Multiple mutations in BADH2 gene reveal the novel fragrance allele in indica rice (Oryza sativa L.).

The aroma in rice is the most appreciable quality trait, controlled by the loss of function of the betaine aldehyde dehydrogenase 2 (BADH2) gene. In the present study, indica rice cultivars (basmati, nonbasmati aromatic, and nonaromatic) were screened to explore allelic differences in the BADH2 gene using two functional markers (badh2-p-5'UTR and FMbadh2-E7). Notably, the results of the present mutational analysis showed that both markers confirmed a different mutation in indica rice cultivars than earlier reported japonica accessions. It was found that there is 250-bp deletion in the promoter region of aromatic Kagesali and Kalakrishna as compared to nonaromatic Kolamb. The results of FMbadh2-E7 showed 8-bp deletion and six SNPs in exon 7 of the Kalakrishna cultivar. Interestingly, the nonbasmati aromatic Lalbhat rice cultivar did not harbour any reported mutation and showed a novel BADH2 allele carrying 1-bp deletion in exon 7. Among the selected aromatic rice cultivars, eight cultivars showed mutation in the 5' UTR region and interestingly 23 rice cultivars carried the mutation in both 5' UTR and exon 7 of a BADH2 gene. The 2-acetyl-1-pyrroline (2AP) biosynthesis related metabolites, enzyme assay and gene expression supported mutation in BADH2 gene and expression of 2AP in aromatic rice cultivars under study.

Genome sequencing and protein modeling unraveled the 2AP biosynthesis in Bacillus cereus DB25.

2-Acetyl-1-pyrroline (2AP) is an important and major flavor aroma compound responsible for the fragrance of basmati rice, cheese, wine, and several other food products. Biosynthesis of 2AP in aromatic rice and a few other plant species is associated with a recessive Betaine aldehyde dehydrogenase 2 (BADH2) gene. However, the literature is scant on the relationship between the functional BADH2 gene and 2AP biosynthesis in prokaryotic systems. Therefore, in the present study, we aimed to explore the functionality of the BADH2 gene for 2AP biosynthesis in 2AP synthesizing rice rhizobacterial isolate Bacillus cereus DB25 isolated from the rhizosphere of basmati rice (Oryza sativa L.). Full-length BcBADH2 sequence was obtained through whole genome sequencing (WGS) and further confirmed through traditional PCR and Sanger sequencing. Then the functionality of the BcBADH2 gene was evaluated in-silico through bioinformatics analysis and protein docking studies and further experimentally validated through enzyme assay. The sequencing and bioinformatics analysis results revealed a full-length 1485 bp BcBADH2 coding sequence without any deletion or premature stop codons. Full-length BcBADH2 was found to encode a fully functional protein of 54.08 kDa with pI of 5.22 and showed the presence of the conserved amino acids responsible for enzyme activity. The docking studies confirmed a good affinity between the protein and its substrate whereas the presence of BcBADH2 enzyme activity confirmed the functionality of BADH2 enzyme in B. cereus DB25. In conclusion, the findings of the present study suggest that B. cereus DB25 is able to synthesize 2AP despite a functional BADH2 gene and there may be a different molecular mechanism responsible for 2AP biosynthesis in bacterial systems, unlike that found in aromatic rice and other eukaryotic plant species.

Evidence of polyamines mediated 2-acetyl-1-pyrroline biosynthesis in aromatic rice rhizospheric fungal species Aspergillus niger.

Rhizosphere soil of aromatic rice inhabits different fungal species that produce many bioactive metabolites including 2-acetyl-1-pyrroline (2AP). The mechanism for the biosynthesis of 2AP in the fungal system is still elusive. Hence, the present study investigates the role of possible nitrogen (N) precursors such as some amino acids and polyamines as well as the enzymes involved in 2AP synthesis in the fungal species isolated from the rhizosphere of aromatic rice varieties. Three fungal isolates were found to synthesize 2AP (0.32-1.07 ppm) and maximum 2AP was synthesized by Aspergillus niger (1.07 ppm) isolated from rhizosphere of Dehradun Basmati (DB). To determine the N source for 2AP synthesis, various N sources such as proline, glutamate, ornithine putrescine, spermine, and spermidine were used in place of putrescine in the synthetic medium (Syn18). The results showed that maximum 2AP synthesis was found with putrescine (1.07 ppm) followed by spermidine (0.89 ppm) and spermine (0.84 ppm). Further, LC-QTOF-MS analysis revealed the mobilization of spermine and spermidine into the putrescine, indicating that putrescine is the key N source for 2AP synthesis. Moreover, higher enzyme activity of DAO, PAO, and ODC as well as higher content of methylglyoxal metabolite in the A. niger NFCCI 5060 as compared to A. niger NFCCI 4064 (control) suggests the prominent role of these enzymes in the synthesis of 2AP. In conclusion, this study showed evidence of the polyamines mediated 2AP biosynthesis in A. niger NFCCI 5060.

Chloroplast genome sequence of Nothapodytes nimmoniana (Icacinaceae), a vulnerable reservoir of camptothecin from Western Ghats.

Nothapodytes nimmoniana belongs to family Icacinaceae and is a major source of compound Camptothecin. The global demand for Camptothecin has caused large-scale exploitation of N. nimmoniana from its wild habitat in Western Ghats of India, thereby making it vulnerable. The species is known to exhibit genetic diversity among the populations in Western Ghats. In this study, we report plastome sequence of N. nimmoniana, first for the genus. For the study, the species was collected from Western Ghats of Maharashtra. The plastome of N. nimmoniana was 150,726 bp in length and exhibited typical quadripartite structure with 83,771 bp LSC, 18,513 bp SSC and 24,221 IR region. The plastome was characterized by presence of 124 unique genes, 87 protein coding genes, 29 tRNA genes and four rRNA genes. Further, the plastome was compared with the available basal lamiid plastomes for gene order and composition. N. nimmoniana plastome exhibited SSC region in an inverted configuration. Phylogenomic study placed N. nimmoniana sister to Mappia mexicana. The SSR markers identified in this study, might help to distinguish genetically diverse populations, prioritizing the populations which need immediate conservation effects as well as for checking adulteration.

Understanding parasitism in Loranthaceae: Insights from plastome and mitogenome of Helicanthes elastica.

Loranthaceae is the largest family of the order Santalales and includes root and stem hemiparasites. The parasites are known to exhibit reductions in the genomic features as well as relaxed or intensified selection shifts. In this study, we report plastome and mitogenome sequence of Helicanthes elastica (subtribe Amyeminae, tribe Lorantheae), an endemic, monotypic genus of Western Ghats, India growing on remarkably diverse host range. The length of plastome sequence was 1,28,805 bp while that of mitogenome was 1,65,273 bp. This is the smallest mitogenome from Loranthaceae reported till date. The plastome of Helicanthes exhibited loss of ndh genes (ψndhB), ψinfA, rps15, rps16, rpl32, trnK-UUU, trnG-UCC, trnV-UAC and trnA-UGC while mitogenome exhibited pseudogenized cox2, nad1 and nad4 genes. The comparative study of Loranthaceae plastomes revealed that the pseudogenization or loss of genes was not specific to any genus or tribe and variation was noted in the number of introns of clpP gene in the family. Several photosynthetic genes have undergone relaxed selection supporting lower photosynthetic rates in parasitic plants while some respiratory genes exhibited intensified selection supporting the idea of host-parasite arm race in Loranthaceae. The plastome gene content was found conserved in root hemiparasites compared to stem hemiparasites. The atp1 gene of mitogenome was chimeric and part of it exhibited similarities with Lamiales members. The phylogenetic analysis based on plastid genes placed Helicanthes sister to the members of subtribe Dendrophthoinae.

Exploring the core microbiota in scented rice (Oryza sativa L.) rhizosphere through metagenomics approach.

Rice is a major food crop cultivated around the globe. Specially scented rice varieties are of commercial importance but they are low-yielding. The rhizospheric microflora plays a significant role in improving yield and aroma. However, the core microbiome of the scented rice rhizosphere is comparatively less explored. Here, we analyzed the core microbiome associated with the rhizosphere of the scented (Ambemohar-157 and Dehradun basmati) in comparison with non-scented rice (Kolam and Arize 6444 Gold) cultivated at two different geoclimatic zones of India (Maharashtra and Uttarakhand) using the metagenomics approach. The alpha and beta diversity analysis showed that the microbial communities associated with scented and non-scented varieties significantly changes with respect to richness, diversity, and evenness. The taxonomic profiling revealed the variation in composition, diversity, and abundance of the microbiome in terms of phyla and genera associated with scented rice varieties over non-scented. The cluster analysis distinguishes the microbial communities based on their geographical positions. The core microbiome analysis revealed that scented rice rhizosphere shelters distinct and unique microbiota. 28.6 % of genera were exclusively present only in the scented rice rhizosphere. The putative functional gene annotation revealed the high abundance of genes related to the biosynthesis of 2-acetyl-1-pyrroline (2AP) precursors in scented rice. The precursor feeding analysis revealed proline as a preferred substrate by 2AP synthesizing bacteria. The 2AP precursor proline and proline metabolism genes showed a positive correlation. The scented rice-specific rhizobacteria pointed out in this study can be used as bio-inoculants for enhancing aroma, yield, and sustainable rice cultivation.

Co-functioning of 2AP precursor amino acids enhances 2-acetyl-1-pyrroline under salt stress in aromatic rice (Oryza sativa L.) cultivars.

Aromatic rice (Oryza sativa) fetches a premium price due to the pleasant aroma. The major aroma compound 2-acetyl-1-pyrroline (2AP) has been found to be enhanced under stress. This condition can be considered to study the genes, precursors, enzymes, and metabolites involved in elevated levels of 2AP biosynthesis. In the present study, 100 mM salt treatment was given to two aromatic rice cultivars Ambemohar-157 (A-157) and Basmati-370 (B-370) at the vegetative stage (VS3). After salt treatment, in the leaves, 2AP contents were elevated by 2.2 and 1.8 fold in A-157 and B-370, respectively. Under these elevated 2AP conditions, the precursor amino acids (glutamate, putrescine, ornithine, and proline), their related genes, enzymes, and metabolites (methylglyoxal and γ-aminobutyric acid (GABA) related to 2AP biosynthesis were analyzed. In addition, agronomic characters were also studied. It was observed that the proline content was enhanced in both the cultivars by 29% (A-157) and 40% (B-370) as compared to control. The Δ1-pyrroline-5-carboxylate synthetase (P5CS) enzyme activity was increased in salt-treated plants leaf tissue by 31% (A-157) and 40% (B-370) compared to control. The P5CS gene expression was enhanced by A-157 (1.8 fold) and B-370 (2.2 fold) compared to control, putrescine content in A-157 and B-370 decreased by 2.5 and 2.7 fold respectively as compared to control. The ornithine decarboxylase (ODC) activity was enhanced in A-157 (12%) and B-370 (35%) over control. Further, ODC gene expression was enhanced in both the cultivars A-157 (1.5 fold) and B-370 (1.3 fold). The diamino oxidase (DAO) enzyme activity was increased by 28% (A-157) and 35% (B-370) respectively over control. The GABA content marginally increased over control in both the cultivars namely, A-157 (1.9%) and B-370 (9.5%). The methylglyoxal levels were enhanced by 1.4 fold in A-157 and 1.6 fold in B-370. Interestingly, the enhancement in 2AP in the vegetative stage also helped to accumulate it in mature grains (twofold in A-157 and 1.5 fold in B-370) without test weight penalty. The study indicated that the ornithine and proline together along with methylglyoxal contribute towards the enhancement of 2AP under salt stress.

Understanding aquaporin regulation defining silicon uptake and role in arsenic, antimony and germanium stress in pigeonpea (Cajanus cajan).

Understanding of aquaporins (AQPs) facilitating the transport of water and many other small solutes including metalloids like silicon (Si) and arsenic (As) is important to develop stress tolerant cultivars. In the present study, 40 AQPs were identified in the genome of pigeonpea (Cajanus cajan), a pulse crop widely grown in semi-arid region and areas known to affected with heavy metals like As. Conserved domains, variation at NPA motifs, aromatic/arginine (ar/R) selectivity filters, and pore morphology defined here will be crucial in predicting solute specificity of pigeonpea AQPs. The study identified CcNIP2-1 as an AQP predicted to transporter Si (beneficial element) as well as As (hazardous element). Further Si quantification in different tissues showed about 1.66% Si in leaves which confirmed the predictions. Furthermore, scanning electron microscopy showed a higher level of Si accumulation in trichomes on the leaf surface. A significant alleviation in level of As, Sb and Ge stress was also observed when these heavy metals were supplemented with Si. Estimation of relative water content, H2O2, lipid peroxidation, proline, total chlorophyll content and other physiological parameters suggested Si derived stress tolerance. Extensive transcriptome profiling under different developmental stages from germination to senescence was performed to understand the tissue-specific regulation of different AQPs. For instance, high expression of TIP3s was observed only in reproductive tissues. Co-expression network developed using transcriptome data from 30 different conditions and tissues, showed interdependency of AQPs. Expression profiling of pigeonpea performed using real time PCR showed differential expression of AQPs after Si supplementation. The information generated about the phylogeny, distribution, molecular evolution, solute specificity, and gene expression dynamics in article will be helpful to better understand the AQP transport system in pigeonpea and other legumes.

Fragrance in Pandanus amaryllifoliusRoxb. Despite the Presence of a Betaine Aldehyde Dehydrogenase 2.

Pandanus amaryllifoliusRoxb. accumulates the highest concentration of the major basmati aroma volatile 2-acetyl-1-pyrroline (2AP) in the plant kingdom. The expression of 2AP is correlated with the presence of a nonfunctional betaine aldehyde dehydrogenase 2(BADH2) in aromatic rice and other plant species. In the present study, a full-length BADH2 sequence was reconstructed from the transcriptome data of leaf tissue from P. amaryllifolius seedlings. Based on this sequence, a 1509 bp coding sequence was defined that encoded a 54 kD PaBADH2protein. This revealed the presence of a full-length BADH2 protein in P. amaryllifolius. Moreover, quantitative real-time PCR analysis, combined with BADH2 enzyme activity, confirmed the expression and functionality of the PaBADH2 protein. To understand the apparent structural variation, docking analysis was carried out in which protein showed a good affinity with both betaine aldehyde (BAD) and γ-aminobutyraldehyde (GAB-ald) as substrates. Overall, the analysis showed the presence of a functional BADH2, along with substantial 2AP synthesis (4.38 ppm). Therefore, we conclude that unlike all other plants studied to date, 2AP biosynthesis in P. amaryllifolius is not due to the inactivation of BADH2.

Rhizobacterial consortium mediated aroma and yield enhancement in basmati and non-basmati rice (Oryza sativa L.).

Basmati and non-basmati rice varieties are commercially important. Aromatic rice varieties are low yielding and recently depletion in aroma is observed due to the shift towards modern agriculture. Therefore, it is necessary to restore the aroma and increase the yield through sustainable agriculture. The use of microbial bioinoculants is one of the promising ways to achieve these targets. With these objectives, rhizospheric bacterial strains Enterobacter hormaechei (AM122) and Lysinibacillus xylanilyticus (DB25) having the property of synthesizing 2-acetyl-1-pyrroline (2AP) were isolated from the rhizosphere of two aromatic rice varieties, Ambemohar-157 and Dehradun Basmati respectively and their effect on plant growth, aroma and yield enhancement under mono-inoculation and consortium conditions was analyzed. The bacterial inoculum in consortium resulted in significant improvement in vegetative growth, yield and 2AP content over mono inoculation and control. The study highlights the potential of E. hormaechei and L. xylanilyticus in plant growth, yield and aroma enhancement in basmati and non-basmati rice varieties. These strains can be taken up further for developing a commercial bioformulation.

Genome Editing in Cereals: Approaches, Applications and Challenges.

Over the past decades, numerous efforts were made towards the improvement of cereal crops mostly employing traditional or molecular breeding approaches. The current scenario made it possible to efficiently explore molecular understanding by targeting different genes to achieve desirable plants. To provide guaranteed food security for the rising world population particularly under vulnerable climatic condition, development of high yielding stress tolerant crops is needed. In this regard, technologies upgradation in the field of genome editing looks promising. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a rapidly growing genome editing technique being effectively applied in different organisms, that includes both model and crop plants. In recent times CRISPR/Cas9 is being considered as a technology which revolutionized fundamental as well as applied research in plant breeding. Genome editing using CRISPR/Cas9 system has been successfully demonstrated in many cereal crops including rice, wheat, maize, and barley. Availability of whole genome sequence information for number of crops along with the advancement in genome-editing techniques provides several possibilities to achieve desirable traits. In this review, the options available for crop improvement by implementing CRISPR/Cas9 based genome-editing techniques with special emphasis on cereal crops have been summarized. Recent advances providing opportunities to simultaneously edit many target genes were also discussed. The review also addressed recent advancements enabling precise base editing and gene expression modifications. In addition, the article also highlighted limitations such as transformation efficiency, specific promoters and most importantly the ethical and regulatory issues related to commercial release of novel crop varieties developed through genome editing.

RNAi-mediated down regulation of BADH2 gene for expression of 2-acetyl-1-pyrroline in non-scented indica rice IR-64 (Oryza sativa L.).

2-acetyl-1-pyrroline (2AP) is a principal aroma compound in scented rice and a mutation in betaine aldehyde dehydrogenase 2 (OsBADH2) is responsible aroma in scented rice. The present study was aimed at inducing 2AP production in non-scented indica rice cultivar IR-64 by silencing OsBADH2 via RNAi technique. A vector pBSK was used for the construction of RNAi cassette and pRI101ON as a binary vector. Agrobacterium (GV3101)-mediated transformation was done using embryogenic calli of IR-64. The resultant transgenic lines showed up to 14-fold reduction in expression of OsBADH2 gene and 50% inhibition in enzyme activity. Gas chromatography (GC-MS) analyses showed a significant amount of 2AP production in RNAi callus, leaves, and seeds of IR-64. A total 39 volatile compounds were identified from the control and RNAi seeds of IR-64. Among them, octanal and 2-pentylfuron were found to be increased (30-40%) in RNAi seeds of IR-64. The content of precursors, proline, and methylglyoxal increased substantially, whereas GABA content reduced up to 25% in transgenic IR-64 lines. The study demonstrated that RNAi approach could be successfully used for imparting pleasant aroma character in non-scented indica rice cultivars.

Integrative omics analysis in Pandanus odorifer (Forssk.) Kuntze reveals the role of Asparagine synthetase in salinity tolerance.

Pandanus odorifer (Forssk) Kuntze grows naturally along the coastal regions and withstands salt-sprays as well as strong winds. A combination of omics approaches and enzyme activity studies was employed to comprehend the mechanistic basis of high salinity tolerance in P. odorifer. The young seedlings of P. odorifer were exposed to 1 M salt stress for up to three weeks and analyzed using RNAsequencing (RNAseq) and LC-MS. Integrative omics analysis revealed high expression of the Asparagine synthetase (AS) (EC 6.3.5.4) (8.95 fold) and remarkable levels of Asparagine (Asn) (28.5 fold). This indicated that salt stress promoted Asn accumulation in P. odorifer. To understand this further, the Asn biosynthesis pathway was traced out in P. odorifer. It was noticed that seven genes involved in Asn bisynthetic pathway namely glutamine synthetase (GS) (EC 6.3.1.2) glutamate synthase (GOGAT) (EC 1.4.1.14), aspartate kinase (EC 2.7.2.4), pyruvate kinase (EC 2.7.1.40), aspartate aminotransferase (AspAT) (EC 2.6.1.1), phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) and AS were up-regulated under salt stress. AS transcripts were most abundant thereby showed its highest activity and thus were generating maximal Asn under salt stress. Also, an up-regulated Na+/H+ antiporter (NHX1) facilitated compartmentalization of Na+ into vacuoles, suggesting P. odorifer as salt accumulator species.

Identification of aroma volatiles and understanding 2-acetyl-1-pyrroline biosynthetic mechanism in aromatic mung bean (Vigna radiata (L.) Wilczek).

Mung bean having high food value and easily digestible proteins, is one of the socioeconomically important crop of India. Among the varied cultivars, Sona mung is having aroma and hence popularly cultivated in the pockets of Ganga river basin at Bhutnir char village of Malda District in the West Bengal state. In the present study, aroma volatiles with special reference to 2-acetyl-1-pyrroline (2AP) were analyzed using HS-SPME-GCMS from Sona mung bean and compared with non-scented mung bean (PHULE M-9339). 26 volatiles in seeds of Sona mung and 20 in non-scented mung bean were identified, in which 3,7-dimethyl-6-octenal, (2E)-2-decen-1-ol, 2-ethyl-1-dodecanol and 3,5,5-trimethyl-2-cyclohexene-1-one are first time reported. 0.19 ± 0.001 ppm 2AP was recorded in Sona mung seeds whereas it was not detected in non-scented mung bean. PCA analysis indicated that 2AP, octanal, 1 pentanol, decanal, phenylmethanol and 2-nonen-1-ol were the major contributors in the aroma of Sona mung bean. The significantly higher level proline, methylglyoxal and lower level of BADH2 transcript were detected in Sona mung than non-scented mung, suggesting similar 2AP biosynthesis mechanism in Sona mung bean as reported in scented rice, sorghum and soybean.

Thirty-three years of 2-acetyl-1-pyrroline, a principal basmati aroma compound in scented rice (Oryza sativa L.): a status review.

Rice is the staple food of around 3 billion people, most of them in Asia which accounts for 90% of global rice consumption. Aromatic rices have been preferred over non-aromatic rice for hundreds of years. They have a premium value in national as well as international market owing to their unique aroma and quality. Many researchers were involved in identifying the compound responsible for the pleasant aroma in aromatic rice in the 20th century. However, due to its unstable nature, 2-acetyl-1-pyrroline (2AP) was discovered very late, in 1982. Buttery and co-workers found 2AP to be the principal compound imparting the pleasant aroma to basmati and other scented rice varieties. Since then, 2AP has been identified in all fragrant rice (Oryza sativa L.) varieties and a wide range of plants, animals, fungi, bacteria and various food products. The present article reviews in detail biochemical and genetic aspects of 2AP in living systems. The site of synthesis, site of storage and stability in plant systems in vivo is of interest. This compound requires more research on stability to facilitate use as a food additive. © 2016 Society of Chemical Industry.

Aroma volatile analyses and 2AP characterization at various developmental stages in Basmati and Non-Basmati scented rice (Oryza sativa L.) cultivars.

BACKGROUND: Rice plant growth is comprised of distinct phases, such as vegetative, reproductive, grain filling and maturity phases. In these phases synthesis and availability of primary and secondary metabolites including volatile organic compounds (VOC's) is highly variable. In scented rice, aroma volatiles are synthesized in aerial plant parts and deposited in mature grains. There are more than 100 VOCs reported to be responsible for flavor in basmati rice. It will be interesting to keep track of aroma volatiles across the developmental stages in scented rice. Therefore, the aroma volatiles contributing in aroma with special reference to the major compound 2 acetyl-1-pyrroline (2AP) were screened at seven developmental stages in scented rice cultivars Basmati-370 and Ambemohar-157 along with non-scented rice cultivar IR-64 as a control following HS-SPME-GC-MS method. In addition, the expression levels of key genes and precursor levels involved in 2AP biosynthesis were studied. RESULTS: The study indicated that volatilome of scented rice cultivars is more complex than non-scented rice cultivar. N-heterocyclic class was the major distinguishing class between scented from non-scented rice. A total of 14 compounds including, 2AP were detected specifically in scented rice cultivars. Maximum number of compounds were synthesized at seedling stage and decreased gradually at reproductive and maturity. The seedling stage is an active phase of development where maximum number green leaf volatiles were synthesized which are known to act as defense molecules for protection of young plant parts. Among the 14 odor active compounds (OACs), 10 OACs were accumulated at higher concentrations significantly in scented rice cultivars and contribute in the aroma. 2AP content was highest in mature grains followed by at booting stage. Gene expression analysis revealed that reduced expression of betaine aldehyde dehydrogenase 2 (badh2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elevated level of triose phosphate isomerase (TPI) and Δ1-Pyrolline-5-carboxylic acid synthetase (P5CS) transcript enhances 2AP accumulation. CONCLUSIONS: Most diverse compounds were synthesized at seedling stage and OACs were accumulated more at flowering followed by seedling stage. Distinct accumulation pattern exists for 2AP and other aroma volatiles at various developmental stages. The study revealed the mechanism of 2AP accumulation such that 2AP in mature grains might be transported from leaves and stem sheath and accumulation takes place in grains.

Determination of some minerals and ß-carotene contents in aromatic indica rice (Oryza sativa L.) germplasm.

39 aromatic indica rice (Oryza sativa L.) cultivars were characterized for Iron, Zinc, Calcium, Magnesium, Copper and ß-carotene contents. The ß-carotene contents were ranging from 1.23 to 9.9µg/g in brown and 0.08 to 1.99µg/g in milled rice. Among the mineral contents, Magnesium was found ranging from 855µg/g (Gham) to maximum of 1636µg/g (Badshahbhog) followed by Iron in 32µg/g (Jirga) to 218µg/g (Kalsal), Copper content from 2µg/g (Girga) to 1004µg/g (Gham), Zinc content from 25 (Gham) to 165µg/g (Ambemohar-157) and Calcium ranged from 14µg/g (Ambemohar pandhara) to 67µg/g (Kate chinoor). The study showed that the germplasm assessed is a good source of micronutrients and can be further exploited in breeding programme.

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.

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 Δ(1)-pyrolline-5-carboxylic acid synthetase (P5CS), were studied by real-time PCR. Maximum 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 accumulation in scented over non-scented cultivar.

2-Acetyl-1-pyrroline augmentation in scented indica rice (Oryza sativa L.) varieties through Δ(1)-pyrroline-5-carboxylate synthetase (P5CS) gene transformation.

2-Acetyl-1-pyrroline (2AP) has been identified as a principal aroma compound in scented rice varieties. Δ(1)-Pyrroline-5-carboxylate synthetase (P5CS) gene is reported to regulate the proline synthesis in plants and acts as the precursor of 2AP. Two scented indica rice varieties, namely Ambemohar 157 and Indrayani, were subjected to Agrobacterium tumefaciens-mediated genetic transformation containing P5CS gene. Overexpression of P5CS led to a significant increase in proline, P5CS enzyme activity and 2AP levels in transgenic calli, vegetative plant parts, and seeds over control in both the varieties. 2AP level increased more than twofold in transgenic seeds in both varieties. This is the first report of enhancement in 2AP content through overexpression of using P5CS gene, indicating the role of proline as a precursor amino acid in the biosynthesis of 2AP in scented rice.

HS-SPME-GC-FID method for detection and quantification of Bacillus cereus ATCC 10702 mediated 2-acetyl-1-pyrroline.

A rapid micro-scale solid-phase micro-extraction (SPME) procedure coupled with gas-chromatography with flame ionized detector (GC-FID) was used to extract parts per billion levels of a principle basmati aroma compound "2-acetyl-1-pyrroline" (2-AP) from bacterial samples. In present investigation, optimization parameters of bacterial incubation period, sample weight, pre-incubation time, adsorption time, and temperature, precursors and their concentrations has been studied. In the optimized conditions, detection of 2-AP produced by Bacillus cereus ATCC10702 using only 0.5 g of sample volume was 85 µg/kg. Along with 2-AP, 15 other compounds produced by B. cereus were also reported out of which 14 were reported for the first time consisting mainly of (E)-2-hexenal, pentadecanal, 4-hydroxy-2-butanone, n-hexanal, 2-6-nonadienal, 3-methoxy-2(5H) furanone and 2-acetyl-1-pyridine and octanal. High recovery of 2-AP (87 %) from very less amount of B. cereus samples was observed. The method is reproducible fast and can be used for detection of 2-AP production by B. cereus.