Biology subjects

Rao, B. J.

Publications and source records attributed to Rao, B. J..

6 recordsLinked to original sources

bioRxiv2025

Organellar-specific ROS dynamics drive differential cross-compartmental responses between chloroplast and mitochondria in C. reinhardtii

Reactive oxygen species (ROS) act as key signaling intermediates in plant metabolism, defense, and stress adaptation. In photosynthetic organisms, chloroplast and mitochondria serve as the major hubs of ROS production, thereby coordinating stress responses across cellular compartments. However, the extent of cross-organellar communication following compartmentalized oxidative stress remains poorly understood. Using C. reinhardtii as a model system, we induced compartmentalized oxidative stress in chloroplast and mitochondria to investigate whether the ROS generation in one organelle triggers any functional response in the other organelle. Methyl viologen (MV) was used to induce ROS production in the chloroplast, while menadione (MD) was used to trigger mitochondrial ROS production. Real-time monitoring of compartment-specific roGFP strains showed specific, localized, and reversible ROS production following MV and MD treatment as a function of time within the respective target organelles. Comprehensive functional analyses following compartmentalized ROS perturbations revealed that the mitochondrial ROS remained localized to their site of origin and did not detectably affect chloroplast function, as evidenced by the unchanged chlorophyll a fluorescence parameters and photosystem stoichiometry. In contrast, chloroplast-derived ROS diffused from its site of origin and transiently suppressed the mitochondrial oxygen consumption rates. Together, these findings underscore a dynamically regulated system, where chloroplast ROS leaks out and affects mitochondrial function while the mitochondrial ROS remains localized and does not impact chloroplast function. These compartment-specific ROS responses likely represent an adaptive mechanism for coordinating cellular energy metabolism in response to fluctuating environmental conditions.

microbiology↗

bioRxiv2025

Light-dark dependent rhythmic changes in chloroplast and mitochondrial activity in Chlamydomonas reinhardtii

In photosynthetic organisms, inter-organellar coordination between mitochondria and chloroplast, particularly in synchronous cultures, has been widely appreciated but relatively less understood. Our study investigated the coordination in photosynthetic and mitochondrial activity during (12:12 h) light-dark cycle in Chlamydomonas reinhardtii. Live cell confocal imaging revealed light-dark-dependent mitochondrial morphology transitions from fragmented to intermediate to tubular forms by the end of 12 hr of light period, which reverses sharply through 6 and 12 hr of dark. Concurrently, chloroplast transitions from an intact cup (light) to a distorted and punctured structure (dark), which gets reversed in light phase. Spatial mapping showed tubular mitochondria positioned peripherally to the chloroplast cup in light, whereas fragmented and intermediate mitochondria were diffused around distorted chloroplast in dark, which again gets reversed in light. Functional analysis using 77K spectroscopy and photosynthetic protein levels (PsaA and D1) reflected that PSI/PSII fluorescence ratio remains stable in continuous light condition but increased exceptionally in continuous dark, which led to rhythmic oscillation in fluorescence ratio in light/dark-dependent manner in synchronous cultures. Mitochondrial activity, measured using Seahorse flux analyzer, showed basal oxygen consumption rate in continuous light and a marked reduction in continuous dark condition, resulting in rhythmic changes in light-dark cycle, indicating a coordinated rhythmicity in organellar function. Further, Target of rapamycin (TOR) kinase activity was essential to maintain inter-organellar coupled rhythmicity as evidenced by subdued rhythmicity following TOR kinase inhibition. The study, for the first time, argues for (12:12 h) light-dark cycle-mediated coupled rhythmicity between mitochondria and chloroplast in C. reinhardtii.

physiology↗

bioRxiv2023

IRE1 regulated autophagy and chaperone levels cooperatively modulate acquired thermotolerance in Arabidopsis thaliana

Rapid climate change demands the development of heat-resilient plants. Elevated temperatures perturb cellular protein homeostasis, and its timely restoration is crucial for plant survival after stress. Thermopriming, which involves pre-exposure to sublethal heat stress, has emerged as a promising strategy for enhancing heat stress tolerance. However, the impact of thermopriming on protein homeostasis remains unclear. Here, we demonstrate that priming-mediated acquired thermotolerance involves the dynamic regulation of protein maintenance and clearance mechanisms. Priming facilitates the activation of heat shock response (HSR) via HSFA1, and unfolded protein response (UPR). Simultaneously, priming induces the protein clearance pathway, namely autophagy, potentially through the dynamic modulation of autophagy-negative regulators. Contrastingly, unprimed seedlings fail to mount HSR and UPR, resulting in disrupted proteostasis and the accumulation of aggregates, and ultimately fail to survive. While the loss of UPR was found to have a minimal impact on priming-mediated outcomes, the HSR response proved essential, as its absence led to lethality under heat stress. Additionally, the absence of HSR was found to enhance the autophagy response post-stress. Our results highlight the critical role of protein maintenance mechanisms over clearance pathways in ensuring survival. Taken together, our study demonstrates that thermopriming enhances heat stress resilience by temporally coordinating autophagy, HSR and UPR responses to maintain proteostasis.

plant biology↗

bioRxiv2018

Modulation of TOR kinase activity in Chlamydomonas reinhardtii: Effect of N-starvation and changing carbon pool

Target Of Rapamycin (TOR) kinase is a sensor as well as a central integrator of internal and external metabolic cues. The upstream and downstream signals of this kinase are very well characterized in animals. However, in algae, higher plants and other photosynthetic organisms, the components of the TOR kinase-signaling are yet to be characterized. Here, we establish an assay system to study TOR kinase activity in C.reinhardtii using the phosphorylation status of its downstream target, CrS6K. We further use this assay to monitor TOR kinase activity under various physiological states such as photoautotrophy, heterotrophy, mixotrophy and nitrogen starvation. We observe that autotrophy in light (and not in dark) leads to TOR kinase attenuation during N starvation while the same is not observed in mixotrophy. Importantly, we show that the external carbon source glucose is sensed and uptaken by C.reinhardtii cells only in the presence of light and not in the dark. And such exogenously added glucose, as the photoassimilate carbon mimic, results in enhanced production of ROS, induction of autophagy and concomitant drop in TOR kinase activity, creating N-starvation-like cellular state even in N+ conditions. Interestingly, dose dependent addition of glucose revealed TOR kinase activation in low glucose regime (ROS independent) followed by attenuation of TOR kinase (ROS dependent) at high glucose levels.\n\nSummaryTOR kinase activity in C.reinhardtii is modulated by available carbon source especially glucose, where low levels of glucose cause an increase whereas high levels cause a reduction in TOR kinase activity.

plant biology↗

bioRxiv2017

Somatic developmental defects in dmrad51/spnA mutant show low temperature sensitivity with enhanced genomic damages and cell death

Homologous Recombination (HR) is one of the key pathways to repair Double Strand Breaks (DSBs). Rad51 serves an important function of catalysing strand exchange between two homologous chromosomes in the HR pathway. In higher organisms, Rad51 function is indispensable with its absence leading to early embryonic lethality, thus precluding any mechanistic probing of the system. In contrast, absence of Drosophila rad51 (Dmrad51/spnA) has been associated with defects in female germline causing ventralization of the egg, without any reported detrimental consequences to Drosophila somatic tissues. In this study, we have performed a systematic analysis of somatic development of dmrad51 null mutant flies by using genetic complementation between multiple dmrad51 alleles. Our current study, for the first time, uncovers the requirement of Dmrad51 in somatic tissue maintenance at both larval and pupal stages. Also, we show that dmrad51 mutant exhibit patterning defects in abdominal cuticle in the stripes and bristles, while there appears to be only subtle defects in the adult wing and eye. Interestingly, dmrad51 null mutant and other alleles show discernible phenotype of low temperature sensitivity, suggesting a role for Dmrad51 in temperature sensitive cellular processes, which thus presents an elegant system for probing temperature sensitive cellular/tissue responses that ensue when a mutation leads to the loss of protein expression (null mutant) rather than its altered protein structure.

developmental biology↗

bioRxiv2016

Genome-level parameters describe the pan-nuclear fractal nature of eukaryotic interphase chromosomal arrangement

Long-range inter-chromosomal interactions in the interphase nucleus subsume critical genome-level regulatory functions such as transcription and gene expression. To decipher the physical basis of diverse pan-nuclear patterns of chromosomal arrangement that facilitates these processes, we investigate the scaling effects within disparate genomes and compared their total number of genes with chromosome size. First, we derived the pan-nuclear average fractal dimension of inter-chromosomal arrangement in interphase nuclei of different species and corroborated our predictions with independently reported results. Then, we described the different patterns across disparate unicellular and multicellular eukaryotes. We report that, unicellular lower eukaryotes have inter-chromosomal fractal dimension = 1 at the pan-nuclear scales, which is analogous to the multi-polymer crumpled globule model. Multi-fractal dimensions, corresponding to different inter-chromosomal arrangements emerged from multicellular eukaryotes, such that closely related species have relatively similar patterns. Using this theoretical approach, we could distinguish fractal patterns from human acrocentric versus metacentric chromosomes, implying that the multi-fractal nature of inter-chromosomal geometry facilitates viable large-scale chromosomal aberrations, such as Robertsonian translocations. We report that the nature of such an average multi-fractal dimension for nocturnal mammals is very different in diurnal mammals, which suggests a greatly enhanced plasticity in arrangement across different cell types, for example retinal versus dermal fibroblasts. Altogether, our results substantiate that genome-level constraints have also co-evolved with the average pan-nuclear fractal dimension of inter-chromosomal folding during eukaryotic evolution.

evolutionary biology↗