Suyan Yee

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 C ₄ 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 C ₄ 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 ( ¹ O ₂ )-responsive beta-cyclocitral (β-CC) /dihydroactinidiolide (DHA) signalling and hydrogen peroxide (H ₂ O ₂ )-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 C ₄ 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.

The Arg/N-degron pathway regulates important agronomic traits. In plants, the PROTEOLYSIS 1 (PRT1) protein binds to aromatic amino-terminal residues and recruits ubiquitin (Ub) conjugating enzymes to ubiquitinate substrate proteins. Here, we demonstrate that PRT1 recruits the UBC35-UEV1A complex via RING1 of PRT1. This stimulates UBC35 to produce K63-linked Ub chains. To afford chemical control over Ub signalling, we construct a synthetic substrate of PRT1 with an N-terminal calmodulin-binding peptide (CBP). The addition of the CBP sequence to the substrate creates an off switch for PRT1-dependent ubiquitination that is regulated by calcium and calmodulin. Finally, we produce a set of nanobodies that are recognised by PRT1 and induce the ubiquitination of a reporter protein. Altogether, we introduce and biochemically validate several tools to both redirect and chemically control the Arg/N-degron pathway.

Chloroplasts sense environmental stress and activate chloroplast-to-nucleus retrograde signalling, reprogramming nuclear gene expression for plant acclimation. One such pathway is regulated by the phosphatase, SAL, which hydrolyses the nucleotide signal 3'-phosphoadenosine 5'-phosphate (PAP), a by-product of secondary sulfur metabolism. In Arabidopsis thaliana, genetic loss of AtSAL1 elevates PAP and enhances stress tolerance but causes pleiotropic growth defects. 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 3-fold greater potency than the known Li+ inhibitor. Structural analoguing of V20 and biochemical assays defined key pharmacophore features required for inhibition. Accelerated molecular dynamics simulations revealed two previously uncharacterized 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. 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 oxidative stress signalling and sulfur metabolism in plants.

Wheat is a major staple crop for over one-third of the world’s population, crucial for global food security, economic stability and cultural traditions. Recently, single-cell and spatial omics approaches have transformed biological discovery, primarily in medical and animal sciences, and they are now beginning to be applied in plant research. Here we summarize the technical innovations and feasibility of spatial omics applications in wheat research, particularly for understanding developmental and environmental responses, thereby potentially enhancing wheat breeding. We highlight how these tools can reveal spatial and temporal patterns in gene expression, cellular heterogeneity and tissue organization in wheat. Furthermore, we propose developing a spatially resolved single-cell atlas of wheat across its life cycle to facilitate breakthroughs in basic research and potential applications in breeding. To achieve these goals, we advocate for a Wheat Spatial Omics Consortium to foster worldwide collaboration for overcoming barriers and developing sustainable and climate-resilient wheat.

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 π–π 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 Statement: We 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 AtSAL1’s 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.

This article is a Commentary on Sun et al . (2026), 249 : 24–38 .

PROTON GRADIENT REGULATION 5 (PGR5) plays a critical role in generating proton motive force across thylakoid membranes and supporting photoprotection under fluctuating light in C3 plants. 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, PGR5 abundance in leaves has increased, coinciding with an enhancement in cyclic electron flow rate. To investigate the role of PGR5 in C4 photosynthesis and photoprotection, we generated Setaria viridis (a model C4 monocot) lines with null pgr5 alleles. We demonstrate that loss of PGR5 severely impairs the establishment of proton motive force, photosynthetic control, and energy-dependent non-photochemical quenching at high irradiances, leading to a loss of Photosystem I activity under light stress. Furthermore, plants lacking PGR5 exhibit drastically reduced growth and photosynthesis when grown under fluctuating daylight; however, they are less severely affected than C3 pgr5 mutants. This relative tolerance arises from the ability of S. viridis lacking PGR5 to maintain significant levels of photosynthetic control, in contrast to C3 mutants. Additionally, in the absence of PGR5 and qE, a slower-relaxing, zeaxanthin-dependent form of non-photochemical quenching supports survival under fluctuating light, albeit at the cost of reduced photochemical efficiency and assimilation. Our findings highlight the essential role of PGR5 in enabling efficient C4 photosynthesis under fluctuating light by regulating photosynthetic control and energy-dependent non-photochemical quenching. This study also uncovers the interplay between multiple photoprotective mechanisms safeguarding C4 photosynthesis under light stress.

PROTON GRADIENT REGULATION 5 (PGR5) is essential for generating proton motive force across thylakoid membranes in C ₃ 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 C ₃ to C ₄ 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 C ₄ photosynthesis, we generated model C ₄ 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 C ₄ 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 C ₄ photosynthesis and emphasises the role of PGR5 in the evolution of C ₄ plants.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO₂-concentrating mechanism. BCT1 could enhance photosynthetic CO₂ assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO₂-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO₂ compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO₂-concentrating mechanism components to improve plant photosynthesis.

The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacteria CO ₂ concentrating mechanism, could enhance photosynthetic CO ₂ assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO ₂ -dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana , but the transformed plants did not have higher carbon assimilation rates or decreased CO ₂ compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO ₂ concentrating mechanism components to improve plant photosynthesis.