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5.44.5Antioxidative Defense,SuppressedNitric Oxide Accumulation,andSynthesis of Protective Proteins inRoots and Leaves Contribute to theDesiccation Tolerance of theResurrection Plant HaberlearhodopensisKatya Georgieva,Gergana Mihailova,Liliana Gigova,Antoaneta V.Popova,Maya Velitchkova,Lyudmila Simova-Stoilova,Mt Sgi-Kazr,Helga Zelenynszki,Katalin Solymosi and dm SoltiSpecial IssueDesiccation/Salinity Tolerance and the Crosstalk ThereinEdited byProf.Dr.Jill M.Farrant and Dr.Mariam AwliaArticlehttps:/doi.org/10.3390/plants12152834Citation:Georgieva,K.;Mihailova,G.;Gigova,L.;Popova,A.V.;Velitchkova,M.;Simova-Stoilova,L.;Sgi-Kazr,M.;Zelenynszki,H.;Solymosi,K.;Solti,.AntioxidativeDefense,Suppressed Nitric OxideAccumulation,and Synthesis ofProtective Proteins in Roots andLeaves Contribute to the DesiccationTolerance of the Resurrection PlantHaberlea rhodopensis.Plants 2023,12,2834.https:/doi.org/10.3390/plants12152834Academic Editors:Jill M.Farrant,Mariam Awlia and Dayong ZhangReceived:19 May 2023Revised:27 July 2023Accepted:28 July 2023Published:31 July 2023Copyright:2023 by the authors.Licensee MDPI,Basel,Switzerland.This article is an open access articledistributedunderthetermsandconditions of the Creative CommonsAttribution(CC BY)license(https:/creativecommons.org/licenses/by/4.0/).plantsArticleAntioxidative Defense,Suppressed Nitric Oxide Accumulation,and Synthesis of Protective Proteins in Roots and LeavesContribute to the Desiccation Tolerance of the ResurrectionPlant Haberlea rhodopensisKatya Georgieva1,*,Gergana Mihailova1,Liliana Gigova1,Antoaneta V.Popova2,Maya Velitchkova2,Lyudmila Simova-Stoilova1,Mt Sgi-Kazr3,4,Helga Zelenynszki3,4,Katalin Solymosi5and dm Solti31Institute of Plant Physiology and Genetics,Bulgarian Academy of Sciences,Academic Georgi Bonchev Str.,Building 21,1113 Sofia,Bulgaria;gmihailovabio21.bas.bg(G.M.);(L.G.);lsimovamail.bg(L.S.-S.)2Institute of Biophysics and Biomedical Engineering,Bulgarian Academy of Sciences,Academic GeorgiBonchev Str.,Building 21,1113 Sofia,Bulgaria;popovabio21.bas.bg(A.V.P.);mayavbio21.bas.bg(M.V.)3Department of Plant Physiology and Molecular Plant Biology,Institute of Biology,ELTE Etvs LorndUniversity,Pzmny Pter Stny 1/C,H-1117 Budapest,Hungary;sagi.kazar.matettk.elte.hu(M.S.-K.);helga.zelenyanszkittk.elte.hu(H.Z.);adam.soltittk.elte.hu(.S.)4Doctoral School of Biology,Institute of Biology,ELTE Etvs Lornd University,Pzmny Pter Stny 1/C,H-1117 Budapest,Hungary5Department of Plant Anatomy,Institute of Biology,ELTE Etvs Lornd University,Pzmny Pter Stny1/C,H-1117 Budapest,Hungary;katalin.solymosittk.elte.hu*Correspondence:katyabio21.bas.bg or ;Tel.:[phone]Abstract:The desiccation tolerance of plants relies on defense mechanisms that enable the protectionof macromolecules,biological structures,and metabolism.Although the defense of leaf tissuesexposed to solar irradiation is challenging,mechanisms that protect the viability of the roots,yetlargely unexplored,are equally important for survival.Although the photosynthetic apparatusin leaves contributes to the generation of oxidative stress under drought stress,we hypothesizedthat oxidative stress and thus antioxidative defense is also predominant in the roots.Thus,weaimed for a comparative analysis of the protective mechanisms in leaves and roots during thedesiccation of Haberlea rhodopensis.Consequently,a high content of non-enzymatic antioxidantsand high activity of antioxidant enzymes together with the activation of specific isoenzymes werefound in both leaves and roots during the final stages of desiccation of H.rhodopensis.Among others,catalase and glutathione reductase activity showed a similar tendency of changes in roots and leaves,whereas,unlike that in the leaves,superoxide dismutase activity was enhanced under severe butnot under medium desiccation in roots.Nitric oxide accumulation in the root tips was found to besensitive to water restriction but suppressed under severe desiccation.In addition to the antioxidativedefense,desiccation induced an enhanced abundance of dehydrins,ELIPs,and sHSP 17.7 in leaves,but this was significantly better in roots.In contrast to leaf cells,starch remained in the cells ofthe central cylinder of desiccated roots.Taken together,protective compounds and antioxidativedefense mechanisms are equally important in protecting the roots to survive desiccation.Sincedrought-induced damage to the root system fundamentally affects the survival of plants,a betterunderstanding of root desiccation tolerance mechanisms is essential to compensate for the challengesof prolonged dry periods.Keywords:antioxidant enzymes;drought stress;non-enzymatic antioxidants;nitric oxide;photosyn-thesis;protective proteins;root anatomyPlants 2023,12,2834.https:/doi.org/10.3390/plants12152834https:/ 2023,12,28342 of 221.IntroductionIn the 21st century,evidence becomes clear on climate change,the primary challengefor humankind presently and in the future.Since extremities in the weather such as pro-longed drought are among the greatest threats to agriculture,the predicted increasingperiods of intense and extended drought as a consequence of global warming will havea deep impact on food production 1.The vegetative tissues of higher plants,includingcrops,are sensitive to water deficiency:depending on the species,loss of 4070%of totalwater content leads to permanent damage or death of the tissues 2.Thus,prolongeddrought stress and critical dehydration ultimately reduce the crop yield.However,resur-rection plants are able to survive up to 95%loss of tissue water content and thus surviveprolonged drought periods,whereas upon rehydration they recover to full metabolicactivity 3.Therefore,they are an optimal model to study and understand vegetativedesiccation tolerance.Such understanding may contribute to the breeding of crops withimproved tolerance 4.A considerable number of studies pointed out that foliar tissuesof resurrection plants are able to tolerate desiccation through a special set of mechanisms,including the alteration of the metabolism and the biosynthesis of novel antioxidants tominimize free radical-induced damages,subcellular reorganization in order to minimizemechanical stress associated with turgor loss,and the accumulation of specific proteins,disaccharides,and other hydrophilic metabolites to maintain the structure and the opera-tion of cell constituents 59.Since,in photosynthetically active tissues,suppression of thephotosynthetic functions and avoiding light-induced damages are primarily challenges tobe resolved in the desiccating foliar tissues for effective survival,the majority of studieson resurrection plants focused on the protective mechanisms that protect mesophyll cells.Nevertheless,in the survival of individual plants,responses of root tissues that are equallyor even better exposed to desiccation have remained an unexplored field.In foliar desiccation tolerance studies,homoiochlorophyllous resurrection plants thatmaintain the structure of the photosynthetic apparatus during dehydration are generallyapplied.Since,in these plant taxa,chlorophyll molecules retained in the desiccated stagecould be sources for harmful singlet oxygen production in mesophyll cells,protectionmechanisms,including rapid repair of the photosynthetic apparatus upon rehydration,are required.It has been proposed that switching off photosynthesis in homoiochloro-phyllous plants is likely to be a programmed process that involves specific protectivemechanisms 10.While the production of reactive oxygen species associated with photo-synthesis is reduced during desiccation in photosynthetically active tissues,the oxidativestress associated with metabolic processes of mitochondria and particularly peroxisomes isexacerbated with acute water loss 11.Thus,oxidative stress is not exclusively associatedwith the photosynthetic apparatus even in the foliage.Moreover,the upregulation ofantioxidant activity is generally acknowledged as one of the key protective mechanismsinduced during desiccation.It has been shown that resurrection plant individuals cansurvive desiccation without damage to the leaves as long as their foliar antioxidant systemis functional 12.In contrast to desiccation-sensitive plants,desiccation-tolerant plantscan maintain foliar antioxidant activity in the desiccated state 5.Accumulation of non-enzymatic antioxidants and enhanced activity of antioxidant enzymes have been observedduring desiccation 6,7.Although a considerable variation was detected in the antioxi-dant responses of different resurrection taxa under desiccation and rehydration 13,theirlongevity in the desiccated state was generally dependent on antioxidative protection 14.Nevertheless,these statements were not taken into a more general context,i.e.,how theprotective mechanisms of the root system contribute to the individuals survival 15,16.Haberlea rhodopensis Friv.is a poikilohydric,perennial,herbaceous plant that,as a preglacialrelict taxon,primarily inhabits shady,northern limestone slopes of relatively high humidityin mountainous regions of the Balkan Peninsula,South-East Europe.It is a generallyapplied homoiochlorophyllous resurrection plant model,in which antioxidative protectionwas previously found to be crucial to the survival of foliar desiccation 7.Although foliarPlants 2023,12,28343 of 22responses of H.rhodopensis to desiccation are well documented,the contribution of the rootsystem to desiccation tolerance is much less studied.Regarding the desiccation tolerance mechanisms in roots,studies on the amount of car-bohydrates reported an increased sucrose content in the root system upon the desiccation ofCraterostigma plantagineum,Pitcairnia burchellii,and Tripogon loliiformis 1719.Dehydratedroots of Tripogon loliiformis were found to contain more sucrose and trehalose-6-phosphatecompared to shoots,and the plant continued to use roots as a carbohydrate sink duringdrought stress,activating the bidirectional sugar transporter SWEET genes to translocatesucrose from shoots to roots 19.Investigation on glycerolipids present in root and leaftissues during the desiccation of Xerophyta humilis revealed an increase in species containingpolyunsaturated fatty acids and a decrease in saturated species 20.Our previous studiesshowed that the plasticity of adaptation in H.rhodopensis leaves and roots during extremewater stress conditions is different 21,22.In general,roots are more sensitive and respondfaster to water stress than leaves.The specific leaf area is reduced by 60%as a result ofsevere desiccation,whereas the root area decreased by 40%.A linear relationship betweenroot respiration and water content is established.In addition,analysis of H.rhodopensisleaves and roots revealed that jasmonic acid,along with and even earlier than abscisic acid,serves as a signal that triggers the response of this resurrection plant to desiccation 23.Inthe biosynthesis of abscisic acid,nitric oxide(NO)generation in root tips was found to bean early response in Triticum aestivum 24.Thus,NO production in plants is among thefirst reactions to drought 25.NO contributes to the regulation of multiple cell processes,including antioxidative defense and the formation of reactive nitrogen species,but alsothe synthesis of defense proteins 26.Antioxidative protection is an important process inroots.Investigating the activity of the antioxidant enzymes superoxide dismutase(SOD),catalase(CAT),ascorbate peroxidase(APX),and glutathione reductase(GR),as well asascorbate and glutathione content in the roots of Xerophyta viscosa during desiccation,roottissues appear to retain their antioxidant potential during drying for use in recovery uponrehydration,similar to leaf tissues but also to leaves of other resurrection taxa 27.Tounderstand the overall strategy of plant desiccation tolerance,holistic studies focusingsimultaneously both on root and leaf systems are required.Although the photosynthetic apparatus in leaves contributes to the generation ofoxidative stress under drought stress,we hypothesized that oxidative stress and thusantioxidative defense is also predominant in the roots.In order to get a holistic view,weperformed a comparative analysis of the desiccation tolerance mechanisms in leaves tothose operating in the root system in the homoiochlorophyllous model plant H.rhodopensis.The anatomy and ultrastructure of the roots and NO production of the root tips werestudied.Antioxidant capacity in leaves and roots from the same plant was assessedand compared by measuring the total antioxidant activity,free-radical scavenging activity,content of non-enzymatic antioxidants,and activity of antioxidant enzymes.The alterationsin stress-induced proteins(dehydrins,ELIPs,sHSPs)in leaves and roots during desiccationand after recovery on rehydration were compared.We have characterized,for the first time,the protective mechanisms of the root system in the Gesneriaceae resurrection plant modelH.rhodopensis.2.Results2.1.Changes in the Water Content of Leaves and Roots during DesiccationControl leaves(LC)of H.rhodopensis were characterized by higher water contentscompared to control roots(RC)(Table 1).The first sampling point during dehydration wasat a moderately dehydrated state,which is very characteristic of H.rhodopensis with itswilting drooping leaves becoming elastic.In moderately dehydrated plants,the WC ofleaves(LD1)and roots(RD1)was reduced by 56%and 70%,respectively.In the D2 stageof desiccation,the WC of leaves(LD2)and roots(RD2)was equal and further water lossled to a faster drop of WC in leaves than in roots(LD3 and RD3,respectively).The leavesand roots were completely desiccated in the D4 stage(Figure S1B).H.rhodopensis leavesPlants 2023,12,28344 of 22and roots in well-hydrated and air-dry states were compared in Figure S1.During therehydration of plants,leaves(LR)and roots(RR)recovered their WC.Table 1.Water content(WC)of control leaves(LC)and roots(RC)during desiccation to differentextent(LD1LD4;RD1RD4)and after 6 days of rehydration(LR;RR).VariantLeavesWC of Leaves(g H2O g DW1)VariantRootsWC of Roots(g H2O g DW1)LC4.960.08 aRC3.820.07 bLD12.160.20 dRD11.140.09 efLD21.300.14 efRD21.300.06 efLD30.880.16 fRD31.430.01 eLD40.220.03 gRD40.350.01 gLR4.100.14 bRR3.230.30 c2.2.Structural Changes in Root Tissues during DesiccationWe observed no striking structural alterations in the roots upon dehydration;however,a massive accumulation of dense accretions occurred in the cytoplasm of the exodermis andendodermis layers of the cortex of the desiccated roots(Figure 1).Transmission electronmicroscopy showed that these cells contained electron-dense dark deposits(not shown).Careful comparison of the root ultrastructure showed that in addition to heterochromati-nous nuclei,several small vacuoles were already present in both cortex and central cylindercells in the RC stage(Figure 2A,B).However,the number of vacuoles seemed to increaseupon desiccation in both regions,but especially in the cor