PUBLICATIONS Publications

A. Original research papers

1. Vidya Hinge, Hemant Patil & Altafhusain Nadaf. (2016). Comparative Characterization of Aroma Volatiles and Related Gene Expression Analysis at Vegetative and Mature Stages in and Non-Basmati ( L.) Cultivars. Applied Biochemistry and Biotechnology, 2016 Feb; 178(4):619-39. DOI 10.1007/s12010-015-1898-2 Epub 2015 Oct 19. 2. Vidya Hinge, Hemant Patil & Altafhusain Nadaf (2016). Aroma Volatile Analyses and 2AP Characterization at Various Developmental Stages in Basmati and Non- Basmati Scented Rice (Oryza sativa L.) Cultivars. Communicated to Rice.

B. Review article

1. Kantilal Wakte, Rahul Zanan, Vidya Hinge, Kiran Khandagale, Altafhusain Nadaf & Robert Henry (2016).”33 Years of 2-Acetyl-1-Pyrroline, a Principle Basmati Aroma Compound in Scented Rice (Oryza sativa L.): A Status Review” Communicated to Journal of Agricultural and Food Science.

C. Book Chapters

1. Vidya Hinge, Rahul Zanan, Deo Rashmi & Altafhusain Nadaf (2016) “Aroma Volatiles as Biomarkers for Characterizing Rice (Oryza sativa L.) Flavor Types and Their Biosynthesis”. In Science and Technology of Aroma, Flavour and Fragrance in Rice, Apple academic press. 2. Rahul Zanan, Vidya Hinge, Kiran Khandagale & Altafhusain Nadaf (2016) “HS- SPME-GCMS: An Efficient Tool for Qualitative and Quantitative Rice Aroma Analysis”. In Science and Technology of Aroma, Flavour and Fragrance in Rice, Apple academic press.

D. Research Papers presented in Symposia / Seminars / Conference

1. Altafhusain Nadaf, Vidya Hinge & Hemant Patil. (2015). Deciphering aroma accumulation and gene expression analysis at developmental and mature stages in basmati and non‐basmati scented rice (Oryza sativa L.) cultivars. [P200]. Theme: 4. Science Driven Solutions. In Tropical Agriculture conference 2015. 16-18 November 2015, Brisbane, Australia. 2. Vidya Hinge & Altafhusain Nadaf. (2014). Aroma volatile characterization and related gene expression analysis at Vegetative and mature stages of Basmati and Non- basmati scented rice (Oryza Sativa L.) cultivars. In XII conference of society of cytologists and Geneticists and National Symposium on Challenges for

88

Publications

Biologists in 21st century held at Shivaji University, Kolapur on December 22-24, 2014. Awarded Prof. Askell Love Cash Prize. 3. V.R. Hinge, A. B. Nadaf & H. B. Patil. (2014). Aroma volatile and fgr gene (badh2) expression analysis in scented rice cultivrs Ambemohar-157.” P505-96. In 35th Annual Meeting of PTCA (India) & National Symposium on Advances in Plant Molecular Biology & Biotechnology held at IISER, Pune on March 10-12, 2014.

89

Appl Biochem Biotechnol (2016) 178:619–639 DOI 10.1007/s12010-015-1898-2

Comparative Characterization of Aroma Volatiles and Related Gene Expression Analysis at Vegetative and Mature Stages in Basmati and Non-Basmati Rice (Oryza sativa L.) Cultivars

Vidya Hinge 1,2 & Hemant Patil2 & Altafhusain Nadaf1

Received: 4 September 2015 /Accepted: 9 October 2015 / Published online: 19 October 2015 # Springer Science+Business Media New York 2015

Abstract Aroma volatiles in Basmati-370, Ambemohar-157 (non-basmati scented), and IR- 64 (non-scented) rice cultivars were qualitatively and quantitatively analyzed at vegetative and maturity stages to study their differential accumulation using headspace solid-phase microextraction, followed by gas chromatography mass spectrometry (HS-SPME-GCMS) with selected ion monitoring (SIM) approach. In addition, expression analysis of major aroma volatile 2-acetyl-1-pyrroline (2AP)-related genes, betaine aldehyde dehydrogenase 2 (badh2) and Δ1-pyrolline-5-carboxylic acid synthetase (P5CS), were studied by real-time PCR. Max- imum number of volatiles recorded at vegetative (72–58) than at mature stage (54–39). Twenty new compounds (12 in scented and 8 in both) were reported in rice. N-containing aromatic compounds were major distinguishing class separating scented from non-scented. Among quantified 26 volatiles, 14 odor-active compounds distinguished vegetative and mature stage. Limit of detection (LOD) and limit of quantification (LOQ) for 2AP was 0.001 mg/kg of 2AP and 0.01 g of rice, respectively. 2AP accumulation in mature grains was found three times more than in leaves of scented rice. Positive correlation of 2AP with 2-pentylfuran, 6-methyl- 5-hepten-2-one, and (E)-2-nonenal suggests their major role as aroma contributors. The badh2 expression was inversely and P5CS expression was positively correlated with 2AP accumu- lation in scented over non-scented cultivar.

Keywords HS-SPME-GCMS . 2-Acetyl-1-pyrroline . P5CS . badh2 . Growth stages

Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1898-2) contains supplementary material, which is available to authorized users.

* Altafhusain Nadaf [email protected]

1 Department of Botany, Savitribai Phule Pune University, Pune 411007, India 2 Department of Plant Biochemistry and Molecular Biology, Vilasrao Deshmukh College of Agricultural Biotechnology, Latur, VNMKV, Parbhani 413512, India 620 Appl Biochem Biotechnol (2016) 178:619–639

Introduction

The rice aroma volatiles constitute more than 100 volatiles belonging to hydrocarbons (13), acids (14), alcohols (13), aldehydes (16), ketones (14), esters (8), and phenols (5) categories [1]. Among these, 2-acetyl-1-pyrroline (2AP), 2-pyrrolidone, pyridine, 2-methoxyphenol, 1H-indole, p-xylene, and 1-octen-3-ol are 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. The interactive effects of these volatile compounds collectively influence the aroma character among different scented rice varieties [2]. Thirteen odor-active compounds; 2AP, hexanal, (E)-2-nonenal, octanal, heptanal, nonanal, 1-octen-3-ol, (E)-2-octenal, (E,E)-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 [3]. As compared with the conventional methods (steam distillation continuous extraction, steam distillation under reduced pressure, solvent extraction, acid and basic phase solvent extraction, static headspace) for quantification of less stable compound like 2AP and other aroma volatiles in rice, HS-SPME followed by GC-FID or GCMS was proved as very effective, rapid, simple, solvent-free technique. HS-SPME has integrated sampling, extraction, concentration, and sample introduction of volatile compounds into gas chromatography (GC) in a single step resulting in high sample throughput [4, 5]. Maraval et al. [4] developed HS- SPME/SIDA with GC-PCI-MS-MS method for quantification of 2AP. However, it requires 2AP and its stable isotope analog and GCMS with electronic impact (EI) and positive chemical ionization (PCI) mode. In present attempt, HS-SPME followed by GCMS with SIM mode was developed for quantification of 2AP and other aroma volatiles in rice leaves and grains [6–8]. The volatile aroma compounds in general and 2AP in particular are synthesized in aerial parts of rice seedling [9]. The 2AP synthesis starts in the early vegetative stage, and final accumulation takes place in seeds. The volatile compounds are synthesized to its maximum number during early stages of development, i.e., when leaves are young and not fully expanded and further decrease or remains constant thereafter [10]. For the major aroma compound 2AP, proline has been identified as a precursor regulated by the enzyme Δ1-pyrolline-5-carboxylic acid synthetase (P5CS)[9]. The 2AP synthesis in scented rice is attributed to the non- functionality of betaine aldehyde dehydrogenase (badh2) gene [11]. As far as scented rice cultivars are concerned, no information in relation to volatile profile and gene expression analysis during developmental stages is available. Therefore, in the present work, the amount and category of aroma volatiles that are synthesized in the early vegetative stage and in mature grain and relative expression of P5CS and badh2 genes in two Indian scented rice cultivars Basmati-370 (BA-370) and Ambemohar-157 (AM-157) has been studied.

Materials and Methods

Method Development for Extraction of Aroma Volatiles Using HS-SPME and GCMS-SIM

Plant Material

The seeds of AM-157 and BA-370 were procured from Rice Research Station, Vadgaon Maval, Maharashtra, India. The seeds of non-scented rice cultivar IR-64 were procured from Appl Biochem Biotechnol (2016) 178:619–639 621

Balashaeb Sawant Kokan Krishi Vidyapeeth (BSKKV), Dapoli, Maharashtra, India. The seeds were sown in earthen pots in greenhouse under ambient conditions at Vilasrao Deshmukh College of Agricultural Biotechnology (VDCOAB), Latur, Maharashtra, India. Fresh leaves of 1-month-old seedlings of cultivar AM-157 were used for method standardization. Subsequent- ly, for volatile and gene expression analysis, leaves of 1-month-old seedlings (vegetative stage) and seeds (mature stage) of three rice cultivars were used.

Optimization of HS-SPME and GC-MS Conditions

For extraction of aroma volatiles HS-SPME coupled with GCMS was used. The conditions of extraction were optimized as follows. Fresh leaves collected from randomly selected five plants of AM-157 were ground in liquid nitrogen, and leaf powder was immediately trans- ferred to 4-ml screw cap vials (15×45 mm) with PTFE silicon septa (Chromatography Research Supplies, Louiseville, KY, USA). The vials were heated in oven at 150 °C for 1 h prior to use for elimination of unintended volatile compounds. One-centimeter-long fiber coated with Carboxen/polydimethylsiloxane with manual holder was used for extraction of 2AP and other volatiles [5, 6]. Optimization of extraction conditions for maximum 2AP recovery for leaf sample was carried out with respect to the sample weight (0.25 to 1.25 g with an increment of 0.25 g), extraction temperature (40 to 100 °C with an increment of 20 °C), equilibration time (10 to 50 min with an increment of 10 min), and adsorption time (10 to 40 min with an increment of 5 min) in triplicate. Area count was used as a measure of quantity. For volatile analysis in rice seed samples, previously optimized HS-SPME conditions were used [5]. Separation and analysis of 2AP and other volatiles was done using GC-MS (Varian 430-GC and 210-MS, Japan) with a factor four capillary column VF5-MS (30 m×0.25 mm×0.25 μm) (Varian, Inc., Palo Alto, CA) of 5 % diphenyl, 95 % dimethyl polysilosane. The research grade helium (99.999 %) was used as the carrier gas under a constant flow of 28.6 cm/s (1 ml/min). Volatiles were extracted and concentrated by using preconditioned SPME fiber at 250 °C for 30 min attached to the SPME manual holder (57330-U) (Supelco, Bellefonte, PA, USA). The SPME fiber was desorbed for 5 min in GC injector having temperature at 260 °C. Optimum performance of SPME fiber was monitored after every ten extractions with the standard fiber maintained separately. The fiber showing comparable performance was continued in further analysis. Blanks were run following every fourth sample as a control. The GC oven program was optimized for the separation of 2AP from seed and leaf samples. Initially, oven temper- ature was kept at 45 °C for 1 min and ramped to 55 °C at the rate 5 °C/min, then at the rate of 9 °C/min up to 120 °C, further ramped to 240 °C at the rate 15 °C/min with final hold of 1 min. The total GC cycles consisted of 19.22-min runs and 1min restabilization time. Blank run of GCMS was also performed after every six samples to remove traces (if any) from earlier runs in GC column. The injector temperature was 260 °C, and the transfer line was held at 230 °C. The detection was performed by a Saturn III mass spectrometer in the EI mode (ionization energy, 70 eV; source temperature, 180 °C). The MS was operated in the scan mode from m/z 20 to 400.

Identification and Quantification of Volatile Compounds

The volatile compounds were identified by comparing their mass spectra with the mass spectra from MS libraries (NIST 05, WILEY 7.0). When available, the MS identifications were 622 Appl Biochem Biotechnol (2016) 178:619–639 confirmed by comparing the GC retention times of the analytics with pure standards. The linear retention indices (RI) of the compounds were calculated using a series of n-alkanes (C8– C20, Sigma-Aldrich, Germany) injected under the same conditions. When standard chemicals were not available, tentative identification was achieved by matching the mass spectra and RI (Table 1). Analytical grade standards of pentanal (Merk, Hohenbrunn, Germamy), octanal, 1-tetradecene, 2,4,6-trimethylpyridine, (E)-2-nonenal, 1-pentanol, phenylacetaldehyde, nonadecane, heptadecane, pentadecane, decanal, 6-methyl-5-hepten-2- one, 2-pentylfuran, 3-carene, 2-hexyl-1-octanol, benzaldehyde, phenylacetaldehyde, 1H- indole, 2,2,6,trimethylcyclohexanone, 3,7-dimethyl-1-octanol, allylcyclohexane, valencen, longifolene, 1-isopropyl-2-methylbenzene, aromadendrene, ethyl laurate, β-cyclocitral, 2- phenoxyethanol, methyl 2-aminobenzoate, (Z)-3-dodecene (Sigma-Aldrich, Germany), hep- tanal, (E)-3-octen-2-one, 1-octen-3-ol, (E, E)-2,4-nonadienal, benzoic acid (SAFC Supply Solutions, St. Louis, MO, USA),1-hexanol (Supelco Analytical, PA, USA), 2-methoxy-4- vinylphenol, (Alpha Aesar, Karlsruhe, Germany), hexanal, nonanal, vanillin, 2-heptanone, linalool, phenol, (Aldrich, Steinheim, Germany), and 2-methoxyphenol, (E, Z)-2,6- nonadienal (Fluka, Steinheim, Germany) were used for confirmation and quantification. The external standard method with 2,4,6-trimethylpyridine (TMP) as a reference standard was used for quantification 2AP [6, 12]. Quantification was performed by following standard addition approach [5]. The optimized HS-SPME extraction conditions and GCMS analysis with SIM mode method were used for quantification of volatiles in leaves and seed samples. The HS-SPME extraction was performed in triplicate. For estimating LOD and LOQ, lowest amount of volatile compound yielding a response higher than or equal to a signal-to-noise ratio (S/N) of 3 and 10, respectively, were used as measure. The S/N ratio for series of measurements was estimated using the following formula:

ðÞS=N ¼ Xave=SD where Xave is the average of analytical signals (peak areas) for the replicates and SD is the sample standard deviation for the replicates. The relative area was plotted against the relative concentration. The linear model was adjusted by the least-squares method. For each volatile compound, repeatability was measured by six replicates of samples and the relative standard deviation (RSD) was calculated. The average percent recovery was calculated using the following formula:

Ave % R ¼ ðÞÂXave=spike level 100 where Xave is the average concentration of the samples and spike level is the initial spike concentration. The standard curve for leaves and seeds samples were developed separately for 2AP and other 25 volatile compounds. LOD and LOQ values for 2AP were calculated as mentioned above using rice samples ranging from 0.05 to 1.0 g and further expressed as amount of 2AP mg/kg. The linearity, correlation coefficient, and recovery % of 2AP was estimated based on TMP as a standard. The validation range, LOD, LOQ, and correlation coefficient (r2) values for each compound are shown in Table 2.TheSIMmethodwas developed by injection of volatile standards under the full MS scan mode to select the quantification and confirmation ions for each volatile (Table 2). The quantitative results were obtained by interpolation of the relative areas into the calibration curves. Appl Biochem Biotechnol (2016) 178:619–639 623

Table 1 List of volatile compounds identified at vegetative (leaves) and mature stages (seeds) in three rice cultivars (Oryza sativa L.)

Sr. no. RT Name of compound RI AM-157 BA-370 IR-64

ERLSLSLS

1. Alkane 5 6 5 6 5 5 1 6.19 Nonane 901 900 × × × √ ×× 2 8.52 4-Methyldecaneb 1022 1023 × √ ×××× 3 11.13 Dodecane 1196 1200 √√√√√√ 4 12.78 Tetradecanea 1390 1400 √√√√√√ 5 14.79 Pentadecanea 1501 1500 √√√√√√ 6 16.44 Heptadecanea 1698 1700 √√√√√√ 7 17.89 Nonadecanea 1898 1900 √√√√√√ 2. Alkene 4 3 7 4 4 2 1 6.91 Pinene 935 930 × × √ × √ × 2 7.47 Allylcyclohexaneb 965 969 × × √ ××× 3 9.64 (E)-5-Methyl-4-deceneb 1094 1100 √√√√×× 4 10.17 (Z)-3-Undeceneb 1116 1123 × × √√×× 5 11.42 (Z)-3-Dodeceneb 1194 1195 √ × √√√√ 6 13.34 7-Tetradecene 1362 1367 √√√× √ × 7 13.76 1-Tetradecenea 1392 1385 √√√√√√ 3. Ketone 9 7 8 9 7 7 1 6.05 2-Heptanonea 896 889 √√√√√√ 2 7.26 6-Methyl-2-heptanonea 956 957 √√√√√√ 3 7.89 6-Methyl-5-hepten-2-onea 987 988 √√√√√√ 4 8.87 (E)-3-Octen-2-onea 1040 1036 √√√√√√ 5 8.9 2,2,6-Trimethylcyclohexanoneb 1043 1047 √ × √ × √ × 6 9.83 2-Nonanone 1094 1093 √ × √√√× 7 10.68 (E)-5-Ethyl-6-methyl-3-hepten-2-one 1147 1144 √√× √ × √ 8 10.72 4-Cyclopentylidene-2-butanoneb 1151 1158 × √ ×××× 9 10.83 2,6,6-Trimethyl-2-cyclohexene-1,4-dione 1156 1152 √ × √√√√ 10 13.89 6,10-Dimethyl-2-undecanone 1404 1398 × √√√× √ 11 14.74 β-lonone 1495 1493 √ ××√ ×× 4. Aromatic hydrocarbon 3 1 3 0 0 0 1 6.12 p-Xylene 897 907 √ × √ ××× 2 8.01 Toluene 995 1005 × √ ×××× 3 8.54 1-Isopropyl-2-methylbenzeneb 1025 1025 √ × √ ××× 4 8.62 1-Isopropyl-4-methylbenzene 1027 1023 √ × √ ××× 5. Non-aromatic cyclic hydrocarbon 8 1 6 2 6 0 1 8.38 3-Carenea 1014 1015 √ × √ × √ × 2 8.76 L-Limonene 1038 1040 √ ××××× 3 13.14 Azulenea,b 1324 1323 × √ ×××× 413.87β-Elemene 1405 1403 √ × √ × √ × 5 14.03 Isolongifolene 1420 1416 √ × √ × √ × 6 14.19 Longifoleneb 1437 1432 √ × √√√× 714.25β-Caryophyllene 1443 1444 √ × √√√× 624 Appl Biochem Biotechnol (2016) 178:619–639

Table 1 (continued)

Sr. no. RT Name of compound RI AM-157 BA-370 IR-64

ERLSLSLS

8 14.52 Aromadendrenea,b 1447 1439 √ ×××√ × 914.97Valencenb 1472 1477 √ × √ ××× 6. Alcohol 10 11 10 12 11 9 1 4.04 1-Pentanola 803 792 √√√√√√ 2 5.4 (Z)-3-Hexen-1-ola 880 872 √ × √√√× 3 5.65 1-Hexanola 866 860 √√√√√√ 4 7.75 1-Octen-3-ola 972 969 √√√√√√ 5 8.67 1-Hexanol, 2-ethyl- 1030 1033 × √ × √ ×× 6 9.45 1-Octanola 1073 1073 √√√√√√ 7 9.99 Linaloola 1102 1100 √√√√√√ 8 10.24 3,4-Dimethylcyclohexanolb 1123 1126 × × √√√√ 9 10.47 2-Nonen-1-ola 1134 1135 √√√√√√ 10 10.91 Carveol 1161 1188 √√√√√√ 11 11.57 3,7-Dimethyl-1-octanolb 1204 1196 × √ ×××× 12 15.65 2-Hexyl-1-octanola 1600 1591 √√√√√√ 13 17.18 2-Hexadecanol 1798 1774 √√× √√× 7. Aliphatic Aldehyde 14 14 16 12 13 10 1 2.92 Pentanala 710 707 √√√√√√ 2 4.41 Hexanala 816 820 √√√√√√ 3 5.38 (E)-2-Hexenal 869 860 × × √ ××× 4 6.28 Heptanala 904 905 √√√√√√ 5 7.35 (Z)-2-Heptenal 961 960 × √√√× √ 6 8.21 Octanala 1007 1005 √√√√√√ 7 8.44 (E,E)-2,4-Octadienala 1017 1021 √√√√√× 8 9.25 (E)-2-Octenala 1063 1068 √√√√√√ 9 10.06 Nonanala 1108 1104 √√√√√√ 10 10.8 (E,Z)-2,6-Nonadienal 1155 1153 √√√××× 11 10.98 (E)-2-Nonenal 1165 1162 √√√√√√ 12 11.63 Decanala 1210 1204 √√√√√√ 13 11.75 (E,E)-2,4-Nonadienal 1219 1217 √√√× √ × 14 11.94 β-Cyclocitralb 1234 1237 √√√√√√ 15 12.39 (2,6,6-Trimethyl-1-cyclohexen-1-yl)acetaldehydeb 1269 1261 √ × √ × √ × 16 13.58 (E,E)-2,4-Decadienal 1325 1318 √√√√√× 8. Aromatic aldehyde 3 3 3 3 2 3 1 7.51 Benzaldehydea 970 965 √√√√√√ 2 9.04 Phenylacetaldehydea 1052 1048 √√√√√√ 3 13.99 Vanillina 1415 1403 √√√√× √ 9. N-containing aromatic/cyclic 3 2 3 2 0 0 1 6.66 2-Acetyl-1-pyrrolinea 925 930 √√√√×× 2 9.3 1-(1H-pyrrol-2-yl)ethanone 1043 1035 √√√√×× 3 12.86 1H-indolea 1306 1304 √ ××××× 4 13.37 Methyl 2-aminobenzoateb 1365 1372 × × √ ××× Appl Biochem Biotechnol (2016) 178:619–639 625

Table 1 (continued)

Sr. no. RT Name of compound RI AM-157 BA-370 IR-64

ERLSLSLS

10.Ester 605151 1 8.13 Ethyl hexanoate 989 984 √ × √ × √ × 2 9.91 Ethyl heptanoate 1088 1083 √ × √ × √ × 3 11.48 Ethyl octanoate 1193 1183 √ × √ × √ × 4 11.54 Methylsalicylate 1201 1206 √ × √√√√ 5 12.71 Acetic acid, 1285 1277 √ ××××× 1,7,7-trimethyl-bicyclo(2,2,1)hept-2-yl, esterb 6 15.62 Ethyl laurateb 1586 1581 √ × √ × √ × 11. Phenol-containing 3 2 4 2 3 1 1 7.65 Phenola 981 980 √ × √ × √ × 2 9.79 2-Methoxyphenola 1087 1090 √√√√√√ 3 11.8 2-Phenoxyethanolb 1224 1226 × × √ ××× 4 13.01 2-Methoxy-4-vinylphenola 1320 1313 √√√√√× 12.Carboxylicacid 101010 1 11.19 Benzoic acida 1179 1174 √ × √ × √ × 13.Furans 111111 1 7.99 2-Pentylfurana 992 996 √√√√√√ Total 705172545839

RT retention time, RI retention index, E experimental RI value, R reference RI value, L leaves, S seeds, × absence of compound, √ presence of compound a Compounds confirmed with authentic standard b Compounds first time reported in rice

Estimation of Odor-Active Values

Odor-active values were estimated by taking a ratio of the concentration of compound and the reported odor threshold value (Supplementary-1) of that compound [13, 14].

Statistical Analysis

Descriptive analysis of volatiles was performed to determine the mean, standard deviation, standard error, and % coefficient of variation (CV %). Duncan’s multiple range test was performed on the mean values for each volatile compound in seeds and leaves of three rice cultivars, to identify compounds exhibiting significant variation. Principle component analysis (PCA) was performed to study variations in the composition of 14 odor-active volatiles among seeds and leaves of rice cultivars under study using XLSTAT software (version 2014) (Addinsoft™).

RNA Extraction and cDNA Synthesis

RNA was extracted from the leaves of rice cultivars under study using TRIzol reagent following the manufacturer’s protocol (Invitrogen, Carlsbad, CA) and treated with DNase 626 Table 2 Validation range, LOD, LOQ, and recovery % of volatiles in leaves and mature grains of three rice cultivars (Oryza sativa L.)

Sr. no. Compound Ions scanned Validity range LOD LOQ Recovery Seed samples Leaf samples

(m/z) (mg/kg) (mg/kg) %±RSD Linear equation R2 Linear equation R2

1 Pentanal 58+57+45 0.002–0.6 0.001 0.002 89.5±5.6 y=4.576x+354.2 0.982 y=17.39x+111.5 0.979 2 1-Pentanol 42+55+41 0.05–1.0 0.005 0.01 90±8.7 y=17.48x+1688 0.988 y=137.3x+192.8 0.995 3 Hexanal 44+56+41 0.02–4.0 0.001 0.008 92.2±3.7 y=25.10x+30381 0.978 y=12.69x+5346 0.985 4 (Z)-3-Hexen-1-ol 67+41+82 0.005–0.4 0.001 0.005 84±4.2 NE NE y=332.5x+12751 0.991 5 1-Hexanol 56+43+41 0.07–0.6 0.01 0.07 79.6±9.3 y=91.70x+1233 0.995 y=70.64x+2490 0.991 6 2-Heptanone 43+58+71 0.01–0.8 0.001 0.009 87.6±6.4 y=14.99x+1784 0.983 y=161.9x+1031 0.989 7 Heptanal 70+41+55 0.03–0.8 0.01 0.03 92.3±7.3 y=35.05x+1987 0.98 y=59.52x+6346 0.976 8 2AP 111+83+82 0.007–1.0 0.001 0.005 82.7±4.1 y=34.56x+1611 0.998 y=44.32x+1176 0.995 9 Benzaldehydes 77+106+105 0.01–0.6 0.005 0.01 91.6±3.2 y=45.22x+1354 0.989 y=61.15x+6425 0.993 10 1-Octen-3-ol 57+43+72 0.01–0.6 0.005 0.01 89.3±13.2 y=24.51x+2871 0.977 y=105.4x+2029 0.994 11 6-Methyl-5-hepten-2-one 69+55+108 0.009–1.0 0.005 0.008 87.9±6.8 y=95.11x+3428 0.991 y=198.9x+1957 0.991

12 2-pentylfuran 81+82+138 0.01–0.4 0.001 0.007 88.3±7.2 y=199.1x+2881 0.994 y=196.3x+982.2 0.997 178:619 (2016) Biotechnol Biochem Appl 13 Ethyl hexanoate 88+43+99 0.01–0.8 0.005 0.01 75.3±8.3 NE NE y=275.6x+972.4 0.998 14 Octanal 43+56+84 0.05–0.8 0.001 0.02 86.3±9.1 y=6.597x+859 0.986 y=64.4x+1726 0.995 15 3-carene 93+91+79 0.01–0.6 0.005 0.01 85.4±9.2 NE NE y=108.1x+961.3 0.997 16 (E,E)-2,4-Octadienal 81+67+124 0.009–0.6 0.005 0.008 93.6±2.4 y=86.9x+1498 0.976 y=67.5x+614.28 0.988 17 (E)-3-Octen-2-one 55+43+111 0.05–1 0.005 0.02 78.4±5.6 y=29.58x+2788 0.989 y=13.26x+1298 0.986 18 Phenylacetaldehyde 91+92+120 0.01–0.8 0.008 0.01 87.2±8.3 y=76.64x+1104 0.995 y=25.54x+2700 0.989 19 (E)-2-Octenal 41+55+29 0.04–0.8 0.01 0.03 77.4±8.3 y=48.18x+3326 0.988 y=48.18x+3326 0.986 20 1-Octanol 56+55+41 0.05–0.8 0.01 0.04 89.5±3.2 y=169.3 x+242 0.995 y=218.3x+692.8 0.983 21 Linalool 71+93+55 0.01–1 0.001 0.007 87.3±3.8 y=215.7x+2065 0.996 y=146.1x+663 0.995 22 Nonanal 57+41+43 0.05–0.8 0.001 0.008 90.3±8.5 y=363.4x+12838 0.995 y=251.0x+16676 0.987 –

23 (E)-2-Nonenal 41+43+70 0.005 0.8 0.001 0.004 88.1±4.4 y=102.6x+513 0.991 y=171.4x+732.0 0.996 – 639 24 Decanal 43+57+55 0.01–0.8 0.005 0.009 84.6±7.1 y=348.8x+2539 0.996 y=505.3x+3894 0.995 plBohmBoeho 21)178:619 (2016) Biotechnol Biochem Appl Table 2 (continued)

Sr. no. Compound Ions scanned Validity range LOD LOQ Recovery Seed samples Leaf samples

(m/z) (mg/kg) (mg/kg) %±RSD Linear equation R2 Linear equation R2

25 Pentadecane 57+85+71 0.01–0.8 0.006 0.01 91.3±6.9 y=84.88x+1634 0.994 y=131.9x+5098 0.997 26 Heptadecane 57+43+71 0.01–1 0.005 0.008 92.5±5.2 y=27.9x+2213 0.979 y=34.01x+10110 0.991

NE not evaluated – 639 627 628 Appl Biochem Biotechnol (2016) 178:619–639

(Fermentas, Germany). RNA was extracted from ~100 mg seeds using extraction buffer

(100 mM Tris-HCl, 150 mM LiCl2, 50 mM EDTA, 1.5 % SDS, 1.5 % β-mercaptoethanol), and phenol/chloroform extraction followed by TRIzol reagent. RNA concentration in samples was determined using a Nanodrop ND-1000 (Nanodrop Technologies, Wilmington, DE, USA). First-strand cDNA synthesis was done using ~1 μgtotalRNAfromeachsampleand RevertAid first strand cDNA synthesis kit (Thermo Scientific). cDNA was then aliquoted and stored at −80 °C.

Quantitative Real-Time PCR (qRT-PCR)

For qRT-PCR, primers for badh2(E6Badh2F1-5′-TGTGCTAAACATAGTGACTGGA-3, E7 Badh2R1-5′ -CTTAACCATAGGAGCAGCT) and P5CS ( P5CS F-5′ - GAAGTGGTAATGGTCTTCTC-3′; P5CS R-5′-AGCAAATCTGCGATCTCATC-3′) were de- signed based on badh2andP5CS sequences available in NCBI gene bank, EF1α gene primers (5′-TTTCACTCTTGGTGTGAAGCAGAT-3′;5′-GACTTCCTTCACGATTTCATCGTAA-3′) were used as control All the three genes were amplified from cDNA samples of the leaves of BA-370 and cloned into pJET cloning vector (Life Technologies, Carlsbad, CA, USA). The purified plasmid containing gene of interest was used as standard DNA for developing standard curve [30]. The real-time quantitative PCR was carried out in a total volume of 25 μl containing 12.5 μlVeriQuest™ Fast SYBR® Green qPCR Master Mix with ROX (2X), 1 μlofeachprimer

(10 pmol/μl), 1.0 μl of cDNA, and 9.5 μlDDH2O. Thermal cycling consisted of a hold at 94 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The PCR reactions were performed on the Mastercycler® ep realplex PCR system (Eppendorf, Hamburg, Germany) in triplicate. Expression analysis in terms of copy number of Badh2 (R2=0.996, e= 1.16) and P5CS (R2=0.983, e=1.14) were done in seed and leaf samples of all rice cultivars and EF1aplha (R2=0.995, e=1.11) as a control gene. The transcript abundance was expressed as copy number/10 ng of cDNA.

Results and Discussion

HS-SPME Parameters for Extraction of Volatiles at Vegetative Stage

The extraction process in HS-SPME is influenced by various parameters such as volatility of the analyte, type of matrix (viscosity, lipophilicity, diffusion constant), extraction conditions (incubation time, extraction time, temperature, fiber chemistry, agitation, sample volume), and the concentration of other constituents present in the sample [15]. Therefore, in the present study, sample weight, extraction temperatures, and extraction time were optimized for maxi- mum recovery of 2AP. Maximum 2AP peak area was recorded for 0.5 g leaf sample (Fig. 1a). Therefore, 0.5 g sample weight was considered as optimum weight and used for further analysis. It was observed that, the 2AP peak area was significantly increases with increase in temperature from 40 to 80 °C. The maximum 2AP peak area was observed at 80 °C (Fig. 1b). Similar observations were recorded by Mathure et al. [5], Grimm et al. [7], and Maraval et al. [4]. Maximum 2AP abundance was recorded at 20-min preincubation time and 20-min adsorption time (Fig. 1c). Fifteen to 20 min of preincubation and 30 to 35 min of adsorption time was reported earlier for maximum recovery of 2AP [5, 7]. The longer extraction time reduces the adsorption of 2AP over other volatiles. Thus, extraction time of Appl Biochem Biotechnol (2016) 178:619–639 629

Fig. 1 Recovery of 2AP under varied sample weight (a), extraction temperature (b), and adsorption and preincubation time (c)

40 min (20-min preincubation followed by 20 min adsorption) was determined as an optimum extraction time for maximum recovery of 2AP.

Method Validation for Quantification of 2AP

HS-SPME coupled with GC-FID or GCMS analysis method was proved as effective technique for quantification of 2AP in rice by several researchers [4–6, 14, 16]. HS-SPME followed by GCMS with SIM mode was developed for quantification of 2AP and other aroma volatile in leaves and grains of rice. The method was validated with respect to linearity, range, sensitivity (LOD and LOQ), and precision. A non-fragrant rice variety IR-64 was subjected to analysis by HS-SPME GCMS with SIM mode using the optimized conditions. It yielded a volatile component profile that closely matched that of the fragrant rice AM-157 and BA-370 except for the absence of 2AP. This revealed that no additional 2AP was formed by heating the rice samples at the optimum conditions. The effective linear concentration range for 2AP was observed from 7 to 1000 ng of 2AP/g of rice sample. Sensitivity is reflected by the LOD and LOQ. The LOD was found 0.001 mg/kg of 2AP with RSD of 3.1 %, and LOQ determined was 0.01 g of rice or 0.005 mg/kg of 2AP with RSD of 4.7 % for both leaves and grain samples. The recovery of 2AP from matrix was found 82.78 % using HS-SPME GCMS (SIM) mode. This shows that the recovery of 2AP was lower than those of HS-SPME GCMS-PCI-MS/MS, but it was higher as compared to HS-GC-NPD and HS-SPME GC-FID detector [4, 17]. The validation data confirmed higher sensitivity and usefulness of this method over HS-SPME GC- FID and HS-GC-NPD methods [17]. The recovery % for other volatiles was ranging from 75.3 630 Appl Biochem Biotechnol (2016) 178:619–639 to 92. 2 % (Table 2). For screening of large number of germplasm for 2AP content during breeding program, the developed method can be utilized. As it is simple, accurate, and sensitive, it does not require stable isotope of 2AP and can be done in single MS within an hour.

Qualitative Analysis of Volatile Compounds

The volatile compounds identified in leaves at vegetative stages and at mature stages in three rice cultivars along with their RI are depicted in Table 1. It was observed that, in general, scented rice cultivars recorded more number of compounds at both the stages than non-scented rice cultivar. Among the two stages, scented rice cultivars contained significantly more number of compounds at vegetative stages than at mature stages. In vegetative stage, 72 volatile compounds were detected in BA-370, 70 in AM-157, and 58 in IR-64. In seeds, also the same trend was followed, viz., 54 volatile compounds were recorded in BA-370 and 51 in AM-157 against 39 in IR-64. Among the identified compounds, 20 (12 in scented and 8 in both) were detected for the first time in rice cultivars (Table 1). The volatile compounds were classified based on their chemical nature (Fig. 2).The scented group represented total 13 classes of volatile compounds, and non-scented represented 10 classes in vegetative stage. Among these classes, aliphatic aldehydes and alcohols contributed their maximum share in all three rice cultivars in both the stages. N-containing aromatic/cyclic compounds were detected in scented ones and were absent in non-scented rice cultivars at vegetative and mature stages, indicating their prominent role in the aroma of scented rice cultivars. Seeds of non-scented rice cultivars were also deficient in aromatic hydrocarbon compounds. The occurrence of category-wise volatiles in both the stages is discussed below.

Alkane, Alkene, Ketone, and Hydrocarbons

In all three cultivars, total 7 alkanes were identified in seeds as well as leaves. The percent range of alkanes in seeds was more (11–13 %) than in leaves (7–9%)(Fig.2a–e,Table1). Alkane 4-methyldecane in leaves of AM-157 was detected for the first time in rice. Alkene contributed 6 to 7 % share in seeds and 6 to 10 % share in leaves of AM-157and BA-370, respectively. In non-scented IR-64, 5 and 7 % alkenes were present in seed and leaves (Fig. 2e, f). (E)-5-Methyl-4-decene (in seed and leaves of scented), (Z)-3-undecene (in seeds and leaves of BA-370), (Z)-3-dodecene (in seeds of BA-370 and IR-64 and in leaves of three rice cultivars), and allylcyclohexane (in leaves of BA-370) are newly reported in rice (Table 1). Bryant and McClung [18] recorded up to 15 alkanes in the grains of . As far as aroma character is considered, alkanes have no contribution [13]. In the present study, maximum content of alkane in leaves of non-scented IR-64 confirmed the same. The percent ketone content was found less in vegetative (11–13 %) stage than in mature stage (14–18 %) in all three rice cultivars. Among 11 ketones, 2,2,6-trimethylcyclohexanone (in leaves of three rice cultivars) and 4-cyclopentylidene-2-butanone (in seeds of AM-157) were newly identified in rice. Four ketones, 2-heptanone, 6-methyl-2-heptanone, 6-methyl-5- hepten-2-one, and (E)-3-octen-2-one, were present in seeds and leaves of all three rice cultivars. Similar compounds were detected previously in freshly harvested aromatic rice. They give banana type, fruity, odor to rice [18]. Aromatic hydrocarbons were found more in vegetative stage (4 %) than in mature stage (2 %). Among four aromatic hydrocarbons, 1-isopropyl-2-methylbenzene in the leaves of both Appl Biochem Biotechnol (2016) 178:619–639 631

Fig. 2 Total volatile compounds detected in vegetative and mature stages of three rice cultivars (Oryza sativa L.). a, b Volatile compounds in AM-157. c, d Volatile compounds in BA-370. e, f Volatile compounds in IR-64 scented cultivars was reported for the first time in rice. In the seeds of scented rice cultivars, 0– 2 % aromatic hydrocarbons were present. Seeds of scented BA-370 and non-scented rice IR- 64 were totally deficient in aromatic hydrocarbon compounds (Fig. 2d–f). Non-aromatic cyclic hydrocarbon compounds were significantly more at vegetative stages (9–12 %) than in mature stages (0–4%)(Table1,Fig.2). Among these compounds, azulene (in seeds of AM-157), longifolene (in seeds of BA-370 and in the leaves of three rice cultivars), valencen (in leaves of AM-157 and IR-64), and aromadendrene (in the seeds of both scented cultivars) were reported for first time in rice. These compounds are cyclic sequiterpenes and occur in many plants having typically high flavour thresholds and likely to have little contribution in the odor of rice. However, some of them are present in high concentrations, particularly 3-carene, toluene, and azulene and may play roles in the overall flavor of rice. Many hydrocarbons (1-isopropyl- 632 Appl Biochem Biotechnol (2016) 178:619–639

2-methylbenzene, 1-isopropyl-4-methylbenzene, 3-carene, β-elemene, isolongifolene, etc.) among this class were terpene and sesquiterpene formed via mevalonic acid (MVA) and methylerithriotol 4-phosphate (MEP) pathway in plants. Terpenes play an important role as fragrances in perfumery and as constituents of flavors for spicing foods [19]. So, their presence in scented rice might be associated with the aroma.

Aldehydes, Alcohol, and Phenols

Among 19 aldehydes, 16 were aliphatic and 3 were aromatic in nature. The number of aldehyde compounds in vegetative and mature stages of scented rice was not significantly different. In volatile profile of seeds, aliphatic aldehydes contributed 27 % in AM-157, 26 % in IR-64, and 23 % in BA-370. Aliphatic aldehydes in leaves was 20 % (AM-157) and 23 % (BA-370 and IR-64). (E)-2-Hexenal was detected only in BA-370 leaves. Aliphatic aldehyde (E,E)-2,4-octadienal and (E,E)-2,4-decadienal were not detected in the seeds of IR-64. (E,E)- 2,4-Nonadienal was detected only in the seeds of AM-157 and in the leaves of all three rice cultivars. (E,Z)-2,6-Nonadienal was detected in the seeds of AM-157 and leaves of AM-157 and BA-370 only. The β-cyclocitral and (2,6,6-trimethyl-1-cyclohexen-1-yl)acetaldehyde were newly reported in rice (Table 1). Aromatic aldehydes contributed 4–5 % in seeds and leaves of scented rice. Benzaldehyde and phenylacetaldehyde were detected in both rice cultivars (Table 1). Vanillin was detected in the seed of three rice cultivars and leaves of scented rice only indicating its role in rice aroma. Similar results were reported by several researchers previously [14, 20] The aliphatic aldehyde compounds are associated with lipid breakdown products and can be linked with flavors like grassy, fatty, and soapy [21, 22]. The alcoholic compounds were found to be more in mature seeds (24 %) than in vegetative stage (19 %) in scented and non-scented rice cultivars (Fig. 2). Besides leaves leaf alcohol ((Z)-3-hexen-1-ol) was detected only in seed of BA-370. Aliphatic alcohol 3,7-dimethyl-1- octanol was detected only in seeds of AM-157 and newly identified in rice. Alcohol 3,4- dimethylcyclohexanol was detected in seed and leaves of BA-370 and IR-64 and first time reported in rice. Among phenol-containing compounds, phenol was detected only in the leaves of three rice cultivars. Newly reported 2-phenoxyethanol was present only in seed of BA-370. 2-Methoxyphenol was detected in three rice cultivars at both the stages. 2-Methoxy-4- vinylphenol contributing in aroma was not detected in the seeds of non-scented IR-64 (Table 1). Alcohols are generally formed by the decomposition of secondary hydroperoxides of fatty acids [21]. Alcohols and phenols can give sweet, floral, or fruity flavor to rice. Bryant and McClung [18] previously detected 11 to 17 alcohols in rice cultivars Wells and JES. In the present study, relatively more number of alcohols were detected in BA-370 at maturity.

N-Containing Aromatic/Cyclic Compounds

N-containing aromatic and cyclic compounds were representing 4 % of total volatilomics in seeds and leaves of scented rice and were absent in non-scented rice suggesting their major role as aroma volatiles in scented rice cultivars (Fig. 2). Besides, the 2AP, N-containing aromatic compound 1-(1H-pyrrol-2-yl) ethanone was also identified in seeds and leaves of scented rice cultivar (Table 1). 1H-indole was detected only in leaves of AM-157 while methyl 2-aminobenzoate was identified only in leaves of BA-370 and first time reported in rice (Table 1). 1H-indole is reported to contribute in the aroma of white glutanous rice [13]and cooked [3]. In the present study, it was not detected in the seeds of scented cultivars. Appl Biochem Biotechnol (2016) 178:619–639 633

Ester, Carboxylic Acid, and Furans

Maximum esters were detected in leaves (7 to 9 %) and a few in the seeds (2–3%)ofallthree cultivars. Four esters (ethyl hexanoate, ethyl heptanoate, ethyl octanoate, and ethyl laurate) and carboxylic acid (benzoic acid) were detected only in the leaves of all cultivars (Table 1). Ester acetic acid, 1,7,7-trimethyl-bicyclo(2,2,1)hept-2-yl, ester (in the leaves of AM-157) and ethyl laurate (in leaves of three rice cultivars) were first time identified in rice (Table 1). 2- Pentylfuran associated with floral, fruity, and nutty flavor was detected in the seeds and leaves of all three rice cultivars.

Quantitative Analysis of Aroma Volatiles

The results of quantitative analysis of 26 major volatile compounds are displayed in Table 3. The 2AP content in seeds of scented rice was found to be 0.662 mg/kg in AM-157 and 0.451 mg/kg in BA-370. 2AP was not detected in the seeds and leaves of IR-64. In vegetative stage, 2AP content was three times less than in seeds (in AM-157 and BA- 370). 2AP content in seeds of AM-157 was significantly higher than in the seeds of BA- 370 and leaves of both cultivars but 2AP content in the leaves of AM-157 was not significantly different than the leaves of BA-370. The 2AP content was found varying from cultivars, environment conditions, genotypes, planting sites and planting time, harvesting time, levels of fertilizer, in the aromatic rice plant [9, 14]. Poonlaphdecha et al. [23] reported that 2AP content in grains was ranging from 0.592 ppm and increased in leaves upto 4.4 mg/kg in Aychade rice cultivars. Yoshihasi and others [9]recorded 0.407 ppm 2AP in shoot of rice cultivar Khao Dawk Mali 105. In Kyeema rice cultivars, 2AP content was found increased three times in mature grains (295 ppb) than in leaves at tillering stage (93 ppb) [24]. The 2AP content in seeds of AM-157 and BA-370 was found similar to the previous report [14]. In both cultivars, we found three times more 2AP accumulation in grains than in leaves. The maximum accumulation of 2AP in grain might be associated with the bound form of 2AP in rice grain. The synthesized 2AP in rice plant was bound with starch granule complex in developing grains and accumulated into seeds. This bound form of 2AP may be released during cooking or boiling process where rice starch collapses and 2AP is released [25]. 2-Pentylfuran and 6-methyl-5-hepten-2-one followed similar trend of accumulation as that of 2AP across the stages. The contents of aldehydes, alcohols, alkanes, and 2 ketone com- pounds 2-heptanone and (E)-3-octen-2-one were significantly more in vegetative stage than in the mature seeds of scented rice. In the seeds of scented rice cultivars, aldehydes, alcohols, and alkane and ketone compounds accumulate significantly in higher quantities than in seeds of IR-64. 1-Pentanol, 1-hexanol, and 1-octanol contents in mature seeds (0.005–0.064 ppm) are in agreement with the previous reports [3]. 1-Octen-3-ol content in seeds of BA-370 is in agreement with the previous reports [14]. The leaves of IR-64 contained significantly higher amount of pentanal, hexanal, octanal, nonanal, and (Z)-3-hexen-1-olthan the leaves of scented rice cultivars. The leaves of AM-157 contained significantly highest amount of (E)-2-octenal, decanal, phenylacetaldehyde, (E)-3- octen-2-one, heptadecane, ethyl hexanoate, and 3-carene than the seeds and leaves of BA-370 and IR-64. The aldehyde contents (0.262–0.006 ppm) in mature seeds of AM-157 and BA-370 are in agreement with previous reports [14]. 634 Appl Biochem Biotechnol (2016) 178:619–639

Table 3 Duncan’s multivariate analysis for 26 volatiles in seeds and leaves of three rice ultivars (Oryza sativa L.)

Volatile compound Mean content of volatile in mg/kg (n=3) CV %

AS BS IS AL BL IL

2-Acetyl-1-pyrroline 0.662 a 0.451 b ND 0.198 c 0.191 c ND 98.08 Aldehydes Pentanal 0.096 d 0.103 d 0.039 e 0.132 c 0.240 b 0.295 a 60.30 Hexanal 0.105 d 0.208 d 0.212 e 1.264 c 1.144 b 1.525 a 79.98 Heptanal 0.157 d 0.191 d 0.062 e 1.687 b 1.726 a 1.359 c 87.89 Octanal 0.247 c 0.260 c 0.110 d 0.378 a 0.297 b 0.383 a 34.13 (E,E)-2,4-Octadienal 0.009 d 0.007 d ND e 0.064 c 0.272 b 0.465 a 132.05 (E)-2-Octenal 0.013 e 0.025 d 0.005 e 0.180 a 0.153 b 0.122 c 87.99 Nonanal 0.055 c 0.065 c 0.032 d 0.399 a 0.323 b 0.413 a 79.95 (E)-2-Nonenal 0.008 c 0.010 b 0.005 d 0.007 c 0.014 a 0.006 c 37.96 Decanal 0.014 d 0.013 d 0.012 d 0.036 a 0.031 b 0.025 c 45.08 Benzaldehyde 0.025 d 0.045 c 0.018 d 0.080 b 0.175 a 0.085 b 76.57 Phenylacetaldehyde 0.027 d 0.022 d 0.006 e 0.129 a 0.113 b 0.085 c 77.22 Alcohols 1-Pentanol 0.012 b 0.013 b 0.005 c 0.020 a 0.019 a 0.018 a 38.82 (Z)-3-Hexen-1-ol ND ND ND 0.341 c 0.382 b 0.421 a 103.78 1-Hexanol 0.016 de 0.022 d 0.012 e 0.144 a 0.130 b 0.097 c 81.46 1-Octen-3-ol 0.086 d 0.158 a 0.053 f 0.076 e 0.107 c 0.147 b 37.45 1-Octanol 0.056 e 0.064 d 0.042 f 0.079 a 0.085 b 0.076 c 22.74 Linalool 0.018 d 0.012 de 0.005 e 0.148 a 0.108 b 0.044 c 99.21 Ketone 2-Heptanone 0.021 c 0.013 d 0.011 d 0.038 b 0.058 a 0.012 d 69.94 6-Methyl-5-hepten-2-one 0.045 b 0.063 a 0.019 d 0.019 d 0.027 c 0.016 d 56.45 (E)-3-Octen-2-one 0.005 d 0.011 d 0.002 d 0.185 a 0.153 b 0.122 c 98.55 Alkane Pentadecane 0.025 d 0.017 e 0.014 e 0.148 c 0.174 b 0.283 a 94.12 Heptadecane 0.077 c 0.039 d 0.012 e 0.475 a 0.415 b 0.401 b 85.43 Furan 2-Pentylfuran 0.040 a 0.029 b 0.011 d 0.014 d 0.024 c 0.012 d 42.89 Ester Ethyl hexanoate ND ND ND 0.019 a 0.011 b 0.010 b 113.75 Non-aromatic cyclic hydrocarbon 3-carene ND ND ND 0.025 a ND 0.009 b 167.71

Difference in lowercase letter indicates significant difference among the mean value presented, a - most significant ND not detected, AS and AL seeds and leaves of AM-157, BS and BL seeds and leaves of BA-370, IS and IL seeds and leaves of IR-64

Contribution of Volatile Compounds in Rice Aroma

Among the several volatiles detected in three rice cultivars, 26 volatile compounds were selected on the basis of their role in aroma contribution and quantified in both the stages (Table 3). Among them, 14 compounds exceeded odor-active value (OAV) 1 in both stages (Supplementary-1). 2AP Appl Biochem Biotechnol (2016) 178:619–639 635 recorded maximum OAV (6617.26) in seeds of AM-157. Thus, 2AP was found to be the major contributor in aroma at vegetative and mature stages of AM-157 and BA-370 cultivars. The odor of 2AP is described as pandan, cooked rice, sweet, pleasant, and popcorn-like. The study also confirmed the role of 2AP as a major aroma volatile in rice as reported by several researchers [2, 3, 20]. Along with 2AP, 1-octanol (3186.848), 1-octen-3-ol (157.79), (E)-3-octen-2-one (157.09), and aliphatic aldehydes octanal (371.31), (E)-2-nonenal (126.36), nonanal (64.75), heptanal (63.66), hexanal (46.19), decanal (6.8), and (E)-2-octenal were major contributors in the aroma of seeds of scented rice cultivars. Aldehydes were also reported previously as contributors into rice aroma because of its low odor threshold value [3]. Lam and Proctor [21] concluded that, based on OAV, hexanal (grassy flavor) probably contributed more to flavor in milled rice in early storage. Aliphatic aldehydes (E)-2-nonenal, octanal, and nonanal were considered to likely contribute to the aroma due to their lowest odor threshold [22]. 2-Pentylfuran exceeded the odor threshold value in seed of scented rice only. In vegetative stage, 1-octanol (4238.60), (E)-3-octen-2-one (2285.84), heptanal (575.44), octanal (547.55), nonanal (398.89), hexanal (338.83), 1-octen-3-ol (106.71), (E)- 2-nonenal (174.64), (E)-2-octenal (59), phenylacetaldehyde (32), and pentanal (24.58) were con- tributing for flavor. Phenylacetaldehyde and (E)-2-octenal, recorded OAV more than their reported values in seeds of scented rice only and in leaves of all cultivars. Pentanal, 1- pentanol, 1-hexanol, 2-heptanone, benzaldehyde, and 6-methyl-5-hepten-2-one showed OAV less than its odor threshold and thus did not contribute toward aroma in AM-157 and BA- 370. Pentanal and 1-hexanol also reported to contribute in aroma in rice [2, 14], but in this study, their contribution toward aroma in the seeds of AM-157 and BA-370 was not recorded. The odor threshold values of remaining compounds were not available; therefore, their contributions in aroma were not determined.

Principle Component Analysis

Principle component analysis based PCA plot of 14 OAV compounds in vegetative and mature stages in three rice cultivars shows clear-cut segregation of scented and non-scented rice category at mature stage (Fig. 3). Among the two stages of development, seeds were separated at negative side from the vegetative stage of three rice cultivars in PC1, suggesting differential accumulation of aroma volatiles in seed and leaves in the scented and non-scented rice cultivars. Yang and others [3] found that 13 odor-active compounds, 2-AP, hexanal, (E)-2- nonenal, octanal, heptanal, nonanal, 1-octen-3-ol, (E)-2-octenal, (E,E)-2,4-nonadienal, 2- heptanone, (E,E)-2,4-decadienal, decanal, and 2-methoxyphenol, could segregate six different flavor types of cooked rice. Mahattanatawee and Rouseff [26] distinguished Jasmine, Basmati, and rice cultivars based on sulfur volatile pattern. The study showed that AM-157 and BA-370 flavors appeared to be closely related with respect to selected 14 odor-active compounds.

Correlation Among 26 Major Volatiles

Correlation analysis of 26 compounds revealed 86 significant correlations in leaves and seeds of three rice cultivars (Supplementary-2). 2AP was positively correlated with 2-pentylfuran and 6-methyl-5-hepten-2-one. Pentanal correlated positively with hexanal, (Z)-3-hexen-1-ol, (E,E)-2,4-octadienal, 1-octanol, nonanal, and pentadecane. Hexanal, (Z)-3-hexen-1-ol, 1- hexanol, and heptanal correlated positively with (E)-3-octen-2-one, pentadecane, heptadecane, ethyl hexanoate, and all aldehydes except (E)-2-nonenal. 2-Heptanone was correlated 636 Appl Biochem Biotechnol (2016) 178:619–639

Fig. 3 PCA plot of 14 OAV compounds in vegetative and mature stages in three rice cultivars (Oryza sativa L.). AS and AL seeds and leaves of AM-157, BS and BL seeds and leaves of BA-370, IS and IL seeds and leaves of IR-64

positively with benzaldehyde, linalool, and (E)-2-nonenal. There was no significant correlation observed with 1-octen-3-ol and any other volatile in both stages. Major aroma volatiles alcohols and aldehydes were representing significant correlations as these are the products derived from either oxidation or degradation of lipids [22]. The similar results were reported by Mathure and others [14] in seeds of various scented cultivars of Indian scented rice except 1-octen-3-ol. The correlation analysis among major aroma volatiles suggests that along with 2AP, 2-pentylfuran, 6-methyl-5-hepten-2-one, and aliphatic aldehydes could be selected as target compounds for metabolic engineering to improve the rice aroma contents.

Expression Analysis of badh2 and P5CS Genes

The expression analysis of badh2 and P5CS genes at vegetative and mature stages in three rice cultivars is presented in Fig. 4.Thebadh2 transcript of scented rice cultivars was found to be 9 to 20 fold less than non-scented cultivar IR-64 in seeds and leaves. The badh2 expression at

Fig. 4 badh2 and P5CS transcription levels in leaves and seeds of three rice cultivars (Oryza sativa L.). AS and AL seed and leaves of AM-157, BS and BL seed and leaves of BA-370, IS and IL seed and leaves of IR-64) Appl Biochem Biotechnol (2016) 178:619–639 637 transcriptional level was found significantly higher in leaves than in the mature seeds of scented rice. Among the scented category, 23–27-fold reduced badh2expressionwasrecordedinseedsthanin leaves. Similar transcriptional downregulation of badh2 gene in aromatic rice cultivars over non- aromatic rice has been previously reported [8, 27, 28]. The accumulation of 2AP is associated with transcriptional expression of badh2andΔ1-pyrolline-5-carboxylic acid synthesis (P5CS) genes [29]. Previous reports showed that badh2 transcript recorded higher abundance in young, healthy leaves than in other tissues of fragrance rice [9]. In fragrant rice, cultivar KDML105 and its mutants varying in aroma levels showed higher badh2 transcript level with lower aroma content [28]. Similar reduction in 2AP level was detected in a transgenic aromatic rice line when it was transformed with functional badh2 gene [8]. On the other hand, suppression of badh2transcript, namely Os2AP transcript by RNAi in a non-aromatic callus (Oryza sativa japonica cv. Nipponbare) increased 2AP level [27]. The 2AP content in seeds of scented rice (AM-157, 0.662 mg/kg; BA-370, 0.451 mg/kg) were found to be two to three times higher than in leaves (AM-157, 0.198 mg/kg; BA-370, 0.191 mg/kg). The seeds of AM-157 and BA-370, with 23 to 27 fold lower badh2 transcripts were found to accumulate two to three times more 2AP as compared to leaves. This confirms the recessive nature of fgr (badh2) gene in scented rice cultivars. In the leaves of AM-157 and BA-370, P5CS expression levels were elevated from 8 to 14 times than in seeds. The P5CS expression in the seeds of scented rice cultivars (AM-157, 104.20 copies/10 ng cDNA; BA-370, 42.00 copies/10 ng cDNA) was found to be 3 to 6 fold higher than in the seeds of IR-64. The leaves of scented rice (AM-157, 890.58 copies/10 ng cDNA; BA-370, 596.42 copies/10 ng cDNA) also expressed 3 to 5 fold higher level of P5CS than IR-64. Huang et al. [29] reported 135 and 137 % higher P5CS gene expression in aromatic rice cultivars Tainung 71 and Tainung 72 than in non-aromatic rice. Our results are in agreement with this report. P5CS is involved in the regulation of proline synthesis. Proline has been identified as one of the precursors of 2AP [9, 29]. In the present study, negative correlation between P5CS expression and 2AP contents in leaves and seeds was recorded. P5CS is reported to have more than one isoform, and the additional isoform is located in the chloroplast; expression of P5CS was found to be upregulated under photosynthetically active stage of plant life [30]. Therefore, increased expression of P5CS in leaves might be due to additional isoform existing in chloroplast. Similar observations were reported by Poonlaphdecha et al. [23] where they found high levels of proline with low levels of 2AP in leaves and vice versa in seeds of scented rice. Keyghobad [31]also demonstrated more than 2-fold enhancements in 2AP after overexpressing P5CS in AM-157 and hybrid scented cultivar Indrayani. In our studies, in general, higher levels P5CS expression was recorded in scented cultivars over non-scented ones confirming its role as precursor of 2AP. However, its higher expression in leaves with less 2AP contents can be justified as follows. Even though proline is one of the precursors for 2AP synthesis, it is equally involved in other primary metabolisms like plant growth and development [32 ]. Since 2AP is serving as secondary metabolite, the left over proline through primary metabolism might be diverted for 2AP synthesis; hence, 2AP contents might be low in leaves. As mentioned elsewhere, 2AP synthesized in rice plant bounds with starch granule complex in developing grains and gets accumulated in seeds [25]; hence, its content might be higher in seeds.

Conclusions

The study confirms vegetative stage as a biosynthetic origin of 2AP and other aroma volatiles. The vegetative stage is actively growing phase of development where maximum number of 638 Appl Biochem Biotechnol (2016) 178:619–639 green leaf volatiles are synthesized via the hydroperoxidelyase (HPL) branch of the oxylipin pathway. Further, the study found that, among the aroma volatile compounds synthesized at vegetative stage, only selected compounds were accumulated in seeds at maturity stages. Among the array of volatiles, 2AP and 13 other aromatic compounds found contributing in the aroma, and thus distinguishing vegetative and mature stages between scented and non-scented rice cultivar. The method developed for analysis of aroma volatiles could be effectively used to screen the rice genotypes and breeding material. Since aroma is an important quality character, for its enhancement, along with 2AP, pathways related to 2-pentylfuran, 6-methyl-5-hepten-2- one, and aliphatic aldehyde biosynthesis could be targeted through metabolic engineering. The study further opens door to explore source to sink relationship of 2AP and other aroma volatiles in scented rice.

Acknowledgments This study was supported by the Department of Science and Technology, New Delhi, India, under WOS-A program grant number SR/WOS-A/LS433/2011(G). The authors are thankful of Rice Research Station, Vadgaon Maval and BSKKV, Dapoli, for providing seed material.

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