Molecular analysis of aroma gene (BADH2) in Biriyanicheera: a tropical aromatic rice genotype from Kerala, India.

BACKGROUND: The aromatic rice cultivars sometimes show variation in aroma when they are grown in regions other than their normal traditional growing regions. An early maturing selection from a Kerala aromatic local landrace with short grains, named 'Biriyanicheera', when grown in normal tropical conditions was sufficiently fragrant. The present study focused on the analysis of aroma in 'Biriyanicheera' rice genotype through molecular methods. METHODS AND RESULTS: The seeds of two aromatic rice varieties viz., Biriyanicheera and Gandhakasala (aromatic check) along with one non-aromatic rice variety Triveni (control) were used for the study. The BADH2 gene was amplified in all the three rice varieties. Upon sequencing the amplified PCR products of genomic DNA, the mutation in BADH2 gene was detected. The sequencing results of aromatic rice varieties revealed the presence of 8 base pair mutation in exon 7 in Biriyanicheera and Gandhakasaala, whereas this mutation was absent in the non-aromatic variety Triveni. CONCLUSIONS: Aroma production in Biriyanicheera variety is observed to be due to the similar mutation in BADH2 gene as that of the popular scented rice Basmati.

Development of transgenic okra (Abelmoschus esculentus L. Moench) lines having RNA mediated resistance to Yellow vein mosaic virus (Geminiviridae).

Begomovirus Yellow vein mosaic virus causes severe yield losses in okra and even the resistant lines developed through conventional breeding show susceptibility at various levels. This paper describes the development of YVMV resistant lines through RNAi strategy. A universal ihpRNA construct harbouring ßC1 ORF from the ß-satellite of the begomovirus was designed using pRNAi-LIC plasmid. Complementarity checks in sequence databases had shown no off-target effects by the target region and the success of siRNA in interference was proven using Custom Dicer-Substrate siRNA analysis. The ßC1 ORF of the begomovirus was PCR amplified and sequenced using the primer combination designed. The pRNAi-LIC vector, a derivative of pCAMBIA2300 containing duplicated CaMV 35S promoter and Nos terminator from pYL44, was SmaI digested and the amplified sense and antisense strands of the ßC1 region were cloned. E. coli transformed with the plasmid were screened for antibiotic resistance, and the plasmids confirmed for the sense and antisense regions through sequencing, were transferred to Agrobacterium tumefaciens strain GV3101. In planta transformation strategy was followed to transform a highly susceptible okra cv. Salkeerthi with ihpRNA-ßC1 cassette. Transformation success, confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5, was 11.42 %. Transcription of siRNA from the ßC1 ORF in the transgenic lines was confirmed by its PCR amplification from the cDNA, using the stem loop primers designed (68 bp). When the transformed and healthy wild-type plants were co-grown with infected wild-type plants, inside an insect cage released with whiteflies and maintained within a containment facility, three of the four transgenic plants remained completely healthy throughout the crop span.

GTR1 and GTR2 transporters differentially regulate tissue-specific glucosinolate contents and defence responses in the oilseed crop Brassica juncea.

GTR1 and GTR2 transporters are components of the source to sink translocation network of glucosinolates, which are major defence metabolites in the Brassicaceae. These transporters can be genetically manipulated for reduction of seed-glucosinolates without inhibiting glucosinolate biosynthesis, thereby maintaining the inherent defence potential of plants. However, the different roles of GTRs in influencing tissue-specific distribution of glucosinolates in agriculturally important Brassica crops are yet unknown. Here, we report functional characterization of two groups of glucosinolate transporters (GTR1 and GTR2) from Brassica juncea based on gene expression data, biochemical analysis, gene-complementation studies in GTR-deficient mutants and RNAi-based knockdown followed by insect feeding experiments. Although both GTRs showed ubiquitous expression patterns and broad substrate specificity, the single-gene knockdown lines displayed different phenotypes. The GTR2-knockdown plants showed a significant reduction of glucosinolates in seeds and a higher accumulation in leaves and pods, while the GTR1-knockdown plants displayed a smaller reduction of glucosinolates in seeds and significantly lower glucosinolate levels in leaves. Consequently, knockdown of GTR2 resulted in higher resistance towards the generalist pest, Spodoptera litura. Overall, our study highlights the distinctive roles of B. juncea GTRs in tissue-specific accumulation of glucosinolates and the potential for manipulating GTR2 for enhanced nutrition and plant defence.

A comprehensive Vis-NIRS equation for rapid quantification of seed glucosinolate content and composition across diverse Brassica oilseed chemotypes.

The globally cultivated Brassica crops contain high deliverable concentrations of health-promoting glucosinolates. Development of a Visible-Near InfraRed Spectroscopy (Vis-NIRS) calibration to profile different glucosinolate components from 641 diverse Brassica juncea chemotypes was attempted in this study. Principal component analysis of HPLC-determined glucosinolates established the distinctiveness of four B. juncea populations used. Subsequently, modified partial least square regression based population-specific and combined Vis-NIRS models were developed, wherein the combined model exhibited higher coefficient of determination (R2; 0.81-0.97) for eight glucosinolates and higher ratio of prediction determination (RPD; 2.42-5.35) for seven glucosinolates in B. juncea populations. Furthermore, range error ratio (RER > 4) for twelve and RER > 10 for eight glucosinolates make the combined model acceptable for screening and quality control. The model also provided excellent prediction for aliphatic glucosinolates in four oilseed Brassica species. Overall, our work highlights the potential of Vis-NIR spectroscopy in estimating glucosinolate content in the economically important Brassica oilseeds.

Targeted editing of tomato carotenoid isomerase reveals the role of 5' UTR region in gene expression regulation.

KEY MESSAGE: A deletion created by CRISPR/Cas9 system in the 5' UTR of the carotenoid isomerase gene in tomato leads to downregulation of the gene resulting in the low conversion of prolycopene to lycopene. CRISPR/Cas9 based genome editing is an effective and useful tool adopted from the bacterial immune response system for altering specific, pre-determined DNA sequences in eukaryotes. Such targeted changes are finding wide application in human health as well as in precision breeding of crop plants for improved traits. Mutations in the coding and regulatory regions can have varying impacts on the function of the gene. In the current study, we demonstrate this on tomato carotenoid isomerase, a key gene in the carotenoid biosynthesis pathway. Mutations were generated in the 5' UTR and exon 1 of the carotenoid isomerase gene using CRISPR/Cas9 expression via Agrobacterium-mediated transformation of tomato variety Periyakulam 1 (PKM1). Molecular and biochemical studies demonstrate that CRISPR-mediated point mutations in the exon sequence lead to complete knockout of protein function whereas deletion in 5' UTR region lowers the expression of the gene leading to changes in plant phenotype.

Molecular Basis of the Evolution of Methylthioalkylmalate Synthase and the Diversity of Methionine-Derived Glucosinolates.

The globally cultivated Brassica species possess diverse aliphatic glucosinolates, which are important for plant defense and animal nutrition. The committed step in the side chain elongation of methionine-derived aliphatic glucosinolates is catalyzed by methylthioalkylmalate synthase, which likely evolved from the isopropylmalate synthases of leucine biosynthesis. However, the molecular basis for the evolution of methylthioalkylmalate synthase and its generation of natural product diversity in Brassica is poorly understood. Here, we show that Brassica genomes encode multiple methylthioalkylmalate synthases that have differences in expression profiles and 2-oxo substrate preferences, which account for the diversity of aliphatic glucosinolates across Brassica accessions. Analysis of the 2.1 Å resolution x-ray crystal structure of Brassica juncea methylthioalkylmalate synthase identified key active site residues responsible for controlling the specificity for different 2-oxo substrates and the determinants of side chain length in aliphatic glucosinolates. Overall, these results provide the evolutionary and biochemical foundation for the diversification of glucosinolate profiles across globally cultivated Brassica species, which could be used with ongoing breeding strategies toward the manipulation of beneficial glucosinolate compounds for animal health and plant protection.

Heterotic patterns of primary and secondary metabolites in the oilseed crop Brassica juncea.

Heterosis refers to the superior performance of F1 hybrids over their respective parental inbred lines. Although the genetic and expression basis of heterosis have been previously investigated, the metabolic basis for this phenomenon is poorly understood. In a preliminary morphological study in Brassica juncea, we observed significant heterosis at the 50% flowering stage, wherein both the growth and reproduction of F1 reciprocal hybrids were greater than that of their parents. To identify the possible metabolic causes or consequences of this heterosis, we carried out targeted LC-MS analysis of 48 primary (amino acids and sugars) and secondary metabolites (phytohormones, glucosinolates, flavonoids, and phenolic esters) in five developmental tissues at 50% flowering in hybrids and inbred parents. Principal component analysis (PCA) of metabolites clearly separated inbred lines from their hybrids, particularly in the bud tissues. In general, secondary metabolites displayed more negative heterosis values in comparison to primary metabolites. The tested primary and secondary metabolites displayed both additive and non-additive modes of inheritance in F1 hybrids, wherein the number of metabolites showing an additive mode of inheritance were higher in buds and siliques (52.77-97.14%) compared to leaf tissues (47.37-80%). Partial least regression (PLS) analysis further showed that primary metabolites, in general, displayed higher association with morphological parameters in F1 hybrids. Overall, our results are consistent with a resource-cost model for heterosis in B. juncea, where metabolite allocation in hybrids appears to favor growth, at the expense of secondary metabolism.

Targeted silencing of genes in polyploids: lessons learned from Brassica juncea-glucosinolate system.

KEY MESSAGE: Intron-spliced hairpin RNAi construct targeting the exonic region of BjuMYB28 driven by the native promoter is the best suited strategy for developing viable low glucosinolate lines in polyploid Brassica juncea. Targeted silencing of specific homolog(s) of a multigene family in polyploids through RNA interference (RNAi) is a challenging effort. Indian oilseed mustard (Brassica juncea), a natural allotetraploid, is expected to have 4-6 copies of every Arabidopsis gene ortholog. In the current study, we have attempted to establish the best gene silencing system suitable for BjuMYB28, a transcription factor gene, with the objective of developing low seed glucosinolate lines in B. juncea. After comparing multiple combinations of BjuMYB28 gene homologs, promoters, target regions (exon and 3' UTR) and silencing strategies (RNAi and antisense), we suggest that the intron-spliced hairpin RNAi construct targeting the specific exonic region of the BjuMYB28 gene under the control of native promoter, whose peak activity synchronises with the highest glucosinolate accumulation phase of the plant, is the best suited strategy for developing viable low glucosinolate lines in polyploid B. juncea.

Feeding behaviour of generalist pests on Brassica juncea: implication for manipulation of glucosinolate biosynthesis pathway for enhanced resistance.

Differential accumulation of plant defence metabolites has been suggested to have important ecological consequence in the context of plant-insect interactions. Feeding of generalist pests on Brassica juncea showed a distinct pattern with selective exclusion of leaf margins which are high in glucosinolates. Molecular basis of this differential accumulation of glucosinolates could be explained based on differential expression profile of BjuMYB28 homologues, the major biosynthetic regulators of aliphatic glucosinolates, as evident from quantitative real-time PCR and promoter:GUS fusion studies in allotetraploid B. juncea. Constitutive overexpression of selected BjuMYB28 homologues enhanced accumulation of aliphatic glucosinolates in B. juncea. Performance of two generalist pests, Helicoverpa armigera and Spodoptera litura larvae, on transgenic B. juncea plants were poor compared to wild-type plants in a no-choice experiment. Correlation coefficient analysis suggested that weight gain of H. armigera larvae was negatively correlated with gluconapin (GNA) and glucobrassicanapin (GBN), whereas that of S. litura larvae was negatively correlated with GNA, GBN and sinigrin (SIN). Our study explains the significance and possible molecular basis of differential distribution of glucosinolates in B. juncea leaves and shows the potential of overexpressing BjuMYB28 for enhanced resistance of Brassica crops against the tested generalist pests.

Arsenic affects the production of glucosinolate, thiol and phytochemical compounds: A comparison of two Brassica cultivars.

Arsenic (As), a non-essential metalloid, severely affects the normal functioning of plants, animals and humans. Plants play a crucial role in metabolic, physiological and numerous detoxification mechanisms to cope up with As induced stress. This study aimed to examine the differential response in two Brassica juncea cultivars, Varuna and Pusa Jagannath (PJn) exposed to different doses of As (50, 150, 300 µM) for 48 h duration. Change in morphological traits, concentration of individual as well as total GSL, sulfur related thiol proteins, sulfur content, and phytochemicals were analyzed in both cultivars. Accumulation pattern of As showed dose dependent accumulation in both the cultivars, being more in PJn. Our finding revealed that both cultivars were tolerant at low concentrations of As, while at higher concentration Varuna excelled over PJn. The increased tolerance of Varuna cultivar exposed to 150 and 300 µM concentration of As, correlated with its increased thiol related proteins, sulfur content and phytochemicals, which serves as defence strategy in the plant against oxidative stress. Differential pattern of total as well as individual GSLs content was observed in both Varuna and PJn cultivars. Varuna cultivar showed higher level of total and aliphatic GSLs, which serves as defence compound with other detoxification machineries to combat As stress. Our findings provide foundation for developing metalloid tolerant crops by analyzing the role of different genes involved in GSL mechanism and signaling pathways in different organs of plant.

Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1-5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses.

Biofortification of oilseed Brassica juncea with the anti-cancer compound glucoraphanin by suppressing GSL-ALK gene family.

Glucosinolates are amino acids derived secondary metabolites, invariably present in Brassicales, which have huge health and agricultural benefits. Sulphoraphane, the breakdown product of glucosinolate glucoraphanin is known to posses anti-cancer properties. AOP (2-oxoglutarate-dependent dioxygenases) or GSL-ALK enzyme catalyzes the conversion of desirable glucoraphanin to deleterious gluconapin and progoitrin, which are present in very high amounts in most of the cultivable Brassica species including Brassica juncea. In this study we showed that B. juncea encodes four functional homologs of GSL-ALK gene and constitutive silencing of GSL-ALK homologs resulted in accumulation of glucoraphanin up to 43.11 µmoles g(-1) DW in the seeds with a concomitant reduction in the anti-nutritional glucosinolates. Glucoraphanin content was found remarkably high in leaves as well as sprouts of the transgenic lines. Transcript quantification of high glucoraphanin lines confirmed significant down-regulation of GSL-ALK homologs. Growth and other seed quality parameters of the transgenic lines did not show drastic difference, compared to the untransformed control. High glucoraphanin lines also showed higher resistance towards stem rot pathogen Sclerotinia sclerotiorum. Our results suggest that metabolic engineering of GSL-ALK has huge potential for enriching glucoraphanin content, and improve the oil quality and vegetable value of Brassica crops.

Biotic elicitors and mechanical damage modulate glucosinolate accumulation by co-ordinated interplay of glucosinolate biosynthesis regulators in polyploid Brassica juncea.

Glucosinolates are nitrogen and sulfur containing secondary metabolites found mainly in the Brassicaceae. They function as plant defense compounds against a broad spectrum of pathogens and pests. Since these molecules form part of the plant defense mechanism, glucosinolate biosynthesis may be modulated by environmental signals leading to activation of a biological stress response. In the current study, we have mimicked such conditions by exogenously applying biotic elicitors such as methyl jasmonate, salicylic acid, glucose and mechanical injury in Brassica juncea seedling over a time course experiment. We found that total glucosinolates over-accumulated under these stress conditions with maximum accumulation observed 24h post treatment. Indole glucosinolates like 1-methoxy-indol-3-ylmethyl and its precursor indol-3-methyl glucosinolates showed a more significant induction compared to aliphatic glucosinolates thereby suggesting a prominent role of indole glucosinolates during plant defense response in B. juncea seedlings. In contrast, the higher amounts of aliphatic glucosinolates were less regulated by the tested biotic elicitors in B. juncea. Expression profiling of multiple homologs of key transcriptional regulators of glucosinolate biosynthesis further showed that a complex interplay of these regulators exists in polyploid B. juncea where they exert co-ordinated and overlapping effects toward altering glucosinolate accumulation. This study has a significant role toward understanding and augmenting plant defense mechanisms in B. juncea, a globally important oilseed crop of genus Brassica.

Genomic origin, expression differentiation and regulation of multiple genes encoding CYP83A1, a key enzyme for core glucosinolate biosynthesis, from the allotetraploid Brassica juncea.

MAIN CONCLUSION: The multiple BjuCYP83A1 genes formed as a result of polyploidy have retained cell-, tissue-, and condition-specific transcriptional sub-functionalization to control the complex aliphatic glucosinolates biosynthesis in the allotetraploid Brassica juncea. Glucosinolates along with their breakdown products are associated with diverse roles in plant metabolism, plant defense and animal nutrition. CYP83A1 is a key enzyme that oxidizes aliphatic aldoximes to aci-nitro compounds in the complex aliphatic glucosinolate biosynthetic pathway. In this study, we reported the isolation of four CYP83A1 genes named BjuCYP83A1-1, -2, -3, and -4 from allotetraploid Brassica juncea (AABB genome), an economically important oilseed crop of Brassica genus. The deduced BjuCYP83A1 proteins shared 85.7-88.4 % of sequence identity with A. thaliana AtCYP83A1 and 84.2-95.8 % among themselves. Phylogenetic and divergence analysis revealed that the four BjuCYP83A1 proteins are evolutionary conserved and have evolved via duplication and hybridization of two relatively simpler diploid Brassica genomes namely B. rapa (AA genome) and B. nigra (BB genome), and have retained high level of sequence conservation following allopolyploidization. Ectopic over-expression of BjuCYP83A1-1 in A. thaliana showed that it is involved mainly in the synthesis of C4 aliphatic glucosinolates. Detailed expression analysis using real-time qRT-PCR in B. juncea and PromoterBjuCYP83A1-GUS lines in A. thaliana confirmed that the four BjuCYP83A1 genes have retained ubiquitous, overlapping but distinct expression profiles in different tissue and cell types of B. juncea, and in response to various elicitor treatments and environmental conditions. Taken together, this study demonstrated that transcriptional sub-functionalization and coordinated roles of multiple BjuCYP83A1 genes control the biosynthesis of aliphatic glucosinolates in the allotetraploid B. juncea, and provide a framework for metabolic engineering of aliphatic glucosinolates in economically important Brassica species.

Four genes encoding MYB28, a major transcriptional regulator of the aliphatic glucosinolate pathway, are differentially expressed in the allopolyploid Brassica juncea.

Glucosinolates are Capparales-specific secondary metabolites that have immense potential in human health and agriculture. Unlike Arabidopsis thaliana, our knowledge about glucosinolate regulators in the Brassica crops is sparse. In the current study, four MYB28 homologues were identified (BjuMYB28-1,-2,-3,-4) from the polyploid Brassica juncea, and the effects of allopolyploidization on the divergence of gene sequence, structure, function, and expression were assessed. The deduced protein sequences of the four BjuMYB28 genes showed 76.1-83.1% identity with the Arabidopsis MYB28. Phylogenetic analysis revealed that the four BjuMYB28 proteins have evolved via the hybridization and duplication processes forming the B. juncea genome (AABB) from B. rapa (AA) and B. nigra (BB), while retaining high levels of sequence conservation. Mutant complementation and over-expression studies in A. thaliana showed that all four BjuMYB28 genes encode functional MYB28 proteins and resulted in similar aliphatic glucosinolate composition and content. Detailed expression analysis using qRT-PCR assays and promoter-GUS lines revealed that the BjuMYB28 genes have both tissue- and cell-specific expression partitioning in B. juncea. The two B-genome origin BjuMYB28 genes had more abundant transcripts during the early stages of plant development than the A-genome origin genes. However, with the onset of the reproductive phase, expression levels of all four BjuMYB28 increased significantly, which may be necessary for producing and maintaining high amounts of aliphatic glucosinolates during the later stages of plant development. Taken together, our results suggest that the four MYB28 genes are differentially expressed and regulated in B. juncea to play discrete though overlapping roles in controlling aliphatic glucosinolate biosynthesis.

Targeted silencing of BjMYB28 transcription factor gene directs development of low glucosinolate lines in oilseed Brassica juncea.

Brassica juncea (Indian mustard), a globally important oilseed crop, contains relatively high amount of seed glucosinolates ranging from 80 to 120 µmol/g dry weight (DW). One of the major breeding objectives in oilseed Brassicas is to improve the seed-meal quality through the development of low-seed-glucosinolate lines (<30 µmol/g DW), as high amounts of certain seed glucosinolates are known to be anti-nutritional and reduce the meal palatability. Here, we report the development of transgenic B. juncea lines having seed glucosinolates as low as 11.26 µmol/g DW, through RNAi-based targeted suppression of BjMYB28, a R2R3-MYB transcription factor family gene involved in aliphatic glucosinolate biosynthesis. Targeted silencing of BjMYB28 homologs provided significant reduction in the anti-nutritional aliphatic glucosinolates fractions, without altering the desirable nonaliphatic glucosinolate pool, both in leaves and seeds of transgenic plants. Molecular characterization of single-copy, low glucosinolate homozygous lines confirmed significant down-regulation of BjMYB28 homologs vis-à-vis enhanced accumulation of BjMYB28-specific siRNA pool. Consequently, these low glucosinolate lines also showed significant suppression of genes involved in aliphatic glucosinolate biosynthesis. The low glucosinolate trait was stable in subsequent generations of the transgenic lines with no visible off-target effects on plant growth and development. Various seed quality parameters including fatty acid composition, oil content, protein content and seed weight of the low glucosinolate lines also remained unaltered, when tested under containment conditions in the field. Our results indicate that targeted silencing of a key glucosinolate transcriptional regulator MYB28 has huge potential for reducing the glucosinolates content and improving the seed-meal quality of oilseed Brassica crops.

Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes), ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.