Aroma in Rice

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aroma in rice

Transcript of Aroma in Rice

  • Molecular Aspects of Fragrance and Aroma in Rice

    APICHART VANAVICHIT*,{,1 AND TADACHI YOSHIHASHI{

    *Rice Gene Discovery, National Center for Genetic Engineering and

    Biotechnology, National Science and Technology Development Agency,

    Kamphangsaen, Nakhonpathom, Thailand{Rice Science Center and Agronomy Department, Faculty of

    Agriculture, Kamphangsaen, Nakhonpathom, Thailand{Postharvest Science and Technology Division, Japan International

    Research Center for Agricultural Sciences, Tsukuba, Ibaraki, Japan

    I. 2-Acetyl-1-Pyrroline, a Potent Flavour Component of Aromatic Rice. . . . 50II. Aromatic Gene Discovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

    A. Mendelian Genetics of Grain Aroma .... .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 51B. Genetic Mapping of Grain Aroma ..... .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 51C. QTL Mapping of 2AP ..... ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 52D. Map-Based Cloning of the Gene Controlling Grain 2AP....... .. .. .. .. 52

    III. Molecular Mechanisms Regulating 2AP Biosynthesis . . . . . . . . . . . . . . . . . . . . . 54A. Isogenic Lines Revealed the Absence of the Os2AP Transcript.... .. .. 54B. Suppressing Os2AP by RNAi Makes Rice Aromatic... .. .. .. ... .. .. .. .. 55C. Overexpression of Os2AP Turns Aromatic Rice to

    Non-Aromatic Rice .... .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 58D. Alternative Mechanism of Regulating 2AP Biosynthesis .... ... .. .. .. .. 58

    IV. Biochemical Functions of Os2AP and BADH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58A. The Os2AP Protein is an AMADH ...... .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 58B. Kinetic and Affinity Studies of Isolated Enzymes,

    Os2AP and BADH ...... ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 59V. Formation Pathway of 2AP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

    A. Chemistry Behind 2-AP Formation .... .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. 60

    1Corresponding author: E-mail: [email protected]

    Advances in Botanical Research, Vol. 56 0065-2296/10 $35.00Copyright 2010, Elsevier Ltd. All rights reserved. DOI: 10.1016/S0065-2296(10)56002-6

  • B. Source of Nitrogen in 2AP...... .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. . 62C. Source of Acetyl Group in 2AP ...... .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. . 62D. Metabolic Disorder Makes Rice More Aromatic .... .. .. .. .. .. ... .. .. .. . 63

    VI. Genetic Diversity and Origin of the Aromatic Gene. . . . . . . . . . . . . . . . . . . . . . . 64A. Genetic Diversity of the Aromatic Rice..... ... .. .. .. .. .. .. .. .. .. ... .. .. .. . 64B. Naturally Occurring Allelic Variation of the Aromatic Gene ..... .. .. . 65C. Origin of the Aromatic Gene ..... .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. . 65D. Ancestors of the Aromatic Gene ..... .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. . 65E. Evolutionary Relationship Among Plant

    BADH/AMADH Family .... .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. . 66F. Deficiency in AMADH Makes Aromatic Plants ... .. .. .. .. .. .. ... .. .. .. . 68

    VII. Environmental Adaptability of Aromatic Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

    ABSTRACT

    Grain aroma is the most attractive characteristic of high-quality rice, and demand forit is not only increasing in the Asian market but is also widely recognized in Europeand all over the world. Aromatic rice is rare and so precious that in some countries,it is considered a national asset and pride. The aromatic compound, 2-acetyl-1-pyrroline (2AP) was discovered in rice in 1983 [Buttery, R. G., Ling, L. C., Juliano,B. O., and Turnbauhg, J. G. (1983). Cooked rice aroma and 2-acetyl-1-pyrroline.Journal of Agricultural and Food Chemistry, 823826.], but the gene controlling theaccumulation of 2AP has only recently been identified by map-based cloning(Os2AP). The molecular genetics, biochemistry, and evolution of the aromatic genehave been elucidated in recent years as a consequence of the gene discovery. Aromaticrice has accumulated several natural mutations in an amino aldehyde dehydrogenase(AMADH) that oxidizes -amino butylaldehyde to -amino butyric acid (GABA).RNA interference against the cloned Os2AP generated aromatic from non-aromaticrice plants. A similar technique was used to achieve new aromatic soybean. Aromaticgene also shed new light on evolutionary and domestication aspects of the mostimportant cereal of mankind. The time has come to review past achievements inthe light of the recent discovery of the functions of aromatic genes in rice and otherplant species.

    I. 2-ACETYL-1-PYRROLINE, A POTENT FLAVOURCOMPONENT OF AROMATIC RICE

    The fragrance of cooked rice consists of more than 200 volatile compounds

    such as hydrocarbons, alcohols, aldehydes, ketones, acids, esters, phenols,

    pyridines, pyrazines, and other compounds (Maga, 1984; Paule and Power,

    1989; Tsugita et al., 1980; Yajima et al., 1978). A comparative study of the

    volatile components of aromatic and non-aromatic rice varieties showed that

    2-acetyl-1-pyrroline (2AP), which contributed to specific flavour in aromatic

    rice and has comparably lower odour threshold among rice volatiles, occurs

    at higher levels in aromatic rice varieties and at significantly lower levels in

    50 A. VANAVICHIT AND T. YOSHIHASHI

  • non-aromatic rice varieties (Buttery et al., 1983). Numerous studies have

    shown that 2AP is the only volatile compound in which the relationship

    between its concentration in rice and sensory intensity has been established

    (Maga, 1984; Paule and Powers, 1989; Tsugita et al., 1980; Yajima et al.,

    1978). The compound 2AP, usually described as a pop-corn or roasted

    flavour compound, was also identified as an important attribute of processed

    foods such as wheat bread crust, rye bread (Buttery et al., 1982, 1983), pop-

    corn (Schieberle, 1991), and wet milled millet (Seitz et al., 1993). Interesting-

    ly, 2AP was also identified in other plants and microbes, including pandan

    leaves (Pandanus amaryllifolius Roxb.) (Buttery et al., 1983), bread flowers

    (Vallaris glabra Ktze.) (Wongpornchai et al., 2003), soybean (Fushimi and

    Masuda, 2001), Bacillus cereus (Romanczyk et al., 1995), Lactobacillus

    hilgardii (Costello and Henschke, 2002), and fungi (Nagsuk et al., 2003).

    II. AROMATIC GENE DISCOVERY

    A. MENDELIAN GENETICS OF GRAIN AROMA

    Grain aroma was reported to be governed by a single recessive nuclear gene

    (Huang et al., 1994; Sood and Siddiq, 1978), with a few exceptions. So far,

    the inheritance of grain aroma has been reported to depend on the genetic

    background of the materials being studied. Grain aroma has also been

    reported to be governed by a dominant gene (Jodon, 1944), or found to be

    di- or trigenic (Dhulappanavar, 1976; Kadam and Patankar, 1938; Nagaraju

    et al., 1975; Reddy and Sathyanarayanaiah, 1980).

    B. GENETIC MAPPING OF GRAIN AROMA

    Several aromatic rice varieties were used for genetic mapping; some examples

    are aromatic japonicas including Della (Ahn et al., 1992), Azucena (Bourgis

    et al., 2008; Lorieux et al., 1996), Suyunuo (Chen et al., 2006; Shi et al., 2008),

    and Wuxianjing (Chen et al., 2006). Quantitative trait locus (QTL) mapping

    was also performed in such aromatic indica rice as Jasmine (KDML105)

    (Lanceras et al., 2000; Tragoonrung et al., 1996), Kyeema (Bradbury et al.,

    2005), and Wuxiangxian (Chen et al., 2006). Those results produced the

    consensus genetic map that confined grain aroma within 3.54.5 cM; this

    region was flanked by two polymorphic SSR markers on chromosome 8.

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 51

  • C. QTL MAPPING OF 2AP

    Genetic mappings of grain aroma were reported as a qualitative trait based

    on sensory tests. However, volatile compounds of different aromatic rice

    varieties, particularly the amount of 2AP, varied quantitatively (Fitzgerald

    et al., 2008; Goufo et al., 2010; Hein et al., 2006; Itani et al., 2004). Due to

    costly analysis of 2AP, only limited QTLmapping experiments for grain 2AP

    content were reported so far. The grain 2AP-density was identified in three

    map locations (Lorieux et al., 1996). The major QTL mapped on chromo-

    some 8 coincided with the consensus genetic map based on sensory test on

    chromosome 8 (Chen et al. 2006; Lorieux et al., 1996). In addition, twominor

    QTLs were localized on chromosomes 4 and 12 (Lorieux et al., 1996).

    Therefore, the 4.5 cM map interval between RG1 and RG28 on the

    chromosome 8 was considered a critical region for map-based cloning.

    D. MAP-BASED CLONING OF THE GENE CONTROLLING GRAIN 2AP

    The first mapping of grain aroma took place in 1992 (Ahn et al., 1992), and

    the gene responsible for grain aroma was identified 12 years later, with the

    first and only successful map-based cloning of the gene controlling 2AP

    (Fig. 1; Vanavichit et al, 2004, 2005). By taking advantage of within-family

    segregation for 2AP from the F6 to the F13 generations of the cross between

    Jasmine rice and a non-aromatic rice, the original 1.13-Mb region flanked by

    RG1 and RG28 was effectively narrowed down to 82.8 kb, where three

    KDML105 Bacterial Artificial Chromosome (BAC) clones were shotgun

    sequenced, and three candidate genes were identified (Vanavichit et al.,

    2005). ORF3, later named Os2AP, was determined to be responsible for

    grain aroma in aromatic rice, because double recombinations within ORF3

    resulted in the disappearance of 2AP. Comparative sequence analysis of

    ORF3 between KDML105 and Nipponbare revealed that the 4.5-kb geno-

    mic sequence contained 15 exons of the 1512-bp coding sequence that trans-

    lated into the 503 amino acid sequence in non-aromatic Nipponbare. In

    aromatic KDML105 and within the exon 7 of Os2AP, two important muta-

    tion events were found at positions 730 (A to T) and 732 (T to A), followed

    by the 8-bp deletion GATTAGGC starting at position 734 (Fig. 1). A

    second map-based cloning approach was also reported; in a cross between

    aromatic Kyeema and a cultivar of non-aromatic rice, grain aroma was

    mapped on chromosome 8 between the SSR markers RM515 and SSRJ07

    (Bradbury et al., 2005). The in silico physical map consisted of four Nippon-

    bare BAC clones spanning the 386 -kb flanked SSR markers RM515 and

    SSRJ07. Re-sequencing one of the BAC clones revealed 17 genes. However,

    52 A. VANAVICHIT AND T. YOSHIHASHI

  • 700 kb

    82.8 kb

    KDML105 contigs

    A

    B

    C

    D

    E

    71J1868L13

    20J11

    70I03155L11

    167M23

    173C14

    10L03

    RM

    342

    2500

    RM

    4213

    790

    KDML105 (control)11H1

    13E128F106H8

    11H94B6

    0 10

    MCCase

    0.0

    67B42W73B

    5.0 10.0 15.0 20.0 25.0

    Hypothetical protein

    27.6 kb

    8 bp deletion in exon 7Non-aromaticAromatic

    TGCATTTACTGGGAGTTATGAAACTGGTAAAAAGATTATGGCTTCAGCTGCTCCTATGGTTAAGTGCATTTACTGGGAGTTATGAAACTGGTATATA--------TTTCAGCTGCTCCTATGGTTAAG

    Os2AP

    20 30n = 1116F12 ISLs

    40 50 60 70 80 82.8

    1610

    InG

    4In

    AAT3

    InTA

    2

    B03_

    129.

    8B0

    3_12

    7.8

    3920

    4510

    3580

    6720

    3540

    0.41

    2AP accumulation (ppm)

    0.34

    0.340.3

    0.09

    0.060

    KDML105

    KDML105Jao Hawn Nin (JHN)

    CT9993Heterozygous

    2AP accumulation (ppm)

    00.28

    0

    KDML105-68L13

    01_5

    UTR

    01_3

    UTR

    In1

    F05_

    21.6

    F05_

    103.

    0

    P045

    6B03

    _127

    .8

    CP04

    133

    E11_

    44.5

    E03_

    92.0

    RM

    223

    1305

    011

    410

    0210

    2500

    3540

    4440

    , 10L

    03_F

    W

    170 kb

    Fig. 1. Map-based cloning of the aromatic gene in rice: (A) fine-scale mapping inthe 700-kb region spanned by a KD BAC contig. Three KD BAC clones spanning a170-kb region where the aromatic gene was expected were shotgun sequenced (B) highdensity mapping using 1116 F12 plants derived from a single F6 plant to narrow downthe critical region to 27 kb in a single BAC. Six segregating F12 ISLs were graphicallygenotyped in the 82.8-kb region enriched by specific indel markers, (C) annotation ofthe genomic sequence of the KD BAC 68L13 found three ORFs similar to methylcrotonyl CoA lyase, hypothetical protein, the AMADH called Os2AP, a candidate

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 53

  • significant sequence variation was identified in only one clone, whichwas later

    identified as BAD2, a betaine aldehyde dehydrogenase (BADH) homologue.

    In the screening of 14 diverse fragrant and 64 non-fragrant rice varieties, the

    sequence variation of exon 7 was perfectly matched with grain aroma, but no

    transgenic evidence was provided. Based on their similarity at both the

    nucleotide and amino acid levels, Os2AP and BAD2 were considered the

    same gene. A third map-based cloning effort was reported using in silico

    physical mapping within the critical region by comparing only genomic

    BAC-end sequences of Nipponbare (Wanchana et al., 2005) and by compar-

    ing genomic sequences between Nipponbare and 93-11 (Chen et al., 2008).

    The restriction map surrounding the region of the three candidate genes,

    carbonic anhydrase (CA), methylcrotonyl CoA carboxylase (MCC), and

    aldehyde dehydrogenase (Os2AP), was used to screen BAC clones of a local

    Chinese aromatic japonica rice cv. Suyunuo and a Chinese non-aromatic

    indica rice cv. Nanjing11; three subclones of each candidate gene were used

    for functional analysis. The conclusion that BAD2 was the best aroma

    candidate locus identified in the Azucena japonica cultivar was also reached

    using fine-scale mapping using Azucena IR64 (Bourgis et al., 2008). Oncethe gene regulating 2AP content in rice was cloned, the next step was to

    understand its functions and the regulation of 2AP accumulation.

    III. MOLECULAR MECHANISMS REGULATING2AP BIOSYNTHESIS

    A. ISOGENIC LINES REVEALED THE ABSENCE OF THE OS2AP TRANSCRIPT

    Grain aroma is recessive to non-aroma. The 8-bp deletion in exon 7 found in

    aromatic rice varieties all over the world is the functional marker of aromatic

    rice. The first approach investigating how the aromatic gene functions was

    achieved by comparing the isogenic lines A117 and NA10, which differ only

    in the 27-kb genomic region containing the aromatic gene Os2AP

    (Vanavichit et al., 2005). Transcription analysis of the Os2AP and flanking

    candidate genes revealed the differential expression of Os2AP in all parts of

    the rice plant. The compound 2AP is naturally expressed starting from young

    gene controlling 2-acetyl-pyrroline, (D) gene models of Os2AP showing a doublerecombinants identified in 177 F6 plants from the cross between KD and JHN thatknock-out the gene function and as a result generating 2AP and (E) the sequence partof the exon 7 where 8 bp deletion causes the early stop codon that disrupts the genefunction.

    54 A. VANAVICHIT AND T. YOSHIHASHI

  • seedling to the grain-filling period and accumulates in mature grains. The

    pattern of 2AP expression was consistent with the constitutive expression of

    functional Os2AP in all plant organs. However, one exception was in the

    roots, where some researchers have reported no expression (Chen et al.,

    2008). Other researchers detected 2AP and Os2AP transcripts at low levels

    from rice roots and culture media (Vanavichit et al., 2005). This inconsistent

    expression result needs detailed studies to explain how the nonsense muta-

    tion causes suppressive expression in the different plant parts. The reduction

    of Os2AP transcripts was highly significant from 10 to 20 days after pollina-

    tion (DAP) (Vanavichit et al., 2005; Fig. 2). In our laboratory, we illustrated

    the effect of reduced expression of Os2AP at the whole-genome level using

    the isogenic lines A117 and NA10 in the rice oligoarray version II Rice

    Array Database, http://www.ricearray.org/nsfarray/nsfarray.shtml, contain-

    ing 20,190 unique gene-specific probes against total RNA isolated from

    plants harvested at 1020 DAP (Fig. 3). The results confirmed that Os2AP

    was overexpressed fivefold, along with 72 other genes, in the non-aromatic

    NA10. On the other hand, only 17 genes were overexpressed in the aromatic

    line A117. In connection with the 8-bp deletion in exon 7 of the aromatic

    allele, the suppressive expression of Os2AP resulted from a premature stop

    codon at position 753, which shortened the full-length peptide to 252 amino

    acids in aromatic rice (Bradbury et al., 2005; Vanavichit et al., 2005). This

    short, incomplete peptide was reported to trigger nonsense-mediated decay

    (NMD) in several cases (Chang et al., 2007). The hypothesis postulated that

    NMD was operative in aromatic isogenic lines and in all aromatic rice.

    B. SUPPRESSING OS2AP BY RNAI MAKES RICE AROMATIC

    To confirm if the reduced expression of Os2AP was the genetic basis for 2AP

    accumulation, two transgenic approaches were applied. First, RNA interfer-

    ence (RNAi) was used to reduce the expression of the non-aromatic allele of

    Os2AP. The RNAi was constructed from the genomic sequence spanning

    exons 6 to 8 in the opposite direction from the corresponding cDNA. This

    allowed the transcript to create double-stranded RNA, resulting in NMD

    and aromatic Nipponbare that could accumulate 2AP in a range of 0.05

    0.20 ppm (Vanavichit et al., 2005). In this experiment, the strongest RNAi

    expression gave the strongest suppression and the highest accumulation of

    2AP, comparable to the 2AP content in Jasmine rice (Vanavichit et al., 2005).

    In an independent study, transgenic rice containing RNAi by an inverted

    repeat of cDNA encoding Os2AP accumulated 2AP in considerable amounts

    (Niu et al., 2008).

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 55

  • Os2AP

    10 15

    ISL ISLAromatic Non-aromatic

    20 10 15 20 DAP

    3-methyl crotonyl CoA carboxylase

    Surface glycoprotein

    NBS/LRR disease resistance gene

    Disease resistance gene

    Hypothetical protein

    Carbonic anhydrase

    Total RNA

    Arom

    atic

    ISL

    Arom

    atic

    ISL

    Non

    -aro

    mat

    ic IS

    L

    Non

    -aro

    mat

    ic IS

    L

    Arom

    atic

    ISL

    Non

    -aro

    mat

    ic IS

    L

    Arom

    atic

    ISL

    Non

    -aro

    mat

    ic IS

    L

    StemsActin

    Os2AP

    Roots Leaves Seeds(15DAP)

    Fig. 2. (A) Expression analysis of the Os2AP and other predicted codingsequences from the genomic sequence of KD. Total RNAs were isolated from 10,15 and 20 days after pollination (DAP), from aromatic versus non-aromatic ISLs. (B)Expression analysis of the Os2AP and actin between the aromatic ISL versus non-aromatic ISL where the total RNAs were isolated from stems, roots, leaves and seeds,15 DAP.

    56 A. VANAVICHIT AND T. YOSHIHASHI

  • Fig. 3. Differential expression profiling using 21K oligonucleotide array (TIGR,2005) against total RNA isolated during 1020 days after pollination. Significant up-regulated genes detected from both isogenic lines were compared. All raw data werelisted in http://rice.kps.ku.ac.th/aroma-rice.html.

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 57

  • C. OVEREXPRESSION OF OS2AP TURNS AROMATIC RICE TO

    NON-AROMATIC RICE

    While RNAi against Os2AP allows non-aromatic rice to accumulate signifi-

    cantly more 2AP, the question remained whether the overexpression of

    Os2AP would revert the aromatic to non-aromatic rice. The overexpression

    of various constructs of Os2AP, driven by a CaMV35S promoter, was

    compared in the transgenic aromatic rice cv. Wuxiangjing 9 (Chen et al.,

    2008). When comparing partial constructs of Os2AP, only overexpression

    from the intact one significantly suppressed the accumulation of 2AP in

    plantlets (Chen et al., 2008). This suggests that the reduced expression of

    the Os2AP is the key regulatory step for 2AP accumulation. Therefore, the

    results from both the RNAi and overexpression of Os2AP confirmed that

    Os2AP determines the accumulation of 2AP in rice.

    D. ALTERNATIVE MECHANISM OF REGULATING 2AP BIOSYNTHESIS

    Some aromatic rice lines from isozyme Groups I and V in our laboratory did

    not show the 8-bp deletion. These aromatic rice lines have half the amount of

    grain 2AP compared to those with the 8-bp deleted lines. A multiple genomic

    sequence alignment among these aromatic lines identified a 3-bp addition in

    exon 13. This insertion is in frame with translation and adds a tyrosine into

    the peptide. In contrast to those 8-bp deleted aromatic lines, in these lines,

    Os2AP is expressed normally during seed development. The predicted three-

    dimensional structure revealed that the additional tyrosine is perfectly

    situated in the NAD-binding pocket of the NAD-binding domain. To un-

    derstand the effect of tyrosine addition, a full-length cDNA of the new

    aromatic allele was overexpressed in Escherichia coli for kinetic and binding

    studies. The isolated enzymes had lower enzyme activities than the wild type,

    perhaps because the proximity of the tyrosine addition may interfere with

    NAD binding. The two mutations, however, made the substrate 1-pyrroline

    more available for 2AP biosynthesis.

    IV. BIOCHEMICAL FUNCTIONS OF OS2AP AND BADH

    A. THE OS2AP PROTEIN IS AN AMADH

    The Os2AP protein was localized immunologically in the cytoplasm with the

    C-terminal serine-lysine-leucine (SKL) signal peptide specific for targeting

    to the peroxisome (Chen et al., 2008). Western blot analysis using immuno-

    detection against the Os2AP peptide revealed a 55-kDa peptide in all

    58 A. VANAVICHIT AND T. YOSHIHASHI

  • non-aromatic rice varieties that was absent in all aromatic rice varieties. Once

    again, this result confirmed that the instability of the Os2AP transcript

    affects the protein stability at the post-transcriptional level in aromatic rice.

    The in vitro prediction of the two Os2AP alleles revealed the intact 55-kDa

    peptide in the non-aromatic allele, while 252 C-terminal residues were deleted

    in the aromatic allele. The significance of the C-terminus was predicted to be

    the entire substrate binding and oligomerization domains of the Os2AP

    protein (Bradbury et al., 2005; Chen et al., 2008; Vanavichit et al., 2005).

    To understand the roles of the missing null allele in 2AP biosynthesis, the

    enzymatic activities and substrate specificity were studied by native gel

    electrophoresis. Extracts from aromatic rice gave only one band at 55 kDa,

    while those from non-aromatic lines gave two major bands at 54 and 55 kDa

    (Unpublished data). The partial amino acid sequences revealed the lower

    band to be amino aldehyde dehydrogenase (AMADH), the product of

    Os2AP; the upper band was the product of BADH, that is the orthologue

    of Os2AP located on chromosome 4. The activity gel confirmed that the 54-

    kDa Os2AP was more specific to the -amino butylaldehyde ABL substrate,

    whereas the BADH had a broader specificity. It is interesting that the two

    orthologues both play roles in 2AP accumulation in rice. Substrate specificity

    was one of the major differences between the Os2AP and BADH, as revealed

    by activity gel electrophoresis. In the activity gel where Abal and Betald were

    used as substrates, the 54-kDa band showed only AMADH activity, while

    the 55-kDa band showed AMADH and BADH activities. As a result, the

    author suggested that BADH2 be renamed AMADH based on the specifici-

    ty, because these results were confirmed by the partial amino acid sequence.

    These conclusions were in contrast with the Western blot results, which

    showed that the 55-kDa band was the product of Os2AP (Chen et al.,

    2008). However, all the non-aromatic rice varieties showed two faint bands

    similar to the activity gel; the upper band was common among several rice

    varieties. Considering the broader specificity of the enzyme, BADH could

    play important roles in interfering with 2AP accumulation in aromatic rice.

    To test this possibility, RNAi against BADH in either the aromatic or the

    non-aromatic background must be developed.

    B. KINETIC AND AFFINITY STUDIES OF ISOLATED ENZYMES,

    OS2AP AND BADH

    To obtain insights into the kinetics of both enzymes, the overexpression

    of the cloned Os2AP and BADH in E. coli was reported (Bradbury et al.,

    2008). The overexpressed Os2AP in E. coli showed moderate affinity to-

    wards Betald but higher affinity towards ABald (Bradbury et al., 2008).

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 59

  • Surprisingly, the BADH showed no affinity towards Betald but moderate

    affinity towards ABald (Bradbury et al., 2008). Similar results were reported

    on the low affinity of both Os2AP and BADH towards Betald, while the

    affinity towards ABald was higher. However, the in vitro and in vivo enzyme

    specificities were quite different. From the native gel activities, the 54 kDa

    Os2AP was expressed only in non-aromatic varieties, and Os2AP bound

    only to ABald. Trossat et al. (1997) (21) reported that transgenically

    expressed BADH from Beta vulgaris showed AMADH activity, and they

    suggested that AMADH from Avena sativa, Pisum sativum, Setaria italica,

    and Vicia faba should be the same enzyme as BADH. However, recent

    studies demonstrated that a homogenous AMADH showed no BADH

    activity (Sebela et al., 2000); overexpressed BADH also showed no

    AMADH activity in higher plants (Hibino et al., 2001). Since BADH and

    AMADH share a high level of similarity at the genomic and amino acid

    levels, AMADH could have been misidentified as BADH even though their

    substrate specificities were different. The differences between in vitro and

    in vivo enzyme specificities must be studied further. One possible experiment

    would be creating a post-transcriptional modification to interfere with the

    substrate binding site.

    V. FORMATION PATHWAY OF 2AP

    A. CHEMISTRY BEHIND 2-AP FORMATION

    The compound 2AP was isolated and characterized from the basic fraction

    of a steam distillation extract of aromatic rice. Thus, 2AP should be consid-

    ered a basic compound. Its six-membered ring analogue, 6-acetyl-1,2,3,4-

    tetrahydropyridine (6-ATHP), which has organoleptic properties similar

    to those of 2AP, is known as its tautomer and is shown in Fig. 4A. Grimm

    et al. (2001) and Yoshihashi (2002) also reported similar tautomerism of

    2AP, by the observation of a tautomer peak in a GC chromatogram. The

    compounds 4-aminobutanal and 1-pyrroline, which are considered

    biological intermediates of 2AP by Os2AP disruption, are in equilibrium

    and can interconvert spontaneously Fig. 4(C). The compound 1-pyrroline

    was reported as 1-pyrroline trimer in neat form; however, it was also

    reported as 1-pyrroline in the gas phase by GC-FTIR analysis (Baker

    et al., 1992). Due to the prototropic tautomerism of these compounds, the

    tautomeric equilibria could affect the results of analysis, especially in aque-

    ous solution. Thus, the quantification of these compounds must be carefully

    investigated, as a result could be influenced by a strong matrix effect of these

    60 A. VANAVICHIT AND T. YOSHIHASHI

  • equilibria. Another problem was the extraction method, as mentioned by

    Adams and De Kimpe (2006) regarding 2AP in B. cereus; they reported

    that the use of a non-thermal extraction method is essential to obtain

    N

    A B

    C

    NNH

    NH

    O O O

    N N

    NNCHOH2N

    H2O

    +H2O

    O

    TCA cycle

    Succinate

    Succinate semialdehyde

    GABA shunt GABAP5C

    Proline

    OrnithinePutrescine

    Polyamine synthesisand catabolism

    Spermidine Arginine

    Aromatic varietiesAMADH (Os2AP) ??

    2AP 4-aminobutanal

    GlutamateProline biosynthesis

    2-Oxoglutarate

    Fig. 4. Tautomeric equilibria of 6-ATHP (A), 2AP (B) and 4-aminobutanal (C).Formation pathway of 2AP in aromatic rice. The pathway drawing is based on theliterature cited. The detailed pathway from P5C to 4-aminobutanal was not reportedyet. Aromatic varieties lack AMADH enzyme activity, which convert 4-aminobutanalto GABA, to yield 2AP.

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 61

  • reliable results on the biological formation of these Maillard flavour com-

    pounds. Therefore, the interpretation of the results must consider these

    constraints.

    B. SOURCE OF NITROGEN IN 2AP

    Yoshihashi (2002) reported that additions of glutamate and its related amino

    acids ornithine and proline induced 2AP formation in rice calli of an aro-

    matic rice variety, Khao DawkMali 105; the addition of proline dramatically

    increased the 2AP content. From tracer experiments with 15N-labelled pro-

    line, it was also concluded that the pyrroline ring of 2AP originated from

    proline. Native AMADH, which is encoded by the Os2AP gene, was consid-

    ered to act in polyamine catabolism because of its substrate specificity. The

    enzyme commonly converts 4-aminobutanal into 4-aminobutyrate (GABA);

    however, this catabolic reaction did not occur in aromatic rice varieties, and

    4-aminobutanal was accumulated. The 4-aminobutanal was formed through

    the oxidation of putrescine by Cu-diamine oxidase via a non-reversible

    reaction. Yoshihashi et al. (2002) reported that the 4-aminobutanal content

    observed by GCMS for aromatic rice callus content was stable even when

    proline was added. Since only 4-aminobutanal was observed as 1-pyrroline

    from the GC analysis, the non-enzymatic formation of 2AP in aromatic rice

    could arise from 4-aminobutanal. Huang et al. (2008) reported the formation

    of 2AP from pyrroline-5-carboxylic acid (P5C) by the up-regulation of P5C

    synthase 1 and 2 (P5CS1 and 2). They mentioned 1-pyrroline as the interme-

    diate to 2AP formation although the detailed formation pathway from P5C

    to 1-pyrroline was not then described. QTL analysis of 2AP (Lorieux et al.,

    1996) determined not only that Os2AP is located on chromosome 8 but also

    that other loci could be related to 2AP formation. Therefore, it can be

    hypothesized that P5CS1 and 2 were the genes controlling 2AP formation

    by controlling the 4-aminobutanal content. In conclusion, both studies of

    nitrogen source point to 4-aminobutanal or 1-pyrroline as the source of the

    pyrroline ring of 2AP in aromatic rice.

    C. SOURCE OF ACETYL GROUP IN 2AP

    Yoshihashi (2002) also performed a tracer experiment using 1-13C labelled

    proline and concluded that the acetyl group of 2AP did not originate from

    proline. Model studies on the thermal formation of 2AP with proline and

    ornithine revealed that the acetyl group of 2AP originated from 2-oxopro-

    panal, which is a sugar degradation product of a deformylation reaction

    (Schieberle, 1995). Further model studies with isotopically labelled

    62 A. VANAVICHIT AND T. YOSHIHASHI

  • compounds also showed that 2-acetylpyrrolidine could be the intermediate of

    1-pyrroline and 2-oxopropanal; however, the reaction also resulted in

    6-ATHP (Hofmann and Schieberle, 1998). The compound 2-oxopropanal

    could react with ornithine and proline under thermal conditions and produce

    2AP and 2AP and 6-ATHP, respectively. Detailed analysis of 2AP formation

    in L. hilgardiiDSM 20176 showed that the catabolism of lysine and ornithine

    led to the formation of 2,3,4,5-tetrahydropyridine and 1-pyrroline, which

    then served to form 6-ATHP and 2AP, respectively (Costello and Henschke,

    2002). Thus, acetyl-CoA or acetoaldehyde was proposed to induce the acyla-

    tion of these intermediates to yield 6-ATHP and 2AP. However, 6-ATHP

    was not reported or observed in aromatic rice flavour; therefore, detailed

    studies of the introduction of the acetyl group into 4-aminobutanal are

    required to understand 2AP formation. In addition, Huang et al. (2008)

    proposed 2-oxopropanal as the precursor of the acetyl group, even in non-

    thermal conditions. In organisms, 2-oxopropanal is common because it is the

    intermediate of glycolysis; the reaction of 4-aminobutanal always produces

    6-ATHP. It is not clear whether the same pathway as thermal formation

    occurred in aromatic rice. We should also mention that 2-oxopropanal is

    known for its high cytotoxicity and high reactivity and also as the most

    important glycation agent to DNA and proteins.

    D. METABOLIC DISORDER MAKES RICE MORE AROMATIC

    The AMADH disorder in aromatic rice disrupts Os2AP and results in the

    formation of 2AP through the accumulation of 4-aminobutanal (Fig. 4B).

    This metabolic disorder in polyamine catabolism can be considered to im-

    prove rice quality. The product of the AMADH reaction, GABA, is accu-

    mulated in plants under stress conditions such as drought, cold, and salinity

    (Aurisano et al., 1995; Kinnersley and Turano, 2000). The accumulation

    pathway, consisting of glutamate decarboxylase (EC 4.1.1.15), GABA trans-

    aminase (EC 2.6.1.19), and succinate semialdehyde dehydrogenase (EC

    1.2.1.16 or 24), is known as the GABA shunt (Fig. 4B), and it bypasses two

    steps of the TCA cycle. The accumulation of GABA through the GABA

    shunt is predominant; however, Turano et al. (1997) reported significant

    GABA flux from putrescine through AMADH. Since rice AMADH did

    not accept betaine aldehyde as its substrate in the native state, aromatic

    rice varieties under stressed conditions could enhance their polyamine con-

    tent, resulting in a higher 2AP content. Yoshihashi et al. (2004) analyzed the

    2AP content of various aromatic rice samples from Thailand and found that

    samples from irrigated areas had a lower 2AP content than those from

    drought-stricken and rain-fed areas. The Os2AP disruption and the

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 63

  • aromatic phenotype could be a genetic predisposition, but the formation

    of 2AP as a phenotype could also be regulated by environmental conditions.

    The genetic difference in Os2APmay be beneficial for breeding new aromatic

    rice varieties. However, detailed studies on environmental conditions, espe-

    cially on stress conditions with potential effects on GABA formation

    through AMADH, are needed to improve the quality of aromatic rice from

    paddy fields.

    VI. GENETIC DIVERSITY AND ORIGIN OF THEAROMATIC GENE

    A. GENETIC DIVERSITY OF THE AROMATIC RICE

    Traditional aromatic rice varieties are classified into three isozyme groups,

    namely Group I (indica), Group V (indica), and Group VI (tropical japonica)

    (Glaszmann, 1987). The aromatic cultivars belonging to Group I are Jasmine

    and include several cultivars from Thailand, Cambodia, Vietnam, and

    Southern China; those in Group V are Basmati and comprise several culti-

    vars from India, Myanmar, Iran, Pakistan, Afghanistan, Bangladesh, and

    China; and those in Group VI are Azucena and encompass several cultivars

    from Indonesia and the Philippines (Khush, 2000). To investigate the allelic

    variation among these diverse aromatic germplasms, 478 aromatic rice vari-

    eties were assessed for variation in the 8-bp deletion in exon 7 and grain 2AP

    content (Fitzgerald et al., 2008). The majority of the aromatic lines contained

    the 8-bp deletion, with approximately 10% of aromatic varieties accumulat-

    ing significant amounts of 2AP containing no 8-bp deletion (Fitzgerald et al.,

    2008). The possibility that the other loci control 2AP accumulation was

    reported in two QTL mapping experiments (Amarawathi et al., 2008;

    Lorieux et al., 1996). The most likely location of the second QTL was

    reported on chromosome 4 but showed a much smaller effect. Data mining

    into the QTL ch4 localized the Os2AP ortholog, BADH, within the region

    (Lorieux et al., 1996). Additional small QTLs were found on chromosomes 3

    (Amarawathi et al., 2008) and 12 (Lorieux et al., 1996). So far, no genetic

    validation has been reported for the existence and roles of these smaller

    QTLs in the biosynthesis and accumulation of 2AP. The discovery of other

    naturally occurring mutations in Os2AP has also been explored recently.

    64 A. VANAVICHIT AND T. YOSHIHASHI

  • B. NATURALLY OCCURRING ALLELIC VARIATION OF THE AROMATIC GENE

    To explore allelic variation in Os2AP, a large collection of aromatic and non-

    aromatic rice with distinct geographic and genetic origins was analyzed for

    Single Nucleotide Polymorphism (SNP) variation within the Os2AP region

    (55 SNP) and across 5.3 Mb (78 SNP) of the flanking region (Kovach et al.,

    2009). The diverse germplasm pool consisted of 280 accessions of Oryza

    rufipogon, 242 cultivated accessions collected from 38 countries, and 26

    aromatic accessions lacking the 8-bp deletion (Fitzgerald et al., 2008).

    Based on gene-specific SNP variations, the rice germplasm was classified

    into 10 haplotypes, where the badh 2.1 haplotype and the 8-bp delection in

    exon 7 were the most common aromatic haplotype (Kovach et al., 2009). The

    less frequent haplotype groups consisted of exon 14 insertions (badh 2.7) and

    exon 13 SNP (badh 2.8) and associated with the highest 2AP content. Four

    out of eight mutations were predicted to cause truncated Os2AP transcripts

    and abolish protein functionality (Kovach et al., 2009). Interestingly, three

    other mutations in found in rice from Bangladesh (Group I) and Myanmar

    (Group V) resulted in one amino acid addition (Kovach et al., 2009). Despite

    several reports of mutations in Os2AP, two other rice varieties exhibiting

    elevated 2AP levels lacked any known non-functional allele. In addition,

    SNP haplotypes were found in BADH gene and it seems to be associated

    with quantitative variation of 2AP content (Singh et al., 2010). Until the new

    gene is found, Os2AP remains the only known major regulator of 2AP in

    aromatic rice.

    C. ORIGIN OF THE AROMATIC GENE

    More aromatic alleles were found in Group V than in any other rice variety,

    indicating that aromatic rice may originate from Group V and might have

    been transmitted to other indica varieties via cross-hybridization. Identifying

    the donor of the aromatic gene to traditional aromatic rice would be very

    interesting. (The presence of MITE at position 51 was associated with

    fragrant japonicas and indicas; Bourgis et al., 2008.)

    D. ANCESTORS OF THE AROMATIC GENE

    Annual wild rice species such as O. rufipogon, Oryza nivara, and Oryza

    spontaneous are believed to be the ancestors of cultivated rice. It may be

    that aromatic gene can be transmitted to cultivated rice. If it is true that these

    are the ancestors and that the aromatic gene came from this source In that

    case, aromatic wild species must be found in natural habitats. Eleven wild

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 65

  • rice species sampled from a germplasm bank were analyzed for the 8-bp

    deletion in Os2AP. The aromatic wild species were first identified in our

    laboratory using the 8-bp functional marker for screening (Vutiyano,

    2009). However, the aromatic marker allele was found in only two wild

    species, O. rufipogon and O. nivara. Moreover, the 8-bp aromatic allele was

    also identified at a low frequency (0.23) in 229 natural wild rice accessions, of

    O. rufipogon, collected in Thailand (Prathepha, 2008). From the latter study,

    the author concluded that the aromatic allele already existed in wild rice.

    However, in 280 accessions of the wild rice speciesO. rufipogon andO. nivara,

    the 8-bp deletion was mostly absent, but one sample was heterozygous

    (Kovach et al., 2009). Because the heterozygous wild rice exhibited several

    characteristics of cultivated species, the author concluded that the aromatic

    allele did not originate from the wild species themselves but from a recent

    introgression of the aromatic allele from cultivated rice. The authors con-

    cluded that the aromatic gene was first domesticated within japonica-type

    cultivars before it was transmitted through indica-type cultivars; this was in

    line with their SNP diversity survey over the 5.8 kb across the Os2AP. To test

    this hypothesis, several hundred aromatic rice varieties from the Southeast-

    ern Asia Greater Mekong Subregion including Myanmar, Thailand, Cam-

    bodia, and Laos were collected.

    E. EVOLUTIONARY RELATIONSHIP AMONG PLANT

    BADH/AMADH FAMILY

    Rice is not the only plant producing 2AP. Pandan, breadflower, soybean,

    coconut, etc., are among well-known aromatic plants. Identification of aro-

    matic gene in rice emerged as a new tool to create desirable aromatic plants.

    Phylogenic relationship among orthologous sequences related to Os2AP

    (AMADH) and BADH plants was recently reported (Arikit et al., 2010).

    Os2AP/BADH homologous sequences retrieved from several dicots, mono-

    cots, and non-flowering plant genomes were analyzed and shown in Fig. 5.

    Two distinct clades, the monocot and dicot, were clearly defined in flowering

    plant genomes (Fig. 5). Within the monocot clade, two orthologous sub-

    groups were distinctively defined as Os2AP-like or BADH-like sequences.

    For most of the dicot clade, two distinct paralogous subgroups were defined

    after speciation. These results suggested a gene duplication event in the

    monocot. Analysis of NAD-dependent aldehyde dehydrogenase protein

    domain, an aldehyde dehydrogenase cysteine active site, revealed two

    major groups according to two concensus domains (Fig. 6).

    66 A. VANAVICHIT AND T. YOSHIHASHI

  • Monocot BADH-like

    Monocot Os2AP-like

    Dicot AMADH

    Fig. 5. BADH gene family was analyzed following a phylogenomic approach.A set of protein sequences homologous to rice Os2AP was obtained by FlowerPowertool (http://phylogenomics.berkeley.edu/cgi-bin/flowerpower/input_flowerpower.py)using Uniprot proteins as database (http://www.pir.uniprot.org). The homologousproteins were aligned following multiple sequence alignments using MUSCLE pro-gram (http://phylogenomics.berkeley.edu/muscle/). A phylogenetic tree was con-structed according to the multiple sequence alignments by using SCI-PHY tool(http://phylogenomics.berkeley.edu/cgi-bin/SCI-PHY/input_SCI-PHY.py). Proteindomains of these homologous proteins were predicted by Prosite program (http://au.expasy.org/prosite/). Sequences of rice BADH (SwissProt O24174), Os2AP (Swis-sProt Q84LK3) and E. coli (SwissProt P77674) were used. The BLAST 2 (BLOSUM62 matrix) search engine was used to create sequence alignments, whereas ClustalW1.83 enabled the alignment of multiple sequences.

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 67

  • F. DEFICIENCY IN AMADH MAKES AROMATIC PLANTS

    As a result of high homology in the protein sequences, it is possible that all

    2AP accumulators may utilize similar mechanism as rice. To prove the

    concept, natural aromatic soybean was used as a case study. Several aromatic

    soybeans such as Yuagari musume and Kaori hime accumulated seed 2AP in

    a range of 300500 ppb. In aromatic soybean, loss of GmAMADH2 activity

    was detected in maturing seeds (Arikit et al., 2010). This suggested that

    BADH (rice)Q6BD95 (Zoysia tenuifolia)Q6BD93 (turf grass)Q94IC0 (barley)BADH (barley)Q5KSN8 (leymus)Q43829 (Sorghum)

    A

    BFaNAGQVCSATSFaNGGQVCSATSFaNGGQVCSATSFfNGGQVCSATSFfNGGQVCSATSFfNGGQVCSATSLpNAGQVCSAAS

    FwTNGQICSATSFwTNGQICSATSFwTNGQICSATSFwTNGQICSATSFwTNGQICSATSFwTNGQICSATSFwTNGQICSATSFwTNGQICSATSFwTNGQICSATSFwTNGQICSATSFwTNGQICSATS

    Os2AP (rice)BADH (spinach)Q6BD3 (turf grass)Q6BD88 (turf grass)Q94IC1 (barley)Q53CF4 (maize)Q6BD99 (zoysia tenuifolia)Q8LGQ9 (wheat)Q4H1G7 (sugar beet)BADH (sugar beet)O9STS1 (Arabidopsis)

    [FYLVA] . x . {GVEP} . {DILV} . G . [QE] . {LPYG} . C . [LIVMGSTANC] . [AGCN] . {HE} . [GSTADNEKR]

    Rice BADH E. coli BADH Rice AMADH/Os2AP

    Fig. 6. Homology modelling of the BADH and Os2AP protein structure andcomparison of structures. The three-dimensional structure of the rice BADH enzymeand Os2AP enzyme were modelled by comparative protein modelling methods usingthe program SWISS-MODEL in the optimizedmode. The structure of the enzyme wasmodelled on the basis of its structural similarity with the E. coli BADH (Protein DataBank entry 1WNB). The degree of identity between the template and theE. coliBADHsequence were 37.23% and 38.65%, respectively, which enabled a preliminary model tobe generated by SWISS-MODEL. The sequence alignment was then improved manu-ally; Swiss-PdbViewer 3.7 was used to produce a structure-based alignment andSWISS-MODEL was used in the optimized mode to minimize energy. The finalmodel was evaluated with PROCHECK, and Swiss-PdbViewer 3.7 was then used toanalyze and visualize the structures.

    68 A. VANAVICHIT AND T. YOSHIHASHI

  • similar suppressive mechanism is similar to rice. The GmAMADH2-RNAi

    was transformed into two non-aromatic soybean varieties, CM60 and Jack.

    The result showed that the expression of GmAMADH1 was not affected by

    GmAMADH2-RNAi. Contents of 2AP in CM60-RNAi and Jack-RNAi

    were detected in a range of 324350 ppb. In conclusion, it is possible to

    generate aromatic plant varieties by suppressing seed-specific Os2AP-like

    gene (Fig. 5).

    VII. ENVIRONMENTAL ADAPTABILITY OFAROMATIC RICE

    Most types of aromatic rice, such as Jasmine, Basmati, and Azucena, are

    landrace varieties. Breeding high-yielding aromatic rice has been attempted

    in many countries, but with little success. So far, the high-yielding aromatic

    rice cultivars developed have shown less intensity of the aromatic compound

    2AP than the traditional ones. Is aromatic rice less productive than non-

    aromatic rice? In light of the biosynthesis of 2AP (Fig. 2), 2AP is the end

    product of the polyamine pathway in aromatic rice. In non-aromatic rice,

    -aminobutyraldehyde is converted to GABA and subsequently back to the

    TCA cycle via succinate. Considering the loss of two nitrogen atoms from a

    molecule of -aminoaldehyde for the biosynthesis of one molecule of 2AP,

    aromatic rice seems plausibly less productive than non-aromatic rice, espe-

    cially under stress conditions. To investigate the latter phenomenon, RNAi

    and wild-type rice were compared for traits related to productivity (Niu et al.,

    2008). The results showed reduction of plant height, 1000-grain weight, and

    overall productivity in aromatic RNAi rice compared to the wild type. Under

    high-salinity conditions, seedling growth rates were more severely affected by

    different salt concentrations compared to the wild type. However, the germi-

    nation of aromatic rice seedlings was unaffected by various salt-stressed

    conditions. To further investigate salt sensitivity, several aromatic and

    non-aromatic cultivars were compared for productivity when grown under

    22-mM salt solution from 11 weeks post-planting (Fitzgerald et al., 2010).

    The seed set was severely affected by such salinity conditions in aromatic rice.

    The authors concluded that Os2AP plays important roles in resistance to salt

    stresses. However, under salt stress conditions, the ratio of BADH to Os2AP

    transcripts was high, suggesting that BADH, and not Os2AP, was responsi-

    ble for salt responses from a molecule of -aminoaldehyde (Fitzgerald et al.,

    2008). At this end, it is a beginning of new chapters for understanding

    functional roles of aromatic gene.

    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 69

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    MOLECULAR ASPECTS OF FRAGRANCE AND AROMA IN RICE 73

    978-0-12-381518-7Front CoverAdvances in: Botanical ResearchCopyrightContentsContributors to Volume 56Contents of Volumes 3555Chapter 1: Nodule Physiology and Proteomics of Stressed LegumesI. IntroductionII. Plant-Microbe Interaction and SpecificityIII. Infection and Nodulation MechanismsIV. Nodule Proteomics: Wet Laboratory and Bioinformatics ProceduresV. Proteomic Response of Nodule to Different StressesVI. Applications of Nodule ProteomicsVII. ConclusionsAcknowledgmentsReferences

    Chapter 2: Molecular Aspects of Fragrance and Aroma in RiceI. 2-Acetyl-1-Pyrroline, a Potent Flavour Component of Aromatic RiceII. Aromatic Gene DiscoveryIII. Molecular Mechanisms Regulating 2AP BiosynthesisIV. Biochemical Functions of Os2AP and BADHV. Formation Pathway of 2APVI. Genetic Diversity and Origin of the Aromatic GeneVII. Environmental Adaptability of Aromatic RiceReferences

    Chapter 3: Miscanthus: A Promising Biomass CropI. IntroductionII. ProductivityIII. PhysiologyIV. Breeding, Genomics and GeneticsV. Environmental ImpactsVI. Technical Challenges to Commercial ProductionAcknowledgmentsReferences

    Author IndexSubject IndexColour Plate

    Aroma in Rice978-0-12-381518-7Front CoverAdvances in: Botanical ResearchCopyrightContentsContributors to Volume 56Contents of Volumes 3555Chapter 1: Nodule Physiology and Proteomics of Stressed LegumesI. IntroductionII. Plant-Microbe Interaction and SpecificityIII. Infection and Nodulation MechanismsIV. Nodule Proteomics: Wet Laboratory and Bioinformatics ProceduresV. Proteomic Response of Nodule to Different StressesVI. Applications of Nodule ProteomicsVII. ConclusionsAcknowledgmentsReferences

    Chapter 2: Molecular Aspects of Fragrance and Aroma in RiceI. 2-Acetyl-1-Pyrroline, a Potent Flavour Component of Aromatic RiceII. Aromatic Gene DiscoveryIII. Molecular Mechanisms Regulating 2AP BiosynthesisIV. Biochemical Functions of Os2AP and BADHV. Formation Pathway of 2APVI. Genetic Diversity and Origin of the Aromatic GeneVII. Environmental Adaptability of Aromatic RiceReferences

    Chapter 3: Miscanthus: A Promising Biomass CropI. IntroductionII. ProductivityIII. PhysiologyIV. Breeding, Genomics and GeneticsV. Environmental ImpactsVI. Technical Challenges to Commercial ProductionAcknowledgmentsReferences

    Author IndexSubject IndexColour Plate