‘vironmental and Experimental Boany 189 2021) 104561 Contents ists available a Slenceirect Environmental and Experimental Botany journal homepage: wor clsovior.comilocatelenvexpbot Hp signaling regulates seed germination in ZnO nanoprimed wheat (Triticum aestivum L.) seeds for improving plant performance under drought stress Prabha Rai-Kalal °, Rupal Singh Tomar, Anjana Jajoo"** * sea of te Sc, De Ai: Une, Ido, a * ha ef neler, Det Aba ret, re, ac ARTICLE INFO ABSTRACT Feprorte [Napopriniag has proved to be an effective technique to protect plans from various suesses. The alm. ofthe hla Dorescenee ‘resent work iw investigate te underlying mechanism of zie oxide (200) aapopining ia inpartig drought Prout soe ‘test tolerance In wheat. Te changes in ined and unprimed whest plants asoelaed dasng drought sess ae ‘were studied by monitoring thei physiological ad biochemeat performance. Results showed that zine oxide pone ee natoputcies (210 NPs) pre‘veatmeat prevented chlorophyll degradation under drought condition thereby —— {improving overall photosyhetic performance and overall plan growth. Study of Chl furesceace induction a ints showed a rie Inecease in vavius photoaynhece parameters in printed drought sessed. plant. ine olde ‘Nanoprining seems to have protete the photoset apparais of plant by ining the fency oF prinoy photoctemistry of PSL under drought sess contins. Furternoce, the acivity of anoxia 2ymes suchas catalase (CAT), peronidase (POD), superoridecsmutase (SOD) and ghrtachione reductase (GR) as ‘well nilondildeliye MDA) conten were deceased significantly in nanoptined dng siesed (NP = DS) plats compared to nprined drt stesed (UP = DS) pants. Generation of mote reactive oxygen species (ROS) mainly hydrogen peroxide (H0;) was observed in nanoprnied geminatng seeds. We propose ole of 0; to act as signaling molecule in improving seed gerrninatin and vigor by enhancing activity of eamlase. ‘ower levels of eetive oxygen species (ROS) production in NP +S plants was obetved whieh ndiates ete toleance to crowght stress in aanoptimed plans. It is concluded that nanoprining facilitates improved seed eimination and increased seedling vigor through HO, signaling necorks, 1. Introduction Sikuku etal, 2012), ‘Wheat (Triticum esvun 1) is the staple food crop in the worl. Drought stress Is one of the mest important Imting factors to egri- Unlike many other crops, wheat Is mostly rainfed which mikes it the cultural productivity primarily in arid and semi-arid regions ofthe world most threatened erop in increasing drought conditions. On an average (Waraich etal, 2011. Almost half 47 %) ofthe terrestrial land used for agriculture Is severely affected by drought (Karin and Rabin, 2014) Drought stress negatively influences various physiological and biochemical processes resulting in oxidative ses, changed carbon an nitrogen metabolie activites, disturbed water relations and hampered photosynthetic activity whimatly resulting in decreased ctop yield and imextreme conditions, even the death ofthe plant (Nassn2 tal, 2012; 20-30 9% ofthe wheat yield is decreased because of drought each year whieh may have serious consequences on the global food supply in future (Daryanto et al,, 2016; Zhang etal, 2017)-Therefore boosting ‘wheat preduetion in adverse ablotie stress conaitons is a major chal lenge across the globe. To address this issue, the development of new environment friendly approaches for sustainable agriculture become essential, Recently, amongst varions other approaches, use of various Arevains: ABS, absorption per reaction center; hl chlorophyll; Cs, cos section; Dl dsipation; DS, drought sues; PT, electron transport Fin, maxima ‘Auescence; Fo, rinnuum fuoescece;Fo/Pu, teat dsipation; Fv Fo, efcleney of water spliting complex 03 bydogen peroxide, NP, nanopuined PEA, lant ‘fcleney analyzer; PSI, photosystem Ul: Pl, pevfrmance index; RC, rescioneeuer; RC/Abs, active teactin center per chlorophyll molecule; TR, apg, ROS, Teactvecaygen species; UP, unpuied, * Corresponding author at: School of Borechnology, Dev Ahilya University, Indore, Inn, Email adres ssnsjjoohotnal com (A. Jao). haps//dot.org/10.1016/.envexpbot.2021.104561 Received 19 May 2021; Raceived in tevsed fora 16 June 2021; Accepted 19 June 2021 ‘Avaliable online 29 June 2021, (n9472/6 2021 Elsevier BL. lights servedconventional micronutrients has show postive effects in mitigating drought sess effects in agricultural erop plants (Re2ail sind Rahn, 2015). Zine is one of the esential micronutrients that plays a important roles functional, structural and regulatory cofactor of many enzymes. Zine is also required for chlorophyll production, pollen function, fri ization and germination of seeds and thus for increasing plane growth and yield (Cakmak, 2000; Rezanl and Rehman, 2015). tn addition t0 these, application of Zine (Zn) based micronutrients to plants were found to be very efficient in increasing the water use efficiency; main taining coll membrane stability and detoxifying toxic fee radicals that accumulate in plant cells during water scarcity (Cakmak, 2000) Excessive production of ROS is one of the harmful consequences of Arought stress (Upslyy ol, 2011) eventually resulting in oxidative stress damage in plants. Zn plays an important role in lowering ROS generation and defending cells against ROS attack (Cakmak, 2000). Sudies have shown that use of diferent Zn based fertilizers play @ critical role in alleviating wheat plant drought stress by Zn-mediated Increase in photosynthesis pigment, reactive oxygen seavenging st stances and reduetion in ipl peroxidation (sa et al., 2017). Dilfer rthods fr Zn fertilization ae being nsed incding use of foliar spray as well as soil mixing methods (izwan etal, 2018), However, most oF the times, dhe bulk forms ofthese fertilizers get fixed inthe soil making it ‘uuavailable (0 the thizosphere as well as become toxic co the soil mi croorganisms and plants easing a damage tothe ecosystem. At the same time, these expensive fertlizers led corse In ost of primary resources every year (Rizwan et sl, 2017) In this regard, for sustainable age cule, there isa need to find new approaches ta reduce application of chemical fettiizers to minimize nutrient losses and increase erop vield tdyough optimized nutrient management. ‘The use of nanoparticles can help in addressing this issue and have found application in agriulture for use as nanofertilizers,nanosensores, nnanopesticides, as well as other nanomateria-based. formulations (@Mialakdinn el, 2016)- One such novel application of nanoparticles in rgrieulture is nanopriming which uses nanoparticles as seed priming ‘gent, Nanoprining consists of controlled hydation of seeds in presence of nanoparticles followed by te-drying dat allows pre-germination tetabolic activities in seeds to proceed rapidly (Parvoq etal, 2005). Owing 1 their nique properties of having smaller sizo, high surface and slow rae of release, nanoparticles get absorbed rapidly and renovate the seed metabolism, resulting ina higher germination rate and wnformiy in germination (Mahakham eal, 2017) Very few studies have been conducted on use of Zn in nano form as seed pretreatment agent for improving plant growth as well as for enhancing drought tolerance potential in wheat. Present study com: prises of priming of wheat seeds with Zn0 nanoparticles to evaluste the Impact of 200 nanopriming on wheat grown under water deficit (drought) conditions. To the best of our knowledge, most ofthe studies ‘on application of nanopaticles as seed pretreatment agent ate mainly limited to seed germination under regulated conditions (Rai Kala and Jsjoo, 2023), Present study alms co assess possible changes in che physflogical, photosyuthetle and biochemical responses on Za ‘nanoprimed wheat planes subjected to droughe stress. Based on our data, Wwe hypothesize the mechanism deciphering role of #02 as a signaling molecule to proteet wheat plants from drought throngh 210) nanopriming 2. Materials and methods 2.1, Blan material Wheat seeds (Triticum aestivum L}; Hl 1544, a drought sensitive cultivar obtained from Indian Agricultural Research Institute CARD, Regional station Indore, were used as plant material to study effect of 2n0 nawopriming on wheat plants subjected co drought stress ont an penta Raa 189 (2021) 104562 2.2. Seed priming metinod For seed priming, engineered nanoparticle were obtained from nano research lab, Jamshedpur. To study the significance of using Za in ‘nanoform, seeds were primed with Zn in nano form as well sin the form. ‘of bulk 280, sale concentention of 10 ng/L. For priming, the wheat seeds were sterilized by using 0.1 6 sodium hypochlorite (1.3 « 10~* MD for 2 min and then eareflly washed with deionized water to remove all the chloride contents. ZnO NPs were freshly prepared by dispersing the particle in deionized water using ultrasonic vibration (100 w, 40 kHz) for 15 min (Malkbiam et al, 2017), Seeds were soaked in priming so luton inthe dark at 25°C for 18h, with constant geutle agitation, The primed seeds were then washed with distilled water 3-4 times (8 min for ‘ene, and dried back to original moisture content (Rawat ets, 2018). Seeds were ten sealed in polythene bags and stored at room tempera ‘ure until further use. Unprimed seeds (untreated seeds) were consi fered as contol. Thus we had three cypes of samples: unprimed (UP) seeds, nanoprinied (NP) seeds and seeds primed with bulk counterpart of ‘2 Le. salt oF Zn80., All these seeds were used for pot experiments. 23. Pot experiments Pot experiments were carried under natural conditions with 60 596 relative humidity and an average tsuperature of 30/24 °C (Gay nigh). ‘Ten UP, NP an seeds primed with Bulk ZnSOs each were uniformly sow at depth of 2 en: maintaining equal distance between seed (0 seed, in lack polythene pors (20 em diameter and 25 cm height filled with Diack garden sol and steved sand (in the rato 3:1) The experiment was Taid out thrice in three replicates with five plants per pot 2A. Drought stress (DS) Drought stress (DS) was given after 20 days of germination. Before {nating the start of experiment i. before DS, all te plants were fly irrigated co aehleve maximum water capacity, Drought eondicions were ensured by measuring relativesoil water content which was measured as described previously by (Pebriansy el 2012) In DS pants, relative soil water content was 20 % of contol after subjecting the pots 10 10 ays of drought conditions, Experimental setup vns as follows: 1 UP 4+ W: Unprimed wheat plants fully irigated 2NP + We Nanoprimed wheat plants fly irrigated 3 Bulk } W: 2080, primed wheat plants fully irrigated 4 UP + DS: Unprimed wheat plants given drought stress 5 NP 4 DS: Nanoprimed wheat plants given drought sires, 6 Bulk + DS: 2580, primed wheat plants given drought stress. Where, unprimed filly irigated wheat plants (UP + W) were considered as control in all the further analysis to study the effect of ought. Nanoprimed fly inigated wheat plants (NP + W) and ZuSO« primed fully irtigated wheat plants (Bulk + W) were also setup as pos itive controls. Additionally effets of drought were studied in unprimed plants, plants primed with 200 nanoparticles (NP 4. DS) ané plants primed with bulk form of Zn (Bulk + DS). 2.5. Pigment and growth analysis ‘The content of photosynthetic pigments (Chl a and b, carotenoids in ig/g fees leaves) was determined spectro-photometrically (thermo scientific). Leaves samples were collected after 30 days of growth. For the extraction of pigments, 0.1 leaves in 5 mb of 80 8 acetone were used. The calculation was made according to Potrs etal. (1989). EW) and dry weights (DW) of aver the ground plant Teaves) were decermined in3 replicates (plants of nel, set randomly selected) of control and treated plants after 30 days of srosith, Por dry weight estimation individual plants were oven-dried at80°C for 24h CTomar and Jajoo, 2014) 26. Chia fluorescence measurements ‘The chlorophyll a (Chl ) Muorescence induction kinetics was measured at room temperature ising a Plant Bficieney Analyzer (PEA), (Gansateh, England. Excitation ight used ws of 650 nm and intensity 3000 jmole 2 5! which was sfctent to generate maxima fo rescence was used, The energy Mxes were calculated by using Bioly2er [HPS software (the chlorophyll fuorescence analyzing program gifted by Bioenergetis Laboratory, University of Geneva, Switzerland) 2.7. Bxaaction and estimation of enzymes (One hundred mig of laf tissue vnsh homogenized in chilled soda, phosphate buffer (50 mf, p78) containing 1% polyvinyl pyrrolidone (PVP) and 1 mM ethylene-diamine tetraacetic acid (EDTA)The ho- mogenate was centrifuged at 15,000 rpm for 15 min. The resulting s eratant was use asthe enzyme extract for SOD and GR. Superoxide dismutase (SOD) [ECI.1S.11] activity was assayed as described previously by Beauchamp and Fridovieh (1971), The activity was expressed as Units/m protein. One unit of SOD was defined as the antount of enzyme required to cause 90 % inhibition inthe rate of NBT photo reduction. The absorbance was measured at 560 nm. Glutathione reductase (GR) [FC1.6.4.2} setivity was determined a 25 °C as deseribed previously by Rao ex al. (1996). The decrease in lbsorbance was recorded at 340 nna for 10min, Theenzymie activity was calculated using the extinction coefficient (6.2 m/em) and expressed as mumiol NADPH oxidized/min/aug protein, For petoxidase (POD ECI.11.1.7) and catalase (CAT EC1.11.1.6), were assayed by the method previously desribed by Zing ol. (2007) For POD the initial and final absorbance was recorded at 470 nm for 2 tin, The activity was ealeared asthe change in absorbanee/min mg protein, CAT activity measured asthe rate of disappearance of HaQ at 240 nm, Decrease in absorbance was detected for 2 min and one unit of CCAT activity was defined as mM HO, decomposed /min/mg protein Protein was estimate by the method of Lowsyet al. (1051) using BSA as standard 28, MDA content analysis Oxidative damage to leaf lipids, resulting from salt stress, was est sated by the content of total 2-thiobarbituric acid reactive substances (TBARS) expressed as equivalents of malondialdehyde (MDA). TBARS content was estimated by the method of Cana wd Horst (1992) 29, Determbiation of ROS in nanoprimed sees 2.9.1. Determination of peroxide radicals Os Superoxide reical(O;-) was quantified in 12 himbibed seeds as described by Katri eta. (2019) through its ability to reduce nitroblue tetrazolium chloride (NBT) following cerain modifications. Weighed amount (0.5) of seeds were homogenized in sodium phosphate butler (0.2 M, pit 7.2) containing dithylihiocarbamate (102M) to inhibit superoxide dismutase (SOD) activity at 4'C. The homogenate was centeifged at 13,000 rpm at 4° for 10 min and the resting super natant was used for assessment of O3°-The optical density of the end prot wns estimated at S40 nm, Iwas calvlated by 12.80 en Nd shsorption coefficient and defined as ynoleg fresh weight 2.9.2. Detemination of hydrogen peroxide (1:02) Hydrogen peroxide (Hj0,) content in 12h imbibed seeds was measured aecording to the method described by Kania etal. (2020), sing the production of ttaniu-hydroperoxide complex. tn brief 0.5 & of imbibed seeds were ensshed in chilled aeetone. Titanium reagent (2 mil; titanium oxide and potassium sulfate digested in concentrated ont an penta Raa 189 (2021) 104562 sulfuric ae) and ammonium hydroxide solution (2.5 mL) was added ro the flerate for preeipitation of the ttanium-hydroperoxide complex ‘This precipitated complex was centrifiged at 12,000 rpm at 4° for 1S ‘min, The obtained pellet was suspended in concenteated sulfite acid (20 5) and ve-contrifuged. Then the absorbance ofthe resulting super natant was taken at 415 mm, He03 was defined pole ¢~ fresh weight, 2.10, Determination of activity of superowie dismutase (SOD) and peroxidase (POD) in 12 I imbibed seeds Antioxidant enzymes were extracted from wheat seed embryos following method deseribed by Tosiar and Jajoo (2014). Seed embryos (0.2 g) were homogenized in 10 mL of 50 mM potassium phosphate Dufer (pH 7.8), Homogenate was centrifiged at 1,000 rp for 15 mi fat 4-Cand the supematant was sed forthe determination of peroxide dismutase (SOD) and peroxidase (POD) estimation, 2.11, Statistical and dara analysis Graphs and daca were analyzed by using Origin Pros, Staiticel analysis was done using Graphpad Prism 5.01 Software (GraphPad Software In.) Results were analyzed using one-way ANOVA followed by the Dunmets comparison test. Significance was determined at P < 0.001¢, P< 0.05, *, P< 0.01,***<0,001) and results expressed a5 ven valies SD, Different letters indicates significant differesce among ‘nentments. All the experiments were done thrice in replicates of tree, 3 Results and discussion 3.1. Effect of droughe iress on photasytetc pigment content ‘The impact of 200 nanopriming under well watered and drought condition on plant photosynthetic pigment content in wheat plants is shown in Fig 1. A significant increase of 14 9%, 2 % and 16 9 in Chl a (Chl and total Chl content respectively is observed in NP + W plants as compared to UP + W plants. However no such significant inrease was observed in Bulk + W wheat plants. This pronounced inerease in Chl content in NP + W plants miny be stibited to Zn indice increase in (Chl synthesis in wheat planss (Wang et al, 2015). Results obtained showed inerease in Ch content in all the treatments subjected co drought stress. However, in NP + DS wheat plants Chl ¢ Chl b and total Chl content were significantly increased by 62 %, 1259 and 78 % and only bby 1496, 26 96 and 17 % respectively in UP 4. DS plants as compared to UP + W wheat plants while showing almost no ehange in Blk + DS plants. This inerease in Chl pigment content in NP + DS as well as UP DS can be explained as an adaptive response towards drought stress conelitions. Exposure of plants to low-level stress induces increase in the synthesis of chlorophyll pigment as a part of plants adaptive defense neclanism, contrary 10 dhe eatabolie pathway of eloropillase syn. thesis (responsible for ehllorophyl hydrolysis) when subjected ro severe stress conditions (Siddiqui ets, 2019). This mechanism protects the plants from undergoing further reduetion in Chl eontent from any up coming igh levels of stress challenges (gathokeons etal, 2020). Of ll the three treatments subjected ro drought stess, prominent increase in CChl content in NP + DS plants thus explains better plants defense veehianism © cope up with water deficit conditions. Another reason for this ise n Chl content in primed plants may be atributed co the role of Zine not only inducing the synthesis of new chlorophylls molecules but, also in protecting existing chlorophyll from further drought induced oxidative stress damage (Calms, 2000) ‘The above results are further supported by an increase in rota Carotenoid (Car) content of NP 4+ DS wheat plants, Carotenoid content ‘whieh plays a vital role in photo protection mechanisms in plants (Su al, 2018) inereased in NP DS (80 96 increase). Smaller size and larger surface area to volume ratio increase the biomvalability of nanoparticles to get efficiently absorbed by seeds thereby increasingsat, a B 3 2 & ‘ 6 = 5 - g [ec > £. Zu = 4 2 : 5 3 ne EB ow. & & 3 a, 'UP+W NP#W BulkeW"UPSDS NPsDS BulkvDS”—_UPW NP+WBulkew UP+DS NP+DS BulkvOS ont an penta Raa 189 (2021) 104562 (646) @ whydosorys (6/64) yweruo> ploweroreg ig. 1 filet of dough stress on A) Chlovophylla (Ch); )Chloxopbyi b (Chi) total ehovophyl content (tal Chl) an) earoenoi (at conten in coatat (UP + Wy, nnoprined inigated (NP + W), 2080, (Bulk ~ DS), snptimed dough (UP + DS), nanopsined dig (NP + DS) snd ZnO, primed dog sues (Baik +DS) whet plans, Al de experiments weve done thicein3 replicates. Significance was determined at p< 0001("p < 005, "p< 0.01, **p <0.001 and as = now sgnfean) and the resus ate given as mean values SD. Difetent alphabets indicate sgnfcan ference among treatments, ‘Thus, inerease in chlorophyll biosynthesis along with inerease in Car content in NP + DS plants is @ manifestation of edaptation of plans for rough tess ‘heir assimilation and penetration ability (Prasad et, 2012). On the contrary the effectiveness of bulk 2n0 as priming agent slow because of highly water soluble Zn" jons which have difficulty in penetating the seed thereby decreasing its bioavailability to seeds (Rosset, 2018) Phroscence inane at = s § Gah 6 ‘Time ms) “00 ‘000 oO “Time ims) ‘joo 100 Fig. 2. Chl a tansient curves for A) control (UP + W), nanopsimed inigated (NP + W), 2080, primed inigated (Bulk + W) and B) contra (UP + W, unprimed ‘oughr (UP + DS), nanoprimied dtought (NP + DS) and 2n80, pried dough suessed (Bulk + DS) wheat plans. Tvansents have been ploted on logasitunle tine sal,4.2, Rift of drought sess on Chl a inducron Kinetics Chlorophyll fuorescence measurements are used as an indicator of diferent drought responses of photosynthesis (Kelaji et al, 2018), Characteristies of chlorophyll uorescence area critical consideration as Te Is used t0 measure the quantum yield of photosystem Tl PSID and photoinactivation by determining the possfble quantum yield under water limiting conditions (Batra etal, 201). Effect of nanoprinting on photosynthetic efficiency in wheat plants was measured by the study of CChla fluorescence measurements. Chl a fluorescence trasient curves for UD + W, NP + W and Bulk + W wheat plants are shown in (Fg. 2). Although there was no remarkable change in values of Fy (representing inital Auorescence) significant changes in various other Muorescence related parnaeters were observed in NP + W wheat plans as compared {0 UP + W plans (Fable 1), Parameters lke Fy, Fy/Fo, RC/ABS, area, Fo/Py and PI au were significantly enhanced in NP + W wheat plants as compared to UP ++ W plants, Increase in of above parameters ths reveals Zn0 naopriming to be & wore ficient ia enhancing plant photochemistry in comparison to UP + W control and other priming ‘methods thereby improving plants photosynthetic performance (Rai Kalal and Jajoo, 2021) Drought stress results in significant reduction in photosynthesis a ic blocks the transport of energy from PSI to PSI (Sideise et al, 2016), Remarkable changes in various Chl a Avorescence parameters like Fv/Fo, RC/ABS, area, Fo/Fm were observed in UP + DS, NP + DS and Bulk + DS as compared 10 UP + W plants (Fable 1). An alteration in "hese parameters gives an idea about the effect of ZO nanoprining on functioning of Photosystem Il (PSID reaction centers and photosynthetic efficiency of the plant subjected to drought sees. Analysis of Nuores cence parameters showed a significant decrease in Fy/F, (representing lhe effeleney of water splitig complex) and RC/ABS (representing ‘number of active reaction center per Chl molecule) by 21 96 and 27% respectively in UP +-DS followed by a significant decrease in Buk + DS plants. Conversely, the maguitde of damage in FY/Fo and RC/ABS was almost non-significant in ease of NP + DS as compared to control UP + W plants. Further, a prominent decline ia azea over the flotescence transient eurve was observed in UP + DS as compared to UP + W. This drastic decline In area may be because of more retardation of Ox oxidation step which leads to decline in eduction of PQ pool thereby lampairing the effcieney of electron transport (Sis et, 2000). On the contrary, change in aren showed not mitch difference in NP + DS. plants as compared to UP 4- W plants demonstrating that drought stress did not affect the overall poo! size of electron transporters in NP plans ‘This resulted inefficient eleetron transport from active reaction centers In NP plans. The decrease in above parameters was further followed by 1 decrease in Fm (nsaxinmim forescenee) in UP + DS plants. Decrease In Fi values suggested a development of non-radiaive dissipation ofthe excited states of PSI antennse chlorophylls in UP + Ds plants compared tocontrol (Srivastava et al, 2010), Results were further supported by & significant rise in Fo/Fm representing energy loss by heat disipation by 2099 in UP + DS plants, and almost no change in NP + DS plants. The analysis of above parameters suggest that NP plants could cope up with the stress condition by inreasing the overall photochemical efficiency ‘Table 1 ont an penta Raa 189 (2021) 104562 ‘of plant for performing primary photochemistry at PSM reaction centers ‘and minimizing the energy loss by heat dissipation. Overall inerease in plant photosynthetic efficiency thereby resulted in significant increase in performance index (PD in NP + DS plants in comparison to UP ++ DS ‘wheat plants ‘The above results gained more support by studying PT) parame: ters (Table 1). The performance index on absorbance bass represented bby DI (abs) is considered as one ofthe sensitive end insightful indicator for stress and is widely used to compare the whole priniary photo chemical reactions (Richman et al, 2015; Mathur et al, 2018). PI (abs) was significantly reduced by 50 % and 48 % in UP + DS and Bulle + DS plants respectively as compared to UP 4. W. However values of PI (abs) for NP + DS plants was as much as that of UP + W plants, suggesting that 2n0 nanoprining has helped the plants photosynthetic apparatis t0 vaintan its efficiency of light reaction, absorbance per reaetion center land net photosynthesis even under severe drought condition (Strasser etal, 2000), 3.3. Effect of drought stress on energy flow through PSI Pattern of energy flow in PSH was ealeulated in the form of energy, pipeline leef model for UP + W, NP + W and Bulk + W of wheat plants (ig, 3).The flow of energy, photons trapped and used for efficient pr uary photochomistry was estimated by width ofthe arows. The model demonstrates the proportion of aetive reaction centers (represented by ‘open creles) and inactive (represented by closed circles) reaction cen ters of PSII per cross-section (RC/CSm) _(J. Photochem. Photo biol. B. BioL Mathur et al, 2018). Parameters like ABS /CSm, TRo/CSm, ETo/ CSm, indicating the efficiency of light absorption, electron trap ping and electron transport per cross section respectively, increased, ‘while Dlo/Sm (dissipation per exosssetion) was decreased in NP + W plants as compared to UP + W plants. Thus nanopriming enhances the ‘overall plant photochemistry and maintains the efficiency of photo synthetic apparats under well watered conditions. However subjecting these plants to water stress resulted ina significant deerease in RC/CSM\ in UP + DS and Bulk + DS plants by 31 9 and 34 9 respectively while showing almost no change in NP + DS wheat plants. Decrease in RC/ (CSm can be explained asa result of drastic reduction in total Chl content due to drought stress (as discussed in Fig. 1) resulting in an numberof inactive reaction centers (closed) over active (open) reaetion centers. This leads to production of dissipative heatsinks fom excitation, energy which i evident by an inerease in the ratio of Fy/Fy (by 209) in UP + DS plants as discussed earlier (Table 1). Decline in number of active reaction centers also resulted in decline in ABS/CSm (represent ing absorption per eros seetion) subsequently reducing other parame ters representing electron trapping (TRo/CSn» and electron transport (ETo/CSm) per eross section by 22 % and 30 % respectively in UP + DS. In contrast, in NP + DS plants, the numbers of active RC/CSm were ‘observed to be almost similar as that in non-stressed UP ++ W plants Increased numberof ative RC in nanoprimed plants thereby shoved & better absorption and trapping by antennae pigments showing an improved efficiency of electron transport per cross section as compared Various Cha tanslen paramere for unprimed conto (UP + W), nanopsed ligated (NP+W, 2380, pened inigated (Bulk + W), unpelmed drought (UP + DS), nnanoprimed drought (NP + DS), 2080. primed drought (Bulk +DS) wheat plans. Each expeviment was done tice in 3 replicates. Sigufeance was determined at p< D001 p = 005, *p = 001,""%p = 0.001 and ns =non significant) and the results ave given as mean values SD. Difeent alphabets indicat significant tference mong treatments wraw Wiig sire ra oat oo aa os oar saa 20-500" Bukew = dayzem ora az ee anr=" 026 £007" Npips foveieme — ioossarm —avestie —ogptamem agg sore’ Gayton = os atm Baieebs asim inaesageee arta samme Gaetooums —omaemaeont an penta Raa 189 (2021) 104562 Fig. 3. Representative energy pipeline leaf model fr (A) contol (UP + W), (B) nanopslmed ligated (NP+ WD, (© 2u80, primed ungated (Bulk+WND) unprimed ‘roupht(UP + DS), (B) nanoprined drought (NP + DS) and (F) 2280,prmed ought stressed (Bulk + DS) wieat plans The furs wsed represent ight absorbance er ctoss section (ABS/CSa),eaitation energy tapping per ctose section (TRY CS mi) eleeuon transport per eros section (ET/CSim). Empy and fll black cicles Indicate, the percentage of active (Q, reducing) and non-active (non Q, educing) reaction centers of PI respectively toUP + Weven under severe drought conditions. This suggests that Zn0 28 priming agent has Ielped che plants to maintain the efeieney of photosynthetic apparatus by protecting it from undergoing extreme damage ander water stressed conditions 8.4, Effect of drought stress om plant grow Increase in all the above mentioned photosynthetic parameters resulted insignificant increase in biomass in NP + W wheat plants (by 38 9%) as compared to UP W control, However water stress conditions resulted in impairment in growth parameters like fresh weight (FW) and dry weight (DW) in UP + DS plants by as much as by 57 9 and 58 96 Fresh weight (a) Us wos Bubs respectively, followed by a similar reduetion in biomass in Bulk + DS plants when subjected co deonght sires as compared to UP W plants ig. 4. However, in NP 4 DS plants this drought stress.mediated reduction in fresh and dry weight was only by 33 9 and 21 9 respec tively as compared to UP + W plants. NP + DS plant leaves showed very lite signs of necrosis and wilting as well as the inward curling o eaves \hiel is one of the adaptive mechanisms to avoid water loss by tan spiration. The elect of water stress causes Toss in tissue water content Whieh reduces the turgor pressure in cell, thereby inhibiting cell enlargement and cell division resulting in restricting internode elonga tion, leaf expansion, reduced plant growth and reduced dry mass acc ‘mulation (Nami and Alia, 2007). In contrast, a comparative lesser Fig. 4. Elect of droughe stress on sh weight 42 (iD and dey weigh (DW) in contol (LP = W), snaopine igated (NP + W), 280, primed Inrigeted (Bulk + W), unpeimed droughe (UP + DS) tmnprined draught (MP DS) and a ‘ZnSO4piimed drought stessed (Bulk + DS) ‘ ig) west pnts Al the experiment wee doe = ag 9) trie fn relates Siitionce wa ctr § <0001 and ow ~ non sini S Sateae given mean vas SD ite POE pints intone since etree among = |G oe 2 en Bak a 0s Babsextent of biomass reduction in primed plants under stress can be explained probably die to the essential role of Zn as a plant growth nutrient required for the proliferation of ronts and thereby increasing the uptake of water and other plant auteients from the soil and supplying it to plamts aerial parts ultimately enhancing the vegetative growth of plants under water stressed conditions (Poorinis and Kotl, 2019). Moreover, the presence of Zn in drought stressed plants considerably Inhibited chlorophyll degradation thereby increasing their overall photosynthetic efficiency, indirectly affecting plans growth parameters even under water stressed conditions (Hassan et s., 2005; Tavallali eral, 2010). 3.5. Antovidant enzyme estimation These interesting observations stimulated us co investigate the mechanism of action of 20 NPs fm improving drought tolerance in Wheat, Various environmental seresses including drought, lead 10 excessive generation of ROS resulting in detrimental effect on plant (Sharm et al, 2012), Seavenging ofthese ROS is 1 be achieved by an. efficent antioxidative system in plants comprising of non-enzymatie as well ss enzymatic antioxidants (Turan, 2079). In relation to this, 10 study the equilibrium between ROS production and their seavenging, the activity of various antioxidant enzymies such as peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were measured in UP + W, NP + W, Bulk + W, UP +S, NP-+ DS. and Bulk + DS wheat plants (able 2, Activities ofall the above antioxidant enzymes showed significant Aeerease in NP + W wheat plans as compared to UP + W. This decrease in activity thus reveals NP 4 W plants to experience a leser extent oF stress conditions as compared to other priming treatments. However, the activities of POD, SOD and CAT whieh ate often considered as intial tioxidative enzynes to get altered under drought stress (Shari el, 2012) showed prominent increase In UP + DS followed by Bulk + DS tnd NP 4 DS weheat plants as compared to UP W plans. Also, the activity of GR, an non-enzymutic antioxidant known to convert oxidized lcarhione (GSSG) lato free radieal seavenger glutathione (GSH) (Tomar and Jajoo, 2014) was increased significantly in all the three treatments under drought stress in comparison to UP + W plants. This drastic increase in activities of various antioxidant enzymes in UP + DS plants can be explained by the fact that there is an increase in rate of renetive oxygen species (ROS) production as response towards drought stress, To eliminate this increase ia ROS, plants have evolved an ‘Table 2 Specific aetvty oF peroxidase (POD), superoide dlsmtase (SOD), catalase (CAT) and glutahione reductase (GA) in unpined inigated (UP + W), ano rime irgated (NP + W), Za80, primes iugated (Bulk + W), unpsimed ‘ought (UP + DS), sanoptined diought (NP + DS) and 2080, pied dough (Baik + DS) wheat plants. Each experimen was done tice in 3 replicates. Significance was determined at p< 0.001 (1p = 005, **p < O01 and < (001, ns — non-significant) and the results ae given as mean ales SD. Diferene alphabets indleate sigaienne difference among treatments Teme FOD CAT (HmaieHLO, SOD(Unia/ GR mma (nterng —decompeetfnin’ mg pretin) NADPH oein) me preein) risen’ te poe) Bukew OI — aswsor™ oan ak sos ares 2a0n-tor Os im tee ont an penta Raa 189 (2021) 104562 antioxidant defense system whieh up-regulates the activites of antiox ‘dant enzymes to counter act extensive cellar damage caused de (0 overproduction of ROS (Turan, 2019bs Wi et al., 2015). However the increase in the activity of antioxidants was to a mic lesser extent in NP “DS plants as compared to UP + DS plants. This decrease inactivity of antioxidants nay be ditetly correlated to lower levels of ROS produc tion in NP + DS plants compared to UP + DS plants under drought conditions. In general, ROS which are produced as 8 normal product of plant celular metabolism are supposed ro play deleterious as well as beneficial species depending on their concentration in plant (Sharia ct al, 2012). When produced in high concentrations they eause exces sive damage to cellular components resulting in progressive oxidative damage to cell ultimately causing cell death (Malesieari and Dubey, 2009). However, at lower concentrations these ROS ret as intacelllae signaling molecies involved in mediating several plant responses under stresses conditions (iitler, 2002). Thus, inthe present study, lower levels of OS produced in Zn0 NP plants could be atributed towards the role of ROS acting as a second messenger in up-regulaing the genes Involved in sensing the water stress conditions thereby redulng water loss (Burman ea, 2013). These results are in aecordance with Miller cr sl. 2008) who reported the involvement of different zine finger proteins as a key player in up-regulating genes involved in water stress related abscisic acid (ABA) transduction pathway at lower ROS con: centration (Yan etal, 2007). Results from the present study therefore justify an enhanced potential of 2n0 NP plants to modulate the balance Derween oxidant (ROS) production and their emioval by the antioxidant to overcome Water stress conditions. Furthermore, the better perfor ‘inne ofthe photosynthetic apparanis of NP + DS plants in comparison {OUP 4 Ds plants can also be explained as a result of ower levels of OS produced in chloroplast of nano primed plants even under severe drought conditions which otherwise is the major site of oxidative damage caused by excessive production of ROS species (Sharma eta, 2012). The above results showed that nanoprimed plants shove enhanced tolerance against rough stress, Ths the metabolic energy required to resulate the plants defense mechanism to degrade excess ROS usually prodiced during drought stess is conserved, thereby clrecing i to sustain the growth and development of nanoprinied wheat plants 36. MDA content anayss Malondialdehyde (MDA), an end product of lipid peroxidation of cellular membrane, is commonly know as oxidative stress biomarker. ‘The extent 10 which plants membrane is damaged in drought stressed conelition ea be known. by measuring the MDA Tevel of Kpid pero ation (Arona et sl., 2008). Bffee of nanopriming on MDA content of [NP + W showed a significant decreased by 4996 in comparison to UP + ‘W wheat plants followed by decrease in Bulk + W (Fig, 5). Results shove ‘ pronounced increase in MDA content in UP + DS sud Bulk + DS plants by 87 4 and 46 96 respectively as compared to UP + W plants, while NP + DS plants did nor show any such signifieant increase in MDA content as compared fo UP 4 W plants. Studies have shown that there exists a positive correlation between oxidative stress factors (ROS) and MDA (oorninia and Kot, 2019), Therefore a significant rise in MDA content in UP + DS plants may beatributed as arexponse towards drought stress resulting in accumulation of ROS having potentially damaging effects, Teading to inerease in ipid peroxidation (MDA eontent) of the cellular ‘membrane (Sapre and Vekbiaris, 2019). On the contrary, priming with ‘200 nanoparticles imparted an enhanced érought tolerance capacity in ‘nanoprimed plants by limiting the production of ROS species at lower Tevels, resulting in lesser percentage of MDA production in NP + DS thereby maintaining Une integrity of ehloroplasts membrane even under severe drought stress conditions (Calsuak, 2000). ‘Thus, dhe analysis of above results showed the effec of 2n0 nano- priming in enhancing drought rolerance capacity in nanaprined Wheat plants as compared to unprimed plants. Further, the mechanismMDA content (nmol gt FW) Fig. 5. Efe of drought stress on malonnletiye (MDA) conte in control (UP + W>, nanoprned inigaced (NP), ZnS0y primed ingated (Blk +), ‘upuimed drought (UP + DS), aanoptined doughe (NP + DS) and 2180, imc! drnght sessed (Blk + DS) wheat plants subjected to drought ses. Enc experiment ws done shtice in 3 epics. Significance was determined at P< 0001 (p< 05, p< B01," < 0.001, ns ~ nowsigafant) athe results aze given as mean vale SD. Dilferent alphabets intcate sii diterence among treatments. vont and Exe ory 189 (20) 104564 involved in improving plant physiological and biochemical performance through ROS signaling ar seeds biochemical level sa nanoprimed seeds were also sted, 82. Determination of 03° and #202 content tn nanoprimed seeds ‘The contents of two diferent ROS species, Le. hydrogen peroxide (H:0,) and superoxide anions (O;-) were quantified spectrophotomet tically in the exubryos of 12 imbibed waprimed, nanoprimed and Bulk ‘2nprimed wheat seeds (ig. 6). Results showed, a signfiean increase by 13% and 26.9 in bot superoxide radials (05-) and hydrogen peroxide (H:0,) content respectively in nanoprimed seeds as compared to ‘unprimed control. The contents of ;- in Bulk Zn primed seeds showed no significant change with an increase of only 16 % in 0, compared to control, Further the role of nanopriming in regulating enzymatic ant ‘oxidants in soeds wore also studied (Fg. 6). Results shoved significant Inerease in the activity of SOD involved in dismutating O5- to Hs0z radicals, with a prominent inerease in annoprined seeds ns compared (0 Lmprimed seeds. Activity of CAT involved in converting HO, to H,0 molecules thereby maintaining 102 radicals was also increased significantly as compared to unprimed control seeds. Similar increase in the aetivity of SOD was observed in Bulk Zn primed seeds however followed by no significant change in the ativity of CAT as compared to contra ‘The mechanism behind the accumulation of ROS, Ke. hydrogen rs a le 8 2 z t0 . ss os x 2 ¢ o 1 e 3 os & BS os 2 3 os § 5 oa 5 z & £ 02 E 3 02 2 s 90 > oo 8f 2 3 c 2 ° ots & 06. z . 018 = Gol, 3 g 02 2 04 : 2 e | go oj at. 0.03 3 00. 0.00 . UP NP ‘Bulk uP NP Bulk Fig. 6. Antioxidant enzyme activites and ROS contents in unprimed (UP) nanoprmne (NP) and 2380, primed (Bul) wheat sed. A) supeconide radicals » hydiogen pevexide content (H,0,), €) superoxide dismurase (SOD); D)exalase (CAT).AI the experiments were done thrice in 3 replicates. Signifiance was detemnned at p < 001("p < 0.05, significant diference antong eaten, p< 001," p-< 0.001 and ns = non-significant) and the resuls ae glven as mean values SD. Diferent alphabets lndieateperoxide (H,02) and superoxide radicals (5°) acting as signaling molecule in breaking seed dormancy and enhancing dynamics of seed sermination has been explained in Fig. 7. Water uptake during seed imbibition results in initiation of various setobic respiration processes in cellular organelles like mitochondria peroxisomes and the apoplestic space of germinating seeds which ae responsible for superoxide radials (©y7) aceummlation (Anan et a., 2019), The concentration ROS pro duced i tightly regulated by ROS seavenging enzymes such as super oxide dismutase, catalase and peroxidase that allow them to act as cellular messengers by existing atthe critical levels, known as ‘oxidative window'(ally etal, 2008). O3- radicals ere rapidly dismutated 10 ,0; by cellular superoxide dismutase (SOD). HO» are furier main tained within the ‘oxidative window range’ by the activity of catalase (CAT) to-etas signaling molecule. HzO, has a longer shelf life ad easily Aifssble molecule to eros the membranes to reach targets site (em bryo) ftom their production sites (Anand et al, 2019). HO. interact as signaling moleesle with phytobormones like gibberellic ald (GA) and absicsc acid (ABA) thereby activating germination related enzymes ike scamylase breaking seed dormancy (eis sel Or, 2007) A significant inereaso in the activity levels of SOD in nanoprinied seeds after 12 h imbibition ean be atribted to the increase in ace ulation of 05 radicals in nanoprimed seeds, SOD is required for ds ruating Oj radicals HO; molecules. Further, this increase in dismutation of 03 radicals can be further correlated with significant increase inthe concentration of HO, in nanoprimed seeds, Interest ingly, this rise in H30z content was further followed by considerable Increase in CAT activity in nanoprimed seeds involved in converting H,02 0 H,0 molecules thereby maintaining the levels of HO» radicals within the oxidative widow range to et as signaling molecule. Thus, & very precise regulatory mechanism of HO accumulation and removal by the cells antioxidant machinery is maintained in nanoprined wheat seeds wiggering germination process, increased seedling. length, ‘enhancing seedling vigor thereby resulting in iniproved photosynthetic ficiency and improved growth as compared co vnprimed and cher priming treated wheat seeds 3.8, Proposed mechanism for enhanced drought tlerance in nanoprimed wheat plans Although it has been known that seed priming ean enhance seed ‘quality and the effectiveness of stres responses of germinating seeds and Wheat “waters Water uptake seeds ————_ by imbibition ont an penta Raa 189 (2021) 104562 seedings, the exact mechanisms involved in the acquisition of stress tolerance by primed seeds in subsequent plant growth remains poorly inderstood. In present study, we propose two possible mechanisms behind enhanced drought tolerance in nanoprimed when seeds (ig. 8). Firstly, the procedure of seed nanoprining involves soaking of seeds, in priming solution for time duration wherein the germination related tabolie aetivities start without radical protrusion. Primed seeds are then dried back to their original moisture content whieh itself imparts stress like conditions on seeds during the inital gemination stage. Ths priming procedure imprints a primary “stress memory” in seeds which can be recruited upon a subsequent stress exposure and mediates greater stress tolerance during germination of primed seeds. The stress memory thus developed in plant pre-exposure to stress during priming makes the plant more resistant ro the subsequent stress exposure further in thelr Iie eyele (chen and Aron, 2011), Secondly, HO acts as signaling molecule in nanoprimed wheat seeds increasing seed performance by metabolically enhancing seed germination, Nanoprimed seeds are at a developmentally more Advanced physiological state compared to non-primed ones. Various studies have reported ROS species mainly hydrogen peroxide (H:02), nitric exide (NO), hydroxyl radicals (OH") and superoxide redicals (O} ) toe involved in seed germination, Out ofthese, HzO» molecules play a key role as signaling molecule only when maintained within oxidative window range in breaking seed donmaney and inereasing germination related metabolic process in seeds (Anand etal, 2019), Srudies have reported the involventent of Hs molecules in cell wall ‘and endosperm loosening in seeds of Pisum sativum thereby breaking seed dormancy and facilitating seedling growth (Barba-Espin et al, 2010), Similar Work has been reported in germination studies of batley showing the role of as signaling molecule in increasing GA (ibberllic acid) gene expression, resulting in breaking of seed dormancy (Sali tal, 2011; Graebore al, 2010), However, role of HO in seed priming thas been reported forthe first ime. Present study showed significant inerease in the produetion hnydrogen peroxide (H,0;) in nanoprimed seeds at 12 h of imbibition Diirng the initial stages of imbibiions, Za0 NPs could penetrate seed coat by interacting with cll walls of sed coat to create nato pores, thus enliancing seed water uptake capacity In nanoprimed seeds (Ral Kell ‘and Jajoo, 2021). Ineease in water uptake results in initiation of rapid foidative respiration in cellular organelles resulting in accumulation of (O} radicals (oxidative burst). 0” radicals thus produced sre Cellular oxidative Coptens] Dismutation <—=1_ 0:7 _J (50D) Signaling —> BalancedH,0, <—— molecule Ce: ) <— catalase (ca) Reactive oxidative ‘window range Interaction with phytohormone (GA) ‘Triggering Activation = ——> of a-amylase germination Fig. 7. Schematic representation of regulation of HO; within the oxidative window range for signaling in wheat seedsPR ab ont an penta Raa 189 (2021) 104562 Wheat Nanoparticles Soaking seeds Improved water use efficiency Increased chlorophy! Reduced oxidative Improved membrane stability Improved plant performance Fig. 8 Proposed mechanism of enhanced dough tolerance in nanoprimed west plants. (A) Nanopviinginates priming memory in wheat seeds. (B) Nano ming elites sce eration at seraing vigor though generation of ROS in oxidative window range for acing signaling molecules (C) Enbanced doug tolerance in wheat plants developed from nanoprimed seeds by reactivation of priming memorydismtated to HsOs molecules by cellular dismutase. The HOs ths produced are nainttined within the oxidative window range by cellular ‘antioxidants to act as signaling molecule thereby triggering germination related metabolic eveats, Diffusion of HaOat0 the embryo results in imerplay between H;02 and phytohormone gibberellic acid (GA). GA is thereby responsible to fasten the process of starch hydrolysis by act vating e-amylase activity, generating higher soluble sugars to support embryo growth thereby enhancing seedling vigor. Inereased vigor of primed seedlings show beneficial effects on plants by showing increase in chlorophyll content, improved plant photosynthetic efficiency and reduced oxidative stress thereby determining enhanced potential of drought tolerance even under severe drought stress conditions as ‘compared to unprimed plants. Also, drought stress invokes reactivation, of siress memory in plants developed from nanoprimed wheat seeds upon second stress exposure. Thus, primed plants are characterized by faster and stronger activation of different kinds of defense responses such as activation of synthesis of LEAS (late embryogenesis abundant protein), dehiydrins, up-regulation of antioxidants enzyme gene (Wo) Iylea etl, 2016). However more in-depth study of gene expression of related plysiologieal processes modulated by seed nanopriming need 10 be entried out. 4. Conclusion 1 is concluded that nanopriming improves seed germination per formance and could provide enhanced drought stress tolerance potential i wheat plants. Wheat plants grown from 20 nanoprimed seeds shoved tolerance rowards drought stress by inhibiting the chlorophylls degradation ftom oxidative stress damage. Physiologically, the benef lal effects of nanopriming under érought conditions may be atibuted ‘o improving the photosynthetic effcieney whieh is directly correlated with improved biomass accumulation. This is evident from the increase in numberof setive reaction centers, inrease in the efficiency of light absorption, energy trapping and electron transport followed by decrease In production of reserve oxygen species thereby protecting the photo- synthetic apparatus from oxidative damage under drought stressed conditions. I is hypothesized that nanoprining improves seed germ nation and vigor through HO» signaling network. Wheat plants devel ‘ed rom nanoprimed seeds retained a long lasting stress memory that triggered an ameliorated and efficent stress tolerance mechanism i then, Moreover, the present tidy dealt with nanoprimed wheat plants fesponse to drought only at early growth stage. Further research is required to study the drought recovery potential of nanoprimed crops at ‘maturity stage too. Molecular mechanisms and gene expression involved, in priming memory needs extensive research, Author statement Authors state thatthe work described has not been published pre viously, that it snot under consideration for publication elsewhere, that ts publication is approved by all authors and tacitly or explicitly by che responsible authorities where dhe work was carried out, and that, i sccepted, it will not be published elsexchere in the same form. Authors’ contribution Study was designed by A, PRand RST, Experiments were performed tnd analysed by PR and RST. Manuscript was written by PR, RST and edited by Ar Declaration of Competing Interest ‘The authors report no declarations of interest. ont an penta Raa 189 (2021) 104562 ‘Acknowledgements PR thanks University Grants Commission, (UGC), Indiafor UGC- NET Junior Research Fellowship [F:16(DEC-2016)/2017(NED)]. RST, thanks Council of Scientific aid Industrial Research for CSIRRA fellowship (09/301/(0134)/2018.EMRD. 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