.5 1.0 1.5 2.0 2.5 Sh oo t Z n co nc en tr at io n (m g. g -1 D W ) 0 0.5 1.0 1.5 2.0 2.5 ab ab bc c a ab ab bc c a 0 0.3 0.6 0.9 1.2 1.5 Z n tr an sl oc at io n fr om ro ot s to s ho ot s C 1 2 3 4 ZnSO4 (mM) (A) (B) (C) Fig. 2: Shoot and root water content of fenugreek plants grown with dif- ferent ZnSO4 concentrations for 52 days. Values (means ± SE of five replicates) followed by the same letter are not significantly different (Duncan test, P = 0.05). Fig. 3: CO2 assimilation rate (A), stomatal conductance (B), transpiration rate (C) and water use efficiency (D) of fenugreek plants grown with different ZnSO4 concentrations for 52 days. Values (means ± SE of five replicates) followed by the same letter are not significantly dif- ferent (Duncan test, P = 0.05). Tab. 1: Effect of Zn supply on leaf pigment content of fenugreek plants grown with different Zn concentrations for 52 days. Values (means SE of three replicates) followed by the same letter are not significant- ly different (Duncan test, P=0.05). C 1 mM 2 mM 3 mM 4 mM Chla (mg.g-1 FW) 7.99 a 4.54 b 1.97 c 1.61 c 1.21 c Chlb (mg.g-1 FW) 2.80 a 1.96 b 0.79 c 0.70 c 0.53 c Chlt (mg.g-1 FW) 10.7 a 6.50 b 2.76 c 2.31 c 1.75 c Carot (mg.g-1 FW) 1.62 a 0.69 b 0.35 c 0.23 c 0.17 c Chl a/b 2.87 a 2.48 b 2.31bc 2.29 bc 2.25 c Car/Chl 0.15 a 0.10 b 0.13 a 0.10 b 0.10 b Fig. 4: Shoot Zn concentration (A), root Zn concentration (B) and Zn trans- location factor (C) of fenugreek plants grown with different ZnSO4 concentrations for 52 days. Values (means ± SE of three replicates) followed by the same letter are not significantly different (Duncan test, P = 0.05). 26 F. Melki, O.T. Zribi, S. Jeder, F. Louati, I. Nouair, H. Mhadhbi, K. Zribi Proline content Our results showed that increasing Zn concentration has no signifi- cant effect on both shoot and root proline content in fenugreek plants (Fig. 6). symplastic transport. In the present study, fenugreek plants cultivated during 52 days in sand culture supplied with 1 mM ZnSO4 showed 56% reduction in shoot DW compared to control plants. However, Al Khateeb and Al-Qwasemeh (2014) showed a 70% reduction in relative fresh weight of two solanum species: Solanum lycoper- sicum and Solanum nigrum grown in vitro under 1 mM ZnSO4. Furthermore, the tolerance index calculated on the basis of whole plant DW of fenugreek plants cultivated in presence of 1 mM ZnSO4 was 0.38 (Tab. 2) which suggest relative tolerance of this species to Zn toxicity when cultivated under 1mM ZnSO4. Indeed, Lux et al. (2004) pointed out that plants with tolerance index between 0.35 and 0.6 are considered as plants with medium tolerance. However, in presence of a severe Zn stress (4 mM ZnSO4), the tolerance index decreased substantially. Besides Zn vacuolar compartmentalization, cited as a potential mechanism for Zn detoxification, its retention by linking to the molecule of phytic acid in non-vacuolated tissues may also play a key role in Zn detoxification (Andressen et al., 2018). 0 5 10 15 20 25 30 P ro lin e co nt en t ( m ol . g -1 D W ) a a a a a Shoot Root a a a a a C 1 2 3 4 ZnSO4 (mM) 0 5 10 15 20 25 30 P ro lin e co nt en t ( m ol . g -1 D W ) a a a a a Shoot Root a a a a a C 1 2 3 4 ZnSO4 (mM) Fig. 6: Shoot and root proline content of fenugreek plants grown with dif- ferent ZnSO4 concentrations for 52 days. Values (means ± SE of three replicates) followed by the same letter are not significantly dif- ferent (Duncan test, P = 0.05). Fig. 5: Shoot and root phosphorus content of fenugreek plants grown with different ZnSO4 concentrations for 52 days. Values (means ± SE of three replicates) followed by the same letter are not significantly dif- ferent (Duncan test, P = 0.05). Discussion The present study revealed that the increase of ZnSO4 concentration in the medium leads to the reduction of fenugreek growth features. This may be due to the toxic effect of Zn that damages plant growth (Paunov et al., 2018). Indeed, lower levels of ZnSO4 reduced dry weight, shoot length and root length slightly compared to higher levels (Fig. 1). Reduction of growth under excess Zn have already been described for other species and it varies in plants according to their tolerance level and experimental conditions such as concentra- tion of the metal and the duration of the stress (Sinisha and Puthur, 2018). Our results showed that the decrease in root biomass of fenugreek plants cultivated under Zn excess was slightly higher than in shoot biomass and R/S DW ratios decreased significantly with increasing Zn supply. These findings suggest that Zn had more inhibitory ef- fects on the root than on the shoot. Similar results were observed by Sagardoy et al. (2009). Glińska et al. (2016) pointed out that excess Zn decreased shoot and root growth of Triticum aestivum seedlings likely by disturbing cell division and/or elongation. Li et al. (2013) revealed that the reduction of root growth in Triticum aestivum seedlings exposed to Zn excess is linked to a substantial loss of cell viability in the root tips and to an increased level of ligni- fication. Furthermore Feigl et al. (2015) demonstrated that high Zn concentrations caused significant deposition of callose in root api- cal meristem of B. juncea and B. napus, which may contribute to growth inhibition because it lowers cell wall loosening and hampers Tab. 2: Tolerance index of fenugreek cultivated during 52 days in presence of increasing concentrations of ZnSO4 Zn1 Zn2 Zn3 Zn4 Tolerance index 0.38 0.29 0.25 0.16 Our results showed also that shoot and root length are similarly af- fected by Zn supply. In contrast, Feigl et al. (2016) showed no sig- nificant effect of Zn supply on root length in Brassica napus plants cultivated during fourteen days under 50, 150 and 300 μM ZnSO4. Marichali et al., (2016) reported that the repression of root elonga- tion of Nigella sativa L. exposed to Zn excess was explained by the inhibition of cell proliferation and subsequent elongation. Kaur and Garg (2021) pointed out that the decrease in growth under Zn excess is a non-specifc manifestation of alterations in physio-biochemical traits which can result from direct effects (toxicity due to accumula- tion in tissues) and/or from indirect effects. Indirect factors include disturbances in photosynthetic activity, limitation of minerals and water acquisition as well as the induction of oxidative stress through overproduction of ROS. Zhang et al. (2020) reported that excess Zn damages the organization of mitochondria and leads to decrease in nicotinamide levels, and consequently reduced the energy metabo- lism, which may explain for the diminution in overall plant growth. Remarkably, we conclude in this research that increasing ZnSO4 concentration has no significant effect in both shoot and root water content. These results suggest that osmotic adjustment in fenugreek subjected to Zn excess is efficient and that this cultivar may regulate cell osmotic potential to reduce toxic effects of Zn. Contrarily, many studies showed a decrease in both, shoot and root water content of many species under Zn excess exposure (Sagardoy et al., 2009; Rucinska-Sobkowiak, 2016). By maintaining the water content, fenugreek seems capable to avoid the reduction in root and shoot hydraulic conductivity and the reduction in aquaporin activity, which are described as principal causes of water content decrease (Kaur and Garg, 2021). These results suggest also that fenugreek may regulate cell osmotic potential to maintain stable water status under Zn stress exposure. The reduction of growth by Zn toxicity in fenugreek plants may be the consequence of the decrease in photosynthesis. Indeed, it was show that the plant growth is closely, related to the quantity of as- similated CO2. Similarly, it has been shown that photosynthesis ac- tivity depend to Zn supply (Sagardoy et al., 2009 and 2010). Indeed, according to Van Assche and Clijsters (1986), high Zn concen- trations can affect Rubisco activity. The decrease in photosynthesis under Zn excess exposure could be due to many factors such as the decrease in chlorophyll biosynthesis, the inhibition of the activities Zinc effect on physiology of fenugreek 27 of key enzymes of the Calvin cycle, the reduction in chlorophyll a fluorescence and the inhibition of photosynthetic electron transport (Yang et al., 2020). Our data showed also that stomatal conductance and transpiration rate decreased significantly with increasing Zn supply. This may be interpreted as a water saving mechanism, which correlates with changes in shoot water content. Furthermore, the decrease in gs may be ascribed to stomatal closure or decrease of the stomatal aperture size. It was also demonstrated that the decrease in stomatal conduc- tance under Zn excess may be related to an alternation in the K+/ Ca2+ ratio in the guard cells and/or to the abscisic acid concentration, which controls the stomatal movement (Marschner, 1995). Another factor that is important to consider in plants under Zn toxicity is water use efficiency (WUE). We showed in this experiment that in- creasing Zn concentrations in the culture medium up to 3 mM ZnSO4 has no significant effect in WUE in fenugreek plants. Leaf chlorophyll content is an important physiological index directly related to photosynthesis in plants. Chlorophylls and carotenoids are involved primarily in light harvesting and a balance in their amounts is imperious for optimum light energy capture in photosynthesis (Wahid and Ghazanfar, 2006; Polivka and Frank, 2010). In the present experiment, we noted a gradual decrease in the concentra- tions of chlorophyll a, b total and carotenoids with the increase in ZnSO4 concentrations in the soil. Furthermore, Paunov et al. (2018) reported a 55% decrease in Chl a content and 24% reduce in chloro- phyll b content in leaves of durum wheat after 7 days treatment with only 600 μM Zn. Nevertheless, Zhang et al. (2020) showed no significant effect of Zn stress in the content of Chl and Car in to- bacco leaves cultivated during 10 days in presence of only 200 μM Zn. The decline in chlorophyll content in the plants exposed to Zn toxicity is believed to be probably due to (i) inhibition of impor- tant enzymes, such as 6-aminolevulinic acid dehydratase (ALA- dehydratase) (Padmaja et al., 1990) and protochlorophyllide reduc- tase (Van Assche and Clijsters, 1990) associated with chlorophyll biosynthesis; and/or (ii) impairment of the supply of Mg2+ and Fe2+ (Marschner, 1995). However, in this experiment, we showed that shoot Mg and iron concentration of fenugreek plants was not signifi- cantly affected by Zn supply (Fig. 7) which suggest that the decrease in chlorophyll content of fenugreek leaves was mainly due to the de- crease of activities of enzymes related to chlorophyll biosynthesis. Furthermore, many studies suggest that heavy metal ions could inter- fere with Chl biosynthesis through central Mg ion substitution, which therefore impairs the functioning of Light Harvesting Complex II: LHCII (Cenkci et al., 2010). In this study, fenugreek plants culti- vated under ZnSO4 showed a significant decrease in Chl a/b ratio as compared to control plants, suggesting that Chl a was degraded at a higher rate than Chl b. Similar findings are observed in Phasoelus vulgaris plants cultivated under Zn excess (Vassilev et al., 2011). Reduction of the chlorophyll a/b ratio indicate that PSI is degraded faster than PSII (Andersson et al., 2004) or that LHCII remains in- tact longer than the reaction centres (Moy et al., 2015). Similar to chlorophyll, carotenoids content also significantly decreased in response to increasing soil Zn concentrations. Giannakoula et al., (2021) reported that the reduction in carotenoid content ob- served in many plant species during metal toxicity may be due to a protective mechanism that preserves chlorophyll pools at the expense of carotenoids, due to overproduction of reactive oxygen species. Broadley et al. (2007) reported that high levels of Zn could decrease plant growth via the induction of a perturbation of the absorption and the repartition of nutrients or by interfering with metabolic processes and antioxidant defence system. In the present study, control plants showed statistically significantly lower amounts of Zn in both shoots and roots compared to all Zn-treated plants. Furthermore, the strong positive correlation between Zn in plant roots and Zn in the culture medium indicates that the Zn content in roots strongly depend on its concentration in the soil. Marschner (1995) reported that relatively to the highly mobile elements such as K or P and the immobile ele- ment Ca, Zn has an intermediate mobility. Our results showed that under control conditions as well as in pre- sence of ZnSO4, the accumulation of Zn in fenugreek plants was ap- proximately the same between roots and shoots. Rascio and Navari- Izzo (2011) pointed out that Zn repartition and translocation in plants is influenced by the level of Zn amount and plant species. At high exogenous quantities of Zn, the tolerance of plants is expressed by an accumulation of this metal in the root and the leaves. Pearson and Rengel (1995) have suggested that the transpiration stream may be a driving force in translocation of Zn and its accumulation in leaves. Furthermore, fenugreek plants cultivated in presence of ZnSO4 ac- cumulate high shoot Zn concentrations. It seems that fenugreek is likely characterized by an efficient Zn transport from roots to shoots. Indeed, plants tolerant to Zn toxicity can reduce the metal damage and grow optimally under Zn excess (Mateos-Naranjo et al., 2014). However, in the present study, we found that plant biomass production was more closely related to CO2 assimilation rate, stoma- tal conductance and pigment contents than to translocation factor. Indeed, a weak correlation was found between translocation factor and whole plant DW (data not shown). Thus, our findings suggest that the increasing accumulation of Zn in plant leaves subjected mainly to 4 mM ZnSO4 may be attributed to the inability of fenugreek plant to chelate Zn with organic and inorganic acids to make it insoluble and limit its transport from roots to leaves. Accumulated Zn may affect the structure of the thylakoid membranes in chloroplasts and lead to a decrease of electron transport rates. Therefore, the effects of Zn stress on plant growth and biomass production were likely related to the accumulation of Zn in leaves at first and then reduced photo- synthesis. Huang et al. (2019) observed similar results in Zelkova schneideriana plants cultivated during 15 days in presence of in- creasing concentrations of Zn. Several research accomplished in different plant types showed the presence of a reversed relationship between phosphorus (P) and Zn accumulation in plants (Khan et al., 2019). Furthermore, Zn excess is known to interfere with Fe, P, Mg and Mn uptake by competing with these ions for binding at numerous sites, such as principal absorption region or loading region of roots (Tewari et al., 2008). Bazihizina et al. (2014) pointed out that changes in nutrient contents under Zn excess exposure may also be due to alteration in the functioning of membrane transporters and ion channels and to membrane depolari- zation. In this experiment, shoot Fe content of fenugreek plants was not af- fected by Zn excess exposure. Similarly, Yang et al. (2011) reported that Vitis vinifera leaves retained high level of Fe under Zn stress, which was attributed to enhanced translocation of this element from the root to shoots. In our present work, we found that increasing Zn amount has no ab ab b ab a 0 1 2 3 Sh oo t F e (m g. g -1 D W ) ZnSO4 (mM) C 1 2 3 4 ab b b b a 0 10 20 30 40 50 ZnSO4 (mM) C 1 2 3 4 Sh oo t M g (m g. g -1 D W ) Fig. 7: Shoot content of Mg and Fe of fenugreek plants grown with differ- ent ZnSO4 concentrations for 52 days. Values (means ± SE of three replicates) followed by the same letter are not significantly different (Duncan test, P = 0.05). ab ab b ab a 0 1 2 3 Sh oo t F e (m g. g -1 D W ) ZnSO4 (mM) C 1 2 3 4 ab b b b a 0 10 20 30 40 50 ZnSO4 (mM) C 1 2 3 4 Sh oo t M g (m g. g -1 D W ) 28 F. Melki, O.T. Zribi, S. Jeder, F. Louati, I. Nouair, H. Mhadhbi, K. Zribi substantial consequence on root P concentrations. However, shoot P concentrations decreased significantly under Zn excess. Sagardoy et al. (2009) showed that increasing Zn supply up to 300 μM Zn in- creased significantly leaf P content and it has no significant effect on root P content of sugar beet plants. Proline prevents membrane damage and had a protective role in lipid peroxidation induced by metals (Thounaojam et al., 2012). In the present study, we showed that both shoot and root proline content in fenugreek plants did not increase significantly as Zn levels increased. Contrarily, Al Khateeb and Al-Qwasemeh (2014) showed a signifi- cant increase in proline content in both Solanum lycopersicum and Solanum nigrum grown under different levels of CuSO4, ZnSO4 and CdCl2. Parlak and Yilmaz (2012) reported also that proline content increased under Zn toxicity in three tested plants. Conclusion In conclusion, excess Zn in fenugreek plants caused an array of ef- fects related to the Zn levels in the culture medium. Lower levels of ZnSO4 reduced growth and physiological parameters slightly com- pared to higher levels. 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DOI: 10.1080/15226514.2013.828017 ORCID Fadwa Melki https://orcid.org/0000-0002-1004-3691 Ons Talbi Zribi https://orcid.org/0000-0003-4902-6925 Sabrine Jeder https://orcid.org/0000-0001-6101-3292 Issam Nouairi https://orcid.org/0000-0003-1850-1164 Haythem Mhadhbi https://orcid.org/0000-0003-0786-4269 Kais Zribi https://orcid.org/0000-0001-8856-9573 http://dx.doi.org/10.1111/j.0031-9317.2004.0275.x http://dx.doi.org/10.1021/acs.jafc.6b00274 http://dx.doi.org/10.1016/B978-0-12-473542-2.X5000-7 http://dx.doi.org/10.1007/978-94-007-4470-7 http://dx.doi.org/10.1016/s0021-9258(18)51320-x http://dx.doi.org/10.1111/ppl.12331 http://dx.doi.org/19910743084 http://dx.doi.org/10.1016/j.ecoenv.2012.08.023 http://dx.doi.org/10.3390/ijms19030787 http://dx.doi.org/10.1021/ar100030m http://dx.doi.org/10.1016/j.plantsci.2010.08.016 http://dx.doi.org/10.1007/s11738-016-2277-5 http://dx.doi.org/10.1111/j.1438-8677.2008.00153.x http://dx.doi.org/10.1111/j.1469-8137.2010.03241.x http://dx.doi.org/10.1007/s12892-018-0042-0 http://dx.doi.org/10.3389/fpls.2019.00748 http://dx.doi.org/10.1016/j.plaphy.2012.01.006 http://dx.doi.org/10.1002/jpln.200700222 http://dx.doi.org/10.3390/plants9030310 http://dx.doi.org/10.1016/S0176-1617(86)80157-2 http://dx.doi.org/10.1111/j.1365-3040.1990.tb01304.x http://dx.doi.org/10.3390/ijms19030787 http://dx.doi.org/10.1016/j.jplph.2005.07.007 http://dx.doi.org/10.1007/s11738-010-0687-3 http://dx.doi.org/10.1371/journal.pone.0228563 http://dx.doi.org/10.1007/s11738-014-1714-6 http://dx.doi.org/10.1016/j.ecoenv.2020.110856 http://dx.doi.org/10.1002/etc.2389 http://dx.doi.org/10.1080/15226514.2013.828017 https://orcid.org/0000-0002-1004-3691 https://orcid.org/0000-0003-4902-6925 https://orcid.org/0000-0001-6101-3292 https://orcid.org/0000-0003-1850-1164 https://orcid.org/0000-0003-0786-4269 https://orcid.org/0000-0001-8856-9573 30 F. Melki, O.T. Zribi, S. Jeder, F. Louati, I. Nouair, H. Mhadhbi, K. Zribi Address of the corresponding author: Dr. Kais Zribi, Laboratory of Legumes and Sustainable Agrosystems, Centre of Biotechnology of Borj-Cedria, BP 901, 2050 Hammam-Lif, Tunisia. E-mail: kais.zribi@cbbc.rnrt.tn © The Author(s) 2022. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en).