CHAPTER 1 INTRODUCTION Introduction 1.1 Rice Rice (Oryza sativa L.) is the staple food for more than one half of world’s population and nearly 90 % of the world's rice is produced and consumed in Asia (Bhattacharjee et al. 2002). It is an annual crop cultivated for grains as a rich source of carbohydrates, minerals and vitamins. Rice is the major source of human nutrition and it has shaped the culture, diets and economic of thousands of millions of peoples (Rani et al. 2006). World rice production in the year 2015-16 was 469.5 million metric tonnes (MMT) with consumption of 481 MMT and trading 41 MMT in world market. India ranks second after China in rice production and contribute 20 % of world’s rice production (FAO, 2015). India was the world’s largest rice exporting country in 2014-15 followed by Thailand, Vietnam and Pakistan (Workman 2016). India’s Basmati rice occupies prime position in the world market fetching 3 times higher prices than non-Basmati white rice (FAO, 2015). Basmati rice contributed 45 % share in total rice export of India. 1.2 Growth and developmental pattern in rice Fig 1.1 Developmental phases in rice plant Based on developmental pattern, rice plant growth is divided into vegetative (germination to panicle initiation), reproductive (panicle initiation to heading), grain filling or ripening and maturity phases (Fig 1.1). At seedling stage, the plant has clearly defined shoot and root parts. Further, tillers are formed on main shoot and a gradual increase in plant height and leaf emergence at regular intervals takes place during vegetative development. At reproductive stage, plant undergoes culm elongation, a decline in tiller number, booting, emergence of the flag leaf, heading or panicle emergence out from sheath and flowering. Grain filling and ripening or maturation stage is characterized by grain growth (Yoshida et al. 2011). The developmental phases have direct and indirect influences on the yield and quality characteristics of rice grains. As the plant undergo different developmental phases, simultaneous level of endogenous 1 Introduction hormones; primary and secondary metabolites including volatile organic compounds (VOCs) are altered. Photosynthates produced in leaves are transported primarily as sucrose to meristem and developing organs such as flowers. During this phase of development, maximum amino acids and proteins are synthesized in plants and transported to developing organs where they are utilized for flower and embryo development. At grain filling or ripening, grain size and weight increases since starch and sugars are translocated from the culms and leaf sheaths. The carbohydrate is stored in the form of starch in grain endosperm. Initially, when florets on the main stem show milky accumulation, starch is white and milky in consistency. It loses moisture and changes into bread dough or firmer during dough grain stages. At maturity physiological process of grain filling ceases and when the moisture content of the grain on the main stem is 25 to 30 % the plant reaches to physiological maturity (Paul and Foyer 2001). 1.3 Rice aroma volatiles Rice aroma volatiles are the volatile organic compounds (VOCs) synthesized in aerial plant parts during growth developmental stages and deposited in mature grains. VOCs are plant’s natural products, lipophilic in nature with low molecular weight and high vapor pressure. Physical properties of these compounds allow them to freely cross cellular membranes and be released into the surrounding environment (Pichersky et al. 2006a). Over the years, more than 1700 VOCs have been identified from 90 different plant families belonging to both angio- and gymnosperms (Knudsen et al. 2006). Biosynthesis of the wide array of different VOCs branches off from only a few primary metabolic pathways. Based on their biosynthetic origin, all VOCs are divided into several classes viz., terpenoids, phenylpropanoids/benzenoids, fatty acid derivatives and amino acid derivatives etc. (Dudareva et al. 2013). As far as basmati rice aroma is concerned, more than 100 volatile compounds have been reported to be responsible for the flavor. They belongs to classes; hydrocarbons (13), acids (14), alcohols (13), aldehydes (16), ketones (14), esters (8) and phenols (5) (Hussain et al. 1987). Volatile compounds 2-acetyl-1-pyrroline (2AP), 2- pyrrolidone, pyridine, 2-methoxyphenol, 1H-indole, p-xylene, and 1-octen-3-ol were the major contributors favoring the consumer acceptability while lipid oxidation products, such as hexanal, acetic acid, and pentanoic acid, led negative influence on consumer acceptability (Lam and Proctor 2003; Monsoor and Proctor 2004). The interactive effects of these volatile compounds collectively influence the aroma character among different scented rice varieties (Yang et al. 2008). Thirteen odor-active compounds; 2AP, hexanal, (E)-2-nonenal, octanal, heptanal, nonanal, 1-octen-3-ol, (E)-2-octenal, (E,E)- 2 Introduction 2,4-nonadienal, 2-heptanone, (E,E)-2,4-decadienal, decanal, and 2-methoxyphenol have been identified as primary compounds responsible for inducing variations in aroma in six scented rice varieties (Yang et al. 2008). Along with 2AP, other major volatiles contributing in the aroma scented rice cultivars are hexanal, nonanal, octanal, trans-2- nonenal, (E,E)-nona-2,4-dienal, heptanal, pentanal, trans-2-octenal, 4 vinyl phenol, 4 vinyl guaicol, 1-octen-3-ol, decanal, guaiacol, 1H-indole and vanillin (Mathure et al. 2011; Mathure et al. 2014). 1.3.1 2-Acetyl-1-pyrroline (2AP) Fig 1.2 Structure of 2-acetyl-1-pyrroline 2-Acetyl-1-pyrroline (2AP) has been identified as the principal aroma compound imparting ‘nutty’ or ‘popcorn-like’ aroma and recognized as a value added character for rice quality determination (Buttery 1982; Buttery et al. 1983; Yang et al. 2010; Mathure et al. 2014). 2-Acetyl-1-pyrroline (IUPAC name 5-acetyl-3,4-dihydro-2H-pyrrole and 1- (3,4-dihydro-2H-pyrrol-5-yl)ethanone, molecular weight 0.111145 kg/mol, density 1090 kg/m3) is 1-pyrroline in which the hydrogen at position 2 is replaced by an acetyl group (Fig 1.2). 2AP has been detected in all aerial plant parts of scented rice (Yoshihashi et al. 2002a; Yoshihashi et al. 2002b; Maraval et al. 2010). 2AP content in scented rice ranges from 0.122 to 0.411 ppm in basmati type and 0.038 to 0.920 ppm in non-basmati scented type (Mathure et al. 2014). 2AP accumulation have been assessed in different parts of aromatic rice like leaves, grains and stems etc under drought, salinity or other environmental stress conditions (Widjaja et al. 1996; Maraval et al. 2010; Poonlaphdecha et al. 2012; Mo et al. 2015). However, accumulation pattern of 2AP throughout developmental stages (seedlings to maturity) is not reported yet in any scented rice cultivars. Therefore, it is interesting to keep a track of aroma volatiles synthesized and translocated across the various developmental stages in scented rice seedlings. 2AP biosynthesis in scented rice is attributed to the non-functionality of betaine aldehyde dehydrogenase (BADH2) gene (Bradbury et al. 2005). The badh2 in aromatic rice has an 8-bp deletion and 3 SNPs in exon 7 resulting in truncated BADH2 protein of 251 residues and loss of its function (Bradbury et al. 2005). The contribution of mutant 3 Introduction badh2 gene in accumulation of 2AP has been confirmed by several researchers at DNA sequence, transcriptional and protein level (Bradbury et al. 2005; Chen et al. 2008; Fitzgerald et al. 2008; Niu et al. 2008; Vanavichit et al. 2008; Fitzgerald et al. 2010; Chen et al. 2012). Biosynthetic pathway of 2AP has been worked out and proline and methylglyoxal (MG) are regarded as immediate key precursors in 2AP accumulation (Fig 1.3) (Yoshihashi et al. 2002a; Thimmaraju et al. 2005; Huang et al. 2008; Wu et al. 2009). Proline accumulation is regulated by Δ1-Pyrolline-5-carboxylic acid synthetase (P5CS) (Yoshihashi et al. 2002a). Fig 1.3 Biosynthetic pathway of 2AP (Wu et al. 2009) (TPI: Triose phosphate isomerase; OAT: Ornithine aminotransferase; P5CS: 1Pyrroline-5 carboxylate synthetase; PRODH: Proline dehydrogenase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; BADH2: Betaine aldehyde dehydrogenase) The expression of P5CS gene was positively co-related with the 2AP accumulation (Huang et al. 2008). Kaikavoosi et al. (2015) demonstrated more than 2 fold enhancements in 2AP after over expression of P5CS gene in Ambemohar-157 and Indrayani rice cultivars. Methylglyoxal is mainly derived from the triose phosphate intermediates (dihydroxyacetone phosphate and glyceraldehyde-3-phosphate) in the glycolytic pathway through fragmentation or elimination of the phosphate group from enediol intermediate at the active site of triose phosphate isomerase (Richard 1991; Phillips and Thornalley 1993). Triose phosphate isomerase (TPI) catalyzes the reversible inter conversion of dihydroxyacetone phosphate and glyceraldehyde 3- phosphate, whereas glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes 4 Introduction the catabolism of glyceraldehyde 3-phosphate. MG formation was enhanced in presences of TPI and decreased activity of GAPDH (Phillips And Thornalley 1993; Beisswenger et al. 2003; Kaur et al. 2015). In rice increased MG concentration leads to the increased TPI which results in decrease of DHAP (Sharma et al. 2012). The presence of badh2 transcripts was reported in all tissues of rice plant except for roots, where it corresponds to accumulation of 2AP. And transcripts were found abundant in young and healthy leaves (Chen et al. 2008). But the relative expression analysis of badh2 and corresponding 2AP accumulation throughout developmental stages in rice plant has not been studied yet. Developmental stage wise analysis of aroma compounds and related gene expression analysis in scented rice cultivars has not been done so far. Therefore, it would be interesting to trace the aroma volatiles and related genes involved in aroma development at various stages of growth in scented rice cultivars. 1.4 Rice varieties under study 1.4.1 Basmati-370 (BA-370) Basmati-370 is premium export quality basmati cultivar developed through pure line selection form traditional basmati land races (Bhattacharjee et al.