Biology subjects

Chan, K. X.

Publications and source records attributed to Chan, K. X..

8 recordsLinked to original sources

bioRxiv2026

Specialisation of photosynthetic biochemistry in C4 chloroplasts potentiates distinct cell-specific functionality of different chloroplast-to-nucleus retrograde signalling pathways.

Chloroplasts are key environmental sensors for plant cells. Upon exposure to abiotic stresses such as excess sunlight and heat, reactive oxygen species (ROS) in chloroplasts are sensed to trigger chloroplast-to-nucleus signalling pathways for acclimatory nuclear gene expression. There is extensive biochemical- and cell type-specialisation of chloroplasts, such as those in heat-resilient plants performing C4 photosynthesis, but how these specialisations intersect with chloroplast-to-nucleus signalling remains unclear. Here we show that specific chloroplast-to-nucleus signalling networks are functional in specialised leaf cell types of the C4 model species, Setaria viridis. RNA-sequencing, electron paramagnetic resonance (EPR) spectroscopy and metabolomics on cell type-enriched fractions, complemented with mutant analyses provided evidence for cell-specific enrichment of chloroplast-to-nucleus retrograde signalling pathways correlating with the photosynthetic specialisation in these cell types. Singlet oxygen (1O2)-responsive beta-cyclocitral ({beta}-CC) /dihydroactinidiolide (DHA) signalling and hydrogen peroxide (H2O2)-responsive 3'-phosphoadenosine 5'-phosphate (PAP) signalling pathways were specific to mesophyll cells, in line with enrichment of photosystem II and linear electron flow to photosystem I, respectively in this cell type. In contrast, bundle sheath cells were enriched for the rapid, ROS-independent retrograde signalling pathway mediated by the Calvin cycle product, dihydroxyacetone phosphate (DHAP), consistent with the exclusive localisation of the Calvin cycle to C4 bundle sheath cells. Taken together, our results provide insights into how chloroplast- and cell type-specialisation intersect with stress signalling in plant cells and identify photosynthetic biochemistry as a key driver of cell-specific cellular signalling.

plant biology↗

bioRxiv2025

High-throughput screening and structure-guided design of small molecules enable modulation of SAL-PAP stress signaling

Chloroplasts sense environmental stress and activate chloroplast-to-nucleus retrograde signalling, reprogramming nuclear gene expression to drive plant acclimation. One such pathway is regulated by the chloroplastic phosphatase, SAL, which hydrolyses the nucleotide signal 3-phosphoadenosine 5-phosphate (PAP). In Arabidopsis thaliana, genetic loss of AtSAL1 elevates PAP and enhances stress tolerance but causes pleiotropic growth defects, complicating the interpretation of the role of PAP in cellular signalling. To uncouple stress signalling from genetic pleiotropy, we conducted a high-throughput in vitro screen of 13,000 small molecules and identified V20, a competitive inhibitor of AtSAL1 with three-fold greater potency than the known Li inhibitor. Structural analoguing of V20 and biochemical assays defined key pharmacophore features required for inhibition, including an ortho-halogenated aromatic ring, hydrogen-bond donor capacity at the triazole carboxamide, and electron-withdrawing substitutions that enhance {pi}-{pi} stacking with aromatic residues. Accelerated molecular dynamics simulations using a new high-resolution crystal structure revealed two previously uncharacterised V20 binding pockets adjacent to the catalytic site. V20 binding induces conformational changes which restrict substrate access to the catalytic site. Exogenous application of V20 to Arabidopsis led to increased PAP accumulation, activated PAP-responsive gene expression and enhanced oxidative tolerance, demonstrating cellular uptake and in vivo efficacy in the energy organelles, chloroplasts and mitochondria, where AtSAL1 is localised. Collectively, our findings reveal new insights into the regulatory domains of SAL enzymatic activity for control of PAP-mediated signalling and establish a proof-of-concept for targeted chemical modulation of SAL activity, which offers novel strategies to selectively manipulate chloroplast-to-nucleus retrograde signalling in plants. Significance StatementWe applied drug discovery strategies to test 13,000 small molecules for inhibition of AtSAL1, a key regulator of plant stress signalling from chloroplasts. Using biochemical assays, chemical analogues and molecular dynamic simulations, we defined the structural-activity relationship of a lead compound, V20, and elucidated its inhibitory mechanism. Application of V20 to plant leaves elevated the levels of AtSAL1s natural substrate, PAP (a chloroplast stress signalling molecule), activated stress-responsive genes and improved oxidative stress tolerance in Arabidopsis. This on-demand activation of SAL-PAP chloroplast signalling avoids pleotropic effects observed in loss-of-function mutants. Our approach provides a new strategy to study chloroplast-mediated cellular signaling pathways that are crucial for plant acclimation to stressful environmental conditions such as excessive sunlight.

plant biology↗

bioRxiv2025

Priming of retrograde signaling in wheat across multiple natural environments reveal how responses to dynamic stimuli can be integrated to alter yield, yield stability and water productivity

O_LIChloroplast-to-nucleus retrograde signaling enables rapid stress responses in plants, but whether these signals accumulate to affect crop performance across entire growing seasons under field conditions remains unknown. C_LIO_LIWe generated wheat mutants with targeted deletions in specific SAL gene copies from two distinct homeologous groups (TaSAL1 and TaSAL2), creating lines with enhanced stress signal responsiveness. We tested these lines across 15 field trials spanning diverse Australian environments with varying temperatures, rainfall, and irrigation regimes, measuring physiological responses, yield, biomass, and water productivity. C_LIO_LILines with TaSAL2 gene deletions showed 4-8% yield improvements with enhanced water productivity, while TaSAL1 deletions reduced yields. The TaSAL2 mutants maintained superior photosynthetic function under drought stress, showed improved relative water content, and demonstrated enhanced yield stability across environments. Canopy temperature measurements revealed dynamic stomatal regulation, with increased closure during midday stress periods but normal aperture under benign conditions. Significantly, specific SAL modifications enhanced photosynthetic efficiency and stress resilience without traditional yield penalties. C_LIO_LITargeted modification of specific SAL homeologous groups can simultaneously improve both yield and stress tolerance in wheat. This demonstrates that retrograde signaling integrates environmental information across the plant lifecycle, and highlights the importance of locus-specific targeting and multi-environment field validation for crop modifications. C_LI

plant biology↗

bioRxiv2024

PGR5 promotes energy-dependent non-photochemical quenching to enable efficient C4 photosynthesis under fluctuating light

PROTON GRADIENT REGULATION 5 (PGR5) is essential for generating proton motive force across thylakoid membranes in C3 plants and supporting photoprotection under fluctuating light conditions. It is proposed that this function is achieved by regulating cyclic electron flow around Photosystem I. During the evolutionary transition from C3 to C4 photosynthesis, the leaf abundance of PGR5 has increased, coinciding with a rise in the cyclic electron flow rate. To investigate the contribution of PGR5 to photoprotection in C4 photosynthesis, we generated model C4 monocot Setaria viridis with null pgr5 alleles. We show that plants lacking PGR5 struggle to establish proton motive force and energy-dependent non-photochemical quenching (qE) at higher irradiances during instantaneous measurements. This leads to a progressive decline in maximum Photosystem I activity when leaves are exposed to repeated cycles of high irradiance. Additionally, plants without PGR5 exhibit severely reduced growth and photosynthesis compared to wild type plants when grown under fluctuating daylight but not under constant daylight. In the absence of PGR5, a slower-relaxing, zeaxanthin-dependent form of non-photochemical quenching supports growth under fluctuating light, albeit at the cost of reduced photochemical efficiency and assimilation rate. Our findings underscore the role of PGR5 in enabling efficient C4 photosynthesis under fluctuating light by establishing proton motive force for the rapid upregulation of qE and preventing photodamage to the electron transport machinery. This study highlights the importance of various non-photochemical quenching mechanisms for C4 photosynthesis and emphasises the role of PGR5 in the evolution of C4 plants.

plant biology↗

bioRxiv2024

The nuclear sulfenome of Arabidopsis: spotlight on histone acetyltransferase GCN5 regulation through functional thiols

Partial reduction of oxygen during energy generating metabolic processes in aerobic life forms results in the production of reactive oxygen species (ROS). In plants, ROS production is heightened during periods of both abiotic and biotic stress, which imposes a significant overload on the antioxidant systems. Hydrogen peroxide (H2O2) holds a central position in cellular redox homeostasis and signalling, playing an important role by oxidising crucial cysteines to sulfenic acid (-SOH), considered as a biologically relevant post-translational modification (PTM). Until now, the role of the nucleus in the cellular redox homeostasis has been relatively underexplored. The regulation of histone-modifying enzymes by oxidative PTMs on redox-active cysteines or tyrosine residues is particularly intriguing as it allows the integration of redox signalling mechanisms with chromatin control of transcriptional activity. One of the most extensively studied histone acetyltransferases is the conserved GENERAL CONTROL NONDEPRESSIBLE 5 (GCN5) complex. This study investigated the nuclear sulfenome in Arabidopsis thaliana by expressing a nuclear variant of the Yeast Activation Protein-1 (YAP) probe, identifying 225 potential redox-active nuclear proteins subject to sulfenylation. Mass spectrometry analysis further confirmed the sulfenylation of GCN5 at specific cysteine residues, with their functional significance and impact on the protein-protein interaction network assessed through cysteine-to-serine mutagenesis. HighlightProtein cysteine thiols are post-translationally modified under oxidative stress. Through the in vivo capturing of nuclear proteins undergoing sulfenylation in Arabidopsis, we highlight the functionality of particular cysteines in the histone acetyltransferase GCN5.

plant biology↗

bioRxiv2023

Cell-specialized chloroplast signaling orchestrates photosynthetic and extracellular reactive oxygen species for stress responses

Cellular responses to abiotic stress involve multiple signals including secondary messengers such as reactive oxygen species (ROS) and Ca2+, phytohormones such as abscisic acid (ABA) and chloroplast-to-nucleus retrograde signals such as 3-phosphoadenosine 5-phosphate (PAP). Mechanism(s) by which these messengers, produced in different subcellular compartments, intersect for cell regulation remain enigmatic. Previously we showed that the chloroplast retrograde signal PAP, similar to ABA, induces an increase in ROS levels in guard cells (Pornsiriwong et al, 2017). Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate both chloroplast and plasma membrane ROS production. In whole leaves, PAP alters various ROS-related processes including plasmodesmal permeability as well as responses to ozone and the bacterial elicitor flg22, but mainly initiates processes that quench ROS during oxidative stress. Conversely, we show in guard cells, both PAP and ABA induce an increase in ROS levels in both chloroplasts via photosynthetic electron transport, and the apoplast via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). Both subcellular ROS sources were necessary for ABA- and PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD, instead of RBOHF, for apoplastic ROS mediated stomatal closure. We identified three calcium-dependent protein kinases (CPKs) as the post-translational activators of RBOHD-mediated ROS production. CPK13, CPK32, and CPK34 were transcriptionally induced by PAP and concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two Serine (S) residues, including S120 which is also targeted by the core ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP-induced CPKs rescues stomatal closure in ost1. Our data identify stomatal chloroplasts, to be nodes in the multifaceted cellular stress response networks as they are both sources and mediators of ROS and retrograde signals such PAP. Thus, chloroplasts are not just mediators of photosynthesis in response to, for example, excess light, but can serve as critical nodes in the multifaceted cellular stress response networks in specialized cells via retrograde signals, providing support to the concept of sensory plastids. Significance StatementThe chloroplast is an environmental sensor for stresses such as excess light and drought via the activation of photosynthetic-mediated retrograde signals. However, how does it function in specialized cells for which carbon fixation is secondary? Here we show the chloroplast is an important node to coordinate multiple plant signaling pathways in response to stresses such as drought. The chloroplast retrograde signal 3-phosphoadenosine 5-phosphate (PAP) plays multiple roles in reactive oxygen species (ROS) signaling and homeostasis. While PAP suppresses ROS in photosynthetic tissue, PAP instead induces guard cell ROS in chloroplasts and extracellular space to induce stomatal closure. We decipher how PAP-induced proteins activate both extracellular ROS production and anion channels for stomatal closure, thus providing a mechanism by which chloroplasts provide a strategic complement to canonical hormonal pathways in regulating plant physiological responses in specialized cells.

plant biology↗

bioRxiv2020

Condensation of Rubisco into a proto-pyrenoid in higher plant chloroplasts

Photosynthetic CO2 fixation in plants is limited by the inefficiency of the CO2-assimilating enzyme Rubisco (D-ribulose-1,5-bisphosphate carboxylase/ oxygenase)1-3. In plants possessing the C3 pathway, which includes most major staple crops, Rubisco is typically evenly distributed throughout the chloroplast stroma. However, in almost all eukaryotic algae Rubisco aggregates within a microcompartment known as the pyrenoid, in association with a CO2-concentrating mechanism that improves photosynthetic operating efficiency under conditions of low inorganic carbon4. Recent work has shown that the pyrenoid matrix is a phase-separated, liquid-like condensate5. In the alga Chlamydomonas reinhardtii, condensation is mediated by two components: Rubisco and the linker protein EPYC1 (Essential Pyrenoid Component 1)6,7. Here we show that expression of mature EPYC1 and a plant-algal hybrid Rubisco leads to spontaneous condensation of Rubisco into a single phase-separated compartment in Arabidopsis chloroplasts, with liquid-like properties similar to a pyrenoid matrix. The condensate displaces the thylakoid membranes and is enriched in hybrid Rubisco containing the algal Rubisco small subunit required for phase separation. Promisingly, photosynthetic CO2 fixation and growth is not impaired in stable transformants compared to azygous segregants. These observations represent a significant initial step towards enhancing photosynthesis in higher plants by introducing an algal CO2-concentrating mechanism, which is predicted to significantly increase the efficiency of photosynthetic CO2 uptake8,9.

plant biology↗

bioRxiv2020

Variation in relaxation of non-photochemical quenching in a soybean nested association mapping panel as a potential source for breeding improved photosynthesis

Improving the efficiency of crop photosynthesis has the potential to increase yields. Genetic manipulation showed photosynthesis can be improved in Tobacco by speeding up relaxation of photoprotective mechanisms, known as non-photochemical quenching (NPQ), during high to low light transitions. However, it is unclear if natural variation in NPQ relaxation can be exploited in crop breeding programs. To address this issue, we measured NPQ relaxation in the 41 parents of a soybean NAM population in field experiments in Illinois during 2018 and 2019. There was significant variation in amount and rate of fast, energy dependent quenching (qE) between genotypes. However, strong environmental effects led to a lack of correlation between values measured over the two growing season, and low broad-sense heritability estimates (< 0.3). These data suggest that either improvements in screening techniques, or transgenic manipulation, will be required to unlock the potential for improving the efficiency of NPQ relaxation in soybean. Table of Abbreviations O_TBL View this table: org.highwire.dtl.DTLVardef@124cbaborg.highwire.dtl.DTLVardef@b7808eorg.highwire.dtl.DTLVardef@1591822org.highwire.dtl.DTLVardef@3f9d64org.highwire.dtl.DTLVardef@764eef_HPS_FORMAT_FIGEXP M_TBL C_TBL

plant biology↗