Senouwa Segla Koffi DOSSOU ,XU Fang-tao ,Komivi DOSSA ,ZHOU Rong ,ZHAO Ying-zhongWANG Lin-hai

1 Oil Crops Research Institute,Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture and Rural Affairs,Wuhan 430062,P.R.China

2 Laboratory of Plant Biotechnology and Physiology,University of Lomé,Lome 01 BP 1515,Togo

3 French Agricultural Research Center for International Development (CIRAD),Genetic Improvement and Adaptation of Mediterranean and Tropical Plants-UMR AGAP Institute,Montpellier F-34398,France

4 UMR AGAP Institute,University of Montpellier,CIRAD,French National Research Institute for Agriculture,Food and Environment(INRAE),Montpellier F-34398,France

Abstract Sesame (Sesamum indicum L.) is a significantly lucrative cash crop for millions of small-holder farmers.Its seeds are an important source of a highly appreciated vegetable oil globally and two clinically essential antioxidant lignans,sesamin and sesamolin.Accordingly,many countries import millions of tons of sesame seed every year.The demand for lignan-rich sesame seeds has been increasing in recent years due to the continuous discovery of several pharmacological attributes of sesamin and sesamolin.To meet this demand,the sesame breeder’s primary objective is to release sesame cultivars that are enriched in oil and lignans.Thus,it is necessary to summarize the information related to the sesamin and sesamolin contents in sesame in order to promote the joint efforts of specialized research teams on this important oilseed crop.In this article,we present the current knowledge on the sesamin and sesamolin contents in S.indicum L.with respect to the updated biosynthesis pathway,associated markers,governing loci,available variability in sesame germplasm,the in planta potential roles of these compounds in sesame,and the newly discovered pharmacological attributes.In addition,we propose and discuss some required studies that might facilitate genomics-assisted breeding of high lignan content sesame varieties.

Keywords: Sesamum indicum,lignan biosynthesis,antioxidants,molecular breeding,sesamin and sesamolin

1.Introduction

Sesame (Sesamum indicumL.) is regarded as the “queen of oilseed crops” owing to its nutritional and health benefits both for humans and animals.Its seed contains 27.89 to 62.7% oil,16.72 to 27.79% protein,13.5% carbohydrates,minerals,and various antioxidants (Uzunet al.2008;Li Cet al.2014;Pathaket al.2014;Daret al.2015).The main antioxidant components in sesame seeds in terms of value and level are the lignans,followed by flavonoids,phenolics,tocopherol,and melatonin (Table 1) (Shahidiet al.2006;Williamsonet al.2008;Wanget al.2012,2018;Zhouet al.2016;Niuet al.2018).Sesame lignans are clinically important nutraceuticals that exhibit several pharmacological properties (Majdalawiehet al.2017,2020;Majdalawieh and Mansour 2019).Lignans are plant secondary metabolites that are chemically classified as phenyl propane dimers(Fig.1) (Dar and Arumugam 2013).Two classes of lignans,the oil-soluble and glycosylated water-soluble lignans which include a total of 17 compounds,have been isolated from sesame seeds (Fukuda 1985;Dar and Arumugam 2013).Among sesame lignans,sesamin and sesamolin are the primary compounds that have gained the attention of pharmacologists and clinicians due to their health-promoting properties against lifestyle-related diseases (Dar and Arumugam 2013;Majdalawiehet al.2017,2020;Abe-Kanohet al.2019).Sesamolin and sesamin have been detected in more than 40 plant species (Table 2).However,due to the low amounts of sesamin in other plants (Table 3) and the unclear issue of identical conformations,sesame seeds remain the principal source of these two clinically priceless antioxidant lignans.

Table 2 Plants reported to content sesamin and sesamolin

Fig.1 Chemical structures of some lignans.

Table 1 Variations in the major antioxidant components in sesame seed

Table 2 (Continued from preceding page)

Table 3 Variations of sesamin and sesamolin in some species

With regard to their tremendous health benefits,interest in sesame products is increasing continuously,especially the demand for lignan-rich seeds (Daret al.2015;Kim A Yet al.2020).In Korea,the daily intake of total lignans from sesame seeds and oil in males and females is approximately 18.39 and 13.26 mg/person/day,respectively (Kim A Yet al.2020).Owing to their potent antioxidant ability and solubility features,sesamin and sesamolin are responsible for sesame seed and oil resistance to oxidation and rancidity.Accordingly,sesame seed flour and oil are used to improve the oxidation stability and the quality of various products,including chia oil,sunflower oil,corn snacks,soybean oil,halva,and mayonnaise (Sujaet al.2004;Nasirullah and Latha 2009;Elleuchet al.2014;Li Yet al.2014;Hashempour-Baltorket al.2018;Hussainet al.2018;Karshenaset al.2018;Bordet al.2019;El-robyet al.2020).In consideration of all these aspects,the contents of both sesamin and sesamolin are a critical factor in evaluating sesame seed quality (Kimet al.2004).Sesame is cultivated only once a year,and its production is influenced by biotic and abiotic stresses (Kole 2019).Therefore,creating environmentally-stable sesame varieties containing high oil and lignans is the main purpose of sesame breeding.

Tiwariet al.(2011) reported the limitations of the conventional breeding approach in sesame,and noted that only genomic assisted-breeding techniques couldbe useful for the genetic improvement of sesame seed quality.Obtaining the information related to the lignan contents in sesame is an essential prerequisite for any breeder interested in meeting the current sesame breeding objectives.In this review,we present the current state of knowledge on sesamin and sesamolin inS.indicumL.Moreover,we propose and discuss some studies that are needed to pave the way for genomics-assisted breeding of sesame varieties with high lignan content.

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2.Sesamin and sesamolin roles in plants and pharmacological attributes

2.1.Sesamin and sesamolin in planta functions

The potential biological roles of sesamin and sesamolin related to human and animal health have been widely studied.Comparatively,their relevant biological functions in the sesame plant remain elusive.According to Bedigianet al.(1985),sesamin and sesamolin might be involved in seed maturation,germination,and seed protection during storage.They are associated with seed germination since their amounts in seeds decrease significantly during germination (Haet al.2017).In a recent study,Teraet al.(2019) discovered steroleosin B,a sesamin-binding protein,and characterized its roles in transgenicArabidopsis thalianaplants.They found that sesamin in the presence of steroleosin B influencedArabidopsisplant development by suppressing leaf expansion and root elongation.The studies by Miyakeet al.(2005) and Radhakrishnanet al.(2013) showed that sesamin and sesamolin are involved in the plant’s defense against diseases by inhibiting reactive oxygen species (ROS) production and staving off oxidative damage in infected sesame plants.However,the molecular mechanisms remain unclear.Further experiments are needed to clarify the biological functions of sesamin and sesamolin during sesame plant development,and to analyze their potential antioxidant effects during osmotic stress.

2.2.Pharmacological attributes of sesamin and sesamolin

Sesamin and sesamolin possess various pharmacological proprieties,such as antioxidative,anti-cancerogenic,antiinflammatory,anti-proliferative,anti-hypertensive,and antimelanogenesis effects.Majdalawiehet al.(2017,2020),Wuet al.(2019),and Daret al.(2013) have reviewed some pharmacological properties of sesamin and sesamolin.These reviews reported the ability of sesamin to reduce oxidative stress,proliferation,inflammation,angiogenesis,and metastasis,and to induce apoptosis and autophagy in defective human and animal cells.They discussed the anti-hyperlipidemic properties of sesamin and sesamolin.Moreover,they outlined the capacity of sesamin to inhibit fatty acid and cholesterol synthesis and absorption,cause fatty acid oxidation,maintain macrophage cholesterol homeostasis,and control circulating serum and liver lipid levels.In a recent study,Abe-Kanohet al.(2019) have confirmed the anti-inflammatory effects of sesamin.

In addition to the above-reported functions,sesamin and sesamolin have been associated with several other healthpromoting abilities,including anti-hypertensive and antimelanogenesis effects (Srisayamet al.2017),prevention of brain damage (Chenget al.2006),improvement of cardiac function (Fanet al.2017;Thuyet al.2017),auditoryprotective effects (Kim Y Het al.2020),suppression of aging phenotypes (Leet al.2019),alleviation of blood-brain barrier disruption (Liuet al.2017),anti-osteonecrosis and anti-osteoporosis effects (Denget al.2018;Maet al.2019),enhancement of habit learning memory deficits (Zhaoet al.2016),reduction of amyloid-β toxicity (Keowkaseet al.2018),anti-collagen and elastin fiber degradation (Kugoet al.2019),influence on voltage-gated Na+and K+currents(Kuoet al.2020),promotion of natural killer cell cytolysis and migration activity (Lee 2020),protection against lesion-induced degeneration (Li and Lv 2020),extension of lifespan (Nakataniet al.2018),promotion of cartilage repair (Narakornsaket al.2017) and anti-depression and memory loss improvement (Zhaoet al.2019).

3.Variability of sesamin and sesamolin in sesame

3.1.Variations in sesame seeds

Information on the diversity of sesame cultivars regarding their sesamin and sesamolin contents is a prerequisite for selecting and breeding lignan-rich sesame varieties.Accordingly,sesame cultivars have been collected worldwide,and the variability in sesamin and sesamolin contents has been checked in many germplasms that are conserved in China,India,Korea,Japan,USA,and Thailand(Table 4).Sesame seed total sesamin and sesamolin contents vary from one germplasm to another.The sesamin and sesamolin contents in sesame seeds broadly range from 0.05 to 11.05 mg g–1and 0 to 10 mg g–1,respectively(Yasumoto and Katsuta 2006;Rangkadiloket al.2010;Wanget al.2012;Ajitet al.2019).In a core collection conserved in Korea,which included accessions from Russia,Japan,Turkey,Nepal,Afghanistan,Iran,Mexico,Pakistan,Korea,India,China,USA,Egypt,and Vietnam,the total sesamin and sesamolin contents varied from 2.33 to 12.17 mg g–1with an average of 8.18 mg g–1(Kim S Uet al.2014).In China and India,the seed sesamin content mainly varied from 0.88 to 11.05 and 0.08 to 6.45 mg g–1,respectively (Wanget al.2012;Muthulakshmiet al.2017;Ajitet al.2019).In the same countries,the sesamolin content in sesame seeds ranged from 0.2 to 6.98 mg g–1and 0.28 to 3.76 mg g–1,respectively(Wanget al.2012;Kaiet al.2017;Ajitet al.2019;Daret al.2019).All the studies listed in Table 4 reported a significant positive correlation between the contents of sesamin and sesamolin in sesame seeds.In addition to the seeds,sesamin has been detected in sesame leaves in a range of 0.5 to 2.6 μg g–1of dry weight (Hataet al.2010).However,no more information is currently available on the variability of lignan contents in sesame leaves.

Table 4 Variations in sesamin and sesamolin contents in sesame seeds

3.2.lnfluences of various factors

Reports on the variability of the sesamin and sesamolin contents in sesame seeds indicated that these lignans are affected by genetics,cultivars,origin,agronomic conditions,environmental stresses,plant architecture,and other seed traits (Yasumotoet al.2005;Kumazakiet al.2009;Wanget al.2012;Kim S Uet al.2014;Daret al.2019).

Seed coat color and seed biochemistrySeed coat color is a critical agronomic trait in sesame that significantly affects the phytochemical contents of seeds (Wanget al.2012;Meiet al.2013;Kim J Het al.2014;Li Cet al.2014;Dossaet al.2018b;Daret al.2019).Studies conducted on germplasms containing a higher number of accessions with different colors (white,yellow,brown,and black) revealed that white sesame seeds are generally richer in lignans than black,brown,and yellow sesame seeds (Wanget al.2012;Kim S Uet al.2014;Kancharla and Arumugam 2020).In contrast,the studies of Shiet al.(2017),Daret al.(2019),Muthulakshmiet al.(2017) and Ajitet al.(2019) on collections containing 6,4,2,and 6 accessions with a black seed coat,respectively,found that black sesame seeds were the richest in sesamin and sesamolin.Some of the individual black and brown sesame lines exhibited higher contents of sesamin and sesamolin.Among the 215 accessions evaluated by Wanget al.(2012),ZZM3495,a line with a brown seed coat,had the highest sesamin and sesamolin contents of 11.05 and 6.96 mg g–1,respectively.The sample 16NF378-1 (black seeds) had the highest contents of sesamin and sesamolin(9.41 and 3.35 mg g–1,respectively) among the cultivars tested by Kaiet al.(2017).

Most of the precursors of sesamin and sesamolin in the biosynthetic pathway are also involved in the biosynthesis of other seed components (Fig.2).Therefore,there is a need to understand the correlations that exist between the contents of these lignans and other seed components for targeting improvements in sesame seed quality.Daret al.(2019) reported a significant negative correlation between the sesamol content and the contents of sesamin and sesamolin.They also found positive correlations between sesamolin and oleic acid,stearic acid and sesamin,and a negative correlation between the sesamolin and linoleic acid contents.Meiet al.(2013) and Wuet al.(2017) observed that the sesamin and protein contents were negatively correlated.Interestingly,the oil content in sesame seed was positively correlated with the content of sesamolin,supporting the possibility of breeding high oil and high lignan sesame varieties in accordance with the sesame seed market demands (Meiet al.2013;Wuet al.2017;Kancharla and Arumugam 2020).

Fig.2 Complexity of the metabolic pathways in Sesamum indicum L.PEP,phosphoenol pyruvate;E4P,erythrose-4-phosphate;G3P,glyceraldehyde-3-phosphate;Phe,phenylalanine;Tyr,tyrosine;Trp,tryptophan.

Plant architecture and growth conditionsAs mentioned above,in addition to genetics,the growing conditions and plant architecture affect the contents of sesamin and sesamolin through the differences in seed weight (Yasumotoet al.2005;Kumazakiet al.2009).Sesamin and sesamolin contents increase proportionally with the seed dry weight.Yasumotoet al.(2005) observed a variation in the sesamin and sesamolin contents among seeds at different positions in the capsules.They suggested that for improving the selection of lignan-rich sesame varieties,it is necessary to sample seeds from capsules that flowered and were harvested on the same date as the variability analysis.The study of Kumazakiet al.(2009) revealed that in normal growing conditions,seeds from higher capsule positions on the stem are richer in sesamin and sesamolin than those from capsules at lower positions.In addition,they observed that in contrast to a high air temperature (30/23°C) during ripening,a low air temperature (22/15°C) increased the amounts of sesamin and sesamolin in seeds.With regard to the day length and soil temperature,they found no impact of high soil temperature or a short-day length (10 h) on the contents of these lignans.

To gain insight into the effect of the day length (light) on the content of lignans in sesame,Hataet al.(2012,2013)performed several types of experiments.They showed that a continuous light (24 h) photoperiod increased sesame leaf sesamin levels to the 1/200th of that in seeds,while in normal agronomic conditions,the leaf sesamin content was the 1/5 000th or less compared to that in seeds (Hataet al.2010).Concerning the light type,they found that blue LED light increased the leaf sesamin content by 2.0-and 4.5-fold compared to white fluorescent and red LED light,respectively (Hataet al.2013).However,the blue LED light caused unfavorable morphological changes and a reduction in growth.

Abiotic stressesDrought,waterlogging,and salt stresses are the common adverse abiotic stresses that affect sesame yield and quality traits.The sesame plant responses to these stresses have been well studied at the genomic level (Wanget al.2016,2020;Mmadiet al.2017;Dossaet al.2018a,2019a;Zhanget al.2019).However,information related to the effects of these stresses on the sesamin and sesamolin contents is very limited,and only two contradictory reports on the impact of drought stress on the variations of these lignans are available.Kermaniet al.(2019) examined the influence of drought stress on the contents of sesamin and sesamolin in 10 sesame cultivars.They found that the sesamin content decreased while the sesamolin content increased under drought stress.In contrast,Dossaet al.(2017b) reported a significant increase of sesamin in the seeds of five sesame varieties subjected to drought stress.Regarding the content of sesamolin,they found no significant difference between the stressed and unstressed seeds.Therefore,experiments involving large germplasm collections are needed to elucidate the influences of these stresses on lignan biosynthesis in sesame,and to identify the environmentally stable lignan-rich sesame varieties.

Biotic stressesIn contrast to abiotic stresses,biotic stresses were reported to have a clearly positive influence on the lignan content in sesame (Radhakrishnanet al.2013,2014;Horii and Ishii 2014).Radhakrishnanet al.(2013,2014) found that infection by fungiPenicilliumspp.(RDA01,NICS01,and DFC01) andFusariumsp.increased the contents of sesamin,sesamolin,and amino acids in sesame,although the two species showed an antagonistic activity.Horii and Ishii (2014) discovered that arbuscular mycorrhizal fungi (AMF) and their partner bacteria could promote the growth and development of the sesame plant,and sesamin accumulation in seeds.In fact,sesame plants infected byGlomus clarumIK97 andPseudomonassp.(KCIGC01) NBRC-109613 contained higher sesamin levels than the control.No significant difference was observed regarding the sesamolin content.

4.Genetic basis and breeding of high sesamin and sesamolin content varieties

4.1.Genetic basis

The genetic basis of sesamin and sesamolin contents in sesame is poorly understood,with only some biosynthetic genes and loci discovered thus far.The sesamin and sesamolin contents in sesame are polygenic traits,controlled by additive,dominant,and environmental effects (Hataet al.2010;Chandraet al.2019;Usmanet al.2020).These two lignans are genetically very close and are biosynthesized from the same precursors (Katoet al.1998).

Sesamin and sesamolin biosynthesis inS. indicumDeciphering the molecular mechanisms involved in the lignan pathway is a prerequisite for breeding lignan-rich sesame varieties.It will help to efficiently target the genes responsible for lignan variations among sesame germplasms.Sesamin(C20H18O6) and sesamolin (C20H18O7) are biosynthesized in developing sesame seeds,mainly between 10 and 30 days post-anthesis (DPA) (Suhet al.2003;Onoet al.2006;Keet al.2011;Wanget al.2014b).The studies of Katoet al.(1998) and Jiaoet al.(1998) revealed that these two nutraceuticals,sesamin and sesamolin,are biosynthesized in the phenylpropanoid pathway from tyrosine (Tyr) or phenylalanine (Phe) (Fig.3).As a reminder,the Phe and Tyr precursors are biosynthesized in plantsviathe shikimate pathway (Maeda and Dudareva 2012).First,these two amino acids are converted through a series of reactions into coniferyl alcohol,which then goes through a stereo-specific dimerization to yield pinoresinol,the basal lignan (Jiaoet al.1998;Katoet al.1998;Suhet al.2003).Then,pinoresinol is converted into sesaminviapiperitol (Onoet al.2006),and finally,sesamin is metabolized to sesamolin and sesaminol by a specific oxygenation reaction (Murataet al.2017)(Fig.3).Sesaminol is mostly accumulated in its triglucoside form (Onoet al.2020).By comparing expressed sequence tags (ESTs) obtained from the cDNA libraries ofS. indicumL.andA. thalianadeveloping seeds,Suhet al.(2003)shed light on some of the genes and metabolic pathways involved in sesame lignan biosynthesis.The biosynthesis of coniferol alcohol from Phe and Tyr still needs to be clarified,as only cinnamate-4-hydroxylase (C4H),caffeic acidO-methyltransferase (COMT),cinnamoyl-CoA reductase(CCR),4-coumarate-CoA ligase (4CL),and cinnamyl alcohol dehydrogenase (CAD) have been identified by ESTs analysis(Suhet al.2003;Keet al.2011).Likewise,the studies of Davin and Lewis (2000),Suhet al.(2003),Onoet al.(2006),Keet al.(2011),Murataet al.(2017),and Chandraet al.(2019) revealed the importance of some key catalytic genes during the sesamin and sesamolin biosynthetic process.

Fig.3 The general biosynthetic pathway of major lignans in Sesamum indicum L.Key enzymes catalyzing some reactions:PAL,phenylalanine ammonialyase;C4H,cinnamate 4-hydroxylase;4CL,4 coumaroyl-coenzyme A ligase;TAL,tyrosine ammonia-lyase;C3H,p-coumarate 3-hydroxylase;CCR,cinnamoyl-CoA reductase;HCT,hydroxycinnamoyl-CoA shikimate/quinatehydroxycinnoyltransferase;COMT,caffeic acid O-methyltransferase;CCoAOMT,caffeoyl-CoA O-methyltransferase;CAD,cinnamyl alcohol dehydrogenase;DIR,dirigent protein;CYP81Q1,piperitol/sesamin synthase (PSS) and CYP92B14,sesaminol and sesamolin synthase.

Caffeic acidO-methyltransferase (COMT): By competing with 4CL for the substrate,caffeic acid,COMT catalyzes the reaction leading to ferulic acid.Suhet al.(2003) and Keet al.(2011) identified 20 and two ESTs associated with COMT,respectively.They suggested that a higher expression of COMT is needed in developing sesame seeds for coniferyl alcohol formation than caffeoyl-CoAO-methyltransferase (CCoAOMT),which is involved in feruloyl-CoA synthesis from caffeoyl-CoA.None of the ESTs was linked to CCoAMT in the two studies.There are five copies of the COMT gene in sesame (Keet al.2011).

Cinnamyl-alcohol dehydrogenase (CAD): CAD is a committed enzyme for the biosynthesis of lignans and lignin in sesame and other plants (Keet al.2011;Liu Qet al.2018).In the lignan pathway,this enzyme converts coniferaldehyde into coniferyl alcohol by competing with ferulate 5-hydroxylase (F5H).Twelve CAD-like genes are present in the sesame genome (Keet al.2011).

Dirigent protein (DIR): The first committed reaction leading to the sesame lignans is the coupling of two coniferyl alcohol molecules to yield pinoresinol (Katoet al.1998;Satakeet al.2015).This reaction is catalyzed by DIR competing with laccase and peroxidase for the substrate coniferyl alcohol (Davinet al.1997;Davin and Lewis 2000,2005).The geneSiDIR(SiN_1015471) corresponds to DIR in the sesame genome (Wanget al.2014 b).

Piperitol/sesamin synthase (PSS): PSS (CYP81Q1/SIN_1025734) is a committed NADPH-cytochrome P450 oxidoreductase in the biosynthesis of sesamin from pinoresinolviapiperitol by the generation of two methylenedioxy bridges (Onoet al.2006).Pathaket al.(2015) have analyzed the expression of PSS,coupled with sesamin content evaluation during seed development,and found that PSS was negatively favored during domestication.Wanget al.(2014b) examined the genome sequences of 29 sesame accessions and discovered that PSS was especially conserved.Thus,they suggested that sesamin content variation in sesame might be imputed to PSS regulatory genes.There are 25CYP81genes in sesame (Wanget al.2014b).

Sesamolin/sesaminol synthase (CYP92B14): Sesamolin is the second most abundant lignan in sesame seed and oil.CYP92B14is a cytochrome P450 enzyme that catalyzes the formation of sesamolin and sesaminol from sesamin by an oxygenation scheme that is named the oxidative rearrangement of α-oxy-substituted aryl groups (ORA)(Murataet al.2017;Haradaet al.2020).The same study revealed that a deletion of four C-terminal amino acids(Del4C) inCYP92B14was responsible for sesamolin deficiency in sesame.Moreover,they demonstrated thatCYP81Q1andCYP92B14operate in coordination in developing sesame seeds (Murataet al.2017).

CYP reductase1 (CPR1): CPR1 is a committed enzyme in sesamin biosynthesis.Its role is to facilitate the transfer of electrons from NADPH toCYP81Q1andCYP92B14(Murataet al.2017;Chandraet al.2019).Functional characterization demonstrated that in the absence of CPR1,theCYP81Q1enzyme was limited in converting pinoresinol to sesamin (Chandraet al.2019).

Transcriptome analyses revealed that lignan biosynthesis in sesame seed is developmentally regulated (Suhet al.2003;Onoet al.2006;Keet al.2011;Wanget al.2014b).However,the regulatory pathway of sesamin and sesamolin biosynthesis in developing seeds is unclear.Weiet al.(2015) performed an association mapping using 705 global sesame accessions grown in four environments,and found that theSiNST1gene associated with lignification in the seed coat was strongly associated with the variations in sesamin,sesamolin,oil,and protein contents in sesame.Recently,by combining QTL mapping and transcriptome profiling,we identified two candidate regulatory genes(SIN_1005755andSIN_1005756) associated with sesamin and sesamolin biosynthesis in sesame (Xuet al.2021).SIN_1005755encodes a NAC domain protein which might be the key regulatory gene of lignan biosynthesis in sesame by controlling lignin biosynthesis from coniferaldehyde or coniferyl alcohol.Functional characterization of these candidate regulatory genes using advanced genomicsediting tools is needed to shed light on the molecular mechanisms involved in lignan synthesis regulation in developing sesame seeds.

Molecular markers related to sesamin and sesamolinMolecular markers and QTLs have significantly enhanced the genetic gain in crop breeding.Various molecular markers have been developed for use in sesame breeding (Dossaet al.2017a).To date,expressed sequence tags (ESTs) are the only class of molecular markers available for sesamin and sesamolin (Table 5).Suhet al.(2003) have developed 3 328 ESTs from a cDNA library of 5-to 25-day-old developing seeds,among which 58 were involved in the sesame lignan biosynthesis pathway.Keet al.(2011) randomly sequenced a cDNA library constructed from 5 to 30 day-old immature seeds and generated 41 248 ESTs,among which 117 were associated with sesamin and sesamolin biosynthesis.Quantitative trait loci (QTLs) are important genetic resources for studying key agronomic traits and implementing markerassisted breeding of both model and non-model crops.In total,34 and 26 loci for sesamin and sesamolin have been detected by Leiet al.(2014) and Xuet al.(2021),respectively(Table 5).Leiet al.(2014) performed an association analysis using the general linear model (GLM) and identified 26 and eight significant loci associated with sesamin and sesamolin,respectively.By coupling the mixed compositeinterval mapping (MCIM) and the multiple interval mapping(MIM) methods,Wuet al.(2017) mapped six QTLs for sesamin content variation in sesame seed using 224 RILs(recombinant inbred lines) grown in three environments.In a recent study,we used composite interval mapping (CIM)in a 548 RILs population (Xuet al.2021).We detected 16 QTLs for sesamin and 10 QTLs for sesamolin,including one pleiotropic QTL located on chromosome 11 in an interval of 127 to 127.21 cM.By analyzing the QTL interactions,we found that sesame lignan biosynthesis might be governed by a major gene with a large effect.

Table 5 Markers and loci detected for sesamin and sesamolin

4.2.Breeding of sesame varieties with high lignan content

Despite the considerable repertory of sesame germplasms,conventional and biotechnological methodologies have not been successful in developing high-quality cultivars (Pathaket al.2014).The most widely used breeding techniques in sesame have been mutation induction and pedigree selection (Kole 2019).Sesame breeding programs are focused principally on the seed yield,disease resistance,plant architecture,and high oil content.To our knowledge,“Gomazou” is the most successful hybrid created that contains high sesamin and sesamolin levels.“Gomazou”was selected from the progeny of a cross between “Toyama 016”,a broad seed line from Peru,and “H65”,a high-lignancontent line from South China (Yasumoto and Katsuta 2006).The total sesamin and sesamolin content in “Gomazou”seed was 13.1 mg g–1.Recently,by crossing different types of parents using the combining ability tool,Khuimphukhieo and Khaengkhan (2018) and Usmanet al.(2020) created four and 21 hybrids,respectively,with variable sesamin and sesamolin contents.These crosses need further evaluation for the possible isolation of high yielding hybrids that have high sesamin and sesamolin contents.Sesame cultivars with higher lignan levels may allow farmers to produce sesame for various industries (Khuimphukhieo and Khaengkhan 2018).Taken together with the heritability found in a RIL population by Wuet al.(2017),it is clear that lignan-rich sesame lines can be selected from the crosses of the proper parents.However,to save time and labor,genomicsassisted breeding approaches might be used to restructure the sesame plant’s ideotype and to increase its lignan level.

5.Future prospects

5.1.Genome-wide association studies of sesamin and sesamolin variation

The studies of Wanget al.(2014b) and Weiet al.(2015)made a great deal of genome-wide information available for use in GWAS to gain some insight into the genetic architecture of complex quantitative traits in sesame,such as the sesamin and sesamolin contents.This approach was useful in locating loci,SNPs,QTLs,and candidate genes of complex agronomic traits such as oil and protein contents,charcoal rot resistance,oleic acid and linoleic acid concentrations,and salinity and drought tolerance in sesame(Dossaet al.2019a;Kole 2019).Therefore,GWAS could be harnessed to shed new light on the genetic basis of the sesamin and sesamolin contents inS.indicumL.

5.2.Functional characterization of sesamin and sesamolin candidate regulatory genes

Candidate genes are putative causative genes that require functional analysis to validate their effects and functional variants (Weiet al.2015).Since the achievement of the greatest sesame transformation and regeneration efficiencies of 42.66 and 57.33%,respectively (Chowdhuryet al.2014),candidate sesamin and sesamolin regulatory genes should be thoroughly studied in mutant sesame andArabidopsisplants to understand the molecular mechanisms involved in lignan biosynthesis and regulation.

5.3.Overexpression and knockout of PSS in sesame

Knocking out,over-expressing or down-regulating the sesamin synthase gene in sesame using advanced genomics tools may help to unveil the biological functions of lignans during sesame plant development.

5.4.Deciphering the influences of abiotic stresses(drought,waterlogging,and salt) on the sesamin and sesamolin contents in seeds through gene expression and quantification

Due to the adverse effects of climate change on crop production,especially in sesame,there is an urgent need to screen large amounts of germplasm to identify drought,waterlogging,and salt-tolerant lignan-rich sesame varieties.Performing these experiments at the genomics level may help us to discover the potential roles of these lignans in osmotic stress tolerance,and to identify the possible correlations between the regulatory genes of lignans and the core abiotic stress-responsive genes(Dossaet al.2019b).

5.5.Insight into the correlations between seed coat colors and lignan content variations at the genomics level

Tracking the expression of genes involved in the biosynthesis and regulation of flavonoids,anthocyanins,lignin,melanin,and lignans during sesame seed development may be useful for dissecting the influence of seed coat color on sesamin and sesamolin variability.

5.6.Genome-wide analysis of the DlR family in sesame

Dirigent proteins play a critical role in lignan biosynthesis.Overexpression ofGmDir22,the homologous gene ofAtDir21/AtDir23,increased the lignan contents in soybean(Liet al.2017).Therefore,a genome-wide analysis of the DIR family in sesame may help us to identify potential candidate DIR genes that contribute to high lignan accumulation in sesame.

5.7.Genomics-assisted breeding of lignan-rich sesame varieties

GWAS will provide numerous markers linked to variations in the sesamin and sesamolin contents in sesame,as well as candidate genes.Functional analysis of those candidate genes will shed light on simple and effective markers that can be targeted for the breeding of high lignan content sesame varieties.Therefore,advanced genome-editing tools may be used to knock out or edit specific sequences of the sesamin and sesamolin candidate genes together with other key genes.Overall,the efficient editing of these genes will generate various sesame materials with different levels of sesamin and sesamolin that might be useful in the process of breeding high-quality knockout mutant sesame varieties.Molecular breeding methods have been developed in rice and maize (Ramsteinet al.2019;Weiet al.2021),and a summary of our suggested ways to accelerate lignan breeding in sesame was shown in Fig.4.

Fig.4 Strategies for breeding high-lignan content sesame varieties.Integration of high-throughput sequencing and phenotyping,GWAS,functional analysis,parent selection,and genomic selection to enhance the development of high-quality sesame varieties in breeding.

6.Conclusion

Sesamin and sesamolin represent the principal lignans in sesame seeds.Human interest in these compounds is increasing due to their multiple biological proprieties.The production of these lignans is limited because sesame seed represents the sole primary source of these nutraceuticals.Besides,the development of nutritionally superior sesame varieties through conventional and biotechnological methodologies has not been successful.Previous studies showed that the sesamin and sesamolin contents in sesame are polygenic quantitative traits.With the availability of sesame genome sequence data and advanced techniques for functional genomic analysis,previously unknown candidate genes and markers linked to sesamin and sesamolin can be identified by GWAS in a massive germplasm sample and multiple environments.These candidate genes will constitute valuable materials for further genomic studies that will lead to teasing out the molecular mechanisms involved in lignan content regulation in sesame and discovering the roles of sesamin and sesamolin in sesame.The integration of high-efficiency genomic editing tools in sesame breeding programs will allow the improvement of sesame quality,especially its sesamin and sesamolin contents.Sesame cultivars with higher lignan contents may also favor the production of sesame for various industries.

Acknowledgements

This study was supported by the Open Project of Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture and Rural Affairs,China(KF2020004 and KF2022002),the Agricultural Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2016-OCRI),the Key Research Projects of Hubei Province,China (2020BBA045 and 2020BHB028),the Science and Technology Innovation Project of Hubei Province,China (2021-620-000-001-035),the China Agriculture Research System of MOF and MARA(CARS-14),and the Fundamental Research Funds for Central Non-profit Scientific Institution,China (Y2022XK11).

Declaration of competing interest

The authors declare that they have no competing interests.