XLG2 and CORI3 function additively to regulate plant defense against the necrotrophic pathogen Sclerotinia sclerotiorum.

The membrane-bound heterotrimeric G-proteins in plants play a crucial role in defending against a broad range of pathogens. This study emphasizes the significance of Extra-large Gα protein 2 (XLG2), a plant-specific G-protein, in mediating the plant response to Sclerotinia sclerotiorum, which infects over 600 plant species worldwide. Our analysis of Arabidopsis G-protein mutants showed that loss of XLG2 function increased susceptibility to S. sclerotiorum, accompanied by compromised accumulation of jasmonic acid (JA) during pathogen infection. Overexpression of the XLG2 gene in xlg2 mutant plants resulted in higher resistance and increased JA accumulation during S. sclerotiorum infection. Co-immunoprecipitation (co-IP) analysis on S. sclerotiorum infected Col-0 samples, using two different approaches, identified 201 XLG2-interacting proteins. The identified JA-biosynthetic and JA-responsive proteins had compromised transcript expression in the xlg2 mutant during pathogen infection. XLG2 was found to interact physically with a JA-responsive protein, Coronatine induced 1 (CORI3) in Co-IP, and confirmed using split firefly luciferase complementation and bimolecular fluorescent complementation assays. Additionally, genetic analysis revealed an additive effect of XLG2 and CORI3 on resistance against S. sclerotiorum, JA accumulation, and expression of the defense marker genes. Overall, our study reveals two independent pathways involving XLG2 and CORI3 in contributing resistance against S. sclerotiorum.

Flavonols affect the interrelated glucosinolate and camalexin biosynthetic pathways in Arabidopsis thaliana.

Flavonols are structurally and functionally diverse biomolecules involved in plant biotic and abiotic stress tolerance, pollen development, and inhibition of auxin transport. However, their effects on global gene expression and signaling pathways are unclear. To explore the roles of flavonol metabolites in signaling, we performed comparative transcriptome and targeted metabolite profiling of seedlings from the flavonol-deficient Arabidopsis loss-of-function mutant flavonol synthase1 (fls1) with and without exogenous supplementation of flavonol derivatives (kaempferol, quercetin, and rutin). RNA-seq results indicated that flavonols modulate various biological and metabolic pathways, with significant alterations in camalexin and aliphatic glucosinolate synthesis. Flavonols negatively regulated camalexin biosynthesis but appeared to promote the accumulation of aliphatic glucosinolates via transcription factor-mediated up-regulation of biosynthesis genes. Interestingly, upstream amino acid biosynthesis genes involved in methionine and tryptophan synthesis were altered under flavonol deficiency and exogenous supplementation. Quercetin treatment significantly up-regulated aliphatic glucosinolate biosynthesis genes compared with kaempferol and rutin. In addition, expression and metabolite analysis of the transparent testa7 mutant, which lacks hydroxylated flavonol derivatives, clarified the role of quercetin in the glucosinolate biosynthesis pathway. This study elucidates the molecular mechanisms by which flavonols interfere with signaling pathways, their molecular targets, and the multiple biological activities of flavonols in plants.

The mysterious non-arbuscular mycorrhizal status of Brassicaceae species.

The Brassicaceae family is unique in not fostering functional symbiosis with arbuscular mycorrhiza (AM). The family is also special in possessing glucosinolates, a class of secondary metabolites predominantly functioning for plant defence. We have reviewed what effect the glucosinolates of this non-symbiotic host have on AM or vice versa. Isothiocyanates, the toxic degradation product of the glucosinolates, particularly the indolic and benzenic glucosinolates, are known to be involved in the inhibition of AM. Interestingly, AM colonization enhances glucosinolate production in two AM-host in the Brassicales family- Moringa oleifera and Tropaeolum spp. PHOSPHATE STARVATION RESPONSE 1 (PHR1), a central transcription factor that controls phosphate starvation response also activates the glucosinolate biosynthesis in AM non-host Arabidopsis thaliana. Recently, the advances in whole-genome sequencing, enabling extensive ecological microbiome studies have helped unravel the Brassicaceae microbiome, identifying new mutualists that compensate for the loss of AM symbiosis, and reporting cues for some influence of glucosinolates on the microbiome structure. We advocate that glucosinolate is an important candidate in determining the mycorrhizal status of Brassicaceae and has played a major role in its symbiosis-defence trade-off. We also identify key open questions in this area that remain to be addressed in the future.

Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low-seed, high-leaf glucosinolate oilseed mustard with uncompromised defence and yield.

Glucosinolate content in the two major oilseed Brassica crops-rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 µmoles/g of seed dry weight, DW), making the protein-rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 µmoles/g DW to as low as 6.21 µmoles/g DW. Detailed analysis of the GTR-edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR-edited lines displayed a defence response at par or better than that of the wild-type line. The GTR-edited lines were equivalent to the wild-type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low-seed, high-leaf glucosinolate lines with an uncompromised defence and yield.

Defense versus growth trade-offs: Insights from glucosinolates and their catabolites.

Specialized metabolites are a structurally diverse group of naturally occurring compounds that facilitate plant-environment interactions. Their synthesis and maintenance in plants is overall a resource-demanding process that occurs at the expense of growth and reproduction and typically incurs several costs. Evidence emerging on different specialized compounds suggests that they serve multiple auxiliary functions to influence and moderate primary metabolism in plants. These new functionalities enable them to mediate trade-offs from defenses to growth and also to offset their production and maintenance costs in plants. Recent research on glucosinolates (GSLs), which are specialized metabolites of Brassicales, demonstrates their emerging multifunctionalities to fine-tune plant growth and development under variable environments. Herein, we present findings from the septennium on individual GSLs and their catabolites (GHPs) per se, that work as mobile signals within plants to mediate precise regulations of their primary physiological functions. Both GSLs and GHPs calibrate growth-defense trade-off interactions either synergistically or directly when they function as storage compounds, abiotic stress alleviators, and one-to-one regulators of growth pathways in plants. We finally summarize the overall lessons learned from GSLs and GHPs as a model and raise the most pressing questions to address the molecular-genetic intricacies of specialized metabolite-based trade-offs in plants.

The multifaceted roles of heterotrimeric G-proteins: lessons from models and crops.

MAIN CONCLUSION: The review summarizes our advanced understanding of the heterotrimeric G-protein research from model plants and their emerging roles in modulating various plant architecture and agronomical traits in crop species. Heterotrimeric G-proteins (hereafter G-proteins), consisting of G-alpha (Gα), G-beta (Gß) and G-gamma (Gγ) subunits, are key signal transducers conserved across different forms of life. The discovery of plant lineage-specific G-protein components (extra-large G-proteins and type-C Gγ subunits), inherent polyploidy in angiosperms, and unique modes of G-protein cycle regulation in plants pointed out to a few fundamental differences of plant G-protein signaling from its animal counterpart. Over the last 2 decades, extensive studies in the model plant Arabidopsis thaliana have confirmed the involvement of G-proteins in a wide range of plant growth and development, and stress adaptation processes. The G-protein research in crop species, however, is still in its infancy, and a handful of studies suggest important roles of G-proteins in regulating plant architectural and key agronomical traits including plant's response to abiotic and biotic factors. We propose that the advancement made in plant G-proteins research will facilitate the development of novel approaches to manage plant yield and fitness in changing environments.

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.

A complex interplay of Gß and Gγ proteins regulates plant growth and defence traits in the allotetraploid Brassica juncea.

KEY MESSAGE: Gene expression analysis coupled with in-planta studies showed that specific Gßγ combination regulates plant growth and defence traits in the allotetraploid Brassica juncea. Plant heterotrimeric G-proteins regulate a wide range of responses despite their limited repertoire of core components. The roles and functional interactions between different G-protein subunits are quite perplexing, which get further complicated with polyploidy. Here, we show that the allotetraploid Brassica juncea comprises multiple homologs of G-protein genes, encoding six BjuGß and ten highly divergent BjuGγ subunit proteins, later being classified into type-A1, type-A2 and type-C Gγ proteins. The encoded BjuGß and BjuGγ proteins shared close evolutionary relationship and have retained distinct spatio-temporal expression patterns during plant developmental stages and in response to the necrotrophic pathogen, Sclerotinia sclerotiorum. RNAi based suppression of BjuGß and BjuGγ genes suggested functional overlap and selectivity of BjuGßs with three distinct BjuGγ type subunits, to regulate plant height (BjuGßγA2 and BjuGßγC), seed weight (BjuGßGγA1 and BjuGßGγC), silique size (BjuGßGγC) and pathogen response (BjuGßGγA1 and BjuGßGγC). Further, the triplicated BjuGß genes, formed due to Brassica specific whole-genome-triplication event, showed differential involvement during pathogen response, wherein overexpression of BjuGß2 displayed higher resistance to Sclerotinia infection. Taken together, our study demonstrates that multiple BjuGß and BjuGγ proteins have retained distinct spatio-temporal expression and functional selectivity to regulate specific plant growth and defence traits in the oilseed B. juncea.

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.

Heterotrimeric Gα subunit regulates plant architecture, organ size and seed weight in the oilseed Brassica juncea.

KEY MESSAGE: Two BjuGα proteins exhibit conserved GTP-binding and GTP-hydrolysis activities, and function in maintaining overall plant architecture and controlling multiple yield-related traits in the oilseed Brassica juncea. Heterotrimeric G-protein (Gα, Gß and Gγ) are key signal transducers, well characterized in model plants Arabidopsis and rice. However, our knowledge about the roles played by G-proteins in regulating various growth and developmental traits in polyploid crops, having a complex G-protein signalling network, is quite sparse. In the present study, two Gα encoding genes (BjuA.Gα1 and BjuB.Gα1) were isolated from the allotetraploid Brassica juncea, a globally cultivated oilseed crop of the Brassicaceae family. BjuGα1 genes share a close evolutionary relationship, and the encoded proteins exhibit highly conserved G-protein activities while showing expression differentiation, wherein BjuA.Gα1 was the highly abundant transcript during plant growth and developmental stages. RNAi based suppression of BjuGα1 displayed compromised effects on most of the tested vegetative and reproductive parameters, particularly plant height (32-58%), flower and siliques dimensions, and seed weight (11-13%). Further, over-expression of a constitutively active Gα, lacking the GTPase activity, produced plants with increased height, organ size and seed weight (7-25%), without altering seed quality traits like fatty acid composition, glucosinolates, oil and protein contents. Our study demonstrates that BjuGα1 proteins control overall plant architecture and multiple yield-related traits in the oilseed B. juncea, suggesting that BjuGα1 could be a promising target for crop improvement.

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.

Molecular cloning and expression profiling of multiple Dof genes of Sorghum bicolor (L) Moench.

DNA binding with one finger (Dof) proteins represent a family of plant specific transcription factors associated with diverse biological processes, such as seed maturation and germination, phytohormone and light mediated regulation, and plant responses to biotic and abiotic stresses. In present study, a total of 21 Dof genes from Sorghum bicolor were cloned, sequenced and in silico characterized for homology search, revealing their identity to Dof like proteins. The expression profiling of SbDof genes using quantitative RT-PCR in different tissue types and also under drought and salt stresses was attempted. The SbDof genes displayed differential expression either in their transcript abundance or in their expression patterns under normal growth condition. Two of the SbDof genes namely SbDof8 and SbDof12 showed comparatively high level of transcript abundance in all the tissue types tested; whereas some of the SbDof genes showed a distinct tissue specific expression pattern. Further a total of 13 SbDof genes showed differential expression when subjected to either of the abiotic stress i.e. drought or salinity. Three of the SbDof genes namely SbDof12, SbDof19 and SbDof24 were found to be up-regulated in response to drought and salt stress. Comparative analysis of SbDof genes expression revealed existence of a complex transcriptional and functional diversity across plant growth and developmental stages.

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.

Differential expression and interaction specificity of the heterotrimeric G-protein family in Brassica nigra reveal their developmental- and condition-specific roles.

Heterotrimeric G-proteins, comprised of α, ß and γ subunits, are important signal transducers across phyla. The G-proteins are well characterized in the model plants Arabidopsis and rice, and their inventories are possible from a few other plant species; however, information about the roles played by G-proteins in regulating various growth and developmental traits particularly from polyploid crops is still awaited. In this study, we have isolated one Gα (BniB.Gα1), three Gß (BniB.Gß1-BniB.Gß3) and four Gγ (BniB.Gγ1-BniB.Gγ4) coding sequences from the paleopolyploid Brassica nigra, a major condiment crop of the Brassicaceae family. Sequence and phylogenetic analysis revealed that whole-genome triplication events in the Brassica lineage had proportionally increased the inventory of the Gß subunit, but not of the Gα and Gγ subunits in B. nigra. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that members of the G-protein subunit genes have distinct temporal and spatial expression patterns and were differentially altered in response to various stress and phytohormone treatments, thereby suggesting differential transcriptional regulation of G-protein genes in B. nigra. Interestingly, specific members of G-protein subunits were co-expressed across plant developmental stages, and in response to different elicitor treatments. Yeast-based interaction screens further predicted that the B. nigra G-protein subunits interacted in most of the possible combinations, although showing a high degree of interaction specificity between different G-protein subunits. Our data on physical interactions coupled with the co-expression pattern of the multiple G-protein subunit genes suggested that tissue- and condition-specific functional combinations of Gαßγ heterotrimers may exist in paleopolyploid B. nigra, to control diverse growth and development processes.

MediatorWeb: a protein-protein interaction network database for the RNA polymerase II Mediator complex.

The protein-protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast Saccharomyces cerevisiae, and model plant Arabidopsis thaliana in a user-friendly web-interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue-level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression.

Two chimeric regulators of G-protein signaling (RGS) proteins differentially modulate soybean heterotrimeric G-protein cycle.

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gß, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1-4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1-4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks.

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.

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.