Browsing by Author "Gershenzon, Jonathan"

Now showing 1 - 4 of 4

Four genes encoding MYB28, a major transcriptional regulator of aliphatic glucosinolate pathway, are differentially expressed in the allopolyploid Brassica juncea
(Oxford University Press, 2013) Augustine, Rehna; Majee, Manoj; Gershenzon, Jonathan; Bisht, Naveen C.
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.
Heterotic patterns of primary and secondary metabolites in the oilseed crop Brassica juncea
(Springer Nature, 2019) Bajpai, Prabodh K.; Reichelt, Michael; Augustine, Rehna; Gershenzon, Jonathan; Bisht, Naveen C.
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.
Isopropylmalate synthase regulatory domain removal abolishes feedback regulation at the expense of leucine homeostasis in plants
(Oxford University Press, 2025) Varghese, Mohan; Kumar, Roshan; Sharma, Aprajita; Lone, Asif; Gershenzon, Jonathan; Bisht, Naveen C.
In the leucine (Leu) biosynthesis pathway, homeostasis is achieved through a feedback regulatory mechanism facilitated by the binding of the end-product Leu at the C-terminal regulatory domain of the first committed enzyme, isopropylmalate synthase (IPMS). In vitro studies have shown that removing the regulatory domain abolishes the feedback regulation on plant IPMS while retaining its catalytic activity. However, the physiological consequences and underlying molecular regulation on Leu flux upon removing the IPMS C-terminal domain remain to be explored in plants. Here, we removed the IPMS C-terminal regulatory domain using a CRISPR/Cas9-based gene editing system and studied the resulting impact on the Leu biosynthesis pathway under in planta conditions. Absence of the IPMS regulatory domain unexpectedly reduced the formation of the end product Leu but increased the levels of Leu pathway intermediates in mustard (Brassica juncea). Additionally, delayed growth was observed when IPMS devoid of the regulatory domain was introduced into IPMS-null mutants of Escherichia coli and Arabidopsis thaliana. Further, a detailed biochemical analysis showed that in the absence of the C-terminal regulatory domain, a Leu pathway intermediate (α-ketoisocaproate) could compete with the native IPMS substrate (2-oxoisovalerate) for the active site. Combining these metabolomic, biochemical, and in planta analyses, we demonstrate that the C-terminal regulatory domain of IPMS is critical for maintaining Leu-Val homeostasis in plants.
Molecular basis of the evolution of methylthioalkylmalate synthase and diversity of methionine-derived glucosinolates
(American Society of Plant Biologists, 2019) Kumar, Roshan; Lee, Soon Goo; Augustine, Rehna; Reichelt, Micheal; Vassão, Daniel G.; Palavalli, Manoj H.; Allen, Aron; Gershenzon, Jonathan; Jez, Joseph M.; Bisht, Naveen C.
Methylthioalkylmalate synthase catalyzes the committed step in the side-chain elongation of methionine-derived aliphatic glucosinolates and likely evolved from the isopropylmalate synthases of leucine biosynthesis. The globally cultivated Brassica species possess diverse aliphatic glucosinolates important for plant defense and animal nutrition; 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 synthase that have differences in expression profiles and 2-oxo substrate preference that account for diversity of aliphatic glucosinolates across Brassica accessions. The 2.1 Å resolution x-ray crystal structure of B. juncea methylthioalkylmalate synthase identifies key active site residues responsible for controlling 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 diversification of glucosinolates profiles across globally-cultivated Brassica species, which could be used with ongoing breeding strategies towards manipulation of beneficial glucosinolates compounds for animal health and plant protection.