24Piriformospora indica, A Root Endophytic Fungus, EnhancesAbiotic Stress Tolerance of the Host PlantManoj Kumar, Ruby Sharma, Abhimanyu Jogawat, Pratap Singh, Meenakshi Dua,Sarvajeet Singh Gill, Dipesh Kumar Trivedi, Narendra Tuteja, Ajit Kumar Verma,Ralf Oelmuller, and Atul Kumar Johri

Piriformospora indica is an endophytic fungus that colonizes the roots of bothmonocotand dicot plants includingmembers of the family Brassicaceae, which are nonhost forarbuscularmycorrhizal fungi (AMF) and canalsobe grown axenically. Like theAMF,P.indica was found to be involved in the enhancement of plant tolerance against abioticstress. Growth promotion in plant is a characteristic effect of the fungal colonization,which canalsobeobservedunder the stress conditions.P. indicamodulates thedefensesystemandalters themetabolism to compensate the loss inphotosynthesis andpreventoxidative damage caused by stress. Primarily, P. indica induces the defense system,especially the ascorbate–glutathione (ASH-GSH) cycle, and maintains a high antiox-idative environment during salt and drought stress. P. indica also induces sevaralantioxidative enzymes during salt and drought stress that are involved in detoxificationof reactive oxygen species (ROS) such as superoxide dismutase (SOD), catalase (CAT),ascorbate peroxidase (APX), glutathione reductase (GR), peroxidase (POD), mono-dehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and soon. P. indica also increases the level of osmolytes such as polyamine and proline inresponse to salinity and drought stress. Interplay of antioxidative environmentmediated by ASH, osmolytes (polyamine, proline, etc.), and strong activity of anti-oxidative enzyme system leads to maintenance of plastid integrity and thereforeenhanced photosynthetic efficiency in colonized plant during abiotic stress. Inaddition, P. indica also induces salt and drought stress-responsive genes of the plant,which may play an important role in enhanced abiotic stress tolerance of crop plants.

24.1Introduction

The unfavorable environmental parameters, such as drought, salinity, cold, freez-ing, high temperature, water logging, high light intensity, UV irradiation, nutrientimbalances, metal toxicities, nutrient deficiencies, climate change, and so on are

Improving Crop Resistance to Abiotic Stress, First Edition.Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

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termed as abiotic stress. Only 10% of the world�s arable land is free of stress. Abioticstresses have become an integral part of crop production. In general, plants sufferfrom dehydration or osmotic stress under drought, salinity, and low-temperaturecondition, which causes reduced availability of water (dehydration) for cellularfunction and maintenance of cellular turgor pressure. Prolonged period of dehy-dration leads to high production of ROS in the chloroplasts, causing irreversiblephotoinhibition and cellular damage. Because of cellular damage, mainly the cellmembrane integrity is disturbed, and therefore plant roots are unable to absorbminerals efficiently, causing nutritional stress [1]. Plants respond to stress asindividual cell and synergistically as a whole organism. Generally, stress signal isfirst perceived by receptors of the plant cells. Following this, the signal informationis transduced, resulting in activation of various stress-responsive genes. Theproducts of these stress genes ultimately lead to a stress tolerance responseor plant adaptation and help the plant to survive and surpass unfavorable condi-tions [2, 3]. The response could also result in growth inhibition or cell death, whichwould depend on the number and type of the genes, those that are up- ordownregulated in response to the stress. The overall stress response of a plant isa coordinated action of several genes encoding signaling proteins/factors, includingprotein modifiers (methylation, ubiquitination, glycosylation, etc.), adapters, scaf-folds, and antioxidative system [2, 3]. Furthermore, plant growth-promoting fungi(PGPF) such as arbuscular mycorrhizal fungi (AMF), ectomycorrhizae, and otherendophytic fungi [4], as well as plant growth-promoting rhizobacteria (PGPR) [5] orplant growth-promoting bacteria (PGPB) [6], confer abiotic tolerance and decreasedyield losses in cultivated crop plants. AMF can act as a biofertilizer, bioprotectant,and biodegrader [7] and, in turn, modulate stress responses and increase the lowestlimit of tolerance of the plant to abiotic stresses. Several studies are available on theimpact of AMF on plant�s abiotic stress tolerance, suggesting that AMF play acomprehensive role in plant�s stress tolerance, and colonization of AMF induces amolecular signaling cascade that affects stomatal conductance, transpiration, pho-tosynthesis, leaf dehydration, root hydration, hydraulic conductivity, growth, nutri-ent uptake, low weight metabolites (e.g., sugars, glycerol, amino acids, and sugaralcohols), and morphology. However, application of AMF in sustainable agricultureis limited due to unavailability of axenic culture and its host specificity, as AMFcannot colonize a group of important crop plants.

A recently discovered root endophytic fungus of the Sebacinaceae family, Pir-iformospora indica, can colonize the roots of many plant species, including Arabi-dopsis [8–19]. Infestation ofP. indica can be intercellular or intracellular (Figure 24.1);however, unlikeAMF, they donot formarbuscular structures in plant cells [10]. Thereare several reports which suggest that P. indica can mimic the effect of AMFcolonization to plants; besides, this fungus is axenically cultivable and has a broadhost spectrum. P. indica can provide several benefits to host plants, such as bettertolerance to biotic (diseases) and abiotic stresses, for example, drought and salinitystress, and improved plant fitness by increasing growth performance under normaland stress conditions [18, 20]. The ability of P. indica to promote plant growth, higherseed yield, seed oil content, and so on of various host plants under conditions that are

544j 24 Piriformospora indica, A Root Endophytic Fungus

not optimal for the plants is well documented [8, 11, 18, 21, 22]. It can act as abioprotector and facilitate hardening ofmicropropagated plants that are transferred tosoil and the natural environment [23, 24]. It also has a stimulatory effect onadventitious root formation in ornamental stem cuttings. There are also studiesshowing that culture filtrates of the fungus are rich in nutrients and can promotegrowth yield [22]. However, the exact nature of plant growth promotional effects is stillunclear [25, 26]. P. indica was reported to activate both nitrate reductase that plays amajor role in nitrate acquisition and a starch-degrading enzyme, glucan-waterdikinase, involved in early events of starch degradation in the plants such as tobaccoandArabidopsis [15]. Root infestation resulted in promotion of plant growth and yield.In this chapter, we have emphasized the effect of P. indica colonization on the abioticstress tolerance and the mechanism for these stress tolerance in colonized plants.Colonizationmay lead to activationof various genes involved in stress acclimation andtheir roles inmaintenance of cell homeostasis and activation of antioxidant systemarealso discussed.

Figure 24.1 Trypan blue staining of maizeplant roots showing intracellular P. indicachlamydospores (black arrow) observed at day10. The fungus forms intercellular andintracellular pear-shaped chlamydospores

within root. Fungus grows into cortex tissue butdoes not colonize vascular tissue of the root andnever colonizes the shoot tissue. Adapted fromRef. [9], reprinted with permission from theSociety for General Microbiology � 2009.

24.1 Introduction j545

24.2Role of P. indica in Salt Tolerance

Soil salinity is a major threat to crop yield and a widespread problem. Around 7% ofthe global land surface is covered with saline soil [27]. Out of 1.5 billion ha ofcultivated land, about 77 million ha (5%) is affected by excess salt content mainlyinduced by irrigation with groundwater of high salt content [28]. At present, it is awell-known fact that crop yield is low in saline soil, mainly due to decrease in efficientnutrient uptake, plant water holding capacity, and adverse effect on photosynthesis.Salinity and drought exert their influence on a cell mainly by disrupting the ionic andosmotic equilibrium [3]. Thus, excess of Naþ ions and osmotic changes in the formof turgor pressure are the initial triggers, leading to a cascade of events that can begrouped into ionic and osmotic signaling pathways, the outcome of which is ionicand osmotic homeostasis, resulting in stress tolerance. Many crop species areextremely sensitive to soil salinity and are known as glycophytes, whereas salt-tolerant plants are known as halophytes. In general, glycophytes cannot grow at100mMofNaCl, whereas halophytes can grow at salinities over 250mMofNaCl. Thesalinity-sensitive plants restrict the uptake of salt and strive to maintain an osmoticequilibrium by the synthesis of compatible solutes, such as amino acids and sugars.The salinity-tolerant plants have the capacity to sequester and accumulate salt in thecell vacuoles, thus preventing the buildup of salt in the cytosol andmaintaining ahighcytosolic Kþ/Naþ ratio in their cells. Recent studies suggested that P. indica couldenhance the ability of plants to cope with salt stress. Waller et al. [18] have shown thatP. indica colonization enhanced salt tolerance of barley plants. The detrimental effectof moderate salt stress was completely abolished by P. indica with a higher biomassgain. This effect of enhancement of salt tolerance by P. indica was similar to that ofAMF. However, AMF-induced salt tolerance is due to improvement in plant nutrientuptake and ion balance, protecting enzyme activity and facilitating water uptake.

Earlier studies suggested salt-induced increase in lipid peroxidation and reductionin metabolic heat production [31] in salt-sensitive plants, while they remainedunchanged in salt-tolerant cultivars. Baltruschat et al. [32] assessed the impact ofP. indica colonization on biochemical markers for salt stress, such as metabolicactivity, fatty acid composition, lipid peroxidation, and marked reduction in meta-bolic heat production in salt-sensitive barley plants. Reduction in plant metabolicactivity is recognized in salt-stressed plants. Salt-induced responses, that is, heatemission and ethane production in P. indica-colonized salt-sensitive plant, resemblethose of salinity-tolerant plants because P. indica increases the metabolic activity inleaves of salt-stressed plant and therefore recompenses the salt-induced inhibition ofmetabolic activity. Calorimetric studies indicated that the rate of metabolic activityincreased in leaves of P. indica-infected plants after salt treatment. Prior studies havealso shown that the extent of natural herbicide resistance ofwild oat biotypes is tightlycorrelated with the rate of heat production upon herbicide exposure, owing to theactivation of metabolic pathways required for defense responses [33]. This suggeststhat enhanced tolerance to salt stress can be associated with highermetabolic activityinP. indica colonized plants [32]. Salt stress can induce ionic stress and osmotic stress

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in plant cell, leading to enhanced accumulation of ROS that are harmful to plant cellat high concentration.On the onehand, ROS accumulation can be toxic to living cells,causing oxidative damage to DNA, lipids, and proteins, and on the other hand, ROScan act as signaling molecules for stress responses. Several studies have demon-strated that tolerance of plants to salt stress is associated with the induction ofantioxidant enzymes [34–36]. Recent reports suggest that P. indica induces antiox-idative environment in the cell by altering the activities of different enzymes involvedin ROS-scavenging system [9, 37]. Exogenously applied unsaturated fatty acids canprotect barley plants during NaCl-induced stress [38]. Lipid desaturation could be animportant component of plant tolerance in response to salt stress. P. indica coloni-zation leads to a significant reduction in the proportion of oleic acid in barley leavesand also induces changes in fatty acid composition similar to those induced bysalinity [32]. Such effects on the fatty acid composition of host plants may display asymbiotic adaptive strategy mediated by the endophyte to cope with salt stress inhostile environments [37]. Owing to its original habitat, P. indica might inducesimilar effects on fatty acid composition of the host plants [32].

It has been shown that P. indica is able to produce auxin when associated withplant roots [39] or changes in phytohormone synthesis and perception in plants [40–42]. Exogenous auxin has been found to transiently increase the concentration ofROS and then prevent hydrogen peroxide (H2O2) release in response to oxidativestress (caused by paraquat) and enhanced APX activity, while decreasing CATactivity [43, 44]. In a recent study, it was found that P. indica increases the level ofosmolytes such as polyamines and amino acid proline (unpublished data) incolonized plants. This increase in polyamine content is due to the upregulationof methionine synthase in colonized plant, which plays a crucial role in biosynthesisof polyamines and ethylene [10]. A well-known adaptive response of plants undersalt stress is the synthesis and accumulation of low molecular weight organiccompounds in the cytosol and organelles, which are collectively known as com-patible osmolytes because they accumulate and function without perturbing intra-cellular biochemistry. Transgenic tobacco plants overproducing polyamines alsohave enhanced tolerance toward salt stress, and salt treatment induces antioxidantenzymes such as APX, SOD, and glutathione S-transferase (GST) more significantlyin these transgenic plants than in wild-type controls [45]. The major function ofosmolytes is osmotic adjustment to counteract higher inorganic salt in vacuole androot medium, protection of membrane, and stabilization of proteins. The osmolyteproline protects membranes and proteins against the adverse effects of highconcentrations of inorganic ions and temperature extremes. Proline may alsofunction as a protein-compatible hydrotrope and as a hydroxyl radical (OH.)scavenger [46].

In a previous study, Waller et al. [18] has reported that P. indica enhances thelevel of antioxidant buffer ascorbate and induces dehydroascorbate reductaseactivity in colonized plant. Ascorbic acid is directly involved in detoxification ofH2O2 by coupling with glutathione cycle or NADH. Moreover, it acts directly toneutralize oxygen free radicals [47]. During early stage of salt exposure, P. indicamaintains the redox balance ascorbate and increases its concentration in colo-

24.2 Role of P. indica in Salt Tolerance j547

nized plant; however, over time, the concentration decreases in both the salt-treated colonized and the control plants. Several studies suggested that thetolerance of plant to salt stress is associated with the capability of detoxificationof ROS, which is directly related to the induction of antioxidant enzymes [32].Overexpression of CAT, APX, or DHAR in transgenic plants enhanced tolerance tosalt stress [48, 49]. However, Arabidopsis double-mutant plants deficient incytosolic and thylakoid APX also show enhanced tolerance to salinity, suggestingthat ROS such as H2O2 could be responsible for activation of an abiotic stresssignal that leads to enhanced stress tolerance [50]. Exposure to NaCl increases theactivities of antioxidant enzymes CAT, APX, DHAR, MDHAR, and GR, but thisinitial induction of activity cannot sustain and decreases over time. However, inthe presence of P. indica, the decrease in the enzyme activity is less pronouncedand delayed. The elevated levels of GR, MDHAR, and DHAR activities affect theascorbate level during salt exposure in P. indica colonized plant. Ratio of ascorbateto DHA decreased in the salt-sensitive Lycopersicon esculentum under salt stressand increased in the salt-tolerant L. pennellii [51]. Earlier, investigations have shownthat ascorbate content decreased in salt-sensitive and salt-tolerant pea cultivars aswell,but thedeclinewas greater in the salt-sensitive plants [34]. The importance of ascorbatein cellular protection under salt stress has also been demonstrated in an ascorbate-deficientArabidopsismutant. Impaired in the ascorbate-glutathione cycle, thismutantaccumulated high amounts of ROS and showed increased sensitivity to salt stress [52].Constant exogenous application of ascorbate increased resistance to salt stress andattenuated the salt-induced oxidative burst [53]. Alternatively, ascorbate can improvethe tolerance of barley to high salinity via processes related to root growth. Ascorbicacid and high ratio of reduced to oxidized ascorbate accelerate root elongation andincrease root biomass [54].

Under salt stress condition, MDHAR activity remained elevated in roots of bothsalt-sensitive and -tolerant plants. P. indica maintains a sustainably higher CAT andAPX activity in salt-sensitive plants during salt exposure. CAT activation is a well-knownmarker of oxidative stress and catalyzes the degradation of hydrogen peroxideinto water and thus reduces the oxidative damage to cell. APX is an integralcomponent of glutathione-ascorbate cycle and it detoxifies peroxides such as hydro-gen peroxide into water using the ascorbate as substrate. Furthermore, it was foundthat during the interaction with P. indica, enhanced glutathione pool was observed inthe plant leaves [18]. As glutathione is a key component in the glutathione-ascorbatecycle, it can suppress the effect of ROS on leaves and, consequently, on photosyn-thesis. The exact mechanism responsible for P. indica-mediated upregulation of theplant antioxidant system is not yet known. The hormonal signaling in the enhance-ment of salt tolerance of the plant in P. indica colonized plant cannot be rule out.Sebacina vermifera, an endophyte closely related to P. indica, downregulates ethyleneproduction in Nicotiana attenuata [40]. During the interaction, induction of methio-nin synthase takes place, and thus an increased level of ethylene has been observed inplant root. Ethylene signaling may be required for the plant salt tolerance and it mayinduce some antioxidant enzyme during heat stress. However, the function ofphytohormones in salt tolerance has not been clear yet.

548j 24 Piriformospora indica, A Root Endophytic Fungus

24.3Role of P. indica in Drought Tolerance

Water deficit stress is known as drought stress, which reduces agricultural produc-tion mainly by disrupting the osmotic equilibrium and membrane structure of thecell. Climate models have indicated that drought stress will become more frequentbecause of the long-term effects of global warming, which highlights the urgent needto develop adaptive agricultural strategies for a changing environment. Actually, thewater stress within the lipid bilayer results in displacement of membrane proteins,which contributes to loss of membrane integrity, selectivity, disruption of cellularcompartmentalization, and loss of membrane-based enzyme activity. The highconcentration of cellular electrolytes due to the dehydration of the protoplasm mayalso cause disruption of the cellularmetabolism. To avoid drought stress, plants closetheir stomata, repress cell growth and photosynthesis, activate respiration, reduceleaf expansion, and start shedding older leaves to reduce the transpiration area [1].The components of drought and salt stress crosstalk as both these stresses ultimatelyresult in dehydration of the cell and osmotic imbalance. Overall, drought stresssignaling encompasses three important parameters [55]: (a) reinstating the osmoticand the ionic equilibrium of the cell to maintain cellular homeostasis underthe condition of stress, (b) control and repair of stress damage by detoxification,and (c) signaling to coordinate cell division to meet the requirements of the plantunder stress.

Recent works suggest thatP. indica is involved not only in salt stress tolerance butalso in drought stress tolerance. Sherameti et al. [56] has shown that P. indicaenhances drought tolerance of Arabidopsis. Furthermore, the authors found thatafter exposure for 84 h to drought stress at the seedling stage, none of theuncolonized plant recovered and survived, while about 50% of the P. indica-colonized plant produced seeds. P. indica is also found to be involved in enhancingthe drought stress tolerance of other plants, such as maize, mustard, cabbages,cress, and tobacco. The primary visible effect ofP. indica-induced drought toleranceon the plant is shoot growth. In another study on Chinese cabbage, no visible effectof drought was seen [57]. Like salt stress, drought stress also induces strongoxidative stress and generates ROS in plant. ROS act upon the polyunsaturatedlipids of membrane and thereby formmalondialdehyde (MDA). The production ofthis aldehyde is used as a biomarker to measure the level of oxidative stress.Drought stress promotes MDA accumulation in the leaves, while P. indica colo-nized plant contains a lower amount ofMDA, suggesting that P. indica prevents thecolonized plant from oxidative stress. Furthermore, P. indica induces the antiox-idant enzyme activity in plant leaf during the drought stress. Three enzymes, SOD,CAT, and POD, were found induced in the colonized plant. The induction of CAT issomewhat different; it is found induced in both control and colonized plants, butthe induction is higher in colonized plant. PODs are a large family of enzymes thatdetoxify hydrogen peroxide, organic hydroperoxides, or lipid peroxides to generatealcohols. PODs contain a heme cofactor in their active sites that is synthesized inthe plastid. In addition, PODs contain redox-active cysteine residues that directly

24.3 Role of P. indica in Drought Tolerance j549

measure the redox potential in cell or organelle. The most important organelle inthe leaf that controls the redox potential in the cell is plastid. These enzymes canplay an important role in the detoxification of ROS.Hence, one can understandwhythe amount of MDA is reduced in colonized plant.

P. indica induces not only the antioxidative enzymes but also the antioxidativemolecules such as ascorbate under the drought stress. The fungus induces theaccumulation of ascorbate in root and shoot, especially leaves of the plant, andmaintains a higher antioxidative environment in plant cell. At the molecular level,fungus induces monodehydroascorbate reductase 2 (MDHAR2) and dehydroascor-bate reductase 5 (DHAR5) in the colonized plant [58]. MDHAR2 andDHAR5 are theimportant part of the ascorbate–glutathione cycle that maintains ascorbate in itsreduced state. MDHAR2 converts MDHA into the ascorbate using NADH, whileDHAR5 converts DHA into ascorbate using its integral part, the glutathione cycle,and finally gives an antioxidative environment to the cell mediated by ascorbate. Theimportance of these two genes was analyzed using the knockdown (KO) lines ofArabidopsis, and it was found that growth, flower development, and seed productionwerenot promoted by fungus andwere inhibited under drought stress. This indicatesthat both the enzymes are crucial for the plant to respond to the fungus and thefungus-mediated growth promotion and cannot be fully replaced by other membersof the gene family [58]. It has been demonstrated that loss of benefits for the plantscould be caused by a shift from mutualism to parasitism, a phenomenon thatoccurred due to an uncontrolled growth of fungal hyphae in the roots [59]. During thedrought stress, KO lines were found over colonized root, indicating that thesemutants were less protected against the fungal colonization. Furthermore, anantifungal protein PDF1.2, which is not expressed in uncolonized root, but expressedat a detectable level in colonized root, is several times upregulated in KO lines, whereinteraction shifts frommutualism to parasitism. This suggests that these two genesof ascorbate-glutathione cycle contribute to the repression of defense gene expressionagainst P. indica under drought stress condition [58]. Taking all the studies intoconsideration, activation of antioxidative system in leaves is a major target of thefungus and plastids are the main targets of drought stress in leaves.

The most crucial adverse effect of drought stress is the reduction in photosyn-thetic efficiency, pigment content, and proteins of the photosynthetic machineryand the biosynthetic pathways in the stroma of the plastids. P. indica does not targetspecific photosynthesis genes or proteins to establish drought tolerance, but createspreventive atmosphere in the cells that stabilizes plastid function by inducingdifferent antioxidative enzymes. However, chloroplast is a major organelle site forantioxidative activity in the cells where SOD, APX, PODs, MDHAR, and DHAR arelocalized [60], and induction of these enzymes in the leaves may stabilize the plastidstructure and detoxify the ROS during the drought-induced oxidative stress. Theultimate/crucial effect of P. indica colonization on leaves under drought condition isthe increase in chlorophyll content. Drought stress also has a strong effect onphotosynthesis. The photosynthetic efficiency (Fvariable/Fmaximum, Fv/Fm) valuesaround 0.83 reflected the potential fluorescence quantum efficiency of photosystemII, which are sensitive indicators of plant photosynthesis performance [61], and the

550j 24 Piriformospora indica, A Root Endophytic Fungus

values lower than 0.83 indicated the exposure of stress [62]. Drought stressdecreases the Fv/Fm values in uncolonized plant, but colonization of funguscompensates the loss in Fv/Fm values and is equal to that of unstressed plant. Thedifference in the Fv/Fm values clearly demonstrates the beneficial effect of thefungus on photosynthetic efficiency under drought stress [56, 57]. Besides, P. indicaimpeded the drought-induced decline in photosynthetic efficiency and degradationof chlorophylls and thylakoid proteins [57]. Interestingly, P. indica does notinfluence any specific photosynthetic genes or protein that may lead to an increasein photosynthetic efficiency, but a plastid-localized CAS protein appears to be aspecific target in the chloroplast during the drought stress. CAS protein is identifiedas a chloroplast-localized Ca2þ -sensing receptor protein that is crucial for properstomatal regulation in response to elevations of external Ca2þ . CAS fulfils this rolethrough modulation of the cytoplasmic Ca2þ concentration under stress conditionand is also involved in signaling [63]. Drought stress induces the mRNA and proteinlevel in the leaves and it is likely that the fungus counteracts drought stress byelevating cytoplasmic calcium transients, which finally results in stomata closure inthe guard cells. A large number of genes involved in drought tolerance are morequickly and strongly upregulated in P. indica-colonized Arabidopsis leaves uponexposure to drought stress. This result was further validated when a P. indica-insensitive Arabidopsismutant (pii) was found less tolerant to drought stress and didnot upregulate the stress-related genes in the presence of P. indica. Hence, P. indica-mediated drought tolerance to Arabidopsis is associated with the priming of theexpression of rather diverse set of stress-related genes in the leaves [42, 56].

These stress-related genes are involved in rather different cellular processes.Phospholipase Dd (PLDd) is involved in phospholipid metabolism at the plasmamembrane; calcineurin B-like protein 1 (CBL1)/CBL-interacting protein kinase 3(CIPK3) is involved in cytoplasmic signaling; histone acetyltransferase (HAT),dehydration response element binding protein 2A (DREB2A), and ANAC072 controlthe gene expression in the nucleus; and other proteins such as salt and drought ringfinger 1 (SDIR1) and early response to dehydration 1 (ERD1) have a role in proteindegradation and response to dehydration 29A (RD29A) with cytoplasmic function.RD29A and ERD1 are the reporters of drought stress responses. ERD1 is a plastid-localized caseinolytic protease that is induced by dehydration or cold stress. PLDd isassociatedwith the plasmamembrane and specifically induced under drought stress.This plasma membrane-bound PLDd is activated in response to hydrogen peroxideand the resulting phosphatidic acid (PA) functions to decrease hydrogen peroxide-promoted programmed cell death [64]. DREB2A is a transcription factor thatspecifically interacts with cis-acting, dehydration-responsive elements involved indrought stress-responsive gene expression inArabidopsis. Intact DREB2Aexpressiondoes not activate downstream genes under normal growth condition, which suggeststhat this transcription factor requires posttranslational modification for activation.However, the activation mechanism has not yet been clarified, but a constitutiveactive formofDREB2Ashowed improveddrought stress tolerance inArabidopsis [65].SDIR1 is an E3 ubiquitin ligase localized in intracellular membranes of all tissues ofArabidopsis and is a positive regulator of ABA signaling, induced by drought and salt

24.3 Role of P. indica in Drought Tolerance j551

stress but not by ABA. Overexpression of SDIR1 is involved in an ABA-dependentpathway leading to stress tolerance [66].

Calcium (Ca2þ ) signaling is an important part of the early signaling system inplantresponse to various stimuli [67, 68]. Alterations occurred in cytosolic-free Ca2þ

([Ca2þ ]cyt) in response to abiotic signals [69], such as drought [70]. Ca2þ acts as asecondary messenger in plant cells and links different input signals to many diverseand specific responses [68]. The Ca2þ signaling system is based on multifactorialprocesses that startwithaspecificCa2þ signatureand theavailabilityofaspecificsetofCa2þ sensorsandendwith targetgenesandproteins that activateprecisedownstreamevents [69, 71]. The source of theCa2þ contributing to the rise in [Ca2þ ]cyt (apoplastsor internal stores or both) is crucial for the physiological responses [70, 72]. Sunet al. [57] found that theexpression levelsof thedrought-relatedgenesDREB2A,CBL1,ANAC072, and RD29A were upregulated in the drought-stressed leaves of P. indicacolonized plants. Furthermore, the CAS mRNA level of the thylakoid membrane-associatedCa2þ -sensingregulatorandtheamountof theCASproteinwerealso foundto be increased. Thus, Ca2þ signaling is involved in P. indica-induced drought stresstolerance inplants.CBL1 isan importantplayer inCa2þ signalingand integratesplantresponses to abiotic stresses, including drought stress ABA-independent pathways.Besides CBL1, a calcium sensor-associated kinase, CIPK3, has multiple functions instress responses and might be a crosstalk node in stress and ABA signaling path-way [73]. Interestingly, CIPK3 primarily modulates cold- and salt-induced geneexpression but not drought-induced gene expression; however, the level was earlierupregulated in P. indica colonized seedlings during the drought stress. Anothertranscription factor DREB1B, involved in dehydration and cold responses, works onthe recruitmentbyHAT.Thus,P. indicamight controlgeneexpressionmoregenerallybyregulatingcrucial factors involvedinhistoneacetylation.Theearlierupregulationofthe genes involved in stress responses in P. indica-colonized plants is themodulationof the defense systembymolecular signaling, whichfinally resulted in the preventionofwater loss,balancedshiftofmetabolism,andfunctionalandstructural integrationofthe organelles and cell.

24.4Conclusions

P. indica is a root endophytic fungus that has a broad host spectrum, including themonocot, dicot, and Brassicaceae family, which are not colonized by the mycorrhizalfungus. This interaction provides a critical linkage between the plant root and the soil.As a result, P. indica colonized plants are often more competitive and better able totolerate environmental stress than the uncolonized plants. Plant responses tocolonization by P. indica can range from growth promotion to multistress tolerance.Growth promotion in plant is the characteristic effect of the fungal colonization andvisible under stress condition. This growth promotion may be due to the nutritionaltransfer by the fungus to the plant and phytohormone signaling mediated by auxinand cytokinine secreted by colonized fungus. On the basis of the reports available, we

552j 24 Piriformospora indica, A Root Endophytic Fungus

proposed a diagrammatic representation of ROS-scavenging system of plant (Fig-ure 24.2), showing how P. indica influences the glutathione–ascorbate cycle thatresulted in enhanced oxidative stress tolerance under the abiotic stresses. P. indicamodulates the defense system and alters the metabolism to compensate the loss inphotosynthesis and prevention of oxidative damage due to stress. As the role ofP. indica in abiotic stress tolerance has been shown under the controlled green houseconditions, therefore we suggest the use of P. indica can also be tested in theagriculture field.We, further, suggest that this endophytic fungus is a good candidatefor the application in sustainable agriculture to help crop plants overcome salt,drought, and other abiotic stresses.

Acknowledgments

We thank Ms. Madhunita Bakshi for critical reading and scientific corrections. We(AKJ,NT) also thank theDepartment of Science andTechnology, Council of Scientific

Figure 24.2 Couples series of redox reactioninvolved with scavenging of H2O2 ofglutathione–ascorbate cycle in the plant cell.P. indica influences nzymes� expression andactivity of the cycle (shown in exploding star)and leads to induction in ROS scavengingcapacity in the cell. SOD (superoxidedismutase), ASC peroxidase (ascorbate

peroxidase), MDHA reductase(monodehydroascorbate reductase), and DHAreductase (didehydroascorbate reductase) arethe important enzymes playing crucial role indetoxification of stress-induced ROS and aredifferentially induced in P. indica-colonizedplant during the abiotic stress.

24.4 Conclusions j553

and Industrial Research, Government of India, for providing the financial help. MK,PS, andAJare also very thankful to the IndianCouncil ofMedical Research andCSIR,Government of India, for granting them research fellowship.

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