Contribution of Glycine Betaine and Proline to Water ...

HORTSCIENCE 54(6):1044–1054. 2019. https://doi.org/10.21273/HORTSCI13955-19

Contribution of Glycine Betaine andProline to Water Deficit Tolerance inPepper PlantsCamilo Escalante-Magana, Luis F. Aguilar-Caamal,Ileana Echevarría-Machado, F�atima Medina-Lara,Lucila S�anchez Cach, and Manuel Martínez-Est�evez1

Unidad de Bioquímica y Biología Molecular de Plantas. Centro deInvestigaci�on Científica de Yucat�an, Calle 43 #130, Col. Chuburn�a deHidalgo, 97200 M�erida, Yucat�an, M�exico

Additional index words. Water potential, relative water content, osmolytes, electrolyteleakage, drought stress, pepper

Abstract. Water stress is the main factor responsible for decreased productivity, whichaffects the growth and development of crops. Plants respond to stress by accumulatingcompatible solutes, which have a key role in osmotic adjustment, thereby resulting inosmoprotection of the plants. The loss of water can increase the concentration ofcompatible osmolytes and molecules that regulate the plant metabolism. These solutescan be metabolized as sugars (sucrose, fructose, trehalosa), amino acids (proline), anamphoteric quaternary amine (glycine betaine), and other low-molecular-weight me-tabolites. However, among all these compatible solutes, proline and glycine betaine occurthe most. Proline is an amino acid that can accumulate in low concentrations underoptimal conditions; however, stress conditions contribute to its increased content. Fewdata are available regarding the levels of endogenous glycine betaine on Solanaceae,which is considered a nonaccumulator under water deficit conditions. The objective ofthis research was to evaluate the role of compatible osmolytes, glycine betaine andproline, in Capsicum sp. plants under different water deficit conditions. In this study, thepresence of endogenous levels of proline and glycine betaine in two species of pepper(Capsicum chinense var. Genesis and Rex and Capsicum annuum var. Padron) werefound. The concentration levels of proline were 362, 292, and 246 mmol·gL1 DW forGenesis, Rex and Padron respectively, and irrigation conditions (rehydration) of prolinelevels increased to 381, 395, and 383 mmol·gL1 DW at 21 days. However, glycine betainelevels were 30–70mmol·gL1 DW. The relative water content, electrolyte leakage, and soilwater potential were also analyzed; therefore, the information suggests that prolinecontributes better to tolerance to water deficit in the genus Capsicum after 14 days ofwater deficit treatment. It seems that the contribution of glycine betaine is less effectivethan that of proline; therefore, it does not have an important role in osmotic adjustment.

In recent years, it has been noted thatextended droughts cause severe damage toseveral important crops in the main produc-ing areas worldwide. Water shortages areresponsible for the greatest crop losses, andthey are expected to worsen (Comas et al.,2019). Water deficit is a critical abiotic factorthat affects the growth and productivity ofplants, especially in arid or semiarid regionsof the planet (Bodner et al., 2015). This typeof stress has become one of the main negativefactors affecting agriculture because it limitsplant growth, development, and productivityin many countries (Gosal et al., 2009; Xuet al., 2010; Zhu, 2002). The effects of waterdeficit are generally presented as inhibition ofcell proliferation and expansion, reduction inleaf size, and reduction in stem length,resulting in an overall negative effect on the

amount of aerial and radicular biomass(Harrison et al., 2013; Sekmen et al., 2014;Shanker et al., 2014; Zdravkovi�c et al.,2013). Under this kind of stress, plants com-monly experience numerous metabolic,physiological, and biochemical changes(Hasanuzzaman et al., 2014). This abioticstress, which arises from water deficiency,decreases the hydric potential of plants,which can induce reductions in gS and pho-tosynthesis, leading to decreased yield andproductivity for most crops (Chen et al.,2012). Plants use water to maintain the flowof nutrients and their turgor; to avoid the lossof water due to evapotranspiration or anyother process, plants have developed severalalternative mechanisms to endure and toler-ate this type of tension (Pimentel, 2004;Verlues et al., 2006). One mechanism thatcontributes to the toleration of water deficit isthe activation of metabolic pathways thatrespond to the low water availability, suchas osmotic adjustment (OA), which consistsof reducing cellular damage by accumulat-ing osmolytes or compatible solutes (Chaves

et al., 2003). The aforementioned osmolytesare metabolites that accumulate inside thecell in high concentrations without causingmetabolic harm such as polyols, which in-clude sorbitol, mannitol, arabitol, and glyc-erol, and amino acids, which include proline(Pro), the quaternary amine glycine betaine(GB), dimethylsulfoniumpropionate, and thedisaccharides sucrose and trehalose (Farooqet al., 2014; Jones et al., 1980; Yancey et al.,1982). Additionally, OA provides plants witha method of sustaining the water content ofthe cell, which is essential for cellular activity(Bartels and Sunkar, 2005; Javot et al., 2003).

The osmolytes Pro and GB have a funda-mental role when higher plants are exposed toconditions such as osmotic, hydric, andoxidative stress because they help with waterconservation and protect proteins and biolog-ical membranes (Ashraf and Foolad, 2007;Farooq et al., 2009a, 2009b). In many plantspecies, free Pro has an important role andaccumulates most under stressful conditions(Bhaskara et al., 2015). The roles of Pro andits metabolism under stressful conditions inseveral species have received considerableattention, and it is currently generally ac-cepted that Pro has a multifunctional rolein the response of plants to stress (Kaurand Asthir, 2015; Mansour and Ali, 2017;Szabados and Savour�e, 2010). In addition toits function as an osmolyte, Pro is a scaven-ger of reactive oxygen species (ROS) andcan stabilize sub-cellular structures, therebymodulating the redox homeostasis of the celland operating as a supply of energy and as asignal molecule interacting with other meta-bolic pathways during periods of stress(Kishor et al., 2005; Sharma et al., 2011;Szabados and Savour�e, 2010; Verbruggenand Hermans, 2008). Therefore, it is veryimportant to understand the regulation of Proat a genetic level as well as its metabolism toproduce plants that could sustain high bio-synthesis of this amino acid to favor itsaccumulation and improve the resistance ofthe plant. Two distinct pathways, the gluta-mate (Glu) and the Ornitine (Orn) pathways,exist in higher plants for the biosynthesis ofPro (Hu et al., 1992; Roosens et al., 1998).Similarly, Pro degradation occurs in themitochondria through the consecutive actionof Pro dehydrogenase (PDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) toproduce P5C and Glu, respectively (Peret al., 2017). An increase in Pro biosynthesisand a decrease in its degradation could implythat its accumulation under stressful condi-tions could lead to great benefits for plants(Chaitanya et al., 2009; Sharma et al., 2011).

GB is synthesized via two distinct path-ways from two distinct substrates, cholineand glycine (Ashraf and Foolad, 2007;Sakamoto and Murata, 2002). The osmolyteGB can accumulate in a several organ-isms such as plants, animals, bacteria, cya-nobacteria, and algae (Rhodes and Hanson,1993); its accumulation provides protectionagainst several environmental factors suchas drought, salinity, and cold (Ashraf andFoolad, 2007; Ashraf and Harris, 2004; Chen

Received for publication 13 Feb. 2019. Acceptedfor publication 27 Mar. 2019.1Corresponding author. E-mail: [email protected].

1044 HORTSCIENCE VOL. 54(6) JUNE 2019

and Murata, 2008). GB is biosynthesized inplants when exposed to diverse environmentalfactors that cause stress, such as salinity. It hasbeen observed that GB can be synthesized andaccumulated; however, some species such asOryza sativa, A. thaliana, and N. tabacumdo not produce GB naturally (Rhodes andHanson, 1993). Other studies suggested thatosmotic stress-inducedGBbiosynthesis occursvia jasmonate signal transduction, which notonly has a key role in osmotic stress resistancebut also contributes to tolerance (Xu et al.,2018). Furthermore, GB maintained a higherphotosynthesis rate, thereby increasing theproduction and translocation of sucrose viaphloem loading to enhance the plant responseto low-phosphate stress (Li et al., 2019).However, Wei et al. (2017) demonstrated thatGB might regulate ion channel and trans-porters, resulting in high potassium and lowsodium levels to enhance salt tolerance intransgenic plants under salt stress conditions.

The goal of this research was to determinewhich of the evaluated osmolytes could havea fundamental role in the water deficit toler-ance of plants of the genus Capsicum (Cap-sicum chinense var. Rex and Genesis andCapsicum annuum var. Padron).

Materials and Methods

Plant material. The species Capsicumchinense (cultivars Rex and Genesis) andCapsicum annuum (cultivar Padron) wereused in this study.

Seed disinfection. One hundred seeds ofeach of the three cultivars were disinfected.Briefly, the seeds were placed in Falcon tubesand washed with 50 mL of ethanol at 80%(v/v) under continuous agitation for 5 min.Then, they were rinsed three times with sterilewater, followed by a simple wash with 50 mLof commercial sodium hypochlorite (Cloralex5% NaOCl) at 30% (v/v) for 15 min and anadditional five washes with sterile water. Theseeds were subsequently maintained in sterilewater for 48 h at 4 �C in darkness.

Seed germination. Disinfected seeds wereplanted in sowing trays with coconut fiber asthe substrate. When germinated, the plantletswere wateredwith Hoagland solution at half itsionic strength once per week using 150 mL ofsolution per tray until the moment of trans-plantation. Hoagland solution (Hoagland andArnon, 1950) contained the following: 1.2 mM

KNO3, 0.8 mM Ca(NO3)2, 0.2 mM KH2PO4,0.2 mM MgSO4, 50 mM CaCl2, 12.5 mM H3

BO3, 1 mM MnSO4, 1 mM ZnSO4, 0.5 mMCuSO4, 0.1 mM (NH4)6Mo7O24, and 10 mMFe-EDTA (pH 6.8; all the reagents used werefrom Sigma-Aldrich, Inc., St. Louis, MO).

Growth conditions. When the plants pre-sented an average of six leaves, they weretransplanted to black polyethylene bags (ca-pacity of 6 kg) with a height of 15 cm andcontaining 4 kg of a mixture of substrate, soil,and organic peat moss–based substrate with aproportion of 3:1 (v:v). Then, they were takento the greenhouse to standardize the size ofthe plants. Planting was performed duringJan. 2013; during this time, plants were

maintained with 1 L H2O until sufficient leaftissue was collected.

Treatment of stress caused by waterdeficit. Twenty-five homogenous plants fromeach of the three pepper cultivars were used.Four plants were used for each of the fivetreatments, and five plants were used ascontrols. Experiments were performed undergreenhouse conditions during the floweringstage. The treatment involved maintainingthe plants without irrigation for 7, 10, 14, 18,and 21 d; at the end of each treatment stage, theplants were watered at field capacity. Beforeapplying the treatments, all pots were wateredto saturation (3 L H2O), after which the plantswere allowed to extract water for a period of2 d. At the end of the third day, the stresstreatment had been established. One group ofplants was watered on day 7, the second groupwas watered on day 10, the third groupwas watered on day 14, the fourth group waswatered on day 18, and the last groupwas watered on day 21. The control plantswere maintained and hydrated with irrigationevery third day (1 L H2O). Leaf tissue collec-tion was performed after each stress treatmentand at 24 h after irrigation (recovery stage).Twelve leaves were taken for each treatmentand for each plant cultivar; these were weighedand divided into four parts to determine therelative water content (RWC), electrolyteleakage, and Pro and GB contents.

Measurement of soil water potential. Soilsamples were collected from plants exposed todifferent treatments (7, 10, 14, 18, and 21 d)and from their respective control (well-hydrated plants). The samples were collectedwith a spatula at a distance of 10 cm from thestem and at a depth of 10 cm, approximatelytwo-thirds of the height of the bags, and wereplaced in trays that were then sealed with self-adhesive paper. These were subsequentlystored in a thermos with ice for transportation.Measurements were performed using a WP4Dewpoint PotentiaMeter (Decagon Devices,Inc.), which was previously calibrated with0.5 M KCl. The reading was adjusted to –2.19MPa, and distilled water (±0.1 MPa) was usedto corroborate the calibration. A soil samplewas collected for each replica of each treat-ment for each cultivar (n = 60); for the control,a sample was collected for each replica foreach cultivar (n = 15).

Determination of relative water content.To determine the RWC, the methodologyreported by Smart and Bingham (1974) wasused. Briefly, leaves were cut into discs witha diameter of 2.5 mm (using three discs perplant), and the fresh weight (FW) was regis-tered. Subsequently, the discs were sub-merged in 20 mL of distilled H2O for 12 hto obtain the turgid weight (TW). Finally, thediscs were dried in an oven at 40 �C, and thedry weight (DW) was obtained. The RWCwas calculated with the following formula:

RWC ð%Þ = ðFW – DWÞ=ðTW – DWÞ· 100

Measurement of electrolyte leakage. Per-meability of the membrane was measured

using electrolyte leakage and a modifiedmethod of Valentovi�c et al. (2006), whichinvolved the following: with the aid of a holepunch, 12 leaf discs with a diameter of 2.5 mmwere cut and transferred to Falcon tubes, towhich 15 mL of deionized water was added;these were left to incubate for 24 h at 25 �C.Subsequently, the electrical conductivity (EC)was measured (L1) with a conductometer(Jenway 4010; Jenway Ltd., Dunmow, Essex,UK) and samples were placed in the autoclaveat 120 �C for 20 min, after which the sampleswere allowed to cool and the ECwasmeasured(L2). Permeability of the membrane wascalculated using the following formula:

Electrolyte leakage ð%Þ = ðL1=L2Þ · 100Quantification of proline content. Quantifi-

cation of free Pro was performed using themethod established by Bates et al. (1973) withmodifications (all reagents were Sigma-Aldrich,Inc.). The ninhydrin acid reactive was preparedby placing 1.25 g of ninhydrin in 30 mL ofglacial acetic acid and 20mL of phosphoric acid6 M and agitating it until homogenized.

Approximately 0.01 g of finely ground leaftissue (DW) was homogenized in 10 mL H2Oat the boiling temperature; after which, theresidue was separated by centrifugation at10,000 rpm (9503 gn) for 15 min using arefrigerated centrifuge (Hettich Mikro 200R,Andreas Hettich GmbH & Co. KG). Then, 2mL of the sample was transferred to a testtube; after which, 2 mL of ninhydrin acid and2 mL of glacial acetic acid were added andvigorously agitated.

The reaction mixture was heated in a waterbath at 100 �C for 1 h (60 min), during whichthe tubes remained covered with a glass sphere(marbles) to avoid evaporation of the mixture.When the incubation period ended, the reactionwas halted by submerging the tubes in ice. Thechromophore–Pro complex was extracted with4 mL of toluene and vigorously agitated. Thephase containing the chromophore was ob-tained, and its absorbance was determined ina spectrophotometer Thermo Spectronic Gen-esys 10 uv Scanning Spectro- photometer;Thermo Fisher Scientific, Madison, WI at awavelength of 520 nm. The absorbance valueswere compared with the standard curve valuesof Pro. A stock was prepared with a concentra-tion of 1 mg/mL. The concentrations used forthe curve were 0–30 mg/mL of L-Pro, andcalculations were performed using the follow-ing formula:

mmoles proline=g

= ½ðmg proline=mL ·mL tolueneÞ= 115:5mg=mmol �= ½ðg sampleÞ=5�

Determination of the glycine betainecontent. The GB content was determinedusing the method of Grieve and Grattan(1983) with modifications (all reagents werefrom Sigma-Aldrich, Inc.). Finely ground drymaterial (0.5 g) was mixed with 20 mL ofdeionized water and maintained with me-chanical agitation for 48 h at 25 �C; sub-sequently, the mixture was centrifuged at

HORTSCIENCE VOL. 54(6) JUNE 2019 1045

10,000 rpm (9503 gn) for 10 min using arefrigerated centrifuge (Zentrifugen hettich,Mikro 200 R). The supernatant was collectedand stored in the freezer until the analysis.Samples were thawed and diluted (1:1 v/v)with 2N of sulphuric acid (H2SO4). An aliquotof 0.5 mL was collected and incubated for 1 hin ice; after which, 0.2 mL of cold potassiumiodide-iodine (KI-I2) was added and mixedgently. The samples were stored at 0 to 4 �Cfor 16 h; after which, they were centrifuged at10,000 gn for 15 min at 0 �C. The supernatantwas discarded while ensuring not to shake it;during this time, the samples were maintainedin ice to allow separation of the periodidecomplexes from the acid medium. The period-ide crystals that formed were dissolved in 9mL of 1,2-dichloroethane, and the mixturewas left to sit for 2 h; after which, theabsorbance (Thermo Spectronic Genesys 10uv Scanning Spectrophotometer) was read at365 nm. For the standard GB curve, a stockcomprising 1 mg/mL diluted in 1 N H2SO4

was prepared; concentrations of 50–200 mg/mL were used.

Statistical analysis. Data were analyzedusing a one-way analysis of variance. Treat-ment averages were compared using Tukey’srange test.WeperformedPearson’s correlationsbetween the different pepper plants. All ana-lyses were conducted using Sigma Stat Version3.1 (Systat Software Inc., San Jose, CA).

Results

Determination of soil water potential. Toidentify the water stress conditions applied,the water potential of the soil samples takenfrom the cultivation area of the plants eval-uated was determined. Figure 1 presents thewater potentials of the samples collected. Theresults showed that as the water deficit in-creased due to the lack of water caused byevaporation or from the intake of plants, thewater potential tended to decrease. This wassimilar to the outcomes of habanero pepperplants that were evaluated after being sub-jected to different stress levels that resulted ina loss of turgor in the leaves and otherphysiological effects, thereby provoking aresponse from the plant.

In soil that was completely hydrated, thewater potential values reached 0 MPa (datanot shown); therefore, plants under theseconditions were not considered during stress.However, in the case of the plants thatreceived water every 7 and 10 d, the soilvalues reached –0.102 and –0.857 MPa. Theplants subjected to 14 d of drought had asevere water deficit, with a value of –2.04MPa, which was similar to the plants sub-jected to 18 d of drought (–5.829 MPa) and21 d of drought (–10.273 MPa). Therefore,we concluded that stress was present on day14 of treatment. These results were in agree-ment with the outcomes of different irrigationtimes and water potential values, thus sug-gesting that 18 and 21 d of drought resulted inthe most negative potential and could beconsidered the most severe treatments forthe two Capsicum species evaluated.

Quantification of relative water contentlevels. RWC quantification of the leaves ofthese plants was performed to identify thestatus of the different plants evaluated re-garding the water relationships. The resultsare shown in Fig. 2. After comparing themeans, no significant difference was ob-served in the RWC among the C. chinense(Fig. 2A and B) and C. annuum (Fig. 2C)control plants; similarly, it was observed thatthese well-hydrated plants had an RWCmorethan 80%. However, under water deficitconditions, the plants had significantly re-duced RWC as the stress reached greaterintensity. During the first 7 d of stress, theRWC levels of Genesis (82.5%), Rex (85%),and Padron (85%) were determined (Fig. 2A–C). At the end of the 21-d treatment, theRWC values were 38.81% and 32.71% forthe varieties Genesis and Rex, respectively.For the Padron cultivar of C. annuum, theRWC percentage on day 21 was 32.6%. Thisindicated that the sensitivity to drought, atleast in relation to the RWC, was dependenton the species and the genotype. As pre-viously mentioned, when these plants aresubjected to water stress, one parameter thatallows us to identify their water status is theRWC, which is indicative of the quantity ofwater present in the plants. Additionally,another parameter of equal importance isthe recovery capacity when subjected tohydration. This capacity of recovery allowsus to predict when the plants will reach theirpermanent wilting point, which is the lowestwater potential at which a plant can accesswater from soil; therefore, the recovery ca-pacity of the pepper plants was evaluated.Furthermore, the plants under stress condi-tions were subjected to rehydration to fieldcapacity; after 24 h, the RWC was measured.

All plants subjected to the different irrigationschemes showed recovery in cell turgor, andthe RWC returned rapidly to values similar tothose of the control. The results of Genesis,Rex, and Padron are shown in Fig. 2D–F.These results demonstrated that the plants at21 d had not reached the point of permanentwilting, which indicated that the two Capsi-cum species evaluated could have a level oftolerance to water deficit under the conditionsused in this study.

Evaluation of membrane damage inplants subjected to water stress. The struc-tural integrity of cell membranes is impor-tant for their survival under droughtconditions or water deficit (Martínez et al.,2004). In addition, the maintenance of in-tegrity and membrane stability are essentialand recognized as important elements forthe tolerance to drought (Bajji et al., 2002).The degree of stability of the cellular mem-brane is considered one of the principalindicators of when a plant is under waterstress, and it has been used as a marker fortolerant or sensitive genotypes (Kochevaet al., 2004). Estimation of this stabilityaccording to electrolyte leakage has beenused to differentiate tolerant and sensitivegenotypes because this loss reflects thedamage that occurs in membranes.

Figure 3 shows that the three controlcultivars showed no significant difference inelectrolyte leakage under conditions of dailywatering.

Electrolyte leakage increased signifi-cantly during 7 d of treatment compared withthe control group for the Genesis and Rexcultivars (Fig. 3A and B). The Padron culti-var did not present damages in the mem-branes at 7 and 10 d of water deficittreatment; however, on day 14, an increase

Fig. 1. Soil water potential of the plants subjected to different irrigation times.


of 63.7% in electrolyte leakage was ob-served (Fig. 3C). On day 21, the electrolyteleakage reached 93.2%. For the Genesiscultivar, electrolyte leakage presented sig-nificant differences on day 7 of treatment,with a value of 45.6 that increased to�93.9% on day 21 (Fig. 3A). Similar be-havior was observed for the Rex cultivar(Fig. 3B), for which electrolyte leakage

reached 83.4% on day 14 of treatment and93.9% on day 21 of treatment.

It was interesting to note that althoughelectrolyte leakage was very high on day 21without watering, the plants were capable ofrecovery when they were rehydrated. More-over, a difference in the behavior of thisparameter was observed in relation to thespecies. C. annuum seemed to have greater

tolerance to the conditions of water deficit atthe membrane level compared with that of C.chinense, for which electrolyte leakage wasmore severe during 14-d and 18-d treatments;the cultivars of both species reached the samepercentage of electrolyte leakage on day 21without watering. Considering the RWC(Fig. 2A–C) and the capacity of the plantsto recover after rehydration (Fig. 2D–F), the

Fig. 2. Effects of water deficit (left panel) and recovery (right panel) on the relative water content (RWC) on Capsicum plantlets: (A, D) Genesis, (B, E) Rex, and(C, F) Padron. The plants were subjected to different stress and irrigation regimens. Control plants were watered daily at field capacity. Means with the sameletter indicate that they are not significantly different (Tukey P # 0.05).


results clearly confirmed that the membranesrecovered their capacity to maintain cellularturgor.

Quantification of the proline content inpepper plants subjected to water deficit. Todetermine if Pro has an important role in the

tolerance to water deficit of Capsicum,quantification of the Pro content was sub-sequently performed to observe its possibleparticipation in the OA of the three peppercultivars subjected to different wateringtreatments.

The control plants of the three cultivars ofpeppers maintained almost constant Provalues between 13 and 31 mmol·g–1 DW(Fig. 4). Under stress conditions, Genesisexhibited a gradual increase in the concen-trations of Pro (Fig. 4A); on day 7, the Prolevel reached a value of 25.83 mmol·g–1 DW,similar to the Pro concentration observed inplants that did not experience stress. Thelevel of Pro had increased 14 times by day14 of treatment, with a concentration of234.77 mmol·g–1 DW (Fig. 4A). The maxi-mum accumulation of Pro was observed onday 21 of treatment, with a value of 362.993mmol·g–1 DW; this value represented an in-crease of 19 times compared with the controlplants.

For the cultivar Rex (Fig. 4B), the Proconcentration on day 14 of treatment was142.12 mmol·g–1 DW, and the maximumlevel was obtained on day 21, with a valueof 292.32 mmol·g–1 DW. This concentrationwas lower compared with that obtained forthe Genesis cultivar.

Finally, for the Padron cultivar (Fig. 4C),the Pro level on day 14 increased up to 7.2times compared with that of the control andup to 6.7 times compared with that on day 7.The maximum concentration of Pro wasfound on day 18 of treatment, with a valueof 304.32 mmol·g–1 DW. This concentrationmay be the quantity required to maintaincellular turgor because on day 21, the leveldecreased to a value of 246.25 mmol·g–1 DW.

After irrigation for rehydration (Fig. 4D–F), the Pro levels on day 14 exhibited amarked reduction and reached basal values.However, for the Genesis cultivar (Fig. 4D),the Pro level on day 18 was reduced to 256mmol·g–1 DW. Subsequently, on day 21, acontrasting effect was observed: the Pro levelincreased to 381.75 mmol·g–1 DW. Finally,for the cultivars Rex and Padron (Fig. 4E andF), after rehydration, it was possible toobserve behavior similar to that of Genesisuntil day 14; however, on day 18, the levelswere similar for both cultivars, with valuesbetween 150 and 200 mmol·g–1 DW. On day21, the highest concentration of Pro wasdetermined, and it was similar to the amountquantified for the Genesis cultivar, reaching�395 mmol·g–1 DW.

Quantification of glycine betaine inpepper plants subjected to water deficit. GBis another osmolyte that has a role in OA andparticipates in the osmoprotection of plantsunder stress conditions. It has been reportedto protect the photosynthetic machinery andalso functions as a stabilizer of enzymes and apurifier of ROS (Anjum et al., 2012; Chaitanyaet al., 2009; Rezaei et al., 2012).

During this study, quantification of thisosmolyte was important to elucidate its con-tribution to the apparent tolerance to droughtof the plants of the genusCapsicum. This wasmainly because Solanaceas are considered tobe non-natural accumulators of GB; in addi-tion, few data are available regarding theendogenous contents of GB for this type ofplant. The quantified results are shown inFig. 5. On day 7 of treatment, the Genesis

Fig. 3. Effects of water deficit on the percentage of electrolyte leakage in Capsicum plants subjected todifferent irrigation regimes: (A) Genesis, (B) Rex, and (C) Padron. Control plants were watered dailyat field capacity. Means with the same letter indicate that they are not significantly different (TukeyP # 0.05).


cultivar (Fig. 5A) showed a GB content of26.94 mmol·g–1 DW, which was increased onday 10 to 44.30 mmol·g–1 DW. On day 21, theGB level was 53.965 mmol·g–1 DW, indicat-ing that drier soils do not provoke significantchanges in the concentration of this osmolyte.On day 7, for the Rex cultivar (Fig. 5B), theGB level was 23.98 mmol·g–1 DW; on day 10,

it was 56.28 mmol·g–1 DW, almost 2.3-timeshigher compared with day 7. This indicatedthat the mechanisms of GB biosynthesis arebeginning to be affected by stress. By day 14of treatment, the concentration of GB de-creased almost 2.12 times compared with thaton day 10, reaching values similar to thosefound on day 7. After day 14, the GB values

remained almost constant until day 21, with aregistered value of 39.18 mmol·g–1 DW.

As was observed for Padron (Fig. 5 C), theresults obtained on day 10 of treatmentshowed increased concentration of GB, witha value of 72.67 mmol·g–1 DW. On day 21, thelevel was 53.69 mmol·g–1 DW; despite theserather notorious increases in GB, statistically

Fig. 4. Effects of water deficit (left panel) and recovery (right panel) on the content of proline in Capsicum plantlets: (A, D) Genesis, (B, E) Rex, (C, F) Padron.The plants were subjected to different stress and irrigation regimens. Control plants were irrigated daily at field capacity. Means with the same letter indicatethat they are not significantly different (Tukey P # 0.05).


speaking, no significant differences werefound for any of the stress treatments, whichcould indicate that GB accumulates as stressbecomes more severe. This erratic behaviorof the GB content suggested that this com-pound is not directly involved in the toleranceof these species to water deficit. An experi-ment consisting of recovery irrigation (re-hydration) was proposed to determine if GBmaintains its endogenous levels or reducesthem when the Capsicum plants are rehy-drated. The results are shown in Fig. 5D–F.These cultivars presented no differences intheir GB contents; however, for Genesis

(Fig. 5D) control plants, the GB valuesranging from 22–32 mmol·g–1 DW wereregistered, and the recently rehydrated plantvalues ranged from 30 to 46 mmol·g–1 DW. Inthe case of the cultivar Rex (Fig. 5E), the GBconcentration was ranged from 25 to 27mmol·g–1 DW and 25–38 mmol·g–1 DW.Finally, for the cultivar Padron (Fig. 5F),the GB concentration ranged from 28 to 39mmol·g–1 DW and 18–40 mmol·g–1 DW. Thiserratic behavior of the GB content couldindicate that this compound is not directlyinvolved in the tolerance of these species towater deficit. These cultivars presented no

differences in their GB content when theywere subjected to rehydration at field capac-ity. Their role of stress in GB accumulationis uncertain because, statistically, the levelsof GB in plants, stressed or not, presentedno difference. Moreover, during recoverystages, the GB levels are similar to those ofplants under stress conditions. However, fewdata are available regarding the effects of GBon peppers, and most reports have focusedmainly on the exogenous application of thiscompound.

Correlation between the relative watercontent and the proline and glycine betaine

Fig. 5. Effects of water deficit (left panel) and recovery (right panel) on the glycine betaine content in Capsicum plantlets: (A, D) Genesis, (B, E) Rex, and (C, F)Padron. The plants were subjected to different stress and irrigation regimens and the control plants were irrigated daily at field capacity. Means with the sameletter indicate that they are not significantly different (Tukey P # 0.05).


contents in pepper plants subjected to waterdeficit. The RWC is a parameter that isconsidered to determine the levels of stressdue to water deficit in plants. The correlationbetween RWC and Pro (Fig. 6A–C) and GB(Fig. 6D–F) concentrations are important toelucidate the function of these osmolytes torespond to different types of stress. It waspossible to observe that Pro decreases as thepercentage of RWC increases in all cultivarsstudied (Fig. 6A–C), indicating a close neg-ative correlation between these variables(P # 0.05). However, for GB (Fig. 6D–F),the correlation coefficients were very low,indicating very clearly that changes in theaccumulation of GB had no relationship withthe tolerance of pepper plants to water deficit.

Discussion

Due to the rapid changes in the environ-ment on a global scale, the ability to cultivateand maintain a crop with good production isbecoming increasingly difficult. Stress willinevitably lead to changes in morphology,physiology, and biochemistry, as well as tomolecular changes resulting from the pro-

ductivity of a crop (Boaretto et al., 2014;Mansori et al., 2015; Medici et al., 2014).Drought is one of the most predominantabiotic factors limiting the global productiv-ity of agriculture (Gholipoor et al. 2012;Ihsan et al. 2016). It also affects growth anddevelopment due to the reduction of turgor inthe leaves, resulting in reduced acquisition ofnutrients from the soil by the plant (Luo et al.2011).

Plants, as sessile organisms, possess strat-egies to mitigate the aforementioned prob-lems and maintain the perpetuation of thespecies. One of these strategies is the synthe-sis of known substances such as compatibleosmolytes, which include Pro and GB, amongmany others.

First, we can report that the plants of thethree cultivars studied were subjected towater deficit from 7 to 21 d of treatmentwithout irrigation, which allowed us to iden-tify the stress conditions to which they weresubjected. As a result of this study, it waspossible to observe that RWC, electrolyteleakage, and water potential are modified asthe water deficit becomes more severe. Re-garding RWC, similar values were found by

Anjum et al. (2012), who evaluated differentcultivars of C. annuum (cultivars Shansshu-2001 and Nongchengjiao-2). They reportedthat after 12 d of treatment with severe waterdeficit, the plants presented a lower percent-age of RWC, with the Shanshu-2001 cultivarpresenting high values of RWC, and greaterresistance to water deficit; they concludedthat the cultivar Shanshu-2001 had greatertolerance to water deficit because it alsocontinued accumulating soluble proteins,high Pro levels, greater turgor, and lessdamage in the membrane. Sahin et al.(2018), while evaluating cabbage plantlets(Brassica oleracea var. Capitata) using dif-ferent irrigation regimens, were able to ob-serve that the plants for which the treatmentwas most severe (close to 60% irrigation)presented less RWC. It was also possible toobserve greater electrolyte leakage with theaforementioned treatment, and their resultsshowed that in addition to electrolyte leak-age, the plants presented higher concentra-tions of lipid peroxidation, which wouldindicate that with this treatment, the mem-branes would be damaged at membrane level,which could be harmful for the growth anddevelopment of cabbage. The RWC valuesfor the three cultivars (Genesis, Rex, andPadron) were 82% to 86%, and by the end ofthis experiment, values of 32% to 39% werereached. After analyzing the behavior of thedifferent cultivars of the two species ofthe genus Capsicum, we determined that theGeneses cultivar was rapidly affected be-cause it had reduced RWC after day 10without irrigation. Plants subjected to slightstress, compared with Rex and Padron sub-jected to severe stress (Fig. 2), indicated thedependence of the genotype on this importantparameter. When we studied types of abioticstress, even within the same species, therewere differences regarding tolerance. Resultssimilar to ours were reported by Ju et al.(2018), who evaluated grape plants (Vitisvinifera L.) with different water deficit times.The RWC showed that from day 15 oftreatment, the plants exhibited lower RWC;however, this did not continue to diminish; atday 30, they continued to present the samevalues as those on day 15 of treatment. Thiscultivar may have greater tolerance to waterdeficit because it loses water more quickly,but it is able to recover its turgor afterrehydration in the same proportion as theother two cultivars studied. Therefore, weconcluded that for these pepper species, thestress of 21 d without irrigation, despitehaving water potential more than –10 MPa,is not stress that could indicate that the plantshave reached their point of permanent wiltingbecause when they are hydrated, they recovertheir turgor and continue their life cycle.

The stress from water deficit has animportant role in the internal state of theplants, thereby causing damage in the cellmembranes, which leads to rapid peroxida-tion of lipids, damage in the transportationsystems, and other problems. Therefore, thepermeability of the membranes of the threecultivars of pepper was evaluated. It was

Fig. 6. Pearson’s correlation between the relative water content and proline content (left panel) andbetween the relative water content and content of glycine betaine (right panel): (A, D) Genesis, (B, E)Rex, and (C, F) Padron. The plants were subjected to different stress regimens. Control plants wereirrigated daily at field capacity.


possible to observe that moderate stress,which appeared on day 14, was sufficient toinitiate the damage of cell membranes, whichwould subsequently lead to the production ofreactive oxygen species and rapid peroxida-tion of lipids in the membrane. Therefore,electrolyte leakage caused by damage in themembrane reflects the magnitude of the cellmembrane lesion, which is why it has beenused to differentiate between tolerant andsusceptible genotypes (Rahman et al., 2004).

Although the measurements showed elec-trolyte leakage more than 80% in plantstreated with 21 d of water deficit, they re-covered satisfactorily when rehydration treat-ments were applied, which agreed with theoutcomes reported by Mihailova et al. (2018)for Haberlea rhodopensis, a chlorophyll-retaining resurrection plant. It is importantto mention that the studies involving Capsi-cum plants examined the leaf, which is theorgan that is first affected during stress due towater deficit.

Although all the cultivars presented mod-erate stress on day 14, Genesis (Fig. 3A)presented lower stability in its membranesfrom day 7, which concurred with the RWClevels presented previously. Regarding theresults presented for Rex (Fig. 3B) on day 7,these may be attributed to climatic changes.The presence of Pro (Fig. 4) increased fromday 14 when the plant presented moderatestress and had water potential of –2.04 MPa.The levels of imposed stress reached at day21 were �240–263 mmol·g–1 DW. However,when these plants were rehydrated, the levelson day 21 continued to increase. This in-crease in Pro levels during the rehydrationstage is comparable with those reported byAn et al. (2013), who found increases of�1.4mg·g–1 DW and 2.0 mg·g–1 DW of Pro inhydrated plants after water stress for 24 h and96 h (–1.0 MPa), respectively. The increasewas also observed in different pepper culti-vars from 6 to 24 d (Anjum et al., 2012) andin tomato cultivars (Moles et al., 2018).Similar studies performed for different culti-vars of potato by Meise et al. (2018) reportthat when the plants were subjected to waterstress for long periods of time (12 d), they hada greater accumulation of Pro and higherRWC. The authors concluded that there was agenetic variation in the cultivars regardingthe treatments they evaluated, and that theroots may have systems that allow the trans-portation of Pro to be accumulated in leavesor roots. They also suggested that the Procould be a target for the improvement ofdrought tolerance in potato.

Moreover, in grapes (Vitis vinifera L.), ithas also been observed that after 10 d oftreatment, the accumulation of Pro increasedfrom 200 mg·g–1 FW to a maximum level of500 mg·g–1 FW on day 20 (Ju et al., 2018);these results coincided with those of in ourstudy. One possible explanation for thisbehavior could be a high accumulation ofenzymes involved in the synthesis of Pro,such as P5CS (Szepesi and Sz}oll}osi, 2018;Yoshiba et al., 1997). Therefore, it would beof significant interest to determine the activ-

ity of the respective enzymes as well as themetabolism of the Pro in the presence ofwater deficit and during periods of recovery.

It has been reported that the exogenousapplication of GB provides tolerance to stressin accumulating and nonaccumulating spe-cies, although evidence suggested that theexogenous application of GB is not effectivefor all crops (Sulpice et al., 2002; Xing andRajashekar, 1999). A toxic effect from theexogenous administration of GB has beenobserved in canola plants (Brassica napusL.), suggesting that it is not a compatiblesolute in all plants because one of the mostimportant characteristics of a compatiblesolute is its accumulation in high concentra-tions without altering cell metabolism (Gibonand Larher, 1997).

Our results indicated that it was possibleto observe that GB (Fig. 5) did not seem to beinvolved in OA or in osmoprotection. Moreimportantly, it did not interfere with theendogenous levels of Pro. Previously, it wasreported that exogenous administration ofGB does not interfere with the metabolismof Pro (Korkmaz et al., 2012).

Based on the data regarding the correla-tion between Pro and RWC (Fig. 6A–C), wecould clearly observe that the correlation wasclose to 1 for all the cultivars, with thecultivar Rex presenting a correlation valueof –0.931; therefore, it can be suggested thatPro contributes to water relationships ofthe Capsicum plants evaluated. These dataare comparable with those presented byChowdhury et al. (2017), who found a strongrelationship between Pro and the damageindex for different cultivars of soya. Theauthors suggested that the synthesis of Proand proteins played a fundamental role in theprotection of cellular structures during stresscaused by water deficit. It has been reportedthat the activation of this synthesis of bothosmolytes and proteins allows plants tosurvive under conditions of stress by de-hydration (Molaei et al., 2012). This benefi-cial protective function of Pro has alsobeen observed in rice plants. Cha-Um andKirdmanee (2010) observed that when riceplants were subjected to different concentra-tions of NaCl (50, 100, and 150 mM) and tofoliar spraying with 50 mM of GB, theaccumulation of Pro and efficient water usewere favorably maintained. A close relation-ship between these two parameters wasobserved; therefore, the authors concludedthat the foliar application of GB in optimaldoses could be a favorable alternative used toimprove the tolerance to salinity in riceplants. However, our results indicated thatGB is not correlated with RWC (Fig. 6D–F)because its values were between –0.259 and0.0127. Therefore, based on other studies(Gibon and Larher, 1997; Korkmaz et al.,2012), we concluded that GB is not anosmolyte, as reported by Asma et al.(2006), who observed that the foliar applica-tion of GB to corn did not have the expectedeffect. Although it has been reported that GBacts as an osmoprotectant in some plantspecies, it is important to mention that these

studies have only been performed in vitro(Saini et al., 2012).

In addition, few data regarding the effectsof osmoprotection and endogenous presenceof GB observed during the winter are avail-able. Agboma et al. (1997) indicated that GBwas exogenously applied to soya plants(Glycine max L.), thereby improving theeffects of dehydration. Regarding pepper,which is considered a nonaccumulating spe-cies, in contrast to the report by Ponce et al.(1996), who did not find GB in pepper plants(Capsicum annum L.; Variedad Jupiter), ourresults indicated that it was possible to de-termine the endogenous presence of GB inCapsicum chinense (cultivars Genesis andRex) and Capsicum annum L. (cultivarPadron) in the range of 20–80 mmol·g–1 DW.

Conclusion

The results of this study suggested that inthe presence of water deficit, pepper plantsexhibit changes in the plasmatic membrane,RWC, and Pro content. In addition, regardingelectrolyte leakage, the Genesis cultivar pre-sented more sensibility and damage in themembrane, because it experienced more loss,whereas the cultivar Padron exhibited lessdamage. Similarly, regarding the RWC forthree pepper cultivars, it was only possible toobserve a significant reduction by applyingstress from 14 to 21 d. Of the three cultivarsstudied, Genesis had lower RWC on day 21after the plants were rehydrated (recovery),and it was found that when all plants weresubjected to recovery, the RWC values weresimilar to those of the control, suggesting thatthe treatment on day 21 does not involvestress that causes permanent wilting anddeath. The Genesis cultivar had the bestresponse to water deficit, and it presentedthe highest Pro content compared with theRex and Padron cultivars. We suggest thatPro is the osmolyte that could have animportant role in the tolerance to waterdeficit; however, we also concluded that GBis not a key osmolyte when plants of theCapsicum genus are subjected to water def-icit. Finally, in general, this study facilitated abetter understanding of the behavior of theCapsicum genus (two species evaluated) inthe presence of one type of abiotic stress andof the role of an amino acid (such as Pro) intolerance. The results of this study will helpfuture research studies that aim to identify theregulation of Pro metabolism and to ascertainthe existence of osmolytes other than Pro thatcould participate in tolerance. Therefore, wesuggest further investigations to respond tothe new questions that will arise.

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