Biology subjects

Schroeder, J. I.

Publications and source records attributed to Schroeder, J. I..

5 recordsLinked to original sources

bioRxiv2023

Forward Genetic Stomatal CO2 Response Screen in Grass Brachypodium distachyon Reveals Central Function of a MAP Kinase in Early Stomatal CO2 Signal Transduction

Plants respond to increased CO2 concentrations through rapid stomatal closure which can contribute to increased water use efficiency. Grasses display faster stomatal responses than eudicots due to dumbbell-shaped guard cells flanked by subsidiary cells working in opposition. However, forward genetic screening for stomatal CO2 signal transduction mutants in grasses has not been reported. The grass model Brachypodium distachyon is closely related to agronomically important cereal crops, sharing largely collinear genomes. To gain insights into CO2 control mechanisms of stomatal movements in grasses, we developed a forward genetics screen with an EMS-mutagenized Brachypodium distachyon M5 generation population using infrared imaging to identify plants with altered canopy leaf temperature at elevated CO2. Among isolated mutants, a "chill1" mutant exhibited cooler leaf temperatures than wildtype Bd21-3 parent control plants after exposure to increased [CO2]. chill1 plants showed strongly impaired high CO2-induced stomatal closure, despite retaining a robust abscisic acid-induced stomatal closing response. Through bulked segregant whole-genome-sequencing analyses followed by analyses of further backcrossed F4 generation plants and generation and characterization of CRISPR-cas9 mutants, chill1 was mapped to a protein kinase, BdMPK5. The chill1 mutation impaired BdMPK5 protein-mediated CO2/HCO3- sensing in vitro. Furthermore, AlphaFold2-directed structural modeling suggests that the identified BdMPK5-D90N chill1 mutant residue is located at the interface with the HT1 Raf-like kinase. BdMPK5 is a key signaling component involved in CO2-induced stomatal movements, potentially functioning as a component of the CO2 sensor in grasses.

plant biology↗

bioRxiv2023

Guard cell epigenomics reveals distinct abscisic acid and CO2 triggered chromatin remodeling during stomatal movements

In plants, epidermal guard cells integrate and respond to numerous environmental signals to control stomatal pore apertures thereby regulating gas exchange. Chromatin structure controls transcription factor access to the genome, but whether large-scale chromatin remodeling occurs in guard cells during stomatal movements, and in response to the hormone abscisic acid (ABA) in general, remain unknown. Here we isolate guard cell nuclei from Arabidopsis thaliana plants to examine whether the physiological signals, ABA and CO2, regulate guard cell chromatin during stomatal movements. Our cell type specific analyses uncover patterns of chromatin accessibility specific to guard cells and define novel cis-regulatory sequences supporting guard cell specific gene expression. We find that ABA triggers extensive and dynamic chromatin remodeling in guard cells, roots, and mesophyll cells with clear patterns of cell-type specificity. DNA motif analyses uncover binding sites for distinct transcription factors enriched in ABA-induced and ABA-repressed chromatin. We identify the ABF/AREB bZIP-type transcription factors that are required for ABA-triggered chromatin opening in guard cells and implicate the inhibition of a set of bHLH-type transcription factors in controlling ABA-repressed chromatin. Moreover, we demonstrate that ABA and CO2 induce distinct programs of chromatin remodeling. We provide insight into the control of guard cell chromatin dynamics and propose that ABA-induced chromatin remodeling primes the genome for abiotic stress resistance. Significance statementSpecialized leaf cells called guard cells integrate environmental cues to optimally control the size of microscopic stomatal pores. The hormone abscisic acid (ABA), a key regulator of plant drought responses, and changes in atmospheric CO2 concentration are signals that control stomatal aperture size, but whether these signals also regulate genome packaging into chromatin is unknown. Using guard cell specific chromatin profiling we uncovered regulatory DNA sequences driving specific gene expression in this cell-type. We also discovered that ABA triggers extensive and persistent changes to chromatin structure in guard cells. Unexpectedly, exposure of plants to elevated atmospheric CO2 had only minimal impact on chromatin dynamics. Furthermore, we identified the specific transcription factors that regulate ABA-induced chromatin dynamics in guard cells.

plant biology↗

bioRxiv2023

MPK12 in stomatal CO2 signaling: function beyond its kinase activity

O_LIProtein phosphorylation by kinases is a major molecular switch mechanism involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf-like kinase HT1 as a required step for stomatal movements by changes in CO2 concentration. However, whether MPK12 kinase activity is required for regulation of CO2-induced stomatal responses warrants in depth investigation. C_LIO_LIWe apply genetic, biochemical, and structural modeling approaches to examining the non-catalytic role of MPK12 in guard cell CO2 signaling that relies on allosteric inhibition of HT1. C_LIO_LIWe show that CO2/HCO3--enhanced MPK12 interaction with HT1 is independent of its phosphor-transfer activity. By analyzing gas exchange of plant lines expressing various kinase-dead and constitutively active versions of MPK12 in a plant line where MPK12 is deleted, we confirmed that CO2-dependent stomatal responses rely on MPK12s ability to bind to HT1 but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO2/HCO3- and present structural modeling that explains the MPK12:HT1 interaction interface. C_LIO_LIThese data add to the model that MPK12 kinase-activity-independent interaction with HT1 functions as a molecular switch by which stomatal guard cells sense changes in atmospheric CO2 concentration. C_LI

plant biology↗

bioRxiv2021

An amiRNA screen uncovers redundant CBF & ERF34/35 transcription factors that differentially regulate arsenite and cadmium responses

Arsenic stress causes rapid transcriptional responses in plants. However, transcriptional regulators of arsenic-induced gene expression in plants remain less well known. To date, forward genetic screens have proven limited for dissecting arsenic response mechanisms. We hypothesized that this may be due to the extensive genetic redundancy present in plant genomes. To overcome this limitation, we pursued a forward genetics screen for arsenite tolerance using a randomized library of plants expressing >2,000 artificial microRNAs (amiRNAs). This library was designed to knock-down diverse combinations of homologous gene family members within sub-clades of transcription factor and transporter gene families. We identified six transformant lines showing an altered response to arsenite in root growth assays. Further characterization of an amiRNA line targeting closely homologous CBF and ERF transcription factors show that the CBF1,2 and 3 transcription factors negatively regulate arsenite sensitivity. Furthermore, the ERF34 and ERF35 transcription factors are required for cadmium resistance. Generation of CRISPR lines, higher-order T-DNA mutants, and gene expression analyses, further support our findings. These ERF transcription factors differentially regulate arsenite sensitivity and cadmium tolerance.

plant biology↗

bioRxiv2018

Cryo-EM Structure of OSCA1.2 from Oryza sativa: Mechanical basis of hyperosmolality-gating in Plants

Sensing and responding to environmental water deficiency and osmotic stresses is essential for the growth, development and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis. Here, we report the cryo-EM structure and function of an ion channel from rice (Oryza sativa; OsOSCA1.2), showing how it mediates hyperosmolality sensing and ion permeability. The structure reveals a dimer; the molecular architecture of each subunit consists of eleven transmembrane helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The transmembrane domain is structurally related to the TMEM16 family of calcium dependent ion channels and scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that is parallel to the plasma membrane. These helical arms are well positioned to sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the transmembrane portion of the molecule to open the channel. Hydrogen-deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. The structure provides a framework to understand the structural basis of hyperosmolality sensing in an important crop plant, extends our knowledge of the anoctamin superfamily important for plants and fungi, and provides a structural mechanism for translating membrane stress to ion transport regulation.

biophysics↗