Biology subjects

Uhrig, R. G.

Publications and source records attributed to Uhrig, R. G..

At least 19 recordsLinked to original sources

bioRxiv2026

Biochemical regulation of Arabidopsis PUB33: a receptor-like cytoplasmic kinase with an integrated U-box domain that ubiquitinates Ralstonia pseudosolanacearum effector protein RipV1

Plant immunity relies on the detection of microbes and the rapid activation of intracellular defense pathways. Catalyzed by protein kinases and E3 ubiquitin ligases, respectively, phosphorylation and ubiquitination are among the most abundant post-translational modifications that regulate immune pathways. It has been well established that members of the receptor-like cytoplasmic kinase (RLCK) and plant U-box E3 ligase (PUB) families are critical components of plant immune signaling. Interestingly, a group of proteins that contain both an RLCK domain and a PUB domain has been conserved throughout plant evolution, referred to as subgroups RLCK-IXb and PUB-VI within their respective families. While very little is known about these proteins, evidence from multiple independent studies indicates that orthologous PUB-VI/RLCK-IXb proteins in potato, tomato, Nicotiana benthamiana, and Arabidopsis thaliana associate with diverse pathogen effectors from the oomycete pathogen Phytophthora infestans, bacterial pathogen Ralstonia pseudosolanacearum, and the mirid bug Apolygus lucorum, suggesting that they may be critical virulence targets or components of the immune response. However, the biochemical activities of these proteins and how they contribute to plant health remain poorly defined. Here, we introduce the PUB-VI/RLCK-IXb clade in Arabidopsis, focusing on PUB32, PUB33, and PUB50. We show that PUB33 exhibits dual kinase and E3 ubiquitin ligase activities that are inversely regulated by autophosphorylation at Thr333. PUB33 forms homomers and heteromers with PUB32 which attenuate PUB33 catalytic activity. Although we did not observe clear defects in innate immune signaling in pub32, pub33, or pub50 mutants, we found that overexpression of PUB33 can suppress cell death triggered by the R. pseudosolanacearum effector RipV1 in N. benthamiana. Moreover, PUB33 directly ubiquitinates RipV1 in vitro and reduces RipV1 accumulation in planta, suggesting that it functions as part of the immune response against R. pseudosolanacearum.

plant biology↗

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

Phosphate resupply differentially impacts the shoot and root proteomes of Arabidopsis thaliana seedlings

Phosphate (Pi) is an essential macronutrient for plant development that is often limited in soil environments. Plants have evolved myriad dynamic biochemical, physiological, and morphological adaptations to cope with nutritional Pi deficiency, collectively known as the Pi starvation response (PSR). While many components of the PSR have been well-characterized, much less is known about how metabolic homeostasis is re-established upon Pi resupply, particularly with respect to tissue- and time-specific adaptations. Here, we applied label-free quantitative proteomics to quantify protein-level changes in Arabidopsis thaliana shoots and roots following Pi resupply after prolonged Pi deprivation, quantifying a total of [~]2,700 differentially abundant proteins (DAPs). Sampling at early (1 h) and late (48 h) time-points, we captured a combination of immediate signaling and metabolic responses, along with longer-term recovery processes. Early responses prioritized metabolic adjustments to restore Pi pools via enhanced glycolysis and energy production, followed by later shifts toward anabolism, including nucleotide production, membrane remodeling, and protein synthesis. Several key enzymes, including ALTERNATIVE OXIDASE 1A, FRUCTOSE- BISPHOSPHATE ALDOLASE 5, and subunits of PHOTOSYSTEM I exhibited unique tissue-specific and time-dependent regulation. Overall, our findings reveal dynamic temporal phases of metabolic reprogramming during recovery from Pi starvation, and identify candidate proteins involved in orchestrating this transition, ultimately identifying potential targets for enhancing Pi uptake- and use-efficiency in crops. While hydroponic liquid culture enabled precise control of Pi availability, soil responses may be further influenced by heterogeneity and other root interactions.

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↗

bioRxiv2025

Prolific release of S-layer units and associated proteins by the methanotrophic bacterium Methylomicrobium album BG8

Some methanotrophs synthesize S-layers that overlay their outer membrane. TEM imaging revealed that Methylomicrobium album BG8 constitutively sheds abundant S-layer units, a phenotype not found in 7 other methanotrophs, even though Methylotuvimicrobium buryatense 5GB1 produces a similar structure. Release of S-layer units occurred regardless of carbon (methane or methanol) or nitrogen (ammonium or nitrate) source, with 50X trace metals, under copper deprivation, and at all growth phases. The released S-layer units were isolated from the culture medium of M. album BG8 by density gradient centrifugation for proteome analysis. The proteome revealed the S-layer protein subunits, transporters for calcium uptake including TolC and Repeats-in-Toxin (RTX) proteins, transporters for uptake for cobalamin and siderophores, cell wall biogenesis proteins, and proteins with Type I secretion system (T1SS) target domains. M. album BG8 adapted to grow at pH 4 lost its S-layer and genome analysis revealed a frameshift mutation plus reduced expression of the S-layer unit gene plus the deletion and almost no expression of an S-layer-associated porin gene. Together, the results suggest that the biogenesis and secretion of M. album BG8 S-layer is mediated by its associated T1SS, the S-layer possesses metal acquisition functions, and low pH adaptation of M. album BG8 results in loss of S-layer, likely due to reduced, or incomplete, expression of S-layer units and loss of an associated porin. The involvement of the T1SS and shedding phenotype of the S-layer in M. album BG8 could be applied towards selective secretion of proteins and other factors of bioindustrial interest. ImportanceThe methanotrophic bacterium M. album BG8 produces and sheds large quantities of S-layer units into the culture medium regardless of carbon or nitrogen source, metal availability or growth phase. Of the 8 methanotrophic bacteria screened, only M. album BG8 possessed this phenotype. Proteomics analysis of density gradient purified culture supernatant identified the S-layer protein units and proteins involved in metal uptake and S-layer biogenesis, some with secretion signals for the T1SS. M. album BG8 adapted to grow at low pH lost production of its S-layer due to mutations in the genes encoding S-layer units and an associated porin. Better understanding of M. album BG8 S-layer production and its shedding phenotype could be harnessed for exporting expressed proteins and bioproducts of industrial interest for ease of collection and downstream processing.

microbiology↗

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

Shaping Kale Morphology and Physiology Using Different LED Light Recipes

Light serves as a fundamental factor in plant development, both as an energy source and as an environmental cue. With the advent of light-emitting diode (LED) technology, light can be precisely manipulated to influence key plant traits. Here, we assess effects of light intensity and spectral composition on the growth and physiology of Kale (Brassica oleracea). Kale is known for its phenotypic plasticity and nutritional composition, making it a crop well-suited for indoor cultivation either as microgreens or as large leafy plants. Here, we employ a combination of advanced phenotyping, computer vision, gas chromatography-mass spectrometry (GC-MS) metabolomics, and liquid chromatography-mass spectrometry (LC-MS)-based quantitative proteomics to characterize the molecular changes that underpin light-dictated differences in the growth and metabolism of two different commercially grown kale cultivars under different light intensities and spectral compositions. We identify time-of-day and cultivar-specific light intensity and spectral composition-induced changes related to growth, shade avoidance, photosynthesis and several aspects of nutritional composition, including amino acids, glucosinolates and carotenoids. Our results offer a key resource to the plant community and demonstrate the translational potential of light manipulation in tailoring kale growth and nutritional content for enhanced crop productivity and/or health benefits, while simultaneously offering a more cost-effective solution for contemporary agricultural challenges. Significance StatementThe effects of light intensity and spectral composition differentially affect the diel molecular responses of Kale (Brassica oleracea). Our results demonstrate the translational potential of light manipulation in tailoring plant growth and nutritional content for enhanced crop productivity.

plant biology↗

bioRxiv2024

Conservation of an immune homeostasis module in land plants

Calcium-dependent protein kinases (CDPKs) decode cellular calcium transients and play diverse roles in plant growth and stress responses, including immunity. In Arabidopsis thaliana (At, Arabidopsis thereafter), AtCPK28 contributes to immune homeostasis by phosphorylating subgroup IV plant U-box proteins AtPUB22/24/25/26, which target the key immune receptor-like cytoplasmic kinase (RLCK) AtBIK1 for turnover. While this module is conserved in multiple angiosperms, it is unclear if the role of CPK28 in immune homeostasis is conserved more broadly across land plants. Here, we took an evolutionary comparative approach to understand the role of CPK28. We identified a single CPK28 ortholog in the liverwort Marchantia polymorpha, MpCPK28, which exhibits Ca2+-dependent kinase activity that is inhibited by calmodulin in vitro. We identified the subgroup IV plant U-box protein MpPUB20e as a substrate of MpCPK28. MpPUB20e is able to ubiquitinate MpPBLa, the functional ortholog of AtBIK1. We also provide preliminary evidence that MpPBLa undergoes proteasomal degradation in Marchantia, suggesting that optimization of MpPBLa protein accumulation is conserved across land plants. Interestingly, while loss of CPK28 function in multiple angiosperm species results in enhanced immune signaling, we find that Marchantia Mpcpk28 mutant alleles do not display enhanced immune-triggered production of reactive oxygen species or resistance to two pathogens. However, transgenic expression of MpCPK28 was able to restore function in Arabidopsis cpk28-1 mutants, suggesting latent functional conservation of MpCPK28. Furthermore, while AtCPK28-mediated phosphorylation of Thr95/94 on AtPUB25/26 is known to contribute to their activation, we could not observe a functional role for the equivalent residue Thr122 on MpPUB20e. Taken together, our results suggest that post-translational fine-tuning by CPK28 is likely to have refined the PUB-BIK1 module in the vascular plant lineages.

plant biology↗

bioRxiv2024

Quantitative proteomic analysis of soil-grown Brassica napus responses to nutrient deficiency

ABSTRACTMacronutrients such as nitrogen (N), phosphorus (P), potassium (K), and sulphur (S) are critical for plant growth and development. Field-grown canola (Brassica napus L.) is supplemented with fertilizers to maximize plant productivity, while deficiency in these nutrients can cause significant yield loss. A holistic understanding of the interplay between these nutrient deficiency responses in a single study and canola cultivar is thus far lacking, hindering efforts to increase the nutrient use efficiency of this important oil seed crop. To address this, we performed a comparative quantitative proteomic analysis of both shoot and root tissue harvested from soil-grown canola plants experiencing either nitrogen, phosphorus, potassium, or sulphur deficiency. Our data provide critically needed insights into the shared and distinct molecular responses to macronutrient deficiencies in canola. Importantly, we find more conserved responses to the four different nutrient deficiencies in canola roots, with more distinct proteome changes in aboveground tissue. Our results establish a foundation for a more comprehensive understanding of the shared and distinct nutrient deficiency response mechanisms of canola plants and pave the way for future breeding efforts.

plant biology↗

bioRxiv2024

Phospho-proteomics identifies D-group MAP kinases as substrates of the Arabidopsis tyrosine phosphatase RLPH2

Despite being one of the few bona fide plant tyrosine phosphatases, RLPH2 has no known substrates. Utilizing phospho-proteomics, we identified the activation loop phospho-tyrosine of several D-group mitogen activated protein kinases (MPKs) as potential RLPH2 substrates. All Arabidopsis D-Group MPKs possess a TDY activation loop phosphorylation motif, whereas other MPKs (Groups A, B and C) contain a TEY motif. Our findings reveal that RLPH2 has a strong preference for aspartate (D) in the TXY motif, providing specificity for RLPH2 to exclusively target and dephosphorylate the D-Group MPKs. Additionally, D-Group MPKs contain a unique activation loop insertion that conforms to a protein phosphatase 1 (PP1) binding motif, with findings presented here confirming Arabidopsis PP1 phosphatases dock at this site. Intriguingly, only D-group MPKs among all identified Arabidopsis protein kinases possess this PP1 recruiting motif. Using multiple RLPH2 deficient plant lines, we demonstrate that RLPH2 represses seed dormancy release. Overall, this work highlights the power of phospho-proteomics in identifying substrates of this novel plant tyrosine phosphatase, while also revealing new complexities in the interactions between MPK activation loops and multiple phospho-mediated cell signaling events. One sentence summaryPhospho-proteomic analysis reveals Arabidopsis tyrosine phosphatase RLPH2 dephosphorylates the activation loop of D-Group mitogen activated protein kinases.

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↗

bioRxiv2024

Catalytically inactive subgroup VIII receptor-like cytoplasmic kinases regulate the immune-triggered oxidative burst in Arabidopsis thaliana

Protein kinases are key components of multiple cell signaling pathways. Several protein kinases of the receptor-like cytoplasmic kinase (RLCK) family have demonstrated roles in immune and developmental signaling across various plant species, making them a family of interest in the study of phosphorylation-based signal relay. Here, we present our investigation of a subfamily of RLCKs in Arabidopsis thaliana. Specifically, we focus on subgroup VIII RLCKs: MAZ and its paralog CARK6, as well as CARK7 and its paralog CARK9. We found that both MAZ and CARK7 associate with the calcium-dependent protein kinase CPK28 in planta, and furthermore that CPK28 phosphorylates both MAZ and CARK7 on multiple residues in areas that are known to be critical for protein kinase activation. Genetic analysis suggests redundant roles for MAZ and CARK6 as negative regulators of the immune-triggered oxidative burst. We find evidence that supports homo- and hetero-dimerization between CARK7 and MAZ, which may be a general feature of this protein family. Multiple biochemical experiments suggest that neither MAZ nor CARK7 demonstrate catalytic protein kinase activity in vitro. Interestingly, we find that a mutant variant of MAZ incapable of protein kinase activity is able to complement maz-1 mutants, suggesting noncatalytic roles of MAZ in planta. Overall, our study identifies subgroup VIII RLCKs as new players in Arabidopsis immune signaling and highlights the importance of noncatalytic functions of protein kinases.

plant biology↗

bioRxiv2023

Twilight length alters growth, flowering time, and clock function in Arabidopsis via LHY/CCA1

The plant circadian clock governs growth and development by syncing biological processes to periodic environmental changes. Decades of research has shown how the clock enables plants to respond to two environmental variables that change at different latitudes and over different seasons: photoperiod and temperature. However, a third variable, twilight length, has so far gone unstudied. The duration of twilight changes across the planet, lengthening with latitude and changing across seasons, and yet most circadian experiments are performed in lab environments with no twilight. Here, using controlled growth setups, we show that twilight length impacts plant growth and changes flowering time via the LHY/CCA1 morning module of the Arabidopsis circadian clock. Using a series of progressively truncated no-twilight photoperiods, we also found that plants are more sensitive to twilight length compared to equivalent changes in solely photoperiod. Further, transcriptome and proteome analyses indicated that twilight length alters the regulation of proteins involved in reactive oxygen species metabolism, photosynthesis, and carbon metabolism. Genetic analyses also implicated a twilight sensing pathway from phytochromes D and E, the morning elements of the circadian clock and modulating flowering time through the GI-FT pathway. Overall, our findings call for more nuanced models of daylength perception in plants and posit that twilight length is an important determinant of plant growth, development, and circadian function.

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↗

bioRxiv2022

Integrated Systems-Level Proteomics and Metabolomics Reveals the Diel Molecular Landscape of Diverse Kale Cultivars

Kale are a group of diverse Brassicaceae species that are nutritious leafy greens consumed for their abundance of vitamins and micronutrients. Typified by their curly, serrated and/or wavy leaves, kale varieties have been primarily defined based on their leaf morphology and geographic origin, despite having complex genetic backgrounds. Kale is a very promising crop for vertical farming due to its high nutritional content; however, being a non-model organism, foundational, systems-level analyses of kale are lacking. Previous studies in kale have shown that time-of-day harvesting can affect its nutritional composition. Therefore, to gain a systems-level diel understanding of kale across its wide-ranging and diverse genetic landscape, we selected nine publicly available and commercially grown kale cultivars for growth under near-sunlight LED light conditions ideal for vertical farming. We then analyzed changes in morphology, growth and nutrition using a combination of phenomics, proteomics and metabolomics. As the diel molecular activities of plants drive their daily growth and development, ultimately determining their productivity as a crop, we harvested kale leaf tissue at both end-of-day (ED) and end-of-night (EN) time-points for all molecular analyses. Our results reveal that diel proteome and metabolome signatures divide the selected kale cultivars into two distinct groups, defined by their amino acid and sugar content, along with significant proteome differences involving carbon and nitrogen metabolism, mRNA splicing, protein translation and light harvesting. Together, our multi-cultivar, multi-omic analysis provides robust quantitative insights into the molecular underpinnings of the diel growth and development landscape of kale, significantly advancing our fundamental understanding of this nutritious leafy green super-food for next-generation horticulture / vertical farming applications.

plant biology↗

bioRxiv2021

Multi-omic analysis reveals the biochemical changes underpinning the varied phenotypes of the Arabidopsis long-period mutant rve 4 6 8

Plants are able to sense changes in their light environments, such as the onset of day and night, as well as anticipate these changes in order to adapt and survive. Central to this ability is the plant circadian clock, a molecular circuit that precisely orchestrates plant cell processes over the course of a day. REVEILLE proteins (RVEs) are recently discovered members of the plant circadian circuitry that activate the evening complex and PRR genes to maintain regular circadian oscillation. The RVE 8 protein and its two homologs, RVE 4 and 6, have been shown to limit the length of the circadian period, with rve 4 6 8 triple-knockout plants possessing an elongated period along with increased leaf surface area, biomass, cell size and delayed flowering relative to wild-type Col-0 plants. Here, using a multi-omics approach consisting of phenomics, transcriptomics, proteomics, and metabolomics we draw novel connections between RVE8-like proteins and a number of core plant cell processes. In particular, we reveal that loss of RVE8-like proteins results in altered carbohydrate, organic acid and lipid metabolism, including a starch excess phenotype at dawn. We further demonstrate that rve 4 6 8 plants have lower levels of 20S proteasome subunits and possess significantly reduced proteasome activity, potentially explaining the increase in cell-size observed in RVE8-like mutants. Overall, this robust, multi-omic dataset, provides substantial new insights into the far reaching impact RVE8-like proteins have on the diel plant cell environment.

plant biology↗