Mapping protein-metabolite interactions in . by integrating chromatographic techniques and co-fractionation mass spectrometry.

Toward characterization of protein-metabolite interactomes, we recently introduced PROMIS, a co-fractionation-based mass spectrometry approach. However, the challenge lies in distinguishing true interactors from coincidental co-elution when a metabolite co-fractionates with numerous proteins. To address this, we integrated two chromatographic techniques-size exclusion and ion exchange-to enhance the mapping of protein-metabolite interactions (PMIs) in .

Transcript profiling of plastid ferrochelatase two mutants reveals that chloroplast singlet oxygen signals lead to global changes in RNA profiles and are mediated by Plant U-Box 4.

Background: In response to environmental stresses, chloroplasts generate reactive oxygen species, including singlet oxygen (O), an excited state of oxygen that regulates chloroplast-to-nucleus (retrograde) signaling, chloroplast turnover, and programmed cell death (PCD). Yet, the central signaling mechanisms and downstream responses remain poorly understood. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant conditionally accumulates O, and Plant U-Box 4 (PUB4), a cytoplasmic E3 ubiquitin ligase, is involved in propagating O signals for chloroplast turnover and cellular degradation.

An evolutionarily conserved metabolite inhibits biofilm formation in Escherichia coli K-12.

Methylerythritol cyclodiphosphate (MEcPP) is an intermediate in the biosynthesis of isoprenoids in plant plastids and in bacteria, and acts as a stress signal in plants. Here, we show that MEcPP regulates biofilm formation in Escherichia coli K-12 MG1655. Increased MEcPP levels, triggered by genetic manipulation or oxidative stress, inhibit biofilm development and production of fimbriae.

Integrated Dual-Channel Retrograde Signaling Directs Stress Responses by Degrading the HAT1/TPL/IMPα-9 Suppressor Complex and Activating CAMTA3.

Unlabelled: The intricate communication between plastids and the nucleus, shaping stress-responsive gene expression, has long intrigued researchers. This study combines genetics, biochemical analysis, cellular biology, and protein modeling to uncover how the plastidial metabolite MEcPP activates the stress-response regulatory hub known as the Rapid Stress Response Element (RSRE). Specifically, we identify the HAT1/TPL/IMPα- 9 suppressor complex, where HAT1 directly binds to RSRE and its activator, CAMTA3, masking RSRE and sequestering the activator.

Transcript profiling of mutants reveals that chloroplast singlet oxygen signals lead to global changes in RNA profiles and are mediated by Plant U-Box 4.

Background: In response to environmental stresses, chloroplasts generate reactive oxygen species, including singlet oxygen (O), an excited state of oxygen that regulates chloroplast-to-nucleus (retrograde) signaling, chloroplast turnover, and programmed cell death (PCD). Yet, the central signaling mechanisms and downstream responses remain poorly understood. The () mutant conditionally accumulates O and Plant U-Box 4 (PUB4), a cytoplasmic E3 ubiquitin ligase, is involved in propagating O signals for chloroplast turnover and cellular degradation.

ZmPILS6 is an auxin efflux carrier required for maize root morphogenesis.

Plant root systems play a pivotal role in plant physiology and exhibit diverse phenotypic traits. Understanding the genetic mechanisms governing root growth and development in model plants like maize is crucial for enhancing crop resilience to drought and nutrient limitations. This study focused on identifying and characterizing ZmPILS6, an annotated auxin efflux carrier, as a key regulator of various crown root traits in maize.

Activation of stress-response genes by retrograde signaling-mediated destabilization of nuclear importin IMPα-9 and its interactor TPR2.

Stress-induced retrograde signal transmission from the plastids to the nucleus has long puzzled plant biologists. To address this, we performed a suppressor screen of the ceh1 mutant, which contains elevated 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP) levels, and identified the gain-of-function mutant impα-9, which shows reversed dwarfism and suppressed expression of stress-response genes in the ceh1 background despite heightened MEcPP. Subsequent genetic and biochemical analyses established that the accumulation of MEcPP initiates an upsurge in Arabidopsis SKP1-like 1 (ASK1) abundance, a pivotal component in the proteasome degradation pathway.

COG-imposed Golgi functional integrity determines the onset of dark-induced senescence.

Plant survival depends on dynamic stress-response pathways in changing environments. To uncover pathway components, we screened an ethyl methanesulfonate-mutagenized transgenic line containing a stress-inducible luciferase construct and isolated a constitutive expression mutant. The mutant is the result of an amino acid substitution in the seventh subunit of the hetero-octameric conserved oligomeric Golgi (COG) complex of Arabidopsis thaliana.

Dosage differences in 12-OXOPHYTODIENOATE REDUCTASE genes modulate wheat root growth.

Wheat, an essential crop for global food security, is well adapted to a wide variety of soils. However, the gene networks shaping different root architectures remain poorly understood. We report here that dosage differences in a cluster of monocot-specific 12-OXOPHYTODIENOATE REDUCTASE genes from subfamily III (OPRIII) modulate key differences in wheat root architecture, which are associated with grain yield under water-limited conditions.

Reciprocity between a retrograde signal and a putative metalloprotease reconfigures plastidial metabolic and structural states.

Reconfiguration of the plastidial proteome in response to environmental cues is central to tailoring adaptive responses. To define the underlying mechanisms and consequences of these reconfigurations, we performed a suppressor screen, using a mutant () accumulating high levels of a plastidial retrograde signaling metabolite, MEcPP. We isolated a revertant partially suppressing the dwarf stature and high salicylic acid of and identified the mutation in a putative plastidial metalloprotease (VIR3).

CamelliA-based simultaneous imaging of Ca2+ dynamics in subcellular compartments.

As a universal second messenger, calcium (Ca2+) transmits specific cellular signals via a spatiotemporal signature generated from its extracellular source and internal stores. Our knowledge of the mechanisms underlying the generation of a Ca2+ signature is hampered by limited tools for simultaneously monitoring dynamic Ca2+ levels in multiple subcellular compartments. To overcome the limitation and to further improve spatiotemporal resolutions, we have assembled a molecular toolset (CamelliA lines) in Arabidopsis (Arabidopsis thaliana) that enables simultaneous and high-resolution monitoring of Ca2+ dynamics in multiple subcellular compartments through imaging different single-colored genetically encoded calcium indicators.

ORA47 is a transcriptional regulator of a general stress response hub.

Transcriptional regulators of the general stress response (GSR) reprogram the expression of selected genes to transduce informational signals into cellular events, ultimately manifested in a plant's ability to cope with environmental challenges. Identification of the core GSR regulatory proteins will uncover the principal modules and their mode of action in the establishment of adaptive responses. To define the GSR regulatory components, we employed a yeast-one-hybrid assay to identify the protein(s) binding to the previously established functional GSR motif, termed the rapid stress response element (RSRE).

A plastidial retrograde signal potentiates biosynthesis of systemic stress response activators.

Plants employ an array of intricate and hierarchical signaling cascades to perceive and transduce informational cues to synchronize and tailor adaptive responses. Systemic stress response (SSR) is a recognized complex signaling and response network quintessential to plant's local and distal responses to environmental triggers; however, the identity of the initiating signals has remained fragmented. Here, we show that both biotic (aphids and viral pathogens) and abiotic (high light and wounding) stresses induce accumulation of the plastidial-retrograde-signaling metabolite methylerythritol cyclodiphosphate (MEcPP), leading to reduction of the phytohormone auxin and the subsequent decreased expression of the phosphatase PP2C.

The eukaryotic MEP-pathway genes are evolutionarily conserved and originated from Chlaymidia and cyanobacteria.

Background: Isoprenoids are the most ancient and essential class of metabolites produced in all organisms, either via mevalonate (MVA)-and/or methylerythritol phosphate (MEP)-pathways. The MEP-pathway is present in all plastid-bearing organisms and most eubacteria. However, no comprehensive study reveals the origination and evolutionary characteristics of MEP-pathway genes in eukaryotes.

Plastidial retrograde modulation of light and hormonal signaling: an odyssey.

The transition from an engulfed autonomous unicellular photosynthetic bacterium to a semiautonomous endosymbiont plastid was accompanied by the transfer of genetic material from the endosymbiont to the nuclear genome of the host, followed by the establishment of plastid-to-nucleus (retrograde) signaling. The retrograde coordinated activities of the two subcellular genomes ensure chloroplast biogenesis and function as the photosynthetic hub and sensing and signaling center that tailors growth-regulating and adaptive processes. This review specifically focuses on the current knowledge of selected stress-induced retrograde signals, genomes uncoupled 1 (GUN1), methylerythritol cyclodiphosphate (MEcPP), apocarotenoid and β-cyclocitral, and 3'-phosphoadenosine 5'-phosphate (PAP), which evolved to establish the photoautotrophic lifestyle and are instrumental in the integration of light and hormonal signaling networks to ultimately fashion adaptive responses in an ever-changing environment.

DHH1/DDX6-like RNA helicases maintain ephemeral half-lives of stress-response mRNAs.

Gene transcription is counterbalanced by messenger RNA decay processes that regulate transcript quality and quantity. We show here that the evolutionarily conserved DHH1/DDX6-like RNA hellicases of Arabidopsis thaliana control the ephemerality of a subset of cellular mRNAs. These RNA helicases co-localize with key markers of processing bodies and stress granules and contribute to their subcellular dynamics.

Retrograde Induction of phyB Orchestrates Ethylene-Auxin Hierarchy to Regulate Growth.

Exquisitely regulated plastid-to-nucleus communication by retrograde signaling pathways is essential for fine-tuning of responses to the prevailing environmental conditions. The plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) has emerged as a stress signal transduced into a diverse ensemble of response outputs. Here, we demonstrate enhanced phytochrome B protein abundance in red light-grown MEcPP-accumulating mutant Arabidopsis () plants relative to wild-type seedlings.

Uncovering the functional residues of isoprenoid biosynthesis enzyme HDS.

The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant () led to the isolation of 3 revertants (designated -) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS).

Jasmonates-Mediated Rewiring of Central Metabolism Regulates Adaptive Responses.

The lipid-derived hormones jasmonates (JAs) play key functions in a wide range of physiological and developmental processes that regulate growth, secondary metabolism and defense against biotic and abiotic stresses. In this connection, biosynthesis, tissue-specific distribution, metabolism, perception, signaling of JAs have been the target of extensive studies. In recent years, the involvement of JAs signaling pathway in the regulation of growth and adaptive responses to environmental challenges has been further examined.

Orthogonal regulation of phytochrome B abundance by stress-specific plastidial retrograde signaling metabolite.

Plant survival necessitates constant monitoring of fluctuating light and balancing growth demands with adaptive responses, tasks mediated via interconnected sensing and signaling networks. Photoreceptor phytochrome B (phyB) and plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) are evolutionarily conserved sensing and signaling components eliciting responses through unknown connection(s). Here, via a suppressor screen, we identify two phyB mutant alleles that revert the dwarf and high salicylic acid phenotypes of the high MEcPP containing mutant ceh1.

The B-box protein BBX19 suppresses seed germination via induction of ABI5.

Seed germination is a fundamental process in the plant life cycle and is regulated by functionally opposing internal and external inputs. Here we explored the role of a negative regulator of photomorphogenesis, a B-box-containing protein (BBX19), as a molecular link between the inhibitory action of the phytohormone abscisic acid (ABA) and the promoting role of light in germination. We show that seeds of BBX19-overexpressing lines, in contrast to those of BBX19 RNA interference lines, display ABA hypersensitivity, albeit independently of elongated hypocotyl 5 (HY5).

Waterlogging tolerance rendered by oxylipin-mediated metabolic reprogramming in Arabidopsis.

Environmental stresses induce production of oxylipins synthesized by the two main biosynthetic branches, allene oxide synthase (AOS) and hydroperoxide lyase (HPL). Here, we investigate how waterlogging-mediated alteration of AOS- and HPL-derived metabolic profile results in modulation of central metabolism and ultimately enhanced tolerance to this environmental stress in Arabidopsis thaliana. Waterlogging leads to increased levels of AOS- and HPL-derived metabolites, and studies of genotypes lacking either one or both branches further support the key function of these oxylipins in waterlogging tolerance.

The MAP4 Kinase SIK1 Ensures Robust Extracellular ROS Burst and Antibacterial Immunity in Plants.

Microbial patterns are recognized by cell-surface receptors to initiate pattern-triggered immunity (PTI) in plants. Receptor-like cytoplasmic kinases (RLCKs), such as BIK1, and calcium-dependent protein kinases (CPKs) are engaged during PTI to activate the NADPH oxidase RBOHD for reactive oxygen species (ROS) production. It is unknown whether protein kinases besides CPKs and RLCKs participate in RBOHD regulation.

ER: the Silk Road of interorganellar communication.

Cellular adaptive responses arise from an array of spatially and temporally distinct biochemical interactions that modulate biological processes and reorganize subcellular structures tailored to the nature of stimulus. As such, cells have evolved elegantly and tightly regulated mechanisms to enable interorganellar communication in part through the dynamic readjustment of physical distance enabling the tethering between two closely apposed membranous organelles and thus formation of Membrane Contact Sites (MCSs). MCSs are dynamic and ubiquitous interorganellar structures that serve as regulatory interfaces to facilitate transmission of signals and to integrate synthesis of metabolic pathways such as lipids required for upholding cellular homeostasis in response to environmental and developmental inputs.

Interplay of the two ancient metabolites auxin and MEcPP regulates adaptive growth.

The ancient morphoregulatory hormone auxin dynamically realigns dedicated cellular processes that shape plant growth under prevailing environmental conditions. However, the nature of the stress-responsive signal altering auxin homeostasis remains elusive. Here we establish that the evolutionarily conserved plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) controls adaptive growth by dual transcriptional and post-translational regulatory inputs that modulate auxin levels and distribution patterns in response to stress.