Browsing by Author "Sharma, Aprajita"

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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.
The mysterious non-arbuscular mycorrhizal status of Brassicaceae species
(John Wiley & Sons, 2023) Sharma, Aprajita; Sinharoy, Senjuti; Bisht, Naveen C.
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.
Pattern of expression and interaction specificity of multiple G-protein beta (Gβ) subunit isoforms with their potential target proteins reveal functional dominance of BjuGβ1 in the allotetraploid Brassica juncea
(Elsevier B.V., 2017) Kumar, Roshan; Sharma, Aprajita; Chandel, Ishita; Bisht, Naveen C.
Heterotrimeric G-protein, consisting Gα, Gβ and Gγ subunits, interacts with various upstream and downstream effector (target) proteins to regulate a large array of conserved and species-specific biological functions. The targets of G-protein components are recently reported in model plant Arabidopsis thaliana; however limited information is available from crop species. In this study, we utilized yeast two-hybrid (Y2H) assay to screen the diversity of interacting partners of multiple Gβ subunit isoforms from allotetraploid Brassica juncea, a globally important oilseed and vegetable crop. The three BjuGβ genes (BjuGβ1-3), resulted from whole genome triplication event in Brassica lineage, showed distinct expression profile during plant developmental stages with maximal transcript abundance during reproductive stages. Protein-protein interaction of three BjuGβ proteins (bait) against the Y2H cDNA library (prey) identified a total of 14 and 1 non-redundant targets for BjuGβ1 and BjuGβ2, whereas BjuGβ3 screening surprisingly did not yield any genuine target, thereby suggesting functional dominance of BjuGβ1. The triplicated BjuGβ isoforms showed a high degree of interaction strength and specificity with the identified target proteins, which are known to be involved in diverse biological functions in plants. qRT-PCR analysis further indicated that the expression of BjuGβ-target genes was developmentally regulated under various tissue types studied and showed a high degree of co-expression pattern with the BjuGβ genes, particularly during flower and silique development in B. juncea. Taken together, our data provides novel insights on pattern of expression and interaction specificity governing functional divergence of multiple Gβ subunit proteins in polyploid B. juncea.