Biology subjects

Sinha, A. K.

Publications and source records attributed to Sinha, A. K..

16 recordsLinked to original sources

bioRxiv2025

A non-canonical MPK3-ACTPK1-RbcS signalling module fine-tunes photosynthesis in rice

Chloroplast protein import is essential for photosynthesis, yet whether and how cytosolic signaling pathways dynamically regulate this process remains largely unknown. Here we uncover a signaling module that links mitogen activated protein kinase (MAPK) activity to fine tune the import of small subunit of Rubisco (RbcS) into the chloroplast. We show that MPK3 negatively regulates the cytosolic Raf-like kinase ACTPK1, which directly phosphorylates the transit peptide of RbcS precursor at Thr12. MPK3 directly phosphorylates ACTPK1, attenuating its kinase activity and thereby limiting RbcS transit peptide phosphorylation. Genetic and physiological analyses demonstrate that loss of MPK3 elevates ACTPK1 activity, increases RbcS phosphorylation and enhances Rubisco accumulation and CO2 assimilation. On the other hand, ACTPK1 deficiency compromises these processes and photosynthetic performance. Phospho-mutant analyses further reveal that reversible phosphorylation of the RbcS transit peptide is required for efficient chloroplast import. Together, our findings establish chloroplast protein import as a signaling-regulated process and identify transit peptide phosphorylation a key check point integrating cytosolic MAPK signaling with photosynthetic capacity.

plant biology↗

bioRxiv2025

Crosstalk Between Phosphorylation and Ubiquitination Controls the Fate and Function of Strigolactone Signal Transducers

Strigolactones (SL) are pivotal plant hormones that sculpt plant architecture by modulating shoot branching, root development, and meristem activity. While transcriptional responses downstream of SL perception have been well explored, the role of post-translational regulation fine-tuning these responses remains less understood. In this study, we identify a dual-layered regulatory module involving MPK4-mediated phosphorylation and MAX2-dependent ubiquitination that synergistically control the stability and function of BRC1, a key SL-responsive transcription factor. Phosphorylation by MPK4 stabilizes BRC1, enhancing its activity and SL sensitivity, whereas loss of phosphorylation leads to BRC1 degradation and functional inactivation. BRC1, in turn, directly activates MPK4 transcription, establishing a positive feedback loop that amplifies SL signaling. Genetic analyses of the brc1-2 x mpk4 double mutant reveals phenotypic defects and SL insensitivity additive to those observed in the brc1-2 and mpk4 single mutants, indicating that MPK4 and BRC1 act in parallel yet converging pathways downstream of SL. Additionally, MAX2 functions as a regulatory checkpoint that degrades non-phosphorylated MPK4 and BRC1, thereby resetting the signaling circuit to ensure accurate and timely response. Together, these findings illuminate a finely-tuned regulatory module integrating phosphorylation and ubiquitination to control the intensity and duration of SL responses, suggesting a model for hormone-driven developmental plasticity in plants.

molecular biology↗

bioRxiv2025

A positive feedback loop of MPK3-PIN1A trafficking-auxin flux trio governs dual gravitropic and wounding response in rice

The auxin flow in plants plays a pivotal role in gravitropic organ movement and tissue regeneration following injury. However, the regulatory aspects of these phenomena are not entirely understood. In this study, we found that hyperactivity of MAP Kinase 3 (MPK3) led to downregulation of PINs and reduction in tissue auxin content, impairing gravitropic response of rice and accelerating tissue senescence following mechanical wounding. The MPK3 was found to phosphorylate PIN1A majorly at its Ser351 residue, mutation of which into its phospho-mimic variant enhanced latters endosomal trafficking and consequently improved auxin flux. Further, overexpression of the phospho-null variant of PIN1A lowered gravitropic response of rice, thus causing wider tiller angle similar to the pin1a knockout lines. Additionally, upon wounding, their tissue senescence was faster in comparison to the overexpression lines of wild-type and phospho-mimic variants of PIN1A, which also displayed improved resistance to wounding by Bipolaris oryzae. The PIN1A trafficking-mediated auxin flux also regulated MPK3 activity via a positive feedback loop. Together, these results uncover a novel feedback loop encompassing "MPK3-PIN1A trafficking-auxin flux" trio that regulates dual gravitropic and wounding response in rice.

plant biology↗

bioRxiv2024

Early Prediction of Preeclampsia Based on Transposable Elements signature in cell-free RNA

Preeclampsia (PE) is a pregnancy-associated hypertension disorder affecting 5-10% of pregnant women each year worldwide, which leads to adverse maternal and child outcomes. PE remains inadequately predicted, prevented and treated. Here, we comprehensively investigated altered transcriptomic and epigenetic changes in the PE placenta and maternal cell-free RNA (cfRNA). We show that many transposable elements (TEs) in sub-families are deregulated in the gestational age-matched PE placenta. Increased expression of endogenous retrovirus (ERV) and long interspersed nuclear element 1 (L1, LINE1) subfamilies of TEs is associated with higher histone acetylation levels at their regulatory elements. A higher TE transcript level correlates with the type I interferon (IFN-I) pathway, suggesting inflammation associated with PE could be due to the sensing of TE transcripts by the antiviral innate immune system. Consistent with higher TE transcript levels in the PE placentas, analysis of maternal cfRNA data revealed differential levels of TE subfamilies in PE compared to healthy controls. Machine learning-based training for TE transcripts for early prediction of PE showed a robust performance in the validation cohort, with an area under the curve (AUC) of 0.88, 81% sensitivity, and 74% positive predictive value (PPV). Overall, we show TE deregulation in the placenta is associated with PE, and maternal cfRNA TE signature accurately predicts early diagnosis of PE, which can improve prophylaxis and obstetric outcomes.

genomics↗

bioRxiv2024

Phosphorylation of KRP3, a KIP-related protein by MPK3 modulates rice tiller and seed number by fine-tuning cell division

During the cell cycle process, multiple inhibitors function as checkpoint regulators ensuring an impeccable duplication of genetic material. Kip-Related Proteins (KRPs) are a group of plant-specific cell cycle inhibitors and are known to regulate plant architecture and yield by differentially regulating cell division. KRPs, though functionally conserved, are dynamic in their amino acid composition. Interestingly, some KRPs are specific to dicots, and some are for monocots. In this study, we have identified that the presence of KRP3 and KRP6 is specific to the Poaceae family of monocots. An in-depth study of KRP3 showed a strict regulation of its expression in actively dividing cells during the G1-S phase progression of cell division. Our study identified KRP3 as a phosphorylation target of MPK3. The phosphorylation enhances KRP3 protein stability, which leads to strong inhibition of cell proliferation. By generating knock-out lines of krp3, mpk3 and double knock-out krp3mpk3, our study demonstrated that the MPK3-KRP3 module regulates rice root and shoot development as well as tiller and seed numbers. It was observed that KRP3 regulate the rate of cell division in a dose-dependent manner. The study, in a nutshell, establishes the role of KRP3 as an important regulator of rice vigor and yield.

plant biology↗

bioRxiv2024

KCNQ2/3 regulates efferent mediated slow excitation of vestibular afferents in mammals

Primary vestibular afferents transmit information from hair cells about head position and movement to the CNS, which is critical for maintaining balance, gaze stability and spatial navigation. The CNS, in turn, modulates hair cells and afferents via the efferent vestibular system (EVS) and its activation of several cholinergic signaling mechanisms. Electrical stimulation of EVS neurons gives rise to three kinetically- and mechanistically-distinct afferent responses including a slow excitation, a fast excitation, and a fast inhibition. EVS-mediated slow excitation is attributed to odd-numbered muscarinic acetylcholine receptors (mAChRs) on the afferent whose activation leads to the closure of a potassium conductance and increased afferent discharge. Likely effector candidates include low-threshold, voltage-gated potassium channels belonging to the KCNQ (Kv7.X) family, which are involved in neuronal excitability across the nervous system and are subject to mAChR modulation. Specifically, KCNQ2/3 heteromeric channels may be the molecular correlates for the M-current, a potassium current that is blocked following the activation of odd-numbered mAChRs. To this end, multiple members of the KCNQ channel family, including KCNQ2 and KCNQ3, are localized to several microdomains within vestibular afferent endings, where they influence afferent excitability and could be targeted by EVS neurons. Additionally, the relative expression of KCNQ subunits appears to vary across the sensory epithelia and among different afferent types. However, it is unclear which KCNQ channel subunits are targeted by mAChR activation and whether that also varies among different afferent classes. Here we show that EVS-mediated slow excitation is blocked and enhanced by the non-selective KCNQ channel blocker XE991 and opener retigabine, respectively. Using KCNQ subunit-selective drugs, we observed that a KCNQ2 blocker blocks the slow response in irregular afferents, while a KCNQ2/3 opener enhances slow responses in regular afferents. The KCNQ2 blockers did not appear to affect resting afferent discharge rates, while KCNQ2/3 or KCNQ2/4 openers decreased afferent excitability. Here, we show pharmacological evidence that KCNQ2/3 subunits are likely targeted by mAChR activation in mammalian vestibular afferents. Additionally, we show that KCNQ3 KO mice have altered resting discharge rate as well as EVS-mediated slow response. These data together suggest that KCNQ channels play a role in slow response and discharge rate of vestibular afferents, which can be modulated by EVS in mammals.

neuroscience↗

bioRxiv2023

Elucidating the role of muscarinic acetylcholine receptor (mAChR) signaling in efferent mediated responses of vestibular afferents in mammals

The peripheral vestibular system detects head position and movement through activation of vestibular hair cells (HCs) in vestibular end organs. HCs transmit this information to the CNS by way of primary vestibular afferent neurons. The CNS, in turn, modulates HCs and afferents via the efferent vestibular system (EVS) through activation of cholinergic signaling mechanisms. In mice, we previously demonstrated that activation of muscarinic acetylcholine receptors (mAChRs), during EVS stimulation, gives rise to a slow excitation that takes seconds to peak and tens of seconds to decay back to baseline. This slow excitation is mimicked by muscarine and ablated by the non-selective mAChR blockers scopolamine, atropine, and glycopyrrolate. While five distinct mAChRs (M1-M5) exist, the subtype(s) driving EVS-mediated slow excitation remain unidentified and details on how these mAChRs alter vestibular function is not well understood. The objective of this study is to characterize which mAChR subtypes drive the EVS-mediated slow excitation, and how their activation impacts vestibular physiology and behavior. In C57Bl/6J mice, M3mAChR antagonists were more potent at blocking slow excitation than M1mAChR antagonists, while M2/M4 blockers were ineffective. While unchanged in M2/M4mAChR double KO mice, EVS-mediated slow excitation in M3 mAChR-KO animals were reduced or absent in irregular afferents but appeared unchanged in regular afferents. In agreement, vestibular sensory-evoked potentials (VsEP), known to be predominantly generated from irregular afferents, were significantly less enhanced by mAChR activation in M3mAChR-KO mice compared to controls. Finally, M3mAChR-KO mice display distinct behavioral phenotypes in open field activity, and thermal profiles, and balance beam and forced swim test. M3mAChRs mediate efferent-mediated slow excitation in irregular afferents, while M1mAChRs may drive the same process in regular afferents.

neuroscience↗

bioRxiv2023

MPK4 mediated phosphorylation of PIF4 controls thermosensing by regulation of H2A.Z deposition in Arabidopsis

Plants have the ability to perceive a slight upsurge in ambient temperature and respond by undergoing morphological changes, such as elongated hypocotyls and early flowering. The dynamic functioning of PHYTOCHROME INTERACTING FACTOR4 (PIF4) in thermomorphogenesis has been well established, although the regulatory pathway involved in thermosensing is not deciphered completely. In our study, we demonstrate that an increase in temperature from 22 to 28 induces the phosphorylation of PIF4 by MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) which shows high expression and activation at 28. Apparently, phosphorylated PIF4 represses the expression of ACTIN-RELATED PROTEIN 6 (ARP6) that is required for mediating histone variant H2A.Z deposition at its target gene loci. We demonstrate that variation of ARP6 expression in PIF4 phosphor -null and phosphor-mimetic seedlings affects hypocotyl growth and flowering at 22 and 28. Further, we show that change in ARP6 expression affects H2A.Z deposition at the loci of genes involved in hypocotyl elongation using PIF4 phosphor -null and phosphor-mimetic seedlings. Interestingly, the expression of MPK4 is also controlled by H2A.Z deposition in temperature dependent manner. Taken together, our findings highlight the cumulative molecular interplay between MPK4, PIF4, and chromatin modification by ARP6-mediated H2A.Z deposition as a regulatory mechanism of thermosensing.

plant biology↗

bioRxiv2023

Substrate availability and dietary fibre regulate metabolism of tryptophan by human gut microbes

Tryptophan is catabolized by gut microbes, resulting in a wide range of metabolites implicated in both beneficial and adverse host effects. However, it remains elusive how the gut microbial tryptophan metabolism is governed either towards indole, associated with adverse effects in chronic kidney disease, or towards indolelactic acid (ILA) and indolepropionic acid (IPA), associated with protective effects in type 2 diabetes and inflammatory bowel disease. Here, we used human fecal cultures in combination with a controlled three-species model to test competition for tryptophan, and measured the resulting metabolites both in vitro and in gnotobiotic mice colonized with the three species. We revealed that the generation of specific tryptophan-derived metabolites was not predominantly determined by the abundance of tryptophan metabolizing bacteria, but rather by substrate-dependent regulation of specific metabolic pathways. In vitro and in vivo, indole-producing Escherichia coli and ILA- and IPA-producing Clostridium sporogenes competed for tryptophan. Importantly, the fibre degrading Bacteroides thetaiotaomicron affected this competition by cross-feeding monosaccharides to E. coli, which inhibited indole production through catabolite repression, and thereby made more tryptophan available to C. sporogenes, increasing ILA and IPA production. We thus present the first mechanistic explanation for why consumption of fermentable fibres suppress indole production but promote the generation of other tryptophan metabolites associated with health benefits. We conclude that the availability of tryptophan and dietary fibre regulates gut microbiome tryptophan metabolism pathways, and consequently influences the balance between the different tryptophan catabolites generated. This balance has implications for host-microbial cross-talk affecting human health.

microbiology↗

bioRxiv2023

Exoribonuclease RNase R protects Antarctic Pseudomonas syringae Lz4W from DNA damage and oxidative stress

RNase R is a highly processive, 3 -5 exoribonuclease involved in RNA degradation, maturation, and processing in bacteria. In Pseudomonas syringae Lz4W, RNase R interacts with RNase E to form the RNA degradosome complex and is essential for growth at low temperature. RNase R is also implicated in general stress response in many bacteria. We show here that the deletion mutant of rnr gene (encoding RNase R) of P. syringae is highly sensitive to various DNA damaging agents and oxidative stress. RNase R is a multidomain protein comprised of CSD, RNB and S1 domains. We investigated the role of each domain of RNase R and its exoribonuclease activity in nucleic acid damage and oxidative stress response. Our results revealed that the RNB domain alone without its exoribonuclease activity is sufficient to protect against DNA damage and oxidative stress. We also show that the association of RNase R with the degradosome complex is not required for this function. Our study has discovered for the first time a hitherto unknown role of RNase R in protecting P. syringae Lz4W against DNA damage and oxidative stress. ImportanceBacterial exoribonucleases play a crucial role in RNA maturation, degradation, quality control and turnover. In this study, we have uncovered a previously unknown role of 3-5 exoribonuclease RNase R of P. syringae Lz4W in DNA damage and oxidative stress response. Here, we show that neither the exoribonuclease function of RNase R, nor its association with the RNA degradosome complex is essential for this function. Interestingly, in P. syringae Lz4W, hydrolytic RNase R exhibits physiological roles similar to phosphorolytic 3-5 exoribonuclease PNPase of E. coli. Our data suggest that during the course of evolution, mesophilic E. coli and psychrotrophic P. syringae have apparently swapped these exoribonucleases to adapt to their respective environmental growth conditions.

microbiology↗

bioRxiv2021

A novel positive feedback mechanism of ABI5 phosphorylation by mitogen activated protein kinase-3 regulates ABA signaling in Arabidopsis

Seed germination is the crucial physiological process regulated by both environmental and endogenous phytohormones. ABA negatively regulates seed germination, post-germination growth and floral transition, however, the cross talk between multiple regulatory pathways are still unclear. Here, we show that ABA activates two MAP kinases, AtMPK3/AtMPK6 and selectively regulates the transcript of AtMPK3 through ABI5, a master regulator of ABA signaling. As a feedback loop, AtMPK3 interacts and phosphorylates ABI5 at the serine-314 position. ABI5 phosphorylation by MAP kinases positively regulates ABI5 nuclear localization and negatively regulates its dimerization. Subcellular localization of ABI5 phospho-null protein further suggests the role of phosphorylation in regulation of its cytoplasmic stability and its nuclear dimerization. Overexpression of phosphor-null ABI5 in abi5-8 mutant restored the ABA sensitivity during seed germination and delayed the floral transition as compared to phospho-mimic ABI5. Additionally, overexpression of constitutive phosphorylated ABI5 in abi5-8 mutants suggest that phosphorylation makes ABI5 partially inactive. Furthermore, phospho-null ABI5 plants showed drought sensitive phenotype whereas, mpk3, mkk4, mkk5, abi5-8 and phosphor-mimic plants showed drought tolerant phenotype. Our findings present a new insight between MAP kinase cascade and ABA signaling which collectively regulates the ABA response through ABI5 phosphorylation.

plant biology↗

bioRxiv2020

The RelA hydrolase domain: a molecular switch for (p)ppGpp synthesis

Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of {gamma}-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114-130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wildtype RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

molecular biology↗

bioRxiv2020

A novel cross talk of AtRAV1, an ethylene responsive transcription factor with MAP kinases imparts broad spectrum disease resistance in plants

Plant diseases pose a serious threat to sustainable agriculture as controlling them in eco-friendly manner remains a challenge. In this study, we establish RAV1 as a master transcriptional regulator of defense genes in model plant Arabidopsis. The overexpression of AtRAV1 provided disease resistance against necrotrophic fungal pathogen (Rhizoctonia solani) infection in A. thaliana. The transgenic lines exhibited enhanced expression of several defense genes including mitogen associated protein kinases (MAPKs) and the amplitude of their expression was further enhanced upon pathogen infection. Conversely, the atrav1 mutant plants were unable to induce the expression of these defense genes and were highly susceptible to infection. Our data suggests that upon pathogen attack, AtRAV1 transcriptionally upregulate the expression of MAPKs (AtMPK3, AtMPK4 and AtMPK6) and AtMPK3 and AtMPK6 are essential for AtRAV1 mediated disease resistance. Further, we demonstrate that AtRAV1 is a phosphorylation target of AtMPK3 (but not AtMPK6) and the phospho-defective variants of AtRAV1 are unable to induce disease resistance in A. thaliana. Considering the presence of AtRAV1 orthologs in diverse plant species, we propose that they can be gainfully deployed to control economically important diseases. In deed we observe that overexpression of tomato ortholog of AtRAV1 (SlRAV1) provides broad spectrum disease resistance against bacterial (Ralstonia solanacearum), fungal (R. solani) and viral (Tomato leaf curl virus) infections in tomato.

plant biology↗

bioRxiv2019

Fatty acid starvation activates RelA by depleting lysine precursor pyruvate

Bacteria experiencing nutrient starvation induce the ubiquitous stringent response, a profound physiological change that reprograms cell physiology from fast to slow growth and stress survival. The stringent response is mediated by the secondary messengers pppGpp and ppGpp collectively referred to as (p)ppGpp or \"alarmone\". In Escherichia coli, two paralogs, RelA and SpoT, synthesize (p)ppGpp. RelA is activated by amino acid starvation whereas SpoT, which can also degrade (p)ppGpp, responds to fatty acid (FA), carbon and phosphate starvation. Here, we discover that FA starvation leads to rapid activation of RelA and reveal the underlying mechanism. We show that fatty acid starvation leads to depletion of lysine that, in turn, leads to the accumulation of uncharged tRNAlys and activation of RelA. SpoT was also activated by fatty acid starvation but to a lower level and with a delayed kinetics. Next, we discovered that pyruvate, a precursor of lysine, is depleted by FA starvation. We also propose a mechanism that explains how FA starvation leads to pyruvate depletion. Together our results indicate that many responses to nutrient depletion may ultimately result indirectly from depletion of amino acids and thereby activating RelA. Interestingly, FA starvation provoked a ~100-fold increase in relA dependent ampicillin tolerance.

microbiology↗

bioRxiv2018

Dynamic regulation of HYL1 provides new insights into its multifaceted role in Arabidopsis

MicroRNAs (miRNAs) are 21 to 24 nucleotide non-coding RNAs that regulate gene expression. Biogenesis of miRNAs is fine-tuned by specialized microprocessor complex, the regulation of which is being continuously understood. Recruitment of HYL1 to the microprocessor complex is crucial for accurate primary-miRNA (pri-miRNA) processing and accumulation of mature miRNA in Arabidopsis thaliana. HYL1 is a double-stranded RNA binding protein also termed as DRB1, has two double-stranded RNA binding domain at N-terminal and a highly disordered C-terminal region. Also, the biological activity of HYL1 is dynamically regulated through transition from hyperphosphorylation to hypophosphorylation state. HYL1 is known to be phosphorylated by a MAP kinase MPK3 and SnRK2. However, the precise role of its phosphorylation are still unknown. Recently, the stability of HYL1 protein has been shown to be regulated by an unknown protease X. However, the identity of the protease and its molecular mechanisms are poorly understood. Here, we describe, three functionally important facets of HYL1, which provide a better picture of its association with molecular processes. First, we identified a conserved MPK3 phosphorylation site on HYL1 and its possible role in the miRNA biogenesis. Secondly, the C-terminal region of HYL1 displays tendencies to bind dsDNA. Lastly, the role of C-terminal region of HYL1 in the regulation of its protein stability and the regulation of miRNA biogenesis is documented. We show the unexplored role of C-terminal and hypothesize the novel functions of HYL1 in addition to miRNA biogenesis. We anticipate that the data presented in this study, will open a new dimension of understanding the role of double stranded RNA binding proteins in diverse biological processes of plants and animal.

plant biology↗

bioRxiv2018

Biochemical Characterization of RecBCD Enzyme from An Antarctic Pseudomonas Species and Identification of Its Cognate Chi (χ) Sequence

Pseudomonas syringae Lz4W RecBCD enzyme, RecBCDPs, is a trimeric protein complex comprised of RecC, RecB, and RecD subunits. RecBCD enzyme is essential for P. syringae growth at low temperature, and it protects cells from low temperature induced replication arrest. In this study, we show that the RecBCDPs enzyme displays distinct biochemical behaviors. Unlike E. coli RecBCD enzyme, the RecD subunit is indispensable for RecBCDPs function. The RecD motor activity is essential for the Chi-like fragments production in P. syringae, highlighting a distinct role for P. syringae RecD subunit in DNA repair and recombination process. Further, the ssDNA-dependent endonuclease activity is notably absent in RecBCDPs enzyme. Here, we demonstrate that the RecBCDPs enzyme recognizes a unique octameric DNA sequence, 5'-GCTGGCGC-3' (ChiPs) that attenuates nuclease activity of the enzyme when it enters dsDNA from the 3'-end. We propose that the reduced translocation activities manifested by motor-defective mutants cause cold sensitivity in P. syrinage; emphasizing the importance of DNA processing and recombination functions in rescuing low temperature induced replication fork arrest.\n\nAbbreviations

biochemistry↗