Barry James Pogson

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.

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.

Plant sulfur metabolism is crucial to the plant acclimation response to abiotic stresses, providing the redox-active compounds cysteine and glutathione for redox buffering as well as the chloroplast-to-nucleus retrograde signal 3′-phosphoadenosine 5′-phosphate (PAP) for activation of gene expression changes. Whilst these processes have been conventionally considered separately, here we review chloroplast retrograde signalling in the context of plant sulfur metabolism, with focus on the biosynthesis and degradation of PAP in secondary sulfur metabolism. We outline mechanisms by which primary sulfur metabolism via cysteine and glutathione contribute to the modulation of chloroplast retrograde signalling. We examine emerging questions in how plant sulfur metabolism is coordinated in different cell types of a plant leaf for synthesis and accumulation of PAP. Finally, while the majority of chloroplast retrograde signalling research has focused on the model Brassicaceae plant species Arabidopsis thaliana, here we outline the opportunities for novel insights from non-Brassicaceae plants to enable an integrated understanding of the intersection of sulfur metabolism and retrograde signalling.

The Rai1/Dxo1/DXO protein family are involved in eukaryotic 5′-end RNA quality control by hydrolyzing non-canonical mRNA cap structures, such as the NAD ⁺ 5′-cap. The plant DXO1 ortholog shows distinct biochemical properties, compared to its fungal and mammalian counterparts, and only minor phenotypic traits can be attributed to its NAD ⁺ -decapping (deNADding) activity. On the other hand, the chloroplast and growth defects observed in dxo1 null mutants appear linked to the presence of a serine-rich domain at its N-terminus. Here, we report that plant DXO1 is a distinct ortholog that likely diverged earlier than its fungal and mammalian counterparts. We attribute this to the presence of a serine-rich N-terminal domain, which is found almost exclusively in plants and likely arose during the emergence of angiosperms. Degradome profiling revealed that Arabidopsis DXO1 contributes to mRNA surveillance, via examination of exon junction complex and ribosome footprints, and this activity is reliant on the presence of the serine-rich N-terminal domain in Arabidopsis. Furthermore, polysome profiling revealed that plants lacking DXO1 have severe ribosome loading defects, which may explain the loss of mRNA surveillance and alterations in pre-rRNA processing. Taken together, our data leads us to propose a model whereby Arabidopsis DXO1 is involved in 5′-end RNA quality control, which is critical for ribosome loading that impacts translational fidelity and translation-dependent mRNA surveillance.

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.

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.

Summary: Chloroplast‐to‐nucleus retrograde signalling enables rapid stress responses in plants, but whether these signals accumulate to affect crop performance across entire growing seasons under field conditions remains unknown. We 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. Lines 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. Targeted modification of specific SAL homeologous groups can simultaneously improve both yield and stress tolerance in wheat. This demonstrates that retrograde signalling integrates environmental information across the plant lifecycle and highlights the importance of locus‐specific targeting and multienvironment field validation for crop modifications.

Burgeoning global demand for crop products and the negative impact of climate change on crop production are driving the need to improve yield by developing new elite crop varieties without expanding planted area or increasing agronomic inputs. Improvement in photosynthesis is critical for enhancing crop productivity. Even though leaf photosynthesis is well-studied, the photosynthetic potential of non-foliar green tissues like pods in Brassicaceae and Fabaceae species remains underexplored. This review emphasizes pod photosynthesis in determining seed yield and quality in Brassicaceae and Fabaceae crops. At present, accurate and efficient phenotyping methods are unavailable, limiting understanding and genetic improvement of pod photosynthesis. Novel approaches like chlorophyll fluorescence and hyperspectral reflectance are promising for high-throughput phenotyping of pod photosynthetic traits. This review further discusses genetic targets and regulatory mechanisms for enhancing pod photosynthesis, including transcription factors like GOLDEN2-LIKE and GATA that may regulate photosynthetic capacity in pods, suggesting potential genetic manipulation strategies to boost crop productivity. In conclusion, unlocking the genetic and physiological bases of pod photosynthesis offers opportunities for advancing crop breeding to ensure sustainable food security amidst climate change and increasing global population pressures. Future research should focus on developing high-throughput phenotyping tools and elucidating genetic pathways to maximize pod photosynthesis in crops.

SUMMARY: Cellular responses to abiotic stress involve multiple signals such as reactive oxygen species (ROS), Ca²⁺, abscisic acid (ABA), and chloroplast‐to‐nucleus retrograde signals such as 3′‐phosphoadenosine 5′‐phosphate (PAP). The mechanism(s) by which these messengers intersect for cell regulation remain enigmatic, as do the roles of retrograde signals in specialized cells. Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate chloroplast and plasma membrane ROS production. Contrary to its role in upregulating processes leading to quenching of ROS in foliar tissue, we show that in guard cells, PAP induces chloroplast ROS accumulation via photosynthetic electron transport and apoplast ROS via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) proteins. Both subcellular ROS sources are necessary for stress hormone ABA‐mediated stomatal closure, as well as PAP‐mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD instead of RBOHF. Three calcium‐dependent protein kinases (CPKs) transcriptionally induced by PAP, namely CPK13, CPK32, and CPK34, concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two SLAC1 serine (S) residues, including S120, which is also targeted by the ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP‐induced CPKs rescues stomatal closure in ost1. Our data identify chloroplast retrograde signals as critical nodes in cellular stress response networks of guard cells.