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1. Responses to Salinity in Four Plantago Species from Tunisia Authors: Hela Belhaj Ltaeif , Anis Sakhraoui , Sara González- Orenga , Anbu Landa Faz , Monica Boscaiu , Oscar Vicente , Slim Rouz Presented by: Muhammad Anees (S2 Pendid.Biologi ) Date: 07/03/2024

2. contents

3. Introduction Brief Overview: Study focused on salt tolerance in Plantago genus. Salinity affects plant due to its two components, osmotic stress and ion toxicity . Compared halophytes (P. crassifolia , P. coronopus ) and glycophytes (P. ovata , P. afra ). Analyzed morphological and biochemical responses . These plants were subjected to salinity for one month. P. afra most susceptible, and the other species showed resistance. Importance: Understanding the mechanisms of salt tolerance in plants is crucial for agricultural and ecological purposes, particularly in regions with high soil salinity. The study of diverse species within the Plantago genus provides valuable insights into the variability of salt tolerance mechanisms in plants. Research Question: How do the salt stress responses vary among different species of Plantago, including halophytes and glycophytes , and what are the underlying biochemical and physiological mechanisms contributing to these variations? Which species within the Plantago genus exhibit the highest and lowest levels of salt tolerance, and what specific adaptive strategies do they employ to cope with salt stress ?

4. Plant species P. crassifolia P. Coronopus P. ovata P. afra

5. Methodology Plant Material: Natural habitat in Tunisia After washing with distilled water, seeds were sown on peat in 1L pots placed in plastic trays (12 pots per tray) The trays were provided with 16 h light. 23C at day and 17C at night. 50% to 80% relative humidity The pots were watered twice per week with deionized water.

6. Plant Growth, Salt Treatments and Plant Sampling Salt treatments were started four weeks after sowing, with concentration0 , 200, 400,600, and 800mM NACL in deionized water. Plant material (the root and the aerial part of each plant) was harvested after four weeks, and several growth parameters were determined. Part of the fresh root and leaf material was weighed (FW), dried in an oven at 65 C for ca. 72 h (until constant weight), Then weighed again (dry weight, DW) to calculate the water content percentage of roots and leaves, as WC % = [( FW-DW ) / FW] 100 .

7. Electrical Conductivity of the Substrate Sample Collection: Samples were collected from five pots per species and treatment. Preparation: The samples were air-dried. Mix Preparation: A mixture of substrate and deionized water in a ratio of 1:5 was prepared by stirring at 600 rpm at room temperature. Filtration: The suspension was filtered through filter paper. Measurement: The electrical conductivity (EC) was measured using a Crison 522 conductivity-meter ( Crison Instruments, Barcelona, Spain). Expression: The EC was expressed in dS m^(-1).

8. Photosynthetic Pigments Determination Pigment Determination: Chlorophyll a ( Chl a), chlorophyll b ( Chl b), and total carotenoid (Caro) contents measured. Sample Preparation: Fresh leaf material (0.1 g) ground with liquid nitrogen. One ml of ice-cold 80% acetone added to the ground material. Sample shaken overnight at 4°C in the dark. Centrifugation and Absorbance Measurement: Extract centrifuged at 13,300 x g, at 4°C. Supernatant collected, and absorbance measured at 470, 645, and 663 nm. Calculation of Pigment Concentrations: Equations used for calculation of pigment concentrations, expressed in mg g^(-1) DW. Chl a ( μ g/mL) = 12.21 * (A663) - 2.81 * (A646) Chl b ( μ g/mL) = 20.13 * (A646) - 5.03 * (A663) Caro ( μ g/mL) = (1000 * A470 - 3.27 * [ Chl a] - 104 * [ Chl b]) / 227 Instrumentation: UV/visible spectrophotometric assays conducted using a UV-1600PC spectrophotometer (VWR, Llinars del Vallès , Barcelona, Spain).

9. Ion Content Measurements Ion Concentration Measurement: Concentrations of sodium (Na+), potassium (K+), and chloride ( Cl -) measured in roots and leaves of plants after salt treatments and in corresponding non-stressed controls. Sample Preparation: Dried material (approximately 0.1 g) ground to a fine powder. Extracted in 15 mL of MilliQ water. Samples incubated for one hour in a water bath at 95°C. Followed by cooling to room temperature and filtration through a 0.45 μ m Gelman nylon filter (Pall Corporation, Port Washington, NY, USA). Quantification: Sodium (Na+) and potassium (K+) quantified using a PFP7 flame photometer ( Jenway Inc., Burlington, VT, USA). Chloride ( Cl -) quantified using a chlorimeter (Sherwood, model 926, Cambridge, UK).

10. Proline and Total Soluble Sugars Quantification Proline (Pro) Content Determination : Proline content measured in fresh tissue using the ninhydrin -acetic acid method. Free Pro extracted in 3% aqueous sulphosalicylic acid. Extract mixed with acid ninhydrin solution, incubated at 95°C for 1 hour, cooled, and then extracted with toluene. Absorbance of the organic phase measured at 520 nm using toluene as a blank. Standard curve prepared using samples containing known amounts of Pro. Pro concentration expressed as μmol g^(-1) DW. Total Soluble Sugars (TSS) Quantification : Fresh leaf material (approximately 0.1 g) extracted in 3 mL of 80% (v/v) methanol on a rocker shaker for 24 hours. Samples vortexed and centrifuged at 13,300 x g for 10 minutes. Supernatants collected and diluted 10-fold with water. Diluted samples supplemented with concentrated sulfuric acid and 5% phenol. Absorbance measured at 490 nm. TSS contents expressed as 'mg equivalent of glucose' per gram of dry weight (mg eq. gluc g^(-1) DW)

11. Oxidative Stress Markers Malondialdehyde (MDA) Content Determination : MDA contents determined following a previously reported procedure with modifications. 80% methanol extracts prepared for TSS quantification were used. Samples mixed with 0.5% thiobarbituric acid (TBA) dissolved in 20% trichloroacetic acid (TCA), incubated at 95°C for 20 minutes. Reactions stopped on ice, samples centrifuged at 13,300 x g for 10 minutes at 4°C. Absorbance of supernatants measured at 440, 532, and 600 nm. MDA concentration calculated using equations previously described, based on the molar extinction coefficient at 532 nm of the MDA-TBA adduct. Hydrogen Peroxide (H2O2) Content Measurement : H2O2 content measured according to a previously published method. H2O2 extracted in a 0.1% (w/v) TCA solution from 0.1 g fresh leaf material. Extract centrifuged at 13,300 x g for 15 minutes, supernatant collected. Supernatant mixed with one volume of 10 mM potassium phosphate buffer (pH 7) and two volumes of 1 M KI. Absorbance of sample measured at 390 nm. H2O2 contents expressed as μ mol g^(-1) DW.

12. Non-Enzymatic Antioxidants Total Phenolic Compounds (TPC) Measurement : TPC measured in the same 80% methanol extracts used for TSS and MDA quantification. Determined by reaction of extracts with NaHCO3 and the Folin-Ciocalteu reagent. Reaction mixtures kept in the dark at room temperature for 90 minutes. Absorbance measured at 765 nm. TPC concentration expressed as equivalents of the gallic acid standard (mg eq. GA g^(-1) DW). Total Flavonoid (TF) Measurement : TF measured in the same 80% methanol extracts used for TSS and MDA quantification. Determined by reaction with AlCl3 under alkaline conditions after nitration of catechol groups with NaNO2. Absorbance of samples read at 510 nm. Catechin used as a standard to plot a calibration curve. Results expressed as catechin equivalents (mg eq. C g^(-1) DW).

13. Statistical Analysis Experimental Design : Assays conducted in a completely randomized design (CRD) with four genotypes and two treatments. Variance analysis performed to determine interaction between treatments and species. Statistical Analysis : Two-way analysis of variance (ANOVA) conducted for measured parameters. Confidence interval calculated at 95% threshold. Mean comparison performed using Tukey test with PLAnt Breeding STATistical software (PLABSTAT), version 3A of 2011-06-14. Data Presentation : Values presented as means of five biological replicas (individual plants) ± standard error (SE). Principal Components Analysis (PCA) : Conducted on correlation matrix using PAST software, version 4.03. PCA applied to data matrix comprising 21 morphological, physiological, and biochemical traits across four Plantago species. Input data included mean values of all parameters measured under different salt stress conditions. Cumulative variability of each parameter, eigenvalues, and principal component scores calculated.

14. Result In this study, morphological, physiological, and biochemical traits were measured to analyse the impact of salinity stress treatments on four Plantago species (P. coronopus , P. crassifolia, P. ovata and P. afra). The 21 traits analysed displayed significant differences.

15. Substrate Analysis Increased with NaCl concentration in irrigation water, showing significant differences between treatments. Highest EC values recorded in pots watered with 800 mM NaCl . EC values: 9.57 dS m^(-1) for P. coronopus and 9.45 dS m^(-1) for P. crassifolia under 800 mM NaCl . No significant differences observed between species for each salt concentration tested. EC not determined ( n.d. ) for treatments resulting in plant death: 800 mM NaCl for P. ovata , and 400 mM and higher salt concentrations for P. afra .

16. Effects of Salt Stress on Plant Growth

17. Effects of Salt Stress on Photosynthetic Pigment Levels

18. Ion accomulation Figure 5. Root ( a,c,e ) and leaf ( b,d,f ) contents of sodium (Na+), ( a,b ), chloride ( Cl -) ( c,d ) and potassium (K+) ( e,f ) in plants of the four selected Plantago species, after four weeks of treatment with the indicated NaCl concentrations. The values shown are means +- SE (n = 5). For each species, different letters over the bars indicate significant differences between treatments , according to the Tukey test (p < 0.5).

19. Discussion Taxonomic and Ecological Classification : The four Plantago species studied belong to two taxonomic groups: P. crassifolia and P. coronopus (halophytes) and P. ovata and P. afra ( glycophytes ). Halophytes are more salt-tolerant than glycophytes , but P. ovata exhibits unexpected salt tolerance, likely due to efficient tolerance mechanisms. Salt Stress Response : Salt stress induces changes in root morphology and growth rate across Plantago species. Relative survival thresholds and stress-induced growth inhibition help rank species' salt tolerance. P. afra is most susceptible to salt stress, while P. crassifolia and P. coronopus are the most stress-tolerant. Chlorophyll Content : Salt stress induces a decrease in chlorophyll levels, corresponding roughly to salt tolerance, with P. ovata being less affected. Ion Concentration and Transport : Glycophytes exhibit lower Na+ content in roots and leaves than halophytes. Chloride accumulation is higher in P. ovata than halophytes, even under low salinity conditions. Leaf K+ content varies across species, with P. ovata showing significant increases under salt stress. Osmotic Adjustment : Proline accumulates significantly in halophytes, contributing to osmotic adjustment. Soluble sugar levels show different accumulation patterns, with P. ovata showing significant increases only at high salinity. Oxidative Stress : MDA content decreases in halophytes but increases in P. ovata and P. afra under salt stress. H2O2 levels increase with NaCl concentration in salt-tolerant species but remain unchanged in P. afra . Antioxidant Compounds : Total phenolic compounds and flavonoids increase in response to NaCl treatments in all species, except flavonoids in P. afra . Flavonoid levels are higher in P. ovata than halophytes at all tested salt concentrations.

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21. Thank you!