PLoS OnePLoS OneplosPLoS ONE1932-6203Public Library of ScienceSan Francisco, CA USA34358230834588810.1371/journal.pone.0248200PONE-D-21-05714Research ArticleBiology and Life SciencesCell BiologyCellular Structures and OrganellesChloroplastsChlorophyllBiology and Life SciencesCell BiologyPlant Cell BiologyChloroplastsChlorophyllBiology and Life SciencesPlant SciencePlant Cell BiologyChloroplastsChlorophyllBiology and Life SciencesCell BiologyCellular TypesPlant CellsChloroplastsChlorophyllBiology and Life SciencesCell BiologyPlant Cell BiologyPlant CellsChloroplastsChlorophyllBiology and Life SciencesPlant SciencePlant Cell BiologyPlant CellsChloroplastsChlorophyllPhysical SciencesMaterials ScienceMaterialsPigmentsOrganic PigmentsChlorophyllBiology and Life SciencesBiochemistryEnzymologyEnzymesDismutasesSuperoxide DismutaseBiology and Life SciencesBiochemistryProteinsEnzymesDismutasesSuperoxide DismutasePhysical SciencesChemistryChemical CompoundsOrganic CompoundsAmino AcidsCyclic Amino AcidsProlinePhysical SciencesChemistryOrganic ChemistryOrganic CompoundsAmino AcidsCyclic Amino AcidsProlineBiology and Life SciencesBiochemistryProteinsAmino AcidsCyclic Amino AcidsProlineBiology and Life SciencesBiochemistryEnzymologyEnzymesPeroxidasesBiology and Life SciencesBiochemistryProteinsEnzymesPeroxidasesPhysical sciencesChemistryChemical compoundsOrganic compoundsVitaminsVitamin EPhysical sciencesChemistryOrganic chemistryOrganic compoundsVitaminsVitamin EPhysical SciencesChemistryChemical CompoundsOrganic CompoundsAmino AcidsAliphatic Amino AcidsGlycinePhysical SciencesChemistryOrganic ChemistryOrganic CompoundsAmino AcidsAliphatic Amino AcidsGlycineBiology and Life SciencesBiochemistryProteinsAmino AcidsAliphatic Amino AcidsGlycineEcology and Environmental SciencesNatural ResourcesWater ResourcesBiology and Life SciencesPlant SciencePlant AnatomyLeavesEffect of exogenous alpha-tocopherol on physio-biochemical attributes and agronomic performance of lentil (Lens culinaris Medik.) under drought stressAmelioration of drought stress tolerance in lentil (Lens culinaris Medik.) using alpha-tocopherolShahWadoodWriting – original draft1https://orcid.org/0000-0002-3177-7360UllahSamiSupervision1*AliSajjadInvestigation2IdreesMuhammadInvestigation3KhanMuhammad NaumanInvestigation2AliKashifSoftware4KhanAjmalSoftware5AliMuhammadWriting – review & editing5YounasFarhanWriting – review & editing6Department of Botany, University of Peshawar, Peshawar, PakistanDepartment of Botany, Bacha Khan University, Charsadda, PakistanDepartment of Chemistry, Bacha Khan University, Charsadda, PakistanSchool of Ecology and Environmental Science, Yunnan University, Kunming, ChinaDepartment of Biotechnology, Bacha Khan University, Charsadda, PakistanCentre of Interdisciplinary Research in Basic Sciences, International Islamic University, Islamabad, PakistanAliBasharatEditorUniversity of Agriculture, PAKISTAN
Competing Interests: The authors have declared that no competing interests exist.
Water being a vital part of cell protoplasm plays a significant role in sustaining life on earth; however, drastic changes in climatic conditions lead to limiting the availability of water and causing other environmental adversities. α-tocopherol being a powerful antioxidant, protects lipid membranes from the drastic effects of oxidative stress by deactivating singlet oxygen, reducing superoxide radicals, and terminating lipid peroxidation by reducing fatty acyl peroxy radicals under drought stress conditions. A pot experiment was conducted and two groups of lentil cultivar (Punjab-2009) were exposed to 20 and 25 days of drought induced stress by restricting the availability of water after 60th day of germination. Both of the groups were sprinkled with α-tocopherol 100, 200 and 300 mg/L. Induced water deficit stress conditions caused a pronounced decline in growth parameters including absolute growth rate (AGR), leaf area index (LAI), leaf area ratio (LAR), root shoot ratio (RSR), relative growth rate (RGR), chlorophyll a, b, total chlorophyll content, carotenoids, and soluble protein content (SPC) which were significantly enhanced by exogenously applied α-tocopherol. Moreover, a significant increase was reported in total proline content (TPC), soluble sugar content (SSC), glycine betaine (GB) content, endogenous tocopherol levels, ascorbate peroxidase (APX), catalase (CAT) peroxidase (POD) and superoxide dismutase (SOD) activities. On the contrary, exogenously applied α-tocopherol significantly reduced the concentrations of malondialdehyde (MDA) and hydrogen peroxide (H2O2). In conclusion, it was confirmed that exogenous application of α-tocopherol under drought induced stress regimes resulted in membrane protection by inhibiting lipid peroxidation, enhancing the activities of antioxidative enzymes (APX, CAT, POD, and SOD) and accumulation of osmolytes such as glycine betaine, proline and sugar. Consequently, modulating different growth, physiological and biochemical attributes.
No funding was given by any source to conduct this study but this study is a part of M.Phil/MS degree of Mr. Wadood Shah for which Botany Department, University of Peshawar, Pakistan provided the laboratory facilities.Data AvailabilityAll relevant data are within the manuscript.Data Availability
All relevant data are within the manuscript.
1. Introduction
Changing climatic condition is becoming an obstacle in fulfilling the demand of food and achieving a sustainable agriculture; climatic changes result in droughts, heavy floods, earthquakes, fluctuation in temperature and other environmental adversities, that ultimately lead to reduce crop productivity [1]. Amongst major abiotic stresses, drought stress has a profound effect on crop growth and yield reduction; though, plants can often withstand limited water condition but at the cost of substantial amount of plant total biomass and yield loss, drought stress condition disturbs vital physiological and biochemical processes ultimately declining plant growth and production [2]. About 50% of the world’s arid and semi-arid regions are being exposed to some kind of drought stresses [3]. With increasing climatic changes crops are losing their yield potential, thus making it hard to fulfil the increasing demand of food around the world [4]. The population of Pakistan is rapidly increasing with a growth rate of 2.1%, which is higher than the growth rate (1.1%) of world population. Keeping in mind the present scenario of climate change, it is predicted that 2.1 million hectares of land of Pakistan will be affected by drought by 2025 [5]. In general, during the spring season crops of Punjab suffer from drought stress due to higher rate of transpiration and elevated temperature. Most parts of the central, southern Punjab and parts of eastern Sindh do not fall under the domain of winter rains, and 50% of the time remains dry, thus considered drought susceptible zones [6].
Abiotic stresses affect photosynthesis, cell growth, development and other vital physiological and biochemical processes [7]. According to previous research findings, it is evident that water shortage in plants prompts oxidative stress in the forms of free radicals and non-radicals; both of the oxidants produced in response to abiotic stress damage biological membranes and other essential biomolecules such as proteins, lipids, chlorophyll and DNA [8]. At the onset of stress conditions, plants tend to accumulate various types of osmolytes such as sugars, proteins, proline and glycine betaine. Osmolytes chiefly accumulate in the cytoplasm preventing cellular degradation and maintain osmoregulation. Owing to their non-toxic nature and high solubility they do not impede other physio-biochemical processes [9].
Drought stress tolerance can be enhanced by using latest techniques like genetic engineering and tissue culture; however, these methods are expensive and inflict long term health concerns.α-tocopherol being a powerful antioxidant, attenuating the negative impacts of drought stress by scavenging free radicals and inhibiting lipid peroxidation, thus shielding biological membranes against oxidative stress. It is suggested that one molecule of a-tocopherol can deactivate 120 molecules of 1O2 under drought stress condition [10]. After encountering drought stress regimes, plants trigger powerful antioxidant systems in the form of vitamins, flavonoids, carotenoids and antioxidant enzymes mainly including peroxidase, catalase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase [11].
Lentil (Lens culinaris Medik.) is annual self-pollinated specie belonging to family Leguminacae (Fabaceae). It is widely grown in South Asia, Middle East, North America, North Africa, and Australia. Protein content of lentil seed ranges from 22% to 34.6% making it the third highest level of protein of any legume or nut after soybeans and hemp. Lentil is one of the major cash crops of Pakistan and it is extensively grown in Punjab and Khyber pakhtunkhwa province of Pakistan [12].
The present research work was aimed to assess growth, physiological and biochemical responses of lentil cultivar (Punjab-2009) to varying levels of exogenously applied α-tocopherol, its potential in alleviating the negative impacts of drought induced stress and to explore the degree of efficacy of α-tocopherol in regulating vital metabolic processes by ameliorating drought tolerance potential of lentil cultivar subjected to varying levels of drought induced stress.
2. Materials and methods2.1. Site description and experimental design
Field experiment was carried out at the Department of Botany, University of Peshawar (34° 1’ 33.3012’’ N and 71° 33’ 36.4860’’ E.) Pakistan, during the growing season 2019. Peshawar is situated in Iranian plateau area having tropical climate. Soil texture was determined as sandy loam as evaluated via hydrometer method by [13].
The seeds of lentil (Lens culinaris Medik.) variety punjab-2009 were obtained from National Agriculture Research Centre (NARC) Islamabad, Pakistan. Surface sterilized seeds were sown 2–4 centimetres deep in the soil-filled earthen pots (20cm height, 18cm upper/lower diameter and 2cm thickness) with each pot containing 3kg sandy loam soil. Experiment was conducted in Randomized Complete Block Design (RCBD). Thinning and weeding were properly maintained and seedlings were exposed to sunlight for better growth. Three replicates were taken for each group. All the groups were normally watered till 60th day of emergence. Different levels (100, 200 and 300 mg/L) of α-tocopherol were prepared by mixing 100, 200 and 300 mg of α-tocopherol separately in 900 ml distilled water and 70% ethanol (9:1) followed by heating at 33°C for 15 minutes. After 60 days of germination, one set of trial was exposed to 20-days of drought-induced stress and sprayed with three levels of α-tocopherol (100, 200, and 300 mg/L) once throughout the growing season. The second set of experiment was subjected to 25 days of drought-induced stress and sprayed with the same levels of exogenously applied α-tocopherol. At the end of drought-induced stress periods, five plants from each replicate were harvested randomly for the determination of various growth and physio-biochemical parameters.
2.2. Soil analysis
Over the last two years average soil chemical properties were as follows: electrical conductivity (EC) 2.67 ds/m, pH 6.3 [14], Nitrogen (N) content 4.02g/kg [15], organic Carbon (C) 23.3 g/kg [16], available potassium (K) 91.4 mg/kg [17] and Phosphorus (P) 8.1 mg/kg [18].
2.3. Growth measurements
Growth parameters: absolute growth rate (AGR), relative growth rate (RGR), coefficient of velocity of germination (CVG) and net assimilation rate (NAR) were calculated by following the formulas suggested by [19].
AGR(plantheight)=H2−H1t2−t1
H1 and H2 denoted plant height (cm) during the time t1 to t2.
RGR=logeW2−logeW1t2−t1
W1 and W2 denoted plant dry weight (gm) at time t1 and t2, loge is natural logarithm.
CVG=N1+N2+N3+⋯+NX100(N1T1+N2T2+N3T3+⋯+NXTX)
The CVG indicates the pace of germination. Hypothetically, the maximum CVG possible is 100. This would happen if all seeds germinate on 1st day.
NAR=W2−W1t2−t1×logeA2−log1A1A2−A1(g/cm/day)
A1 and A2 denoted surface area of leaf and W1 and W2 are plant total dry matter at Time t1 and t2.
Crop growth rate (CGR), leaf area index (LAI), leaf area ratio (LAR) and relative water content (RWC) were calculated using the following formulae suggested by [20]:
CGR=W2−W1T2−T1×1Landarea(g/m2/d)
W1 and W2 are plant dry weights taken at time T1 and T2, respectively.
LAI=leafarea(cm)2landarea(cm)2LAR=leafareafinalplantdryweightRWC=Wf−WdWs−Wd×100(%)
“Wf” represented leaf fresh weight and “Wd” leaf dry weight. “Ws” indicated saturated weight of leaf material determined after floating the leaves in distilled water for 18 hours.
Root-shoot ratio (RSR) was calculated by using the formula proposed by [21]:
RSR=rootdrymassshootdrymass
Seed vigor index (SVI) was measured by the formula proposed by [22]:
SVI=Seedlinglength(cm)xSeedgermination%age
2.4. Photosynthetic pigments (chlorophyll a, b & carotenoids)
Fresh leaf material (0.5 gm) was homogenized in 10 ml 80% acetone solution. Samples containing homogenized solution were kept in centrifuge machine and spun for 5 minutes. After centrifugation, the samples were kept in dark overnight at 4°C. On the following day, optical density of each sample was measured at 470, 645 and 663 nm for carotenoids and chlorophyll a & b quantification by following the protocol of [23].
2.5. Soluble sugar content (SSC)
Fresh foliar material (0.5 gm) was taken and grounded in 5 ml distilled water and homogenized mixture was prepared. The samples containing homogenized mixture were placed in centrifuge machine and spun for 10 minutes; after the process of centrifugation, 1ml supernatant was taken from each sample and 4 ml concentrated (35%) H2SO4 was added. Optical density (OD) was noted at 490 nm by adopting the methodology of [24].
2.6. Total proline content (TPC)
Proline content was quantified by the methodology of [25]. Fresh leaves (0.5 gm) were grounded in 10 ml 3% aqueous sulphosalicylic acid and a homogenized mixture was prepared. The mixture was filtered and 2 ml filtrate was taken. Similarly, 4 ml ninhydrin solution and 4 ml glacial acetic acid (20%) were mixed with 2 ml filtrate taken. The mixture was heated at 100°C for 1 hour and 4 ml toluene was added to it. OD readings were recorded at 520 nm.
2.7. Glycine betaine content (GBC)
Fresh foliar material (0.5gm) was chopped in 10 ml distilled water. The mixture was filtered; filtrate obtained was diluted by adding 2 ml H2SO4 solution. The samples were centrifuged for 10 minutes and Cold potassium iodide (KI–I2) was added to supernatant. 1 ml supernatant was collected from each sample and optical density was measured at 365 nm by using the methodology of [26].
2.8. Soluble protein content (SPC)
Protein content in leaf tissues were investigated by following the protocol of [27]. 0.5 gm fresh leaf tissues were grounded in 5 ml phosphate buffer (pH 7.0) in ice cooled pestle and mortar. After grinding a homogenized mixture was obtained, samples from the prepared mixture were kept in centrifuge machine and spun for 15 minutes. After centrifugation, 0.1 ml supernatant was taken from each sample and 2 ml Bradford reagent was added. Optical density was measured at 595 nm.
2.9. Malondialdehyde content (MDAC)
Fresh leaf material (0.25 gm) was chopped in 3 ml 1.0% (w/v) Trichloro acetic acid (TCA) and mixture was prepared. Samples containing homogenized mixture were kept in centrifuge machine and spun in for 10 minutes. After the process of centrifugation, 1 ml supernatant was taken and 4 ml 0.5% (w/v) 2-thiobarbituric acid was added. Samples were heated at 95°C for 1 hour and then cooled by placing in ice bath for 10 minutes. Optical density was measured at 532 nm by the following the method of [28].
2.10. Hydrogen peroxide content (HPOC)
Fresh foliar material (0.5 gm) was chopped in 5 ml trichloro acetic acid (TCA) and homogenous mixture was prepared. The samples were placed in centrifuge machine and rotated for 15 minutes. After centrifugation, 0.5 ml supernatant was taken from each sample and 0.5 ml phosphate buffer and 1.0 ml potassium iodide (KI) reagent was added to it. Optical density was recorded at 390 nm by following the methodology of [29].
2.11. Endogenous tocopherol content (ETPC)
Methodology of [30] was followed to measure the levels of endogenous tocopherol content in leaf tissues. Leaf material (0.1 g) was grounded in 10 ml solution (petroleum ether and ethanol 2:1.6 v/v) and homogenized mixture was prepared. Samples containing homogenized mixture were placed in centrifuge and spun for 15 minutes. After centrifugation, 1 ml supernatant was taken and mixed with 0.2 ml (2%) 2-dipyridyl in ethanol (v/v). The mixture was poured into cuvete and placed in spectrophotometer. Optical density was measured at 520 nm.
2.12. Ascorbate peroxidase activity (APX)
Ascorbate peroxidase (APX) levels were evaluated by pursuing the method of [31]. Fresh leaf material (0.5 gm) was grounded in 5 ml phosphate buffer (pH 7.0). The samples were placed in centrifuge machine and rotated for 15 minutes. After the process of centrifugation, 0.2 ml supernatant was collected from each sample and 0.1 mM hydrogen peroxide, 0.6 mM ascorbic acid and 0.1 mM ethylenediamine tetraacetic acid (EDTA) was added. Optical density was recorded at 290 nm.
2.13. Catalase activity (CAT)
Catalase activity was measured by following the protocol of [32]. Fresh foliar material (0.5gm) was homogenized in 5 ml buffer solution (pH 7.0). The samples of the mixture were kept in centrifuge machine and rotated at 3000 rpm for 15 minutes. After centrifugation, 0.1 ml supernatant was taken and 1.9 ml phosphate buffer (50 Mm) and 0.1 ml H2O2 (5.9 mM) was added. Optical density readings were noted at 240 nm for 3 minutes.
2.14. Superoxide dismutase activity (SOD)
Fresh foliar material (0.5 gm) was grounded in 5 ml phosphate buffer and homogenized mixture was prepared. The samples of the mixture were placed in centrifuge machine and spun for 15 minutes. After centrifugation, 0.1 ml supernatant was taken from each sample and 5 ml methionine, 150 μl riboflavin and 24 μl nitro-blue-tetrazolium (NBT) were added. Optical density was recorded at 560 nm by pursuing the method of [33].
2.15. Peroxidase activity (POD)
Peroxidase (POD) activity was determined by following the methodology of [32]. Leaf material (0.5 gm) was chopped in 2 ml morpholino ethane sulphonic acid (MES) and homogenized mixture was prepared. The samples were placed in centrifuge machine and spun for 15 minutes. After centrifugation, 0.1 ml supernatant was collected from each sample and 1.3 ml MES, 0.1 ml phenyl diamine and 1 ml hydrogen peroxide (30%) were added. OD was noted at 470 nm for 3 minutes via spectrophotometer.
2.16. Statistical analysis
The experiment comprised of two factors including drought induced stress of 20 and 25 days and α-tocopherol levels, 100, 200 and 300 mg/L. Randomized complete block design (RCBD) was adopted for experiment and three replicates were taken for each group. SPSS Statistic-25 software was used for analysis of variance (ANOVA). By using standard techniques, mean and standard errors were calculated and least significance difference (LSD) test at (p≤0.05) was performed and indicated by letters (A-E). Correlation analysis was performed by using Statistix 8.1 software.
3. Results3.1. Growth responses under drought induced stress and α-tocopherol levels
Statistical analysis revealed a significant increase at (P≤0.05) in AGR, CGR, RGR and LAI with exogenously applied α-tocopherol 200 mg/L in comparison with control group and rest of the treatments under 20-days of drought induced stress. On the contrary, these parameters were affected negatively under 20-days of drought induced stress with no α-tocopherol treatment (Fig 1A–1D). NAR and CVG showed improvement at (P≤0.05) in control group and group with 100 mg/L α-tocopherol treatment (Figs 2A and 3B). Furthermore, RSR showed significant improvement at (P≤0.05) with α-tocopherol 200 mg/L and was affected adversely on exposure to drought induced stress with no α-tocopherol application (Fig 2C). In contrast with other treatments, RWC was recorded maximum in the control group only (Fig 2D). In comparison with the rest of the treatments and control group LAR showed significant results at (P≤0.05) with 200 mg/L α-tocopherol. On the contrary, LAR was adversely affected under induced water stress condition with no α-tocopherol treatment. SVI showed positive response at (P≤0.05) in control group only (Fig 3A and 3B). Same growth parameters were studied under 25-days of drought induced stress sprayed with the same levels of α-tocopherol. Results showed significant improvement at (P≤0.05) in AGR, LAI, and LAR with the application of 200 mg/L α-tocopherol only. On the other hand, AGR, CGR, LAI, LAR and NAR showed negative responses under drought induced stress of 25-days with no α-tocopherol application. Highest NAR and CVG values were calculated for control group and 100 mg/L α-tocopherol treatment in contrast with the rest of the treatments.
10.1371/journal.pone.0248200.g001
Effect of varying levels of exogenously applied α-tocopherol on absolute growth rate (a) crop growth rate (b) relative growth rate (c) leaf area index (d) of lentil (Lens culinaris Medik.) grown under varying drought stress condition (Mean ± standard error.) letters (A–E) indicating least significance difference among the mean values at p≤0.05.
10.1371/journal.pone.0248200.g002
Effect of varying levels of exogenously applied α-tocopherol on net assimilation rate (a) coefficient of velocity of germination (b) root-shoot ratio (c) relative water content (d) of lentil (Lens culinaris Medik.) grown under varying drought stress condition (Mean ± standard error.) letters (A–E) indicating least significance difference among the mean values at p≤0.05.
10.1371/journal.pone.0248200.g003
Effect of varying levels of exogenously applied α-tocopherol on leaf area ratio (a) seed vigor index (b) of lentil (Lens culinaris Medik.) grown under varying drought stress condition (Mean ± standard error.) letters (A–E) indicating least significance difference among the mean values at p≤0.05.
RGR and RSR both showed significant enhancement at (P≤0.05) with the application of 200 mg/L α-tocopherol; however, both the parameters were affected negatively on exposure to 25-days of drought induced stress with no α-tocopherol treatments. Subsequently, in comparison with the rest of treatments RWC and SVI values were highest only in the control group.
3.2. Determination of photosynthetic pigments (chlorophyll a, b & carotenoids)
Varying levels of drought induced stress markedly (P≤0.05) reduced chlorophyll “a” content. Application of α-tocopherol enhanced (P≤0.01) chlorophyll “a” content in both the drought induced stress levels. In contrast with control group and rest of the treatments sprayed with 200 mg/L tocopherol showed maximum chlorophyll “a” content (Table 1 and Fig 4A). Drought stress condition significantly (P≤0.01) decreased chlorophyll “b” content; however, α-tocopherol ameliorated (P≤0.05) the levels of chlorophyll “b” content. Among all the treatments and control group application of α-tocopherol 200 mg/L showed better response in improving the levels of chlorophyll “b” content (Table 1 and Fig 4B).
10.1371/journal.pone.0248200.g004
Effect of varying levels of exogenously applied α-tocopherol on chlorophyll “a” content (a) chlorophyll “b” content (b) total chlorophyll content (c) carotenoid content (d) of lentil (Lens culinaris Medik.) grown under varying drought stress condition (Mean ± standard error.) letters (A–F) indicating least significance difference among the mean values at p≤0.05.
10.1371/journal.pone.0248200.t001
Analysis of variance of physio-biochemical attributes of lentil cultivar to varying levels of α-tocopherol under drought induced stress.
Traits
Source of variation
SS
Df
MS
F
P
Chl “a”
Cultivar
0.162
2
0.081
0.664
0.523
Drought
2.023
9
0.225
3.135
0.016*
Drought × Treatment
1.861
7
0.266
3.708
0.001**
Error
1.434
20
0.072
-
-
Chl "b"
Cultivar
0.557
2
0.278
2.993
0.067*
Drought
1.936
9
0.215
3.802
0.006**
Drought × Treatment
1.379
7
0.197
3.482
0.013*
Error
1.131
20
0.057
-
-
TCC
Cultivar
1.226
2
0.613
2.043
0.149
Drought
7.316
9
0.813
8.091
0.000***
Drought × Treatment
6.090
7
0.870
8.659
0.000***
Error
2.009
20
0.100
-
-
CC
Cultivar
0.771
2
0.385
1.134
0.337
Drought
7.765
9
0.863
7.900
0.000***
Drought × Treatment
6.995
7
0.999
9.148
0.000***
Error
2.184
20
0.109
-
-
SSC
Cultivar
8.585
2
4.292
40.732
0.000***
Drought
10.392
9
1.155
22.250
0.001***
Drought × Treatment
1.807
7
0.258
4.975
0.002***
Error
1.038
20
0.052
-
-
SPC
Cultivar
0.676
2
0.338
0.961
0.395
Drought
7.843
9
0.871
7.478
0.000***
Drought × Treatment
7.167
7
1.024
8.785
0.000***
Error
2.331
20
0.117
TPC
Cultivar
8.769
2
4.385
70.578
0.000***
Drought
9.749
9
1.083
31.064
0.000***
Drought × Treatment
0.980
7
0.140
4.014
0.007**
Error
0.697
20
0.035
-
-
POD
Cultivar
4.433
2
2.216
28.347
0.005**
Drought
5.315
9
0.591
9.611
0.002**
Drought × Treatment
0.882
7
0.126
2.051
0.008**
Error
1.229
20
0.061
-
-
SOD
Cultivar
8.353
2
4.177
36.118
0.002**
Drought
9.876
9
1.097
13.720
0.004**
Drought × Treatment
1.523
7
0.218
2.720
0.007**
Error
1.600
20
0.080
-
-
APX
Cultivar
8.394
2
4.197
16.699
0.034*
Drought
10.032
9
1.115
11.512
0.002**
Drought × Treatment
1.638
7
0.234
2.417
0.006**
Error
1.936
20
0.097
-
-
CAT
Cultivar
9.864
2
4.932
59.922
0.005
Drought
11.200
9
1.244
12.849
0.003**
Drought × Treatment
1.336
7
0.191
10.866
0.001**
Error
0.351
20
0.018
-
-
HPOC
Cultivar
1.122
2
0.561
2.070
0.146
Drought
4.910
9
0.546
3.091
0.017*
Drought × Treatment
3.788
7
0.541
3.065
0.023*
Error
3.531
20
0.177
-
-
ETPC
Cultivar
4.227
2
2.113
15.269
0.000***
Drought
5.709
9
0.634
5.629
0.001**
Drought × Treatment
1.483
7
0.212
1.880
0.007**
Error
2.254
20
0.113
-
-
MDAC
Cultivar
1.035
2
0.517
3.927
0.032*
Drought
3.820
9
0.424
11.020
0.000***
Drought × Treatment
2.786
7
0.398
10.331
0.000***
Error
0.770
20
0.039
-
-
GBC
Cultivar
3.628
2
1.814
13.427
0.000***
Drought
3.804
9
0.423
2.435
0.047*
Drought × Treatment
0.176
7
0.025
0.145
0.003**
Error
3.472
20
0.174
-
-
Chl “a” = Chlorophyll “a”, Chl “b” = Chlorophyll “b”, TCC = Total chlorophyll content, CC = Carotenoid content, SSC = Soluble sugar content, SPC = Soluble protein content, TPC = Total proline content, POD = Peroxidase, SOD = Superoxide dismutase, APX = Ascorbate peroxidase, CAT = catalase, HPOC = Hydrogen peroxide content, ETPC = Endogenous tocopherol content, MDAC = Malondialdehyde content, GBC = Glycine betaine content. (* Significant at the P = 0.05, ** Significant at the P = 0.01, *** Significant at the P = 0.001) SS = sum of square, Df = degree of freedom, MS = mean square, F = variation between sample means, P = probability value.
Drought induced stress regimes considerably (P≤0.001) reduced the levels of total chlorophyll content, among all the treatments and control group. α-tocopherol 200 mg/L boosted (P≤0.001) the levels of total chlorophyll content in both (20 and 25 days) of drought induced stress levels (Table 1 and Fig 4C). Drought stress condition reduced (P≤0.001) the levels of carotenoid content to a great extent. As compared to other treatments and control, 100 mg/L of α-tocopherol application showed significant (P≤0.001) response in increasing the levels of carotenoid content (Table 1 and Fig 4D).
3.3. Changes in concentration of soluble sugar content (SSC)
Drought stress condition had a significant influence on the concentration of soluble sugar content. Limited water regimes raised (P≤0.001) the levels of soluble sugar content in both the drought levels. Exogenously applied α-tocopherol further raised (P≤0.001) the levels of soluble sugar content. In comparison with control and other applied treatments, α-tocopherol 200 mg/L proved more effective in ameliorating soluble sugar content (Table 1 and Fig 5A).
10.1371/journal.pone.0248200.g005
Effect of varying levels of exogenously applied α-tocopherol on soluble sugar content (a)soluble protein content (b) total proline content (c) peroxidase content (d) of lentil (Lens culinaris Medik.) grown under varying drought stress condition (Mean ± standard error.) letters (A–F) indicating least significance difference among the mean values at p≤0.05.
3.4. Effects on soluble protein content (SPC)
Drought stress inflicted a profound effect on soluble protein content as it markedly reduced (P≤0.001) the levels of soluble protein content. Among all the treatments, α-tocopherol 200 mg/L was significant in raising the levels of soluble protein content (Table 1 and Fig 5B).
3.5. Fluctuations in the levels of total proline content (TPC) and Glycine betaine content (GBC)
Drought stress condition increased total soluble proline content to a considerable level (P≤0.001). Application of α-tocopherol further enhanced (P≤0.01) soluble proline content under varying drought stress regimes. Among different α-tocopherol levels, 100 mg/L showed better results (Table 1 and Fig 5C).
On exposure to drought induced stressed condition a significant increase was noted in the activities of POD (P≤0.01) (Table 1 and Fig 5D), SOD (P≤0.01), APX (P≤0.01), and CAT (P≤0.01). Foliar application of α-tocopherol further significantly (P≤0.01) enhanced the activities of these enzymes. In case of peroxidase and superoxide dismutase 100 mg/L α-tocopherol treatment proved more effective while in the case of ascorbate peroxidase and catalase 200 mg/L α-tocopherol responded better as compared to the rest of the treatments and control group (Table 1 and Fig 6A–6C).
10.1371/journal.pone.0248200.g006
Effect of varying levels of exogenously applied α-tocopherol on superoxide dismutase content (a) ascorbate peroxidase content (b) catalase content (c) hydrogen peroxide content (d) of lentil (Lens culinaris Medik.) grown under varying drought stress condition (Mean ± standard error.) letters (A–E) indicating least significance difference among the mean values at p≤0.05.
3.7. Changes in the concentration of hydrogen peroxide content (H2O2)
Drought stress condition caused a marked (P≤0.05) increase in hydrogen peroxide concentration. In comparison with control group and rest of the applied treatments α-tocopherol 200 and 300 mg/L showed a significant (P≤0.05) response in alleviating the levels of hydrogen peroxide content under limited water condition (Table 1 and Fig 6D).
3.8. Levels of endogenous tocopherol content (ETPC)
Drought stress condition resulted in the accumulation of endogenous/membrane bounded tocopherol content to a level significant at (P≤0.01). Among all the foliar applied α-tocopherol treatments 100 mg/L tocopherol increased the levels of endogenous tocopherol content to a significant (P≤0.01) level (Table 1 and Fig 7A).
10.1371/journal.pone.0248200.g007
Effect of varying levels of exogenously applied α-tocopherol on endogenous tocopherol content (a) malondialdehyde content (b) glycine betaine content (c) of lentil (Lens culinaris Medik.) grown under varying drought stress condition (Mean ± standard error.) letters (A–E) indicating least significance difference among the mean values at p≤0.05.
3.9. Changes in the concentration of Malondialdehyde (MDAC) content
Water deficit stress significantly (P≤0.001) raised the concentration of malondialdehyde content. On the contrary, α-tocopherol application reduced the concentration of malondialdehyde to a considerable level (P≤0.001). However, in both the drought levels α-tocopherol 100 mg/L showed better response in decreasing the concentrations of malondialdehyde (Table 1 and Fig 7B).
3.10. Fluctuations in the levels of total Glycine betaine content (GBC)
Water deficiency triggered a marked (P≤0.05) increase in glycine betaine content. Foliar applied α-tocopherol further improved the concentration of glycine betaine content. α-tocopherol 200 mg/L showed better response in increasing the concentrations of glycine betaine content (Table 1 and Fig 7C).
3.11. Correlation between drought induced stress, α-tocopherol and physio-biochemical attributes
Positively significant (P≤0.05) correlation was noted between varying drought stressed condition and physio-biochemical attributes of lentil including malondialdehyde, hydrogen peroxide, glycine betaine, soluble sugar, total proline, endogenous tocopherol contents and activities of antioxidant enzymes including peroxidase, superoxide dismutase, catalase and ascorbate peroxidase; whereas, chlorophyll a, b, total chlorophyll content, carotenoid content, and total soluble protein content correlated negatively with both 20 and 25 days of drought induced stressed regimes. A positive and significant correlation was observed between varying levels of exogenously applied α-tocopherol and chlorophyll a, b, total chlorophyll content, carotenoid content, soluble sugar content, soluble protein content, total proline content, endogenous tocopherol content, glycine betaine content and activities of antioxidant enzymes (APX, CAT, SOD and POD); however, a significantly negative correlation was observed between α-tocopherol levels and concentrations of hydrogen peroxide and Malondialdehyde content (Table 2).
10.1371/journal.pone.0248200.t002
Correlation between induced drought stress, α-tocopherol and physio-biochemical attributes of lentil cultivar.
Correlation analysis between drought stress and physio-biochemical attributes
Chl “a”
Chl "b"
TCC
CC
SSC
SPC
TPC
POD
SOD
APX
CAT
HPOC
ETPC
MDAC
GBC
-0.421*
-0.562*
-0.601*
-0.654*
0.862*
-0.854*
0.907*
0.799*
0.885*
0.854*
0.833*
0.968*
0.777*
0.961*
0.882*
Correlation analysis between tocopherol and physio-biochemical attributes
Chl “a”
Chl "b"
TCC
CC
SSC
SPC
TPC
POD
SOD
APX
CAT
HPOC
ETPC
MDAC
GBC
0.893*
0.884*
0.899*
0.903*
0.911*
0.941*
0.993*
0.887*
0.918*
0.889*
0.924
-0.551*
0.889*
-0.647*
0.919*
Chl “a” = Chlorophyll “a”, Chl “b” = Chlorophyll “b”, TCC = Total chlorophyll content, CC = Carotenoid content, SSC = Soluble sugar content, SPC = Soluble protein content, TPC = Total proline content, POD = Peroxidase, SOD = Superoxide dismutase, APX = Ascorbate peroxidase, CAT = catalase, HPOC = Hydrogen peroxide content, ETPC = Endogenous tocopherol content, MDAC = Malondialdehyde content, GBC = Glycine betaine content. (* Significant at the P = 0.05, ** Significant at the P = 0.01, *** Significant at the P = 0.001).
4. Discussion4.1. Growth responses
Oxidative stress and higher production of ROS have been considered the major causes that adversely affect plant growth [34]. Reduction in plant growth parameters such as LAI and LAR have been reported to be mainly caused by stomatal closure during drought stress condition, that eventually leads to leaf senescence [35]. On exposure to drought stress condition, plants tend to close their stomata which slow down the water loss from aerial parts of the plants. As a result, the carbon dioxide (CO2) absorption is reduced and consequently net photosynthesis [36]. Moreover, reduction in growth attributes such as CVG, CGR, AGR, RGR, RSR, SVI and NAR are closely linked with suppression of cell elongation and cell growth under drought stress regimes [37]. In the present study, induced water deficit stress impacted negatively on lentil growth and physio-biochemical mechanisms. On the contrary, foliar applied α-tocopherol curbed the negative consequences of drought induced stress by regulating key metabolic activities and boosted the growth parameters including AGR, CGR, LAI, LAR, NAR and CVG (Figs 1–3). Induced water deficit stress caused a considerable reduction in AGR, CGR, LAI, LAR and NAR of lentil. Likewise, [38,39] also observed reduction in these growth parameters under drought induced stress conditions. It is believed that this reduction is due to stomatal closure, persistent exposure to limited water regimes ultimately leading to shrinkage of leaves [38,40]. Similar variations in growth parameters were recorded by other workers [41,42]. Results from statistical analysis in Figs 1–3 indicated that RSR, RGR, RWC and SVI were improved in the control group and affected negatively in groups subjected to varying drought stress levels with no α-tocophrol treatments, confirming the same investigations made by [43].
4.2. Physiological and biochemical responses
Under drought stress conditions, an increase in chlorophyll degrading enzymes activity prompts the destruction of chlorophyll pigments [44]. Drought stress causes chlorophyll pigments degeneration and hampering the process of photosynthesis [45,46]. Chloroplast is the most sensitive organelle to drought stress exposure, and it has been proved that drought stress damages photosynthetic pigments in various crops [47,48].
It is suggested that the degeneration of chlorophyll is associated with the production of ROS, which lowers down the rate of photosynthesis and increases cellular respiration [49,50]. In the present study, photosynthetic pigments (chlorophyll a, b and carotenoid content) were affected negatively on exposure to drought induced stress. In contrast, exogenously applied α-tocopherol significantly enhanced the levels of photosynthetic pigments (Fig 4A–4D). α-tocopherol protects the chloroplast membranes from photo oxidation and assists to provide suitable environment for the photosynthetic machinery to work efficiently under oxidative stress condition. Accumulation of tocopherol in biological membranes occurs as a response to various types of abiotic stresses including drought, high temperature, salinity and cold [51]. Additionally, tocopherol contributes to membrane integrity and stability by manipulating its permeability and fluidity [52]. Our results were in accordance with the previous research work carried out by [10,53,54].
In response to abiotic stress, plants perceive a disturbance in their physiological activities and respond abruptly by accumulating a variety of osmolytes mainly including glycine betaine and proline. Osmolytes provide suitable environment for various metabolic activities and protecting plants from the damages caused by oxidative stress described by [55]. Compounds like proline, MDA and H2O2 are generally being used as stress markers. Proline is well known for its osmoprotective role. In many plants an increased concentration of proline during drought stress condition indicated to be correlated with drought stress tolerance [56]. It is also evident that proline has the potential to directly act as ROS scavenger and regulator of cellular redox status [57]. The studied lentil cultivar revealed a marked rise in soluble sugar content (Fig 5A), total proline content (Fig 5C), and glycine betaine content (Fig 7C) by experiencing water deficit stress condition. Similarly, foliar applied α-tocopherol further increased the concentrations of these osmolytes. Our results were consistent with the investigation made by [58] in Chinese rye grass (Leymus chinensis) in which a high level of proline was observed in drought stressed seedlings grown from seeds primed with α-tocopherol. The same results were recorded by [59] in soybean (Glycine max) and in faba bean (Vicia faba) by [60].
Water-limited condition causes protein and lipid degeneration and affect plant growth and other vital activities [61]. In case of lentil, it was found that low moisture content in the soil alleviated the soluble protein content to a great extent. Application of α-tocopherol (200 mg/L) was significant in raising the levels of soluble protein content (Fig 5B). [54] Concluded that flax (Linum usitatissimum) plants sprayed with α-tocopherol under salinity stress caused a marked increase in soluble protein content. Similarly, [60] suggested that application of (100 mg/L) of α-tocopherol in faba bean (Vicia faba) plants proved significant in preservation of soluble protein content.
The main role of a-tocopherol is the removal of lipid peroxyl radical prior of its attack to target lipid substrate synthesizing a-tocopheroxyloxyl radicals [62]. Under abiotic stress condition, α-tocopherol deactivates 1O2 in chloroplast; according to an estimate a single molecule of α-tocopherol can deactivate 120 molecules of 1O2 [63]. Membrane bounded tocopherol levels were observed maximum in lentil plants exposed to drought stress regimes. Application of α-tocopherol (100 mg/L) proved more significant in raising the levels of membrane bounded tocopherol content (Fig 7A). Our findings in the case of lentil were supported by the investigations made by [64] who reported high levels of endogenous tocopherol contents in maize plants cultivated under induced water stress condition [65]. Confirmed same results in canna (Canna edulis) cultivars under induced drought stress condition.
Malondialdehyde (MDA) is the product of membrane degradation. Under abiotic stress condition, a rise in MDA concentration marks the disintegration of biological membranes [66]. Accumulation of MDA has been considered an indication of lipid peroxidation in various plants under stress condition [67,68]. In plant tissues peroxidation of free fatty acid could occur both in non-enzymatic and enzymatic ways, producing a number of breakdown products which mainly include alcohol, aldehydes and their esters and this process is considered to be mainly involved in oxidative damage to cellular membranes and other biomolecules [69]. Physiological analysis of lentil cultivar revealed an increase in MDA content under drought stress condition. Whereas, exogenously applied α-tocopherol significantly decreased levels of MDA content (Fig 7B). The same results were obtained by [70] in case of geranium (Pelargonium graveolens) treated with (100 mg/L) α-tocopherol.
Hydrogen peroxide (H2O2) being a prominent reactive oxygen species (ROS) in plants results in cell oxidation, disturbs vital metabolic processes and interrupts membrane stability under stress condition [71]. Plants naturally produce ROS, mainly including H2O2 superoxides, there is a delicate balance between ROS production and it’s scavenging, under drought stress condition this balance is disturbed as plant tend to close their stomata which also limits CO2 fixation [72]. In present study, a significant increase was observed in H2O2 content under drought stress condition. Application of α-tocopherol decreased the concentration of H2O2 to a considerable level (Fig 6D). Parallel to our results, [73] recorded that application of resveratrol and α-tocopherol decreased the levels of H2O2 in citrus plants subjected to salinity stress. The same results were reported by [74] who found that (100 mg/L) α-tocopherol proved better in lowering the levels of H2O2 in wheat plants exposed to salinity stress.
Plants counteract the oxidative stress generated by ROS in a coordinated way both enzymatically and non-enzymatically [75,76]. SOD act as first line of defence as it converts the superoxide radicals to H2O2 [46,77]. Likewise, [78] in their demonstration on Carthamus tinctorius cultivars subjected to drought stress, concluded that enzymatic as well as non-enzymatic antioxidants were involved in the removal of ROS, SOD is needed to scavenge superoxide radical [79], while scavenging H2O2, requires POD, CAT and APX [80]. Natural self-defence systems are well developed in plants. On exposure to stress condition these defence systems are activated both enzymatically (ascorbate peroxidase, catalase, superoxide dismutase and peroxidase) and non-enzymatically (secondary metabolites) scavenging the reactive oxygen species (ROS) formed as result of stress condition [76]. Though, activities and response of these antioxidants varies plant to plant [78]. In the present research study, water stress condition caused a prominent increase in the activities of antioxidant enzymes such as, POD (Fig 5D), SOD, APX, and CAT (Fig 6A–6C). However, foliar applied α-tocopherol further enhanced the activities of these enzymes. Our results are in agreement with the findings made by [58] who observed a marked increase in SOD and POD activities in Chinese rye grass (Leymus chinensis) subjected to induced water stress condition. Similarly, [81] reported an increase in the activities of POD and CAT in sunflower plants under salt stress. Furthermore, [60] reported that, application of α-tocopherol (200 mg/L) enhanced the performance of antioxidant enzymes to a great extent in drought stress plants of faba beans (Vicia faba).
5. Conclusions
From the present study, it was concluded that application of exogenous α-tocopherol under drought induced stress conditions prevented membrane degeneration by hampering lipid peroxidation, improving the levels of osmolytes such as glycine betaine, proline, protein and sugar. Moreover, exogenously applied α-tocopherol ameliorated the activities of antioxidant enzymes including APX, CAT, POD, and SOD. Resultantly, regulating various physio-biochemical and growth attributes. Furthermore, application of α-tocopherol (200 mg/L) followed by (100 mg/L) showed better responses in mitigating the damaging effects of oxidative stress. Importantly, the present scenario of alarmingly increasing changes in climatic conditions calls for a dire need of further research studies to investigate various growth and physiological responses of lentil cultivars to different types of biotic and abiotic stresses.
We are highly acknowledged to Department of Botany, University of Peshawar for providing all facilities regarding this work.
Effect of exogenously applied alpha-tocopherol on vital agronomic, physiological and biochemical attributes of Lentil (Lens culinaris Medik.) under induced drought stress
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Reviewer #1: The authors have presented the effects of drought induced stress by exogenous application of alpha-tocopherol on the agronomic and physiological attributes of lentil. The manuscript shows mechanistic approach and provides extensive results that could be useful in scientific community. However, this manuscript could not be considered for publication in its present form. Some comments are available for revision in order to improve the manuscript:
Major comments:
1. Highlights are missing. Add them substantially.
Minor comments:
Title should be revised. Either change the title by giving the punch line of your findings as a title or keep the same in this form “Effect of exogenously applied alpha-tocopherol on vital agronomic, physiological and biochemical attributes of Lentil (Lens culinaris Medik.) under drought induced stress”
Line 31: Revise it to “climatic conditions”
Line 32: “ill” must be replaced with “drastic”
Line 33: Revise “induced drought” to “drought induced”
Line 34: Mention the abbreviation of “Alpha α” at the first place (line 32) and then use it throughout.
Line 66: Add Comma after “findings”
Line 74: Insert Comma after “plants”
Line 79: Insert Comma after “regimes”
Line 83: Full stop after “Fabaceae”
Line 88: Change “induced drought stress” to “drought induced stress” in the whole manuscript.
Line 113: Revise “potassium available” to “available potassium”
Line 123: Please provide some detail about CVG in materials and methods.
Line 204: Seems like Alpha-tocopherol levels??
Line 205: Inset TAB at the start of the paragraph.
Line 211: Insert comma after “treatments”
Line 227: Add Full stop after “error”. Follow this for all the figures.
Line 239: Delete the word “group” after treatments. You have used this repeatedly. Please revise it carefully throughout as it is not making a good sentence structure.
Line 250: It is better to mention all the figure legends together after the references. Please revise it. Follow this for all the legends.
Line 262: Add comma after treatments.
Line 266: Delete the word “condition”
Line 338: Add Comma after “condition”
Line 338: “slow” must be replaced with “slows”
Line 342: Add comma after “study”
Line 343: Add comma after “contrary”
Line 350: Remove parenthesis after “in”
Line 354: Add Comma after “conditions”
Line 359: Add Comma after “study”
Line 361: Add Comma after “contrast”
Line 362: Add Full-stop after figure number.
Line 384: Add Comma after “condition”
Line 387: Add parentheses (100 mg/L)
Line 392: Add Comma after “condition”
Line 408: Add Comma after “results”
Line 421: Add “such as” after “enzymes”
Line 423: Revise it “Our results are”
Line 426: Add Parentheses (200 mg/L). You did this mistake repeatedly. Please revise it throughout the manuscript.
Line 429: Add Comma after “study”
1. It is better to polish the conclusion section. It is not up to the mark. Substantially revise this portion with justifications and logical statements.
2. For all the figures: you have missed the Alphabetical Letter within the figures. Please insert the letters to indicate the small figures within the main figures.
3. Secondly, all the small figures are not combined properly into one figure. Please revise it carefully.
Reviewer #2: The present study explored the possibility to using alpha-tocopherol as a mitigating agent against drought s tress on Lens culinaris. The manuscript provides an important set of data demonstrating the beneficial effects of alpha-tocopherol. The experiment is well conducted and data analyzed in an intelligible way. In its current state however, the manuscript is not suitable for publication. Major revision is needed. Substantial language editing is needed to reach standard for publication in this Journal. Long sentences should be avoided as much as possible. Besides, there are many inaccurate statements and/or incomplete information that need to be addressed throughout the manuscript.
Below are detailed comments:
The title of the study can be simplified… for ex. “Effect of exogenous of alpha-tocopherol on physio-biochemical attributes and agronomic performance of Lentil (Lens culinaris Medik.) under drought stress”
The introductive sentence of Abstract can be refined to highlight the link between drought stress and oxidative burst, and the potential mitigating effects of alpha-tocopherol. Further, the strategy used to induce drought stress (L33) can be mentioned in the Abstract.
The Abstract is too descriptive and doesn’t provide insights about specific mechanisms underlying the mitigating effects of alpha-tocopherol. A major overhaul is needed. Notably, it is important to highlight the interrelations of obtained data, and their relative relevance in influencing plant performance under drought stress.
An alternative word can be used for “Chaos” (L31). And throughout the manuscript, the term “environmental Chaos” could be avoided, as it seems quite subjective.
The Introduction section can be improved by underlining the regional biophysical context to highlight how severe is drought stress in Punjab. And the reason for choosing Lentil (Lens culinaris) as biological material in this study should be clearly stated.
There is also a need for more background information to justify selection of Alpha-tocopherol as alleviating agent against drought stress… Existing reports on Tocopherol changes under drought stress, or its other potential association with plant responses to stress could be useful. Information in L383-L387, L389-391 can also be explored for the purpose. This may smooth the formulation of the hypothesis of the study, which is not clear in current manuscript.
The “Methods” part needs to be rewritten in a more rigorous way, by providing necessary information required for possible replication of the study:
- L99-100: What the ratio 2:1 stands for, since physico-chemical analyses suggested that the soil is sandy loam. Please clarify this.
- What is the relative proportion of water and 70 % EtOH in the vehicule solution used for preparation of Alpha-tocopherol treatment?
- What was the basis for selecting the different levels of Tocopherol treatments (100, 200, 300 mg/L) and those of drought treatments (20 and 25d drought periods)
- Please rewrite statement for statistical analyses, and integrate the approach used for correlation analysis.
- Please accurately present Dubois methodology for measurement of soluble total content.
- It is apparent that the experiment was designed with two factors (tocopherol level, level of drought stress). However, authors appear to suggest three factors (L198). Please address this matter.
The quality of graphs is rather poor… Please improve their resolution. And for better readability, the legend of different figures can be modified as (within drought stress period): (i) Control, (ii) Drought + 0 mg/L tocopherol, (iii) Drought + 100 mg/L tocopherol, (iv) Drought + 200 mg/L tocopherol, (v) Drought + 300 mg/L tocopherol. Information about statistical inference, including the interaction of studied factors can also be integrated in legends.
It is important to indicate how strong is the coefficient of correlation (Table 2) rather than only mentioning that the correlation is significant. This way, results may be interpreted in a different way, notably drought effects on chlorophyll (a and b) content…
Besides, to gain more insights about the influence of studied factors, it would be interesting to analyze how studied parameters are related one with another, which would allow a deeper discussion of results.
Overall, the discussion didn’t help understanding the specific mode of action of Tocopherol in attenuating harmful effects of drought stress. Since drought stress is commonly related to stomatal closure, resulting in impaired Photosynthesis efficiency, it would have been interesting to clarify which parameter (s) in the photosynthesis apparatus was/were site(s) of Tocopherol beneficial action…
Following minor flaws could also be addressed:
- L303-306. This section is not at its appropriate place… Please check.
- L378: alternative expression can be found for “distinctly significant”.
- L344: “key metabolic activities”…What are they?
- For better readability, at the first occurrence in each section, please mention what different acronyms stand for.
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Reviewer #1: Yes: Shahbaz Atta Tung
Reviewer #2: No
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10.1371/journal.pone.0248200.r002Author response to Decision Letter 0Submission Version1
8 Jun 2021
Author Response to the Reviewer 01 Comments
1. Highlights have been added accordingly.
2. The term “Climatic condition” is replaced with “climatic conditions”
3. The word “ill” is replaced with word “drastic”.
4. The word “induced drought” is revised to “drought induced” throughout the manuscript.
5. Instead of “alpha” abbreviation “α” is used throughout the manuscript.
6. Comma is added after word “findings”.
7. Comma is added after word “plants”.
8. Comma is added after word “regimes”.
9. Full stop is added after word “Fabaceae”.
10. “Induced drought” is revised to “drought induced” throughout the manuscript.
11. “Potassium (K) available” is revised to “available Potassium (K)”
12. Details of “CVG” added as suggested by reviewer#1.
13. “α” is added before tocopherol.
14. TAB is added at the start of paragraph.
15. Comma is added after word “Treatments”.
16. Full stop is added after word “error” throughout the manuscript for all figures
17. Word “group” has been deleted after word “treatments”.
18. All the figure legends are mentioned together below the references.
19. Comma is added after word “treatments”
20. The word “condition” has been removed.
21. Comma has been added after “condition”.
22. The word “slow” is correct according to grammar rules.
23. Comma is added after word “study”.
24. Comma is added after word “contrary”.
25. Parenthesis has been removed after word “in”
26. Comma has been added after word “conditions”
27. Comma has been added after word “study”.
28. Comma has been added after word “contrast”
29. Full stop has been added after (Fig. 4a-d).
30. Comma has been added after word “condition”
31. “100 mg/L” has been written inside parenthesis.
32. Comma has been added after word “condition”
33. Comma has been added after word “results”
34. Word “such as” is added after word “enzymes”
35. Word “were” is revised with word “are”
36. Parenthesis have been added and revised throughout the manuscript.
37. Comma has been added after word “study”
Author Response to the Reviewer 02 Comments
1. Title has been simplified as “Effect of exogenous alpha-tocopherol on physio-biochemical attributes and agronomic
performance of Lentil (Lens culinaris Medik.) Under drought stress”
2. A brief mechanism of mitigating effect of α–tocopherol under drought stress has been added in abstract section by
highlighting the link between drought stress and oxidative burst.
3. The strategy used for inducing drought stress has been added in the abstract section.
4. The term “Chaos” has been replaced with “adversities” throughout the manuscript.
5. The required information has been added in the introduction section.
6. Being rich in protein content, highly exportable to international markets and a major cash crop of Pakistan, were the
main reasons behind selecting lentil as biological material in the present study.
7. Further information has been added in the abstract, introduction and discussion sections.
8. The “Method” part has been improved, where improvement was needed.
9. The ratio of sand and silt was mistakenly written; it has been removed and replaced with accurate statement “with each
pot containing 3kg sandy loam soil”.
10.The relative proportion of water and 70 % EtOH was 9:1 used for preparation of Alpha-tocopherol treatment.
11. Inferences from petri dish experiment revealed better responses in terms of radicles and plumules length with applied
treatments of tocopherol used in pot experiment. Moreover, 20 and 25d drought periods were induced in the original
trial because low levels of drought induced periods (5 to 15 d) did not show obvious physio-morphological effects on
plant growth in the experiment that was designed
12. Statistical analyses statement has been rewritten and the approach used for correlation analysis has also been added.
13. Methodology for soluble sugar content has been rewritten and presented accurately.
14. The experiment actually consisted of two factors, three factors were written mistakenly, and rectification has been
incorporated in the revised manuscript.
15. Quality of graphs has been improved and the legends of graphs have been modified according to the suggestions given
by reviewer#2.
16. A detailed account of tocopherol, in attenuating harmful effects of drought stress and the site where tocopherol shows
maximum activity has been mentioned in the discussion section from previous literature.
17. Rectification has been done by arranging the small figures alphabetically within the main figure (figure #7).
18. The term “distinctly” has been removed from results section, as it seems a bit inappropriate.
19. The key metabolic activities are photosynthesis and respiration.
20. Conclusion section has been improved by adding logical arguments.
21. All the acronyms in the manuscript have already been added below the abstract section.
22. Correlation was significant at P≤0.05. The magnitudes of positivity and negativity of coefficient of correlation for
different parameters have already been added in Table 2.
Effect of exogenous alpha-tocopherol on physio-biochemical attributes and agronomic performance of Lentil (Lens culinaris Medik.) under drought stress
PLOS ONE
Dear Dr. Sami,
Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.
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ACADEMIC EDITOR: Dear authors, plz have a look on the comments raised by reviewers. I agree with both reviewers that some minor changes are still required..
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Reviewers' comments:
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Comments to the Author
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Reviewer #1: (No Response)
Reviewer #2: (No Response)
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Reviewer #1: Yes
Reviewer #2: Yes
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3. Has the statistical analysis been performed appropriately and rigorously?
Reviewer #1: Yes
Reviewer #2: N/A
**********
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Reviewer #1: Yes
Reviewer #2: Yes
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Reviewer #1: Yes
Reviewer #2: Yes
**********
6. Review Comments to the Author
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Reviewer #1: The authors have made good efforts in revising the manuscript according to the comments. But at some points, I still feel that changes should be made. You should have to follow the below mentioned points before acceptance for publication.
1. Even you have revised your paper according to the suggestions but the newly added information is still the same in the Abstract and Conclusion. So, please be rational and make the information unique for both of these sections. They must not have similar sentences.
2. You have added the highlights and the information is good but please follow the standard rules for highlights. Make the bullet points and divide the information in these bullet points.
3. Please arrange the abbreviations in the footnotes of the first page to make them more obvious and precise.
Reviewer #2: The manuscript has been substantially improved, and authors have satisfactorily addressed previous comments, though some minor concerns persist.
For example, the sentence in paragraph L104-L111 is too long! It can be split in shorter sentences as follows: From [In plant (L104)] to [environment (L106)], from [Among (L104) to oxidative stress (109)], from [It is suggested (L109) to condition (L111)]. Please double-check the manuscript for similar flaws.
Further, I am not sure whether the style for presentation of “Highlights” (added section: L61-77) is conform to the Journal instructions…
**********
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Reviewer #1: Yes: Shahbaz Atta Tung
Reviewer #2: No
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10.1371/journal.pone.0248200.r004Author response to Decision Letter 1Submission Version2
14 Jul 2021
Response letter to the Reviewers and Editor's comments has already been attached.
Submitted filename: Response to the Reviewers.docx
Effect of exogenous alpha-tocopherol on physio-biochemical attributes and agronomic performance of Lentil (Lens culinaris Medik.) under drought stress
PONE-D-21-05714R2
Dear Dr. Sami Ullah,
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Effect of exogenous alpha-tocopherol on physio-biochemical attributes and agronomic performance of Lentil (Lens culinaris Medik.) under drought stress
Dear Dr. Ullah:
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