Improving Crop Resistance to Abiotic Stress (TUTEJA:PLANT STRESS OMICS O-BK) || Sesame: Overcoming the Abiotic Stresses in the Queen of Oilseed Crops

48Sesame: Overcoming the Abiotic Stresses in the Queenof Oilseed CropsSuman Lakhanpaul, Vibhuti Singh, Sachin Kumar, Deepak Bhardwaj,and Kangila Venkataramana Bhat

Sesame, one of the important oilseed crops, is valued for its high quality oil rich inpolyunsaturated fatty acids (PUFA) and thus offers excellent health benefits. The oilalso has unique antioxidative property that increases its keeping quality by preventingoxidative rancidity. However, the research efforts for developing improved sesamecultivars having tolerance to biotic and abiotic stresses have been rather meager sofar. Sesame is largely cultivated in marginal lands by resource-poor farmers and isthereby prone to several abiotic stresses. The crop possesses effective tolerance todrought due to its extensive root system. Preliminary studies have been carried out insesame regarding salt, drought, and heavy metal stress. Parameters such as root andshoot morphology, cuticle thickness, antioxidative enzymes, malondialdehyde, pro-line content, and so on have been assessed under stressed and control conditions.Role of stress-associated genes and their products such as lipid transfer proteins,caleosins, steroid dehydrogenase, phytostatins, g-aminobutyric acid, metallothio-neins involved in diverse stresses are under investigation. The presence of phenyl-propanoid compounds, namely, lignans, an innate nonenzymatic antioxidantdefense mechanism against reactive oxygen species in sesame, is a special areabeing researched. However, the areas that still remain untouched include water-logging and chilling stress, both of which are highly detrimental to the crop survival.Inspite of huge repertoire of germplasm collection, limited research efforts on theuse of conventional and biotechnological methodologies have resulted in minimalsuccess in developing abiotic stress-tolerant cultivars. The absence of efficient in vitroregeneration protocols further compounds challenges for development of desirednovel genotypes. The possible strategies that could be helpful in incorporating abioticstress tolerance in plants have been discussed here along with the fundamentalstudies dealing with different stresses and their effects on sesame, followed byinformation on stress-related genes under focus in sesame.

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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48.1Introduction

Global population has increased with the unprecedented rate over the past fewdecades resulting in shortage of resources at all fronts. However, the biggest problemis the shortage of food particularly in the underdeveloped parts of the world.Providing additional land for cultivation is already becoming unrealistic due toincreasing demands of urbanization on the one hand and the need to maintain andrestore the much reduced forest cover on the other hand. In addition, a series ofnatural calamities and global environmental changes have compounded the abioticand biotic stresses that adversely affect the productivity of the crop plants. Abioticstress is, in fact, a general term referring to physical stresses experienced by the plantvis-a-vis the unfavorable conditions posed by the environmental factors or thesubstratum where the plant is growing. The negative influences of abiotic stressessuch as drought, salinity, cold, chilling, flooding, and so on affect survival, biomassproduction and accumulation, and grain yield in most crops [1]. About 85% of cropproductivity losses are due to different kinds of abiotic stresses [2], which is muchmore significant in comparison to the losses that occur due to insects/pests, weeds,and diseases [3].

Plants have evolved several adaptations to survive the harsh environmentalconditions due to innate plasticity in their physiological and metabolic processes.The conquest of nearly the entire planet with the living organisms spanning extremeconditions on both sides of all the physical parameters provides evidence for therange of functional capability of the biological machinery and hope for the incor-poration of such traits in the desired taxa. Theneed for the addition of traits impartingtolerance to abiotic stresses cannot be overemphasized. Both conventional breedingand biotechnological tools, individually or in combination, have yielded superiorgenotypes in major crop plants. However, several other crop plants that not onlycontribute significantly to the food and nutritional requirement but also play animportant role in diversification of the crops are far from researched. Sesame(Sesamum indicum L.) is one such crop that deserves urgent and immediate attentionof the scientific community.

48.2Sesame: an Oilseed Crop

Sesame, an important oil yielding plant, is one of the most ancient crops known andused as oilseed. The high regard it enjoys among the users has earned it the poeticlabel �queen of oilseeds� [4]. Ironically, it has also been considered an �orphan crop�due to lack of research efforts ascribed to the fact that it is not a mandate crop for anyinternational crop research institute [5]. Though sesame is cultivated on a worldwidebasis for its seeds, oil, and protein, it is predominantly an annual crop of warmerareas, particularly Asia and Africa [6] where it is used both as a leafy vegetable and anoilseed crop. The presence of unique antioxidant compounds such as sesamin,

1252j 48 Sesame: Overcoming the Abiotic Stresses in the Queen of Oilseed Crops

sesamolin, and sesaminol makes the sesame oil one of the most stable vegetable oilsin the world. The oil content ranges from 32.5 to 58.8%, which is generally greater inwhite than black seeds [7]. In general, the oil content compareswell with other oilseedcrops (Figure 48.1). The oil is rich in linoleic acid (LA) (Figure 48.2) and has beenrecommended for healthy diets with low LA and high alpha linoleic acid incombination with canola or mustard oil [8]. Seeds as a whole serve as nutritiousfood for humans and are widely used in bakery and confectionery products [9].Sesamemeal contains 35–50% protein, which is rich in tryptophan andmethionine,and is used as poultry feed. Its leaves used as a vegetable are a rich source of carotene,ascorbic acid, iron, and calcium along with adequate quantities of protein [10].

Figure 48.1 Oil and protein content of selected oilseeds (% content per weight basis) (http://www.fao.org/inpho/content/compend/text/ch05-01.htm).

Figure 48.2 Fatty acid composition of common oilseeds. Adapted from [11, 12].

48.2 Sesame: an Oilseed Crop j1253

Sesame cultivation has many advantages as the seed set and yields are relativelywell under high temperature and the crop can be grown even on residual moisturewithout any extra rainfall. Sesame crop also improves water percolation of the soil asits extensive branching systemof feeder roots penetrates very deep into the soil. It canbe grown in pure stands and also as a companion crop [5]. Furthermore, it respondswell to additional inputs in terms of irrigation and fertilization (by at least doublingthe yield capacity), and thus has an important role in intensivemanagement systems,including sequential multiple cropping [13].

Sesame is grown in the tropical to temperate zones from about 40�N latitude to40�S latitude [14]. The plant is adapted to many soil types, but it thrives best on well-drained, fertile soils of medium texture (typical sandy loams) and at neutral pH. InIndia, sesame as a sole crop is mainly cultivated in drier parts of Maharashtra,Madhya Pradesh, Rajasthan, Gujarat, Andhra Pradesh, Uttar Pradesh, and Karna-taka. However, it is grown in almost all states of India either as a mixed crop or inother forms of small-scale cultivation [15]. In Northern India, the crop is taken asrain-fed kharif crop and in Central India as a semi-rabi crop. However, in the Souththe crop is taken in both the seasons and in the Northeast the crop is taken threetimes in a year.

According to the Food and Agriculture Organization of the United Nations (FAO,2002), sesame ranks 6th in the world production as an edible oil seed (2 893 114million ton) and 12th in the overall world production of vegetable oil (754 159millionton). The world production of sesame seed and seed oil is 3.2 million ton and 0.8million ton, respectively (http://www.agmrc.org/agmrc/commodity/grainoilseeds/sesame/sesameprofile.htm). However, the world production fluctuates due to localeconomic crop production pressures and weather conditions. India accounted for7.4% of the world�s edible oil consumption with an estimated production of 28.21million ton of nine cultivated oilseeds in 2007–2008 (http://fcamin.nic.in/dfpd/EventDetails.asp?EventId¼561&Section¼EdibleþOil&ParentID¼0&Parent¼1&check¼0). The major oilseeds responsible are groundnut, soybean, and rape/mustard seeds covering 80% of the total oilseeds grown in India. The consumptionof edible oils in India reached 142.62 lakh ton that exceeded from their netavailability of 86.54 lakh ton from all domestic sources. This gap bridged by importof mainly soybean and sunflower oil, takes them away from the reach of majoritypopulation due to their high costs. Inspite of this lacuna, export of oilmeals,oilseeds, and minor oils from India has increased from 5.06 million ton in thefinancial year 2005–2006 to 7.3 million ton in 2006–2007 in order to hold theagricultural economy. In year 2009, of the 0.6 million ton of sesame seedproduction, 0.194 million ton was exported (http://fcamin.nic.in/dfpd/EventDetails.asp ?EventId¼561&Section¼EdibleþOil&ParentID¼0&Parent¼1&che ck¼0).Highprices of both soybean and sunflower oil are still a major concern. In light of theseaspects from both Indian and global perspective, sesame oil needs to find a strongfoothold tomeet the demands of a healthydiet. In this constrained scenario, increasingthe production of sesame offers some kind of hope, which can be achieved only byovercoming various hurdles limiting the crop yield.


48.3Constraints on Sesame Productivity

The plant architecture of sesame is poorly adapted to modern farming systemsbecause of its indeterminate growth habit causing nonuniform ripening of capsules,sensitivity to wilting under intensive management, and absence of nonshatteringcultivars suited for mechanical harvest [16]. Other physiological and biotic aspectsinclude low yielding varieties and yield losses due to pests. These along with abioticstresses are responsible for the reduced output,making it a small holder�s crop.Moreimportantly, sesame seedlings apparently show slow growth to develop root mass forsoil penetration, the duration in which it is susceptible to various pest infestationsand fluctuations of soil environments.

48.4Abiotic Stress and Sesame

Incorporating abiotic stress tolerance in sesame necessitates first and foremost theidentification of the important stresses that delimit its productivity. Baseline studiesregarding effects of various abiotic stresses have been carried out on sesame, mostlyconcerned with breeding aspects. Of all the abiotic stresses such as drought, salinity,extremes of temperature, and so on, waterlogging and chilling sensitivity are the twospecific abiotic stresses encountered by the cultivated sesame. It does not in any wayundermine the need to address other stresses. Although, it is important to note thatsesame crop is specifically prone to suffer significant losses if faced with waterlogging and its cultivation is restricted to areas and duration that are not subjected tolow temperatures. However, due to its locally adapted drought tolerance the crop isvaluable in many semiarid regions.

48.4.1Waterlogging Stress

Sesame crop is highly susceptible to waterlogging, as the crop undergoes imme-diate senescence and declines within 2–3 days of exposure to flooding stress. Thiscauses excessive devastation in fields (Figure 48.3) where accumulated water needsto be regularly drained out. Increased irrigation has shown to significantly reducethe sesame yield [17]. Even drought-tolerant sesame accessions are very susceptibleto high moisture [18]. Hence, waterlogging is an important abiotic stress on thiscrop and there is a need to develop improved genotypes that could survive theflooding stress.

Studies on any aspect of water logging stress on sesame are entirely lacking.However, other crops showing waterlogging susceptibility such as cotton displaysignificant reductions in stem elongation, shoot mass, root mass, and leaf numberalong with altered expression of 1012 genes (4% of genes assayed) in root tissue 4 h

48.4 Abiotic Stress and Sesame j1255

after flooding [19]. Many of these genes were associated with cell wall modificationand growth pathways, glycolysis, fermentation,mitochondrial electron transport andnitrogenmetabolism.Altered global gene expressionwas also observed in leaf tissuesin response to waterlogging changing 1305 gene expression profiles (5% genesassayed) after 24 h of flooding, mainly involving genes for cell wall growth andmodification, tetrapyrrole synthesis, hormone response, starch metabolism, andnitrogen metabolism [19]. Interestingly, in case of rice cultivars, wide variationexhibited for the ability to tolerate complete water submergence was found to beassociated with just one locus (Sub 1) on chromosome 9. A transcriptional factor ofthe B-2 subgroup comprising ethylene-responsive element binding proteins(EREBP) located in the Sub 1 locus could explain the physiological and developmentalprocesses associated with submergence tolerance in rice [20].

Therefore, recent approaches used to develop flooding-tolerant plants are con-centrating first on the fermentation pathway. The second focus is specifically onmodifying the transcriptional factor gene, AtMYB2, to finally enhance the expressionof fermentation pathway genes [21]. However, none of the developed transgenic linesin rice or cotton using this approach has yielded significant tolerance to hypoxicstress. The identification of novel genes from cDNA library of anaerobically inducedArabidopsis root [22] that alsowould behelpful in developingflooding-tolerant sesamegenotypes.

48.4.2Chilling Stress

Sesame comes under the category of chilling (0–15 �C) sensitive plants that includecrops such as rice,maize, soybean, cotton, and tomato,where the plants are incapableof cold acclimation, that is, are unable to increase their freezing tolerancewhen exposed to low temperatures [23]. There is a spontaneous retardation insesame plant growth observed as drying off of axillary buds and restricted growthof plant organs, namely, leaves, floral buds, and fruits, finally leading to plant death.

Figure 48.3 Field photograph depictingextreme susceptibility of sesame towaterlogging during kharif crop. (a) Well-drained plot (after regular pumping out of

stagnant water) showing normal plant growth.(b) Plots left with stagnant rainwater for morethan 12 h showing growth retardation,senescence, and plant mortality.


Oplinger et al. [24] have reported that the absence of 90–120 frost-free days arerequired for commercial cultivars of sesame where day temperature between 25 and26.7 �C is favorable. The plant shows significant reduction in growth below 20 �Candgrowth and germination are totally inhibited below 10 �C. Sesame seed showsmarked reduction in content of lignans (sesamin and sesamolin) in the oil [25]during frost damage. This reduced strength of the reactive oxygen species (ROS)scavenging machinery that prevents the system from oxidative stress, generated dueto chilling reveals the seriousness of the situation and could be one of the factorsresponsible for the cellular injury and senescence of the sesame plant.

The mechanism of chilling tolerance, although less worked out, is finding way bythe pathways deciphered for cold acclimation as the molecular changes that occurduring latter are also found to play a role in chilling tolerance [26, 27]. The fewcharacterized genes basically include the transcription factors that act upstream incold acclimation or as effector molecules that act to counter the potential damagingeffects of cold stress [28]. These cold-responsive genes aremembers of different low-temperature regulons, as some are regulated by the C-repeat binding (CBF) tran-scription factor while the others are not [29].

Exogenous application of chemicals such as glycine-betaine has proved fruitful inimprovement of tolerance in chilling-sensitive tomato plants [30]. In anotherapproach, chilling-tolerant plants were obtained by increasing levels of trienoic fattyacids [31] or the accumulation of cold-responsive proteins (COR) by increasedexpression of the genes positively regulating their expression such as SCOF-1 fromsoybean [32], CBF-1 [33], and ABI3 [34]. CBF3 is known to integrate variouscomponents of the cold response and its overexpression, in addition to increasingthe tolerance of cold-induced accumulation of proline and soluble sugars (reviewedby Yuanyuan et al. [35]) [36]. Activation of stress-inducible genes by binding of factorssuch as DREB1A on the upstream regulatory elements (DRE, dehydration-respon-sive element) has also led to substantial increase in stress tolerance, including coldstress [37]. Other important category of genes involved in providing cold stresstolerance includes KIN (cold-induced), LTI (low-temperature-induced), and RD(responsive to dehydration) genes. The proteins with multiple hydrophilic icebinding domains termed antifreezing proteins (AFPs), having the ability to inhibitthe growth and recrystallization of ice in intercellular spaces [38], are being workedout. The heterologous overexpression of genes encoding AFPs in freezing-sensitiveplants enhanced the freezing tolerance of host plants as observed in case oftransgenic tobacco plants (made of AFPs of carrot) that survived subfreezingtemperature of �2 �C [39].

The role of cellular metabolic signals and RNA splicing, their export, andsecondary structure unwinding has emerged out to be of central importance inregulating cold-responsive gene expression and chilling tolerance. One of the keyplayers is ubiquitination-mediated proteasomal protein degradation that has a crucialrole in regulating one of the upstream transcription factors, INDUCER OF CBFEXPRESSION 1 (ICE1), and thus in controlling the cold-responsive transcrip-tome [40]. The knowledge of such key players in plants under cold stress is pavingthe way for more efficient tools to make them chilling tolerant.


48.4.3Salt Stress

High salt concentrations limit sesame cultivation especially in arid and semiaridregions. Sesame cultivars show a considerable variation in the degree of salt toler-ance [41]. In response to highNaCl treatments (50 and100mM), two sesamecultivarsOrhangazi and Cumhuriyet showed reduction in root and shoot length, increasedlipid peroxidation while dry weights were affectedminimally [42]. These affects weremore pronounced in the cv. Orhangazi than in cv. Cumhuriyet. Similar studies haveshown that dry weight was less affected in salt-tolerant sugar beet and moderatelytolerant cotton [43].Change in freeproline levels in relation to salinityhavefiguredoutits roles such as balancing capacity as an osmolyte, stabilizing proteins, regulatingcytosolicpH,andscavenginghydroxyl radicals [44].CultivarCumhuriyetwassuperiorto the other one in proline content, which increased with time and concentration ofNaCl treatment as observed in relatively salt-tolerant plants such as Beta vulgaris [45],Brassica juncea [46], and alfalafa [47]. Increase in the activities of ROS scavengingenzymeshasbeen foundclosely related tosalt tolerance incaseofmanyplants [48–52].Constitutive and induced levels of superoxide dismutase (SOD) activity was observedfor cv.Cumhuriyet compared to cv.Orhangazi thatwas accompaniedby an increase inthe activity of major H2O2 scavenging enzymes such as ascorbate peroxidase (APX),catalase (CAT), and peroxidase (POX). This activity declined after 2 weeks that wassuggested to be taken care of by lignans (nonenzymatic antioxidative processes insesame) that also showed increased level of accumulation under stress.

Germinated seedlings of different sesame cultivars grown in the presence ofvarying concentrations of NaCl (30, 50, and 70mM) showed alterations in electro-phoretic patterns of proteins and other metabolites. Analyses revealed cv. RT-46, RT-54, and RT-127 to be salt tolerant, while cv. RT-125 to be sensitive as it showedretarded seedling growth along with low levels of total soluble sugars, sucrose, SODactivity, and higher malondialdehyde (MDA) and proline content in the presence ofmore than 30mM NaCl [53].

During seed germination in sesame, expression of SeMIPS (myo-inositol 1-phosphate synthase) showed downregulation with increase in concentration andduration of exposure to the saline environment. The protein catalyzes glucose-6-phosphate tomyo-inositol 1-phosphate, which is the first product in the biosyntheticpathways of myoinositol, phytic acid, and other essential cellular components[54–56]. The SeMIPS protein was highly homologous with those from other plantspecies (88–94%). It was present in several copies and expressed in an organ-specificmanner. In case of Arabidopsis, Nelson et al. [57, 58] have also shown salinity to affecttranscription of the MIPS gene during biosynthesis of myo-inositol and its deriva-tives. Similar salinity stress studies on Arabidopsis thaliana showed its upregulationin salt-tolerant plants and its reduction in the salt-sensitive ones [59]. Downregulationof ESTcoding forMIPSwas also observed in sunflower (a salt-sensitive crop), undersalt stress [60]. Genes responding to salinity have been reported from rice [61],common ice plant (Mesembryanthemum crystallinum) [62–64], and tomato [65], whichcould be implemented in achieving salt tolerance.


48.4.4Drought Stress

Sesame is known to be endowed with the property of drought tolerance due to itsextensive root system. However, drought severely limits sesame production inmarginal and low rain-fed areas. In addition, accessions from wet areas such asKorea and Bangladesh are very susceptible to drought [18]. Assessment of droughttolerance as a ratio of yield under water stress to that under normal irrigation carriedout for 17 sesame genotypes showed that the seed yield was sensitive to watershortage more than the morphological characters [66], while the mean weight ofindividual seeds escaped such effects. This depicts that postflowering response ofdrought in sesame is production of less seeds, instead of compromising on the seedsize [67]. A survey of 27 sesame genotypes inMoghan region for tolerance to droughtrevealed Karaj1, Naz takshakhen, Varamin237, and Varamin2822 genotypes to bemid-resistant and suitable for cropping under drought stress on the basis of sixdrought tolerance indices [68].

Assessment of enzymes involved in prevention of ROS generated as an outcome ofdrought stress revealed increased activities for SOD, POX, CAT, and polyphenolox-idase (PPO) both in leaves and roots of sesame [69]. On the other hand, fresh and drymass and total protein content of leaves showed a decreasing trend. Assay for MDAcontent was indicative of reduced lipid peroxidation. Yekta cultivar of sesame wasfound to be more resistant than the cultivar Darab14 [69].

Improved tolerance against drought is reported to have an association withincreased content of cuticular waxes per unit leaf surface area in oats, rice, sorghum,alfalfa, and crested wheat grass [70]. However, the increased wax deposition does notalways correlate inversely with transpiration rate. Imposition of water deficit onsesame cultivars caused an increase in wax amount by 30%, with 34% increase inalkanes, 13% in aldehydes, and 28%of the unknown ones [67]. An increase of 49% incuticle thickness due to monomers of alkanes has also been observed in Arabidopsisplants on subjection to water deficit. Under such conditions, the gene ECERI-FERUM1 having role in alkane metabolism showed upregulation [70]. Transcrip-tional factors such as WAX INDUCER 1 (WIN 1) in Arabidopsis have also beenimplicated in increasing wax deposition [71]. Deciphering of drought induction ofalkane metabolic pathway, role of cuticle, and the actual mechanism behind slowingdown of transpiration rate in drought tolerance, which regulates survival of sesameplant, is still awaited.

48.4.5Heavy Metal Stress

Previous investigations have found oil-yielding plants, namely, mustard and sun-flower, to be suitable for rhizoextraction as they accumulatedmore Cr from the soil incomparison to 36 other agricultural plant species [72]. Within oilseeds the pattern ofheavy metal accumulation showed sesame to stand third after peanuts and rapeseedwith sunflower being the last. The distribution pattern in the plant organs of sesame


showed an order of leaves > stem > roots > fruit shell > seeds [73] (Figure 48.4),revealing that the content was very less in the economically important edible organ,the seeds. The respective accumulation of heavymetals after 60 days of growth periodin sesame followed anorder of K>Na> Fe>Zn>Cr>Mn>Cu>Pb>Ni>Cdasdetermined by the technique of sequential extraction using EDTA [7]. In addition, thetranslocation of thesemetalswas found less in upper parts. The potential of sesame toextract heavy metals from contaminated soils has also been determined by chemicalfractionation analysis of S. indicum var. T55, which depicted an increased accumu-lation of toxicmetals (Cr,Ni, andCd)with increase in sludge ratio. The tannery sludgefavored plant growth at lower amendment rates (25%) as depicted by increased freshweight and number of leaves [74].

Increased affinity of sesame seeds toward accumulation of trace elements has alsobeen reported in comparison to corn grains (0.54–1.94 ppm). Range of Cr content insesame seeds was 0.77–2.14 ppm, with recommended daily allowances being 0.05–9.2 ppm [75]. Application of organic waste compost includingmunicipal waste (MW)had no effect on the chemical constituents (oil, carbohydrates, and total protein) ofsesame seeds.With treatment ofMW, lead and cadmiumconcentrations increased inthe plant; however, the amount of metals tested did not exceed the phytotoxiclevel [76]. Seeds of the high-yield sesame cultivar PB-1 showed significant toleranceto lead as shown by accumulation of more dry mass during early growth phase,although fresh weight showed slight inhibition at higher levels (2mM of Pb2þ ). Onthe other hand, cultivar HT-1 was shown to be Pb2þ sensitive [77]. Accumulation ofPb2þ increased with increasing concentration of the heavy metal in root, stem, andleaves. Estimation of in vivo nitrate reductase activity showed inhibition in roots andleaves with less effect on the latter, correlating with the respective accumulation ofPb2þ in the organs. However, in vitro nitrate reductase activity was not inhibited by

Figure 48.4 Organ-wise distribution of heavy metals in sesame at commercial ripeness stage(adapted from Angelova et al. [73]). Error bars represent the standard deviation.


the metal. Total organic nitrogen was higher in young roots proposed due toincreased N translocation from seeds to roots [77]. All these studies suggest thatsesame shows tolerance to the presence of heavy metals in the soil and in fact showssignificant ability to accumulate such elements. Therefore, sesame plant provides aninteresting research system to understand the mechanisms involved in heavy metalaccumulation, an area that is gaining importance for dealingwith themanagement ofdegraded ecosystems due to contamination by undesirable concentration of toxicmetals. However, it is of utmost importance to ensure the complete absence of theseharmful moieties in the seed and seed oil for the safe edible consumption.

48.5Abiotic Stress and Areas of Special Focus in Sesame

Out of the vast repertoire of genes governing abiotic stress tolerance in plants, fewgenes and gene products have gained special focus in sesame.

48.5.1Lipid Transfer Proteins

The transfer of phospholipids betweenmembranes is facilitated by group of proteinscalled lipid transfer proteins (LTPs) [78]. These are small (7–10 kDa), abundant, basicproteins containing eight conserved cysteine residues involved in four disulfidebridges. On the basis of the number of amino acids between the fourth and fifthcysteine residues in the motif�s core, plant LTPs have been categorized into eightgroups (LTP1–LTP8) [79]. Initially observed in spinach leaves, maize coleoptiles, andbarley aleurone layers, they show ubiquitous expression in seedlings, leaves, stamen,tapetum, andmicrospores, as well as in somatic and zygotic embryos [78]. LTP genesare shown to be responsive to environmental changes such as salt, drought, abscisicacid (ABA) or cold treatment [80–84]. A nonspecific LTP gene isolated by subtractivehybridization between drought-tolerant and -sensitive sunflowers showed transcriptinduction by water stress and ABA [85]. cDNA microarray analysis in sunflowerrevealed upregulation of an LTP under chilling stress. However, downregulation ofLTP was exhibited in saline environment as earlier shown in case of Arabidopsis [86].LTPs are proposed to have a function in repairing stress-induced damage inmembranes or changes in their lipid composition, perhaps to regulate their perme-ability to toxic ions and the fluidity [87, 88]. LTPs have also been shown to be secretedin response to NaCl stress and even affect cell wall extensibility [89]. Periodicdehydration stress in tree tobacco (Nicotiana glauca) leaves increased LTP mRNAexpression and cuticular wax deposition. In addition, immunolocalization and LTP::GFP fusion studies have localized LTPs to the cell wall and predominant expressionin the epidermis [90, 91] suggesting their role in cutin and wax assembly [92]. Mostplant LTPs are secreted to the cell wall by unidentified pathways. Although someintracellular LTPs have been observed in glyoxysome matrix of castor (Ricinuscomunis) seed and protein storage vacuoles of cowpea (Vigna unguiculata) [93, 94].

48.5 Abiotic Stress and Areas of Special Focus in Sesame j1261

These LTPs are present in multiple isoforms as detected in Arabidopsis and ricegenome, showing differential expression pattern, whose functions are yet to bediscovered [79, 83].

It is important and pertinent to point out that LTPs constitute one of the mostabundant ESTs of immature sesame seeds of which 21 isoforms were identified [95].Partially purified LTP isoforms of sesame (SiLTP) showed the ability to bindfluorescent fatty acids and transfer fluorescent phospholipids. Five SiLTP isoformswere most abundantly expressed in developing seeds, but also detected in flowertissues. SiLTP3 and SiLTP4 transcripts were also expressed in leaves and seed walls,respectively. SiLTP2 and SiLTP4 isoforms exhibited significantly inducible expres-sion patterns with exogenous application of 300mM NaCl and 300mM mannitol.Exogenous ABA, which has been shown to mediate plant tolerance to water or coldstresses, also significantly induced the SiLTP2 and SiLTP4 isoforms. These werelocalized to plant cell membranes as seen by transient expression in Arabidopsis andwere found to be associatedwith large organelles such as ER, probably being secretedfrom the cells via the classical secretory pathway. However, the biological roles ofSiLTPs in response to salt and osmotic stresses remain to be elucidated, whichmightplay an important role in plant acclimation to water stress during seed development.

48.5.2Caleosins

Caleosins are calcium binding proteins of 27 kDa, ubiquitous among higher plantswith similar candidates in algae and fungi [96]. Thesewere identified in sesame seedsasminor proteins (Sop1) of oil bodies (OB) by immunolabeling [97]. Sop1 was foundto be homologous to a rice protein (OsClo) that expresses abundantly in lateembryogenesis and is responsive to ABA and osmotic stress (dehydration and NaCl)in seedlings and in vegetative tissues [98]. Similarly,mRNAof anArabidopsis caleosinhomologue (AtClo1) was detected in response to ABA during dehydration [99].Sesame caleosin (SiClo1) mRNA accumulates in OBs, withmaximal expression seen2 weeks after flowering, thereafter undetectable in mature seeds [100]. In contrast toOsClo, SiClo1 is therefore apparently seed specific. Recent analyses indicate thatArabidopsis contains at least five caleosin-like genes situated on four of the fivechromosomes (AtClo1–5, [96]). AtClo2–4 are expressed at low levels in varioustissues, including nonoil storage tissues, while At-Clo1 expression is seed specific.Interestingly, AtClo1–4 expression is not responsive to ABA or osmotic stress invegetative tissues, as in case of riceOsClo. Carrot oleosin is responsive only to ABA inembryonic tissue, and not in adult tissue [101]. A better understanding of thisexpression pattern in plant stress responses would be beneficial.

Caleosins have been located on the surface of OBs or associated with an endo-plasmic reticulum (ER) subdomain [96]. Caleosin comprises three distinct structuraldomains: a unique N-terminal hydrophilic domain (containing a single Ca2þ

binding EF hand), a central hydrophobic anchoring domain, and a C-terminalhydrophilic domain containing four possible phosphorylation sites [98, 100].The central hydrophobic domain of SiClo1 is proposed to consist of an amphipathic


a-helix followed by a short anchoring region formed by a pair of antiparallel b-strandsconnected by a proline knot-like motif [100]. The EF hand ofOsClo is known to bindcalcium in vitro [98]. Similar probability exists for SiClo1 to bind calcium as SiCLO1purified from oil bodies or heterologously expressed exhibits EGTA-retarded andcalcium-rescued migration in SDS-PAGE [100].

An Arabidopsis caleosin, RD20 (responsive to dehydration 20) is reported to showenhanced expression by ABA, salt, dehydration, and osmotic stresses [99, 102]. It isamong the early induced genes, one of themost highly expressed and often used as astress marker gene [103, 104]. RD20 is located at the bottom of chromosome 2, aregion that appears to be important in the regulation of plant transpiration efficiencyin Arabidopsis. It is expressed in particular tissues or organs during plant develop-ment. In contrast to other Arabidopsis caleosin genes RD20 promoter sequence isenriched with AtMYC2 binding sites such as the ABRE (ABA-responsive element),ABRE-like, and DRE. Interestingly, AtMYC2 acts as a positive regulator of ABAsignaling under drought stress [105].RD20 is able to bind calcium and also support aputative peroxygenase activity as in case of OsClo and AtClo1 [99, 106]. Suchperoxygenase activity in maize is involved in cuticle and wax synthesis [107], thelatter is enhanced under water deficit conditions and ABA, to prevent waterlosses [70]. Recently, RD20 was shown to be one of the components involved inenhancing tolerance to water deficit mechanisms through the regulation of stomatalaperture, plant growth, andwater use efficiency [108] and in salt stress response, thus,hypothesized to act as a stress signaling hub that sets up multiple abiotic responses.The ability of sesame caleosins to undergo ubiquitination at two sites in the lysineresidues in the C-terminal domain has been reported [109]. As ubiquitination ofproteins has also been observed in stress responses [110], it would be interesting toestablish such a role, if any, for SiClo.

48.5.3Steroid Dehydrogenase

Sesame steroleosin (Sop2), a 39 kDaprotein comprising 348 amino acid residues, is aminor protein of OBs [111]. The Sop2 gene was obtained by immunoscreening thatshowed transcription in the maturing seeds. The protein possesses a hydrophobicanchoring segment preceding a soluble domain homologous to sterol bindingdehydrogenases/reductases known to be involved in signal transduction in diverseorganisms. Structure of the soluble domain consists of a seven-stranded parallelb-sheet with the active site, S-(12X)-Y-(3X)-K, between an NADPH and a sterolbinding subdomain. Its sterol-coupling dehydrogenase activity has been demon-strated both in the overexpressed soluble domain of steroleosin and in purified oilbodies. Southern hybridization suggests the presence of one steroleosin gene andcertain homologous genes in the sesame genome [111]. In contrast, eight hypothet-ical steroleosin-like proteins are present in the Arabidopsis genome with a conservedNADPH binding subdomain, but a divergent sterol binding subdomain. Steroiddehydrogenase-like protein (SDs) has been found to be induced by droughtstress [112] and in response to high light stress and ascorbate deficiency in


Arabidopsis [113]. This indicates role of SDs in protecting against ROS produced byhigh light and in the absence of ascorbate (vitamin C), one of the major antioxidantspecies of chloroplasts that is a cofactor of thylakoid-bound and stromal ascorbateperoxidases that detoxify H2O2 produced by SODs. Transgenic Arabidopsis plantsoverexpressing hydroxylsteroid dehydrogenases (AtHSD1) also provide increasedtolerance to salinity stress [114]. Whether this tolerance is mediated by brassinoster-oid signaling or increased ABA catabolism remains to be elucidated, before gettingutilized in other crops such as sesame.

48.5.4Phytostatins

Phytocystatins, homologues of cystatins of animals, are a small family of plantproteins commonly ranging from 12 to 16 kDa, consisting of more than 80 mem-bers [115]. In general, they possess three conserved residues, a G residue in thevicinity of the N-terminal end, a highly conserved QVVAG motif in a central loopsegment, and a PWdipeptidemotif closer to the C-terminal end of the protein, whichare known to interact with the active site cleft of cysteine proteinases belonging to thepapain family, causing their reversible inhibition [116]. In addition, plant cystatinspossess a unique and conserved LARFAVDEHN sequence at the N-terminal end ofan a-helix segment [117]. Apart from their participation in biotic stresses and seedgermination, their role in abiotic stresses has been proposed. Accumulation ofphytocystatin mRNA (AtCYS1) has been observed in Arabidopsis under high-tem-perature stress [118], in the vegetative tissues of barley plants subjected to anaero-biosis, darkness, and cold shock [119], and in the leaves and roots of chestnut plantletsexposed to cold, saline- or heat-stress [120]. Similarly, in grain amaranth, increasedexpression of cystatins (AhCPI) was seen in roots and stems substantially in responseto water deficit, salinity, cold, and heat stress, whereas heat stress induced a rapid andtransient accumulation in leaves [117].

Low-abundant endogenous cystatin (22 kDa) has been purified to homogeneity viaa papain-coupling affinity column frommature sesame seeds. These were shown toexpress in germinating seeds; however, their ability to inhibit endogenous cysteineproteases was not revealed [121]. These proteins from sesame have been expressedand purified via artificial oil bodies forfinal biotechnological application in protectionof plants and other industrial uses [122]. The role of sesame phytocystatins in abioticstresses requires further research efforts.

48.5.5Lignans

Stresses such as drought, salt, UV radiation, ozone, chilling, heat shock, pathogenattack, and so on increase the production of ROS in plants leading to development ofseveral enzymatic and nonenzymatic defense systems against ROS. Under suchoxidative stress conditions, plants with high constitutive and induced antioxidantlevels are known to have better resistance to damage [123–125].


In contrast to the meager knowledge on enzymatic antioxidative defense systemoperating in sesame, the nonenzymatic antioxidant substances termed lignanseffective in oxidative defense system are well studied. Widely distributed in vascularplants, lignans comprise a large group of natural products characterized by couplingof two p-hydroxyphenylpropane (C6C3) units and possessing ab,b0-linkage. These arerepresented by about 20 compounds in sesame, basically in the form of oil-solubleand glycosylated lignans [126]. Among the oil-soluble ones, the most predominantare sesamin and sesamolin, accompanied by traces of others. Interestingly, the twolignans, sesamolin and sesamolinol, possess an oxygen bridge between theirbenzene and furofuran rings, the feature unique to the genus Sesamum [127].Sesame seeds have on average 0.63% lignans with sesamin and sesamolin contentsranging from 0.07 to 0.61% (mean� SD, 163� 141mg/100 g) and from 0.02 to0.48% (101� 58mg/100 g), respectively [7, 128]. Among sesame cultivars of India,those collected from the Northeastern states are found to be higher in lignan content(18 g sesamin/kg, 10 g sesamolin/kg) [129]. The most frequent technique used forlignan-type detection and quantification in sesame is high-performance liquidchromatography [130–132] that has now been subsequently simplified [133]. LC-NMR-MS has also emerged as the fast screening device for the characterization ofsesame lignans from various sesame oil-based sources, for assessing the antioxidantactivity of the extracts, and especially for modifying the lignan profile of theconventional sesame oil extract to get a sesaminol-enriched extract [134].

Studies on these lignans with respect to stress have displayed their stresscombating ability. Under in vitro experimentation, sesaminol was shown to inhibitCu2þ -induced lipid peroxidation in low-density lipoprotein and was found to bemore effective scavenger than eithera-tocopherol or probucol in reducing the peroxylradicals in aqueous solution [135]. The analysis of sesame oil extracts for antioxidantactivity has revealed the following order: sesamin< sesame oil extract< sesaminol-enriched sesame oil extract< sesamol. The combination of sesaminol and c-tocoph-erol has been proposed to be synergistically responsible for the actual oxidativestability of sesame oil [134].

Sesamin, one of the major furofuran lignan in sesame seeds, has been extensivelystudied by Ono et al. [136]. The gene responsible for the catalysis of sesaminbiosynthesis from pinoresinol (the first lignan in the pathway) is deciphered outto be a cytochrome SiP450, CYP8101. The protein requires NADPH for its activityand is unique due to its dual catalyzing ability leading to the formation of twomethylenedioxy bridges (producing sesamin via piperitol), which is restricted to onlyone such bridge in all known P450 proteins. This biosynthesis has been localized tothe cytoplasmic surface of endoplasmic reticulum as revealed by the expression ofCYP81Q1-GFP protein in onion epidermal peel via transient system. The gene ispresent singly and its functional validation has been obtained by the observation of afunctional homologue isolated from S. radiatum (having sesamin in seeds) and thepresence of a nonfunctional P450 homologue from S. alatum that lacks sesamin. TheP450 proteins are shown to have evolved independently as the CYP81Q1 shows only24% sequence identity with the Ranunculaceae member, Coptis CYP719A1. Themode of action of CYP81Q proteins has also been proposed, the diagrammatic


representation of which is given in Figure 48.5. Genes involved in biosynthesis ofantioxidant lignans and accumulation of storage products have been identified in theform of 3328 ESTs from a cDNA library of immature seeds [137]. When compared tothemodel plantArabidopsis proteome, a total of 62 ESTs from sesame were proposedto be involved in lignan biosynthesis (Figure 48.6).

Apart from being beneficial to human health as anticancerous and anticholestrolagents, studies on rats showed lignans to increase the expression of b-oxidation-associated enzymes in peroxisomes (upregulation of 38 genes, 16 of which areinvolved in lipid metabolizing function) [138]. Several mechanisms of action havebeen proposed to explain the potential physiological role of sesame lignans. Espe-cially in animal systems, namely, cultured liver cells. The role of sesamin on the lipidmetabolism has been studied extensively and the following mechanisms of actionshave been proposed: inhibition of D5-desaturase activity [139], inhibition of HMG-CoA reductase activity [140], and inhibition of acyl-CoA cholesterol acyltransferase(ACAT) activity [141]. However, such gene profiling studies and their potential statusassociating lignans with different stresses are lacking with respect to plants as asystem. Overall, this area needs further attention to decipher the actual mechanismbehind the combating of stress in plants in general and sesame in particular.

48.5.6Geranylgeranyl Reductase

Tocopherols are lipid-soluble antioxidants known collectively as vitamin E. Thesecompounds are the major line of defense against ROS generated during various

Figure 48.5 Diagrammatic representation ofthe two alternative models proposed for themode of action of cytochrome P450 of sesame,CYP81Q (adapted from Ono et al. [136]).(a) Sequential methylenedioxy bridgeformation. Piperitol reverses in the active site

after the first catalysis for formation of secondmethylenedioxy bridge at the opposite end.(b) Piperitol is released after first catalysis andthen recaptured for secondone, a part of it beingconverted to its derivatives.


abiotic stresses. Plants synthesize four isoforms of tocopherols, a-, b-, c-, andd-tocopherol, which differ by the numbers and positions of methyl substituents onthe aromatic rings of themolecules [142]. The highly efficient antioxidant sesame oilcontains about 528 mg/g of total tocopherols [143]. Formation of the first tocopherolintermediate requires reduction of geranylgeranyl diphosphate (GGPP) to phytyldiphosphate [144] that is catalyzed by the enzyme GGPP reductase (Chl P) [145].Sesame GGPP reductase (SiChl P) encoded as a 465 amino acid polypeptide showshigh degree of similarity to the plantChl Ps with the closest evolutionary relationshipto tobacco Chl P [146]. In contrast to the bacterial Chl Ps, the SiChl P contains anaminoterminal extension that resembles a plastid transit peptide sequence, indicat-ing the possible localization of the enzyme in chloroplasts as known for plant ChlPs [147]. The sequence consists of 55 amino acids with the cleavage site locatedbetween A55 and A56 in the sequence NLR ! VAV. This transit peptide predictedfrom SiChl P is rich in serine and threonine but deficient in acidic amino acids [148].GGPP belongs to the family of oxidoreductases that contain a nucleotide cofactor-binding domain for stabilization of theb-strand anda-helix interaction, connected bya short loop in which the ligand binding domain is located [149]. The presence oftypical motif commonly found in oxidoreductases, V/IXGX1- 2GXXGXXXG/A, inthe N-terminus of the mature SiChl P polypeptide clearly indicates its function [146].SiChl P is present as a single gene with its high expressions observed in developing

Figure 48.6 Putative ESTs involved in synthesis of lignan, an innate antioxidant defense systemin plants (a) sesame and (b) Arabidopsis (adapted from Suh et al. [137]). Sectors unique to therespective plant are highlighted with white borders.


seeds and leaves supporting its role in tocopherol biosynthesis in sesame. In additionto factors such as dark and ethylene, SiChl P was shown to be repressed by ABA,which is generally produced upondrought and cold stresswith subsequent inductionof expressions of various subsets of downstream genes [150, 151]. Similar dimin-ished expression was seen for PpChl P in peach leaves in response to cold stress andwounding [152]. Chl P has been identified from soybean, ice plant, and severalphotosynthetic bacteria, although detailed investigations have been performed onlyin a few plant species such as tobacco [145] and Arabidopsis [153].

48.5.7c-Aminobutyric Acid

c-Aminobutryric acid (GABA) is a four carbon nonprotein amino acid found in allprokaryotic and eukaryotic organisms. The metabolic pathway involving synthesisand catabolism of GABA is known as �GABA shunt,� as it bypasses two steps oftricarboxylic acid cycle. It rapidly accumulates in responses to heat, drought, salt andlow-temperature stresses [154–157]. GABA is proposed to be involved in stressperception in sesame, as application of stresses such as drought, salt, heavy metal,and high temperature showed increment in GABA levels, but it was not able tosustain normal plant growth [158]. Highest increment of GABAwas in case of heavymetal treatment, followed by drought, while accumulation rates under salt and high-temperature treatmentswere almost the same.Differential ability of the sesameplantin coping with these stresses was proposed to be the reason behind variation in therate of GABA synthesis [158]. Upregulation of a GABA receptor has been observed intwo tolerant genotypes of barley [159] and increased expression of genes for GABAshunt in response to drought [160]. GABAhas been shown to reduce accumulation ofROS in aluminum and proton-stressed barley [161]. Its other proposed roles underdifferent stresses include maintenance of C:N balance, regulation of cytosolic pH,and osmoregulation and as a signaling molecule [162]. Furthermore, the role ofCa2þ /CaM in GABA-mediated tolerance to oxidative stress, heat shock, and osmoticand salt tolerance through effector molecules has also been suggested [163].

48.5.8Metallothioneins

Metallothioneins (MTs) are ubiquitous low-molecular weight, cysteine-rich cyto-plasmic proteins that can bind metals via mercaptide bonds. On the basis of thenumber and arrangement of cysteine residues, all the plantMTs belong to class II (incontrast to the vertebrate class I) and can be further subdivided into four typesaccording to the distribution pattern of Cys residues [164, 165]. Expression analysishas shown type 1 MTs to express preferentially in roots [166], type 2 mainly inleaves [167, 168], type 3 in ripe fruits and leaves [169], and type 4 in developingseeds [170–172]. In plants, MTs are known to participate in maintaining thehomeostasis of essential copper (Cu) and zinc (Zn) at micronutrient levels and inheavy metal stress for detoxification of nonessential toxic metals such as cadmium


(Cd) and arsenic (As) [173–176]. The expression of MTgenes is affected by oxidativestress, and other abiotic stresses, such as drought and salt [177, 178], hence their roleas protectants from oxidative damages has been proposed [179–182]. MTs fromTamarix hispida (ThMT3) was upregulated by high salinity and heavymetal ions, withpredominant expression in the leaf [183]. Transgenics for MTs in rice (OsMT1a)enhanced its drought tolerance and increased activity of ROS scavengingenzymes [184] showing expression in roots. Tobacco plants overexpressingGhMT3a,a type 3 MT isolated from cotton (Gossypium hirsutum), were shown to have reportedonly half of H2O2 levels in transgenic than those in wild-type plants under salt,drought, and low-temperature stresses, suggesting that changes in ROS signalingmight be the reason for higher stress tolerance [185]. It has been hypothesized thatMTs scavenge the superoxide radicals either independent of SOD or act as anactivator of SOD by supplying metals such as Cu or Zn to apo-SOD [186]. Tran-scriptome analysis of mature sesame seeds has revealed the presence of abundanttranscripts for metallothioneins [171] indicating the presence of a conserved metaltoxicity combating mechanism in sesame. Further research on overcoming thelimitations regarding in vivo protein expression studies would be beneficial indissection of the actual process of scavenging performed by the MTs.

48.6Approaches for Incorporation of Abiotic Tolerance

The process of incorporation of genes imparting tolerance to abiotic stresses wouldinvolve either avoiding the stress or combating the stress by inducing expression ofgenes that directly or indirectly are responsible for the synthesis of requiredmetabolites. The basic strategies initiating synthesis of these metabolites can betargeted either at the functional genes or regulatory genes. Advances in high-throughput techniques have enabled identification of large number of genes thatare differentially regulated in response to a specific environmental stress. Bothconventional and nonconventional methodologies await their utilization for incor-porating abiotic stress tolerance in sesame. The final goal of developing stress-tolerant genotypes can be achieved by different approaches, but the key steps thatneed to be undertaken (Figure 48.7) are as follows:

. Search for useful traits/genotypes.

. Identifying and understanding the precisemechanism and genes responsible forthe stress tolerance.

. Transfer of the trait using conventional or biotechnological tools.

. Screening of the target genotypes for successful transfer of the desired trait.

. Acceptance of the farmers for growing the novel genotypes.

Therefore, irrespective of the approach used for incorporation, the first andforemost requirement is the search of the donor of the desired trait. Conventionalapproaches can be applied only if taxa having the desired trait constitute the primary,secondary, or even tertiary gene pool of the crop plant. In the absence of desired trait

48.6 Approaches for Incorporation of Abiotic Tolerance j1269

within the conventional gene pool, the biotechnological approaches provide therequired tools for the transfer of the traits from the donor taxa to the desiredbackground. Once the transfer has taken place, the screening methodologies wouldremain the same in both the approaches. However, the acceptance of the improvedcultivar by the farmer and the consumer can again have different reactions depend-ing upon the approach used.

48.6.1Search for Useful Genes in the Sesame Germplasm

Natural genetic diversity is a sustainable resource that can enrich the genetic basis ofcultivated genepool with novel alleles that improve productivity and adaptation.

Figure 48.7 Schematic representation of the fundamental steps involved in development of abioticstress-resistant plants using different approaches.


Substantial genetic variation existing in the cultivated genepool of sesame can beexploited in breeding programs addressing abiotic stress tolerance. Furthermore, thewild species of Sesamum have been reported to possess desirable genes for thecharacters ofmajor importance in sesamebreeding including drought resistance andtolerance to heavy rainfall [187]. Desirable characters have been identified for thesesame improvement with aims to maintain and enhance sesame production inaddition to high yield potential coupled with harvest index, seed retention, uniformmaturation through determinate habit, and tolerance to biotic and abiotic stresses. Alist of major characters identified [6, 188–192] with an emphasis on overcomingabiotic stresses are listed below.

Seedling characters: Fast vigorous germination and emergence with strong hypo-cotylar elongation, rapid growth in early stages, and ability to germinate andwithstand lower temperatures.Plant characters: Rapid root growth, deep taproot penetration with well-distributedsecondary root system; leaves with medium to broad base, narrow lanceolate towardapex, short petioles, higher photosynthetic efficiency, and early abscission.Physiological characters: Photo- and thermoinsensitivity, early maturity, and highernutrient uptake under low fertility; tolerance to water logging, drought, salinity;nonlodging under high fertility; and uniform ripening.Yields: High and stable under a wide range of environmental conditions.

Significantly large germplasm collections have beenmade for sesame by differentsesame growing countries and have been characterized for morphoagronomic traitsfollowing the IBPGR (now Biodiversity International) descriptors.

Being the center of origin and diversity for sesame [4], India has particularly richdiversity for economically important traits that are largely underexplored for use incrop improvement programs [5, 193]. However, a systematic screening of thegermplasm or even the core collections identified is entirely lacking. Few frag-mented studies using limited collections have been attempted for tolerance todifferent abiotic stresses. Therefore, there is need to undertake systematic andcomprehensive screening of sesame germplasm for tolerance to individual abioticstresses giving suitable environment. The specific trait-based core collections canbe identified and used as reference collections for individual stresses. The prom-ising germplasm accessions identified can be directly used as the donor in theconventional breeding programs and also subjected to detailed investigations forgene prospecting.

The major challenges in successful incorporation of abiotic stress tolerance in thedesired taxa or genotypes are the gaps in the understanding of the mechanismsresponsible for their expression. First, the precisemetabolic and structural attributesthat impart such valuable adaptations in the plants are far from understood. Nextmajor challenge is the transfer of these traits to the genotypes of our choice. A synergybetween traditional breeding and genomic approach is the need of the hour inmeeting these challenges. Characterization of sesame genetic diversity shouldemploy the tools of functional genomic approaches. Therefore, discovery of genetrait from the diverse genetic resources available coupled with phenotyping and

48.6 Approaches for Incorporation of Abiotic Tolerance j1271

bioinformatics followed by proof of the candidate gene function in planta leading tosuccessful expression of the trait in the desired background is being advocated [194].Growth or yield penalty in the stress-resistant plants under the unstressed conditionsis yet another challenge that can be addressed by driving expression of genes inresponse to stress by an inducible promoter [195].

48.7Conventional Approach

The conventional approaches to develop the genotypes of interest involve creation ofnew gene combinations by either crossing the genotypes having the desired traitsindividually or by introducing new germplasm. The desired variability thus obtainedneeds to be narrowed down to few genotypes. The success of the effort necessitatesexercising high selection intensity (i.e., selection of few genotypes) on genotypeshaving large differences (i.e., having high genetic variance) using accurate methodsto assess characters that are transmitted to subsequent generations (i.e., having highheritability).

Conventional breeding approaches for incorporation of the desired trait havealways been the first choice of the breeders provided the source of the desired geneand the target genotypes do not pose uncircumventable crossability barriers. Thisapproach essentially requires identification of stress-resistant genotype within thegene pool, followed by their crossing with agronomically superior cultivar andrepeated backcrosses and selection of the desired phenotypes in each generation.Although attempts are being made to develop drought-tolerant sesame cultivarsthrough breeding approach [66], the process is challenging because of gaps inunderstanding/information on precise phenotypic traits to be selected in thesegregating generation. This process can be made more specific and targeted bymarker-assisted breeding. However, there are still no such usable validated markersavailable in sesame. Furthermore, the very basic requirement of MAS, that is, asaturated linkagemapbased onmolecularmarker, is still lacking and is a prerequisitefor background selection for recipient�s genotypes.

48.7.1Recent Approaches in Utilization of Genetic Variation

48.7.1.1 Association GeneticsAssociation mapping, a high-resolution method for mapping quantitative trait loci,based on linkage disequilibrium, holds great promise for the dissection of complexgenetic traits. Most traits related to abiotic stress tolerance are controlled by multiplequantitative trait loci. Genetic mapping and molecular characterization of thesefunctional loci would facilitate genome-aided breeding for stress tolerance. Two ofthe most commonly used tools for dissecting complex traits are linkage analysis andassociation mapping. Linkage analysis exploits the shared inheritance of functionalpolymorphisms and adjacent markers within families or pedigrees of known


ancestry. Linkage analysis in plants has been typically conducted with experimentalpopulations that are derived from a biparental cross. Although based on the samefundamental principles of genetic recombination as linkage analysis, associationmapping examines this shared inheritance for a collection of individuals often withunobserved ancestry. As the unobserved ancestry can extend thousands of genera-tions, the shared inheritance will persist only for adjacent loci after these manygenerations of recombination. Essentially, association mapping exploits historicaland evolutionary recombination at the population level [196]. By exploring deeperpopulation genealogy rather than family pedigree, associationmapping offers severaladvantages over linkage analysis such as much higher mapping resolution, greaterallele number, broader reference population, and less research time in establishingan association. Linkage analysis and association mapping, however, are comple-mentary to each other in terms of providing prior knowledge, cross-validation, andstatistical power.

The linkage disequilibrium approach that forms the basis for associationmappingis useful in identifying the genetic mechanisms responsible for abiotic stresstolerance and their fine dissection. Linkage disequilibrium (LD) refers to thenonrandom association of alleles between genetic loci. The core collection of sesamerepresenting the genetic variation in cultivated sesames [197] is a good source foridentifying the basic mechanisms contributing to abiotic stress tolerance in sesame.The process is also useful in detection of molecular markers closely linked to thegenes of interest, thereby providing a mechanism to transfer the genes identifiedfrom donors to recipients through marker-assisted breeding programs.

48.7.1.2 PhenomicsPrecision of association analysis depends to a large extent on the accuracy ofphenotyping. Although enhanced yield of economically important product of a cropis the ultimate target in crop breeding, yield as a trait is highly complex to bedeciphered in terms of effects of a gene or its allelic forms. Furthermore, associationanalysis attempts to relate a product of expression to an allele. Therefore, accurateestimation of gene effects is basic to the success of the approach. Association analysisrequires accurate �phenotyping� of genotypes on a scale larger than any breeding-related analysis; hence, an approach such as �phenomics� is essential for unbiasedestimation of effects of genes [198]. State-of-the-art phenomics facilities such as theAustralian Plant Phenomics Facility with the Plant Accelerator involving the WaiteCampus of the University of Adelaide and the High Resolution Plant PhenomicsCentre involving CSIRO Plant Industry and the Australian National University inCanberra have highlighted the scientific advantages of automation in large-scalephenotyping in cropmodeling. Such facilities include infrared andRGB imaging andother facilities to estimate total biomass, canopy temperature, and other features thatare useful in evaluating a plant response to environmental stress and impulses. Sincethe procedures involved are nondestructive in nature, there is an added advantage forcarrying forward the genotypes selected.

Besides, molecular dissection of tolerance mechanisms in heterologous systemsthat is an integral part of such approaches may lead to isolation of novel genes and

48.7 Conventional Approach j1273

promoters. These genes can be used to improve tolerance in cultivated species oragronomically superior cultivars of sesame. Several genes for abiotic stress tolerancesuch as codA, metD, tps1, mdh, hsp(s), acdS, and sigma factors and antioxidantenzymes have been identified and utilized for development of transgenic crop plants.Isolation of such genes and alleles from indigenous resources will enrich the genepool, which can further enhance both the transgenic and the conventional cropbreeding programs. Phenomics approach also enables us to understand the precisemolecular mechanism involved in conferring tolerance against different kinds ofabiotic stresses.

Breeders conventionally go for early selection for characters such as biotic stresses,desirable plant type, and other yield-related traits. It is only at the advanced testingstages that the entries are tested for abiotic stresses when their numbers have beensignificantly reduced and are far from a complete representation of the initialmaterial. Therefore, the success for breeding abiotic stress-resistant genotypes hasbeen poor due to low selection intensity at this stage. The various reasons for havingapprehensions on selection at early stages range from the presence of fewerdifferences between entries leading to reduction in the selection gain due to lowheritabilities and variance for yield traits under abiotic stresses as the yields fall. Also,there are practical problems in selecting the best germplasm due to experiment-to-experiment variation because of high genotype� environment interaction in stressexperiments. In addition, a general assumption that the high-yield entries in thestress-free environments will have the increased grain yield potential under theabiotic stress condition prevails. Finally but perhapsmost importantly, lack of intereston the part of private seed sector for the resource-poor farmers due to theircommercial interest in favor of rich farmers prevails that in turn unfortunatelyinfluences the public sector scientists also.

In addition to the constraints due to conventional breeding practices, the geneticconsiderations for the lack of abiotic stress tolerance in the cultivated gene poolcannot be ignored. Foremost among the genetic reasons is the founder effect thatresults in the genetic bottleneck during domestication of the crop plants. Occurrenceof wild relatives as natural populations under the harsh environmental conditions incase of most of the crop plants confirms the loss of useful alleles duringdomestication.

Though the traditional/conventional approaches of plant breeding have contrib-uted significantly to the increase in the crop productivity resulting in cultivars havinghigher tolerance to biotic stresses and better plant types, there is need to adoptmodern biotechnological approaches for dealing with abiotic stresses as the successstories in this aspect are rather few. The basic causes responsible for this shiftare [195] as follows:

1) Focus has been on yield rather than on specific traits.2) Difficulties in breeding for tolerance traits as they are subjected to GXE

interactions.3) Relatively infrequent use of simple physiological traits as measures of tolerance.4) Desired traits can be used only from closely related taxa.


48.8Transgenic Approach

Transgenic approaches being employed for the transfer of abiotic stress tolerance toother crops could be helpful in sesame. The use of transgenic approaches forincorporation of useful genes often depends upon the efficient in vitro regenerationprotocols. However, a highly efficient regeneration protocol for sesame has yet to beoptimized. Several attempts have beenmade to develop efficient in vitro regenerationand transformation protocols for sesame in the past decades. The first initiative wastaken by Govil and Singh [199] for haploid production using anthers. Whole plantswere obtained by shoot tip culture by Lee et al. [200]. Further studies using shoot tip asexplants resulted in callus [201], while multiple shoot buds to single plantlet wereobtained when shoot tips used were pretreated [202] or were supplemented withkinetin in addition to BA [203]. Leaf segments as explants showed responses such asadventitious shoot formation and rooting of shoots [204]. Using anther as explantcaused callus generation in most cases [205]. The use of hypocotyls and cotyledongave no callus even under various combinations of media [201], while Lee et al. [206]were successful in attaining the callus stage. Adifferent attempt via protoplast cultureof hypocotyls also resulted in callus production [207]. Although, embryo-like struc-tures and adventitious shoots were observed when reducing the concentration ofboth NAA and BA from the earlier adopted ones [208, 209]. In addition, direct usageof callus in protoplast culture also failed to generate plantlets and terminated atcallusing [210]. Rooting of the regenerated shoots also poses problem. Some successwas achieved by Takebayashi et al. [211], but the use of seedling or seed resulted onlyin callus formation [212] and subsequent variations of explants aswounded cotyledonlamina or deembryonate cotyledon as a whole ended up with multiple shoots [213,214]. There is no report of standardized protocol for indirect regeneration of plantsfrom callus. Xu et al. [215] reported a low plant conversion rate from somatic embyosthat was followed by the report of induction of multiple shoots from nodal segmentswith axillary buds [216]. Further progress in shoot regeneration has been achieved at amaximum frequency of 63% and 4.4 shoots per regenerating explants [213]. Onlyrecently, internodal thin-cell layer culture was reported to produce efficient shootswith rooting and establishment of 80% plantlets [217].

Success stories of Agrobacterium-mediated genetic transformation in sesame arefew. Ogasawara et al. [218] transformed sesame for increasing the yield of naphtho-quinone by the means of Agrobacterium rhizogens ATCC 15834 mediated hairy rootculture. The use of A. tumefaciens by Taskin et al. [219] for expression of carrotcalmodulin gene in another species of sesame (S. schinzianum) has beenreported [220]. Production of recombinant proteins (a fungal protein, phytase) hasbeen achieved by sesame hairy root cultures [221]. Recently, successful recovery offertile transgenic plants was achieved using A. tumaefaciens strain with cotyledonexplants that displayed a transformation efficiency of 1.01% [222]. In view of theabove, any attempt to use transgenic technologies for incorporating abiotic stress insesame should prefer methods where the in vitro regeneration step is not needed.This includes floral dip method where flowering shoots are swirled in a solution of

48.8 Transgenic Approach j1275

Agrobacterium, resulting in the formation of transgenic seeds [223, 224]. Of thevarious biolistic methods that involve physical DNA delivery, microprojectile bom-bardment of tissues has also proven useful for transforming plants that lack goodregeneration systems [225]. Another method involving infection of apical meristemof the differentiated embryo of the germinating seedling with Agrobacterium hasworked well in the recalcitrant cotton resulting in transgenic T1 plants [226].However, in groundnuts infection of Agrobacterium after pricking of embryo axesofmature seedswith one excised cotyledon directly gave transformed seedlings [227].Nevertheless, lack of protocols necessary for transgenic development in sesamemayget compensated with extremely high genetic variability present in the cultivatedgene pool of sesame and its wild allies [5].

High-throughput technologies along with powerful bioinformatics tools areproving useful to meet the challenges in understanding the abiotic stress tolerancein more than one way. The �omics� approaches have opened up the genome, itstranscriptional products, regulatory networks, signaling pathways and their inter-actions, and themetabolites involved in the entire gamut of stress tolerance spanningfrom its perception, differential gene expression, and imparting protection fromdamage caused due to abiotic stress. Therefore, the functional genomics approachesinvolving transcriptome, interactome, and metabolome profiling of the plant sub-jected to specific stress environment are required in sesame (Table 48.1).

Table 48.1 The generalized key steps, activities, and expected outputs involved duringincorporation of abiotic stress tolerance.

Key steps What needs to be done Output

Identification of traitdonor

Large-scale germplasm screen-ing including wild relatives

Core/reference collection forfine scale analysis

Phenomics for fine dissection ofunderlying mechanism

Donors for specific mechanismsof tolerance

Functional genomics Understanding the naturalmechanisms and pathways

Association genetics SNPs for specific traitsGenome-wide approach Allelic variations for specific

mechanismsCandidate gene approach Marker tags for specific traitsAllele mining Superior/elite germplasm for

direct utilizationGene mapping and markertagging

Understanding themechanism of tolerance

Functional genomics Identification of genes/path-ways/networks for tolerance

Systems biology approachIncorporation of thestress tolerance

Conventional back crossbreeding

Abiotic stress resistant varieties

Marker assisted breedingTransgenic approach


Research efforts on incorporation of abiotic tolerance in sesame can greatly benefitfrom the lessons learnt from other crops where considerable progress has alreadybeen made. The studies on large number of crops indicate that pathways and genenetworks between different abiotic stresses overlap significantly. On the other hand,managing tolerance to one type of stress can result inmaking the plants susceptible toother type of stress due to opposingphysiological andmolecular processes. Thoroughinvestigations into the individual stress response may contribute to the understand-ing of the basic mechanisms involved, but would be largely unsuccessful intranslating this knowledge for developing genotypes that exhibit superior perfor-mance in the field. Therefore, there is need to study the multiple stresses simul-taneously in various combinations. In addition, due consideration needs to be givento the phenology-related parameters of the plant also as similar stresses are known toaffect plants with different life cycle differently.

Research efforts and funds to �orphan� crops such as sesame that go beyond themere increase in crop yields deserve special emphasis as they are crucial for food andnutritional security on a regional or local basis. Though gross economic and welfareimpacts of investmentsmade in crops such as sesamemay appear to give low returns,the alternative indicators such as promotion of crop and genetic diversity leading tooverall agricultural stability highlight the urgent need to pay attention to thesecrops [228]. In addition, an understanding of the basic mechanisms of the uniquetraits lacking in major plants and model systems such as extraordinary antioxidativeability of sesame seed oil, drought tolerance, ability to accumulate heavy metals, andso on can be utilized for the improvement of major cops subsequently.

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