Biology subjects

Talasila, M.

Publications and source records attributed to Talasila, M..

7 recordsLinked to original sources

bioRxiv2025

The plant circadian clock exerts stronger control over the diel proteome than the transcriptome

AbstractThe plant circadian clock is a genetic circuit composed of multiple mutually-regulating transcription factors that together synchronize internal biological rhythms to the [~]24-hour period of planetary rotation. While it has been known for over a decade that nearly 40% of the transcriptome in the model plant Arabidopsis oscillates with a circadian rhythm, it is yet unclear to what extent this translates to the proteome. Here, through parallel quantitative proteome and transcriptome time-course profiling of Arabidopsis wild-type plants and a panel of clock deficient plant lines, we show that specific clock genes exercise extensive control over diel proteome rhythmicity, and to a much greater extent than they do the transcriptome. This control results in a clock-dependent synchronization of rhythmic proteins along a bimodal phase distribution that is lost in circadian clock deficient plants. This suggests pervasive post-translational control of gene expression by specific elements of the circadian system, notably the morning expressed LHY/CCA1 module. Our findings imply that the circadian clock exercises much greater control of gene expression through proteostasis mechanisms than previously recognised, necessitating a recalibration of our current understanding of clock proteins as primarily transcriptional regulators.

plant biology↗

bioRxiv2025

TurboID-based proximity labeling enables in vivo mapping of Plasmodiophora brassicae secretome in Arabidopsis

O_LIPlasmodiophora brassicae, the causal agent of clubroot disease, is an obligate biotrophic protist belonging to the poorly characterized Rhizaria. Its intracellular lifestyle and resistance to genetic manipulation have hindered functional analysis of its effector repertoire, leaving mechanisms underlying disease development unresolved. Here, we sought to experimentally define the P. brassicae secretome within infected plant cells and identify effectors targeted to specific host subcellular compartments. C_LIO_LIA proximity labeling approach based on the TurboID biotin ligase was used to capture pathogen-derived proteins within the nucleus, cytosol, endoplasmic reticulum, and plasma membrane of infected Arabidopsis roots during primary and secondary stages of clubroot disease. C_LIO_LIThis strategy yielded the first in planta experimental view of the P. brassicae secretome, identifying both established and previously uncharacterized effectors. The resulting dataset provides a valuable resource and methodological framework for dissecting effector function in this and other intracellular plant pathogens. C_LIO_LIThis study expands our understanding of Rhizarian pathogenicity and provides a methodological template for identifying the secretomes of other obligatory intracellular plant pathogens. C_LI

plant biology↗

bioRxiv2025

Optimization of Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) for effective low-disruption labelling of Arabidopsis proteins in vivo

Plants require robust responses in protein synthesis to adapt to variable environmental conditions. Measurement of newly synthesized proteins has been successfully facilitated with Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) across a multitude of organisms. Here, we use non-canonical amino acids (NCAAs) L-azidohomoalanine (AHA) or L-homopropargylglycine (HPG) incorporation in place of methionine residues into the actively translating Arabidopsis proteome, allowing for that subset of proteins to be enriched for mass spectrometry quantification. Although this technique has seen occasional use in plants, optimization of the protocol to maximize functionality while minimizing organismal stress has not yet been established. Here, we provide evidence for successful implementation through the liquid immersion of seedlings in AHA or HPG-containing media that functions with significantly lower concentrations than the literature standard. Our approach splits acute exposure and incorporation phase of labelling to mitigate potential negative impacts of prolonged NCAA exposure without compromising effective enrichment capacity, and demonstrate that this results in an unperturbed growth phenotype for AHA-treated seedlings. Finally, we demonstrate the capacity of this modified approach to enrich newly synthesized proteins from the whole proteome under standard stress conditions. These improvements allow for a broader use of BONCAT technologies in molecular plant research, affording a deeper understanding of the newly synthesized proteome without negatively impacting plant health.

plant biology↗

bioRxiv2025

Chemical Inhibition of Splicing-Related Protein Kinases Reveals Phosphorylation-Driven Regulation of RNA Alternative Splicing in Arabidopsis Seedlings

Serine / arginine (SR) protein kinases (SRPKs) are capable of transmitting external signals to the spliceosome by phosphorylating SR proteins. In plants, few studies have looked at the regulation of RNA splicing by post-translational modification (PTMs), despite the spliceosome and splicing factor proteins exhibiting extensive phosphorylation. Many of these PTM events have the potential to dramatically change the expression of various genes, such as those involved in abiotic stress. Here, we sought to explore the regulatory function of Arabidopsis thaliana SRPKs in the context of RNA alternative splicing. To do so, we utilized four well-known and well-characterized chemical inhibitors specifically designed to target and inhibit SRPK enzymatic activity. Our data found that SRPK chemical inhibitors SPHINX31 and SRPIN340 induced shorter root development phenotypes and an abolishment of root hair formation. Using a multi-omics approach combining transcriptomics and phosphoproteomics, we find extensive changes in the splicing of genes involved in root development, RNA splicing, cytoskeletal organization, and cell differentiation. We also reveal splicing factors exhibiting differential alternative splicing as well as a down-regulated in their phosphorylation status. Overall, our data indicate that AtSRPKs phosphorylate diverse splicing factors and influence the alternative splicing of genes involved in root development and a wide-range of related pathways.

plant biology↗

bioRxiv2024

Defining the molecular impacts of Humalite application on field-grown wheat (Triticum aestivum L.) using quantitative proteomics

Increasing global food production demands have resulted in increased fertilizer usage, causing detrimental environmental impacts. Biostimulants, such as humic substances, are currently being applied as a strategy to increase plant nutrient-use efficiency and minimize environmental impacts within cropping systems. Humalite is a unique, naturally occurring coal-like substance found in deposits across southern Alberta. These deposits contain exceptionally high ratios of humic acids (>70%) and micronutrients due to their unique freshwater depositional environment. Humalite has begun to be applied to fields based on scientific data suggesting positive impacts on crop growth, yield and nutrient usage; however, little is known about the underlying molecular mechanisms of Humalite. Here, we report a quantitative proteomics approach to identify systems-level molecular changes induced by the addition of different Humalite application rates in field-grown wheat (Triticum aestivum L.) under three urea fertilizer application rates. In particular, we see wide-ranging abundance changes in proteins associated with several metabolic pathways and growth-related biological processes that suggest how Humalite modulates the plant molecular landscape. Overall, our results provide new, functional information that will help better inform agricultural producers on optimal biostimulant and fertilizer usage.

plant biology↗

bioRxiv2024

B4 Raf-like MAPKKK RAF24 regulates Arabidopsis thaliana flowering time through HISTONE MONO-UBIQUITINATION 2

The timing of flowering is a critical agronomic trait governed by a number of external cues. Despite our genetic understanding of flowering time being well established, we have a limited understanding of how these signals are transmitted to different flowering genes through protein phosphorylation. Here, we characterize a novel B4 Raf-like MAPKKK protein kinase called RAF24, whose mutation results in an early flowering phenotype. Comparative analysis to related B4 Raf-like MAPKKKs indicates that RAF24 uniquely affects flowering time, while phosphoproteome analyses found RAF24 impacts the phosphorylation status of proteins involved in distinct flowering pathways. In particular, we found the RING-type ubiquitin ligase HISTONE MONO-UBIQUITINATION 2 (HUB2) to possess the largest phosphorylation change in raf24 deficient plants relative to wild-type Arabidopsis and that RAF24 suppresses ligase activity of HUB2 in order to maintain appropriate levels of H2Bub1. Furthermore, we found that RAF24 regulates HUB2 phosphorylation through subclass I and III SUCROSE NON-FERMENTING KINASE 2 (SnRK2) protein kinases; known substrates of B4 RAF-like MAPKKKs. Lastly, using a combination of phospho-mimetic and -ablative plant lines, we validate the importance of HUB2 phosphorylation at S314 in regulating flowering time. Collectively, our findings implicate RAF24 as a higher-order flowering regulator, while further implicating HUB2 as a centerpiece of flowering regulation.

plant biology↗

bioRxiv2023

Quantitative Time-Course Analysis of Osmotic and Salt Stress in Arabidopsis thaliana using Short Gradient Multi-CV FAIMSpro BoxCar DIA

A major limitation when undertaking quantitative proteomic time-course experimentation is the tradeoff between depth-of-analysis and speed-of-analysis. In high complexity and high dynamic range sample types, such as plant extracts, balance between resolution and time is especially apparent. To address this, we evaluate multiple composition voltage (CV) High Field Asymetric Waveform Ion Mobility Spectrometry (FAIMSpro) settings using the latest label-free single-shot Orbitrap-based DIA acquisition workflows for their ability to deeply-quantify the Arabidopsis thaliana seedling proteome. Using a BoxCarDIA acquisition workflow with a -30 -50 -70 CV FAIMSpro setting we are able to consistently quantify >5000 Arabidopsis seedling proteins over a 21-minute gradient, facilitating the analysis of ~42 samples per day. Utilizing this acquisition approach, we then quantified proteome-level changes occurring in Arabidopsis seedling shoots and roots over 24 h of salt and osmotic stress, to identify early and late stress response proteins and reveal stress response overlaps. Here, we successfully quantify >6400 shoot and >8500 root protein groups, respectively, quantifying nearly ~9700 unique protein groups in total across the study. Collectively, we pioneer a short gradient, multi-CV FAIMSpro BoxCarDIA acquisition workflow that represents an exciting new analysis approach for undertaking quantitative proteomic time-course experimentation in plants.

plant biology↗