Biology subjects

Romanowski, A.

Publications and source records attributed to Romanowski, A..

11 recordsLinked to original sources

bioRxiv2025

PHYTOCHROME INTERACTING FACTOR 7 moderates the activity and the cytosolic-nuclear shuttling of PHYTOCHROME A

In nature, plants perceive and respond to complex vegetative environments through the phytochrome (phy) photoreceptor-signaling system. The canopy adaptation strategy, elicited by phyA, together with the shade avoidance syndrome, regulated by phyB, enables plants to adapt to diverse forms of vegetation shade. The phyA High Irradiance Response (HIR) mode, which involves photocycle-coupled nuclear shuttling, is essential for sensing sustained far-red rich conditions typical of canopy or occlusive shade. Our research identifies PHYTOCHROME INTERACTION FACTOR 7 (PIF7) as a key regulator of phyA function. We show that PIF7ox effectively suppresses phyA action. Under persistent shade, PIF7 and phyA both accumulate and interact within nuclear photobodies, with binding mediated by the C-terminal domain of PIF7 and the PAS-PAS domain of phyA. PIF7 also interacts with FHY1 and FHL transport facilitators, thereby interfering with phyA cytosolic-nuclear shuttling. This coordinated molecular targeting by PIF7 constitutes an effective strategy for modulating phyA function under shaded conditions.

plant biology↗

bioRxiv2025

An oomycete effector that induces shade avoidance like growth and suppresses plant defenses targets the AUX/IAA protein IAA11

Filamentous pathogens, such as oomycetes, employ multiple strategies to suppress plant immunity, one of which involves manipulating phytohormone signaling pathways. The effector HaRxL106 from Hyaloperonospora arabidopsidis (Hpa), known to induce shade avoidance syndrome (SAS)-like growth, has previously been shown to interact with brassinosteroid signaling components. Here, we investigate the role of HaRxL106 in altering auxin signaling, revealing that HaRxL106 targets the AUX/IAA protein IAA11, a key negative regulator of auxin responses. Our data suggest that HaRxL106 impedes the repressive function of IAA11, enhancing auxin signaling and promoting SAS, which concurrently suppresses plant immune responses. These findings provide insights into how pathogen effectors manipulate plant growth and immunity to facilitate infection.

plant biology↗

bioRxiv2024

Downy mildew effector HaRxL106 interacts with the transcription factor BIM1 altering plant growth, BR signaling and susceptibility to pathogens

Hyaloperonospora arabidopsidis (Hpa) is an oomycete pathogen that causes downy mildew disease on Arabidopsis. This obligate biotroph manipulates the homeostasis of its host plant by secreting numerous effector proteins, among which are the RxLR-effectors. Identifying the host targets of effectors and understanding how their manipulation facilitates colonization of plants is key to improve plant resistance to pathogens. Here we characterize the interaction between the RxLR effector HaRxL106 and BIM1, an Arabidopsis transcription factor (TF) involved in Brassinosteroid (BR) signaling. We report that HaRxL106 interacts with BIM1 in vitro and in planta. BIM1 is required by the effector to increase the host plant susceptibility to (hemi)biotrophic pathogens, and thus can be regarded as a susceptibility factor. Mechanistically, HaRxL106 requires BIM1 to induce the transcriptional activation of BR-responsive genes and cause alterations in plant growth patterns that phenocopy the shade avoidance syndrome. Our results support previous observations of antagonistic interactions between activation of BR signaling and suppression of plant immune responses and reveal that BIM1, a new player in this crosstalk, is manipulated by the pathogenic effector HaRxL106. SIGNIFICANCE STATEMENTOur study reveals that the Arabidopsis transcription factor BIM1 plays a key role as a susceptibility factor that is manipulated by the oomycete effector HaRxL106 to induce the Brassinosteroid (BR) signaling pathway and suppress plant immunity. HaRxL106 induces BIM1-dependent expression of BR-responsive genes which affects normal plant growth generating a shade avoidance-like syndrome that impacts negatively on plant defenses against bacterial and oomycete pathogens.

plant biology↗

bioRxiv2023

Phytochrome-dependent responsiveness to root-derived cytokinins enables coordinated elongation responses to combined light and nitrate cues.

Plants growing at high densities can detect competitors through changes in the composition of light reflected by neighbours. In response to this far-red-enriched light, plants elicit adaptive shade avoidance responses for light capture, but these need to be balanced against other input signals, such as nutrient availability. Here, we used a combination of transcriptomics, growth assays and dedicated genetic and pharmacological interventions to demonstrate how Arabidopsis integrates light and nitrate signalling. We unveiled that nitrate modulates shade avoidance via a previously unknown shade response pathway that involves root-derived transZeatin (tZ). Under nitrate-sufficient conditions, tZ promotes hypocotyl elongation specifically in the presence of supplemental far-red light. This occurs via PIF transcription factors-dependent inhibition of type-A ARRs cytokinin response inhibitors. Our data thus reveal how plants co-regulate responses to shade cues with root-derived information about nutrient availability, and how they restrict responses to this information to specific light conditions in the shoot.

plant biology↗

bioRxiv2023

Cooperativity and additivity in plant enhancers

Enhancers are cis-regulatory elements that shape gene expression in response to numerous developmental and environmental cues. In animals, several models have been proposed to explain how enhancers integrate the activity of multiple transcription factors. However, it remains largely unknown how plant enhancers integrate transcription factor activity. Here, we use Plant STARR-seq to characterize three light-responsive plant enhancers--AB80, Cab-1, and rbcS-E9--derived from genes active in photosynthesis. Saturation mutagenesis reveals mutations, many of which cluster in short regions, that strongly reduce enhancer activity in the light, in the dark or in both conditions. When tested in the light, these mutation-sensitive regions do not function on their own; rather, cooperative interactions with other such regions are required for full activity. Epistatic interactions occur between mutations in adjacent mutation-sensitive regions, and the spacing and order of mutation-sensitive regions in synthetic enhancers affects enhancer activity. In contrast, when tested in the dark, mutation-sensitive regions act independently and additively in conferring enhancer activity. Taken together, this work demonstrates that plant enhancers show evidence for both cooperative and additive interactions among their functional elements. This knowledge can be harnessed to design strong, condition-specific synthetic enhancers.

plant biology↗

bioRxiv2022

COR27/28 Regulate the Evening Transcriptional Activity of the RVE8 and LNK1/2 Circadian Complex

The timing of many molecular and physiological processes in plants occurs at a specific time of day. These daily rhythms are driven by the circadian clock, a master timekeeper that uses daylength and temperature to maintain rhythms of approximately 24 hours in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT INDUCIBLE AND CLOCK REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover new connections within the clock and between other pathways, here we use affinity purification coupled with mass spectrometry to discover time-of-day-specific protein interactors of the RVE8-LNK1/2 complex. Among the interactors of RVE8/LNK1/LNK2 were COLD REGULATED GENE 27 (COR27) and COR28, which were coprecipitated in an evening-specific manner. In addition to COR27/28, we found an enrichment of temperature-related interactors that led us to establish a novel role for LNK1/2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach identified time-of-day-specific protein interactions and a novel RVE8-LNK-COR protein complex that implicates a new regulatory mechanism for circadian and temperature signaling pathways.

plant biology↗

bioRxiv2021

PIF7 controls leaf cell proliferation through an AN3 substitution-repression mechanism

Plants are agile, plastic organisms, able to adapt to ever-changing circumstances. Responding to far-red (FR) wavelengths from nearby vegetation, shade-intolerant species elicit the adaptive Shade Avoidance Syndrome (SAS), characterised by elongated petioles, leaf hyponasty and smaller leaves. We utilised end-of-day FR (EODFR) treatments to interrogate molecular processes that underlie the SAS leaf response. Genetic analysis establishes PHYTOCHROME INTERACTING FACTOR 7 (PIF7) is required for EODFR-mediated constraint of leaf blade cell division, while EODFR mRNAseq data identified ANGUSTIFOLIA3 (AN3) as a potential PIF7 target. We show PIF7 can suppress AN3 transcription through a sequestering mechanism that prevents AN3 activation of its own expression. We also establish PIF7 and AN3 impose antagonistic control of gene expression via common cis-acting promoter motifs in several cell cycle regulator genes. EODFR triggers the molecular substitution of AN3 to PIF7 at G-box/PBE-box promoter regions, and a switch from promotion to repression of gene expression.

plant biology↗

bioRxiv2020

NPRL3: Direct Effects on Human Phenotypic Variability, mTOR Signaling, Subcellular mTOR Localization, Cortical Lamination, and Seizure Susceptibility

Nitrogen Permease Regulator Like 3 (NPRL3) variants are associated with malformations of cortical development (MCD) and epilepsy. We report a large (n=133) founder NPRL3 (c.349delG, p.Glu117LysFS) pedigree dating to 1727, with heterogeneous epilepsy and MCD phenotypes. Whole exome analysis in individuals with and without seizures in this cohort did not identify a genetic modifier to explain the variability in seizure phenotype. Then as a strategy to investigate the developmental effects of NPRL3 loss in human brain, we show that CRISPR/Cas9 Nprl3 knockout (KO) in Neuro2a cells (N2aC) in vitro causes mechanistic target of rapamycin (mTOR) pathway hyperactivation, cell soma enlargement, and excessive cellular aggregation. Amino acid starvation caused mTOR inhibition and cytoplasmic mTOR localization in wildtype cells, whereas following Nprl3 KO, mTOR remained inappropriately localized on the lysosome and activated, evidenced by persistent ribosomal S6 and 4E-BP1 phosphorylation, demonstrating that Nprl3 loss decouples mTOR activation from metabolic state. Nprl3 KO by in utero electroporation in fetal (E14) mouse cortex resulted in mTOR-dependent cortical dyslamination with ectopic neurons in subcortical white matter. EEG recordings of these mice showed hyperexcitability in the electroporated hemisphere. NPRL3 variants are linked to a highly variable clinical phenotype likely as a consequence of mTOR-dependent effects on cell structure, cortical development, and network organization.

neuroscience↗

bioRxiv2020

BrphyB is critical for rapid recovery to darkness in mature Brassica rapa leaves

Crop biomass and yield are tightly linked to how the light signaling network translates information about the environment into allocation of resources, including photosynthates. Once activated, the phytochrome (phy) class of photoreceptors signal and re-deploy carbon resources to alter growth, plant architecture, and reproductive timing. Brassica rapa has been used as a crop model to test for conservation of the phytochrome-carbon network. B. rapa phyB mutants have significantly decreased or absent CO2-stimulated growth responses in seedlings, and adult plants have reduced chlorophyll levels, photosynthetic rate, stomatal index, and seed yield. Here, we examine the transcriptomic response of adult wild-type and BrphyB leaves to darkening and recovery in light. Three days of darkness was sufficient to elicit a response in wild type leaves suggesting a shift from carbon fixation and nutrient acquisition to active redistribution of cellular resources. Upon a return to light, wild-type leaves appeared to transcriptionally return to a pre-darkness state restoring a focus on nutrient acquisition. Overall, BrphyB mutant plants have a similar response with key differences in genes involved in photosynthesis and light response which deviate from the wild type transcriptional dynamics. Genes targeted to the chloroplast are especially affected. Adult BrphyB mutant plants had fewer, larger chloroplasts, further linking phytochromes, chloroplast development, photosynthetic deficiencies and optimal resource allocation.

plant biology↗

bioRxiv2019

Global transcriptome analysis reveals circadian control of splicing events in Arabidopsis thaliana

O_LIThe circadian clock of Arabidopsis thaliana controls many physiological and molecular processes, allowing plants to anticipate daily changes in their environment. However, developing a detailed understanding of how oscillations in mRNA levels are connected to oscillations in post-transcriptional processes, such as splicing, has remained a challenge. C_LIO_LIHere we applied a combined approach using deep transcriptome sequencing and bioinformatics tools to identify novel circadian regulated genes and splicing events. C_LIO_LIUsing a stringent approach, we identified 300 intron retention, 8 exon skipping, 79 alternative 3 splice site usage, 48 alternative 5 splice site usage, and 350 multiple (more than one event type) annotated events under circadian regulation. We also found 7 and 721 novel alternative exonic and intronic events. Depletion of the circadian regulated splicing factor AtSPF30 homolog, resulted in the disruption of a subset of clock controlled splicing events. C_LIO_LIAltogether, our global circadian RNA-seq coupled with an in silico, event centred, splicing analysis tool offers a new approach for studying the interplay between the circadian clock and the splicing machinery at a global scale. The identification of many circadian regulated splicing events broadens our current understanding of the level of control that the circadian clock has over this posttranscriptional regulatory layer. C_LI

plant biology↗

bioRxiv2019

HEXOKINASE 1 Glycolytic Action Fuels Post-Germinative Seedling Growth

O_LIIn darkness, PHYTOCHROME INTERACTING FACTOR (PIF)-induced skotomorphogenic seedling growth, is exemplified by increased hypocotyl elongation. HEXOKINASE1 (HXK1), which is also implicated in seedling establishment, can operate as a glycolytic enzyme or as a glucose-activated sensor signalling molecule. Under light and nutrient limiting conditions, the HXK1 sensor-signalling has been shown to control hypocotyl elongation. Little is known of whether HXK1 glycolytic function, or HXK1 and PIF cross-talk, is required to control hypocotyl growth. C_LIO_LIWe demonstrate HXK1 glycolytic activity is critical for cell expansion, and hypocotyl growth, post-germination. Notably, application of glucose-6-phosphate, the HXK1 enzymatic product, can restore short gin2-1/hxk1-1 mutant hypocotyls to wild-type length. Further, HXK1 sensor-signalling complex components, VHA-B1 and RPT5B, do not contribute to this response, for unlike gin2-1/hxk1-1, the vha-B1 and rpt5b alleles only disrupt hypocotyl growth following exogenous glucose application. C_LIO_LImRNA-seq analysis illustrates that HXK1 and PIF signalling converge at genes with known roles in light signalling. HXK1 imposes strong regulation on chloroplast and mitochondrial encoded genes, also branched chain amino acid catabolism pathway genes, which can provide a source of respiratory substrates in starvation conditions. C_LIO_LIOur study establishes the importance of HXK1 enzymatic function in supporting cell expansion and hypocotyl growth. We demonstrate a degree of cross-talk between HXK1 and PIFs through common target gene set. C_LI

plant biology↗