Biology subjects

Furbank, R. T.

Publications and source records attributed to Furbank, R. T..

At least 19 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↗

bioRxiv2026

PsbS confers limited adaptive benefit to C4 photosynthesis under fluctuating light

Adaptation of plant photosynthesis to dynamic light conditions experienced in natural environments is achieved through specific protective mechanisms. Energy-dependent non-photochemical quenching (qE), regulated by Photosystem II Subunit S (PsbS), is a key process facilitating acclimation to fluctuating light in C3 plants, which operate conventional photosynthesis. C4 plants, which include some of the worlds most productive and agriculturally important crops, have evolved a distinct high-efficiency photosynthetic pathway. Little is known about the role of specific processes, like qE, in acclimation of C4 plants to dynamic light environments. We generated gene-edited lines of the model C4 grass Setaria viridis lacking PsbS, which were found to be deficient in qE. This deficiency resulted in a modest increase in PSII photoinhibition and a CO2 assimilation penalty under light stress in short-term experiments, but photosynthesis and growth under fluctuating light were unaffected. Instead, keeping Photosystem I oxidised through photosynthetic control, negative feedback regulation of the Cytochrome b6f complex, was critical. Therefore, unlike in C3 plants, qE does not provide a significant adaptive advantage to C4 plants under dynamic light conditions. These findings provide important insights into the biology of C4 plants and help prioritise future strategies for improving the productivity and resilience of C4 crops.

plant biology↗

bioRxiv2026

C4 photosynthetic pathway fluxes in transgenic rice plants

Most land plants photosynthesize using the C3 pathway, in which ribulose bisphosphate carboxylase/oxygenase (Rubisco) fixes CO2 into 3-carbon acids. The C4 pathway, a biochemical CO2-concentrating mechanism that operates in the context of specialized leaf anatomy to concentrate CO2 around Rubisco, is more efficient. Introduction of the C4 pathway into the C3 crop rice could increase yield by 50%. Expression of five C4 enzymes in transgenic rice previously led to flux through the first step. However, there was no evidence for flux later in the cycle. Here we developed new transgenic rice lines and novel protocols to detect C4 cycle activity: CO2 fixation into C4 acids by carboxylation of a C3 compound, decarboxylation, refixation of CO2 by Rubisco, and regeneration of the C3 donor. We demonstrate that these four core C4 reactions are operating in rice, establishing the in vivo flux framework needed to progress towards a functional carbon-concentrating mechanism

plant biology↗

bioRxiv2025

Fitting photosynthetic carbon dioxide and irradiance response curves for C4 leaves

Gas exchange measurements provide crucial insights into the complex mechanisms of photosynthesis. Responses of CO2 assimilation rate (A) to intercellular CO2 partial pressure (Ci) and irradiance (I) link gas exchange measurements to the underlying photosynthetic biochemistry of a leaf. The unique biochemistry and leaf anatomy which distinguish C4 photosynthesis make it necessary to apply models and fitting routines which appropriately parameterise and incorporate these characteristics. Here we provide updates to the C4 photosynthesis model by improving the parameterisation of cyclic electron flow in C4 photosynthesis using experimentally derived values from S. viridis. We additionally describe two fitting routines for assessing C4 photosynthesis based on the updated model. Fitting of a CO2 response curve (A/Ci) provides estimates of maximum PEP carboxylase activity (Vpmax) and maximum Rubisco activity (Vcmax), and calculates the electron transport rate (J) needed to sustain the measured CO2 assimilation rate (A). Fitting of an irradiance response curve (A/I) provides estimates for the maximum electron transport rate (Jmax), day respiration rate (Rd), the quantum yield ({phi}CO2) and light compensation point ({Gamma}light). Values of the above output parameters are provided at both the measurement temperature and at 25 {degrees}C for ease of comparative reporting. The fitting tool has been designed in Microsoft Excel to minimise barriers to entry and enable simplicity of fitting while simultaneously catering to individuals with diverse expertise and experience in C4 gas exchange modelling.

plant biology↗

bioRxiv2024

A novel PGRL1 paralog refined the control of photoprotection in grasses and facilitated cell specialisation in C4 photosynthesis

The PGR5-PGRL1 pathway protects plants from photodamage by regulating electron flow to maintain Photosystem I in an oxidised state. Grasses possess two PGRL1 paralogs, but their functional roles have remained unknown. Here, we show that the ancestral PGRL1 paralog, PGRL1, which is conserved across algae and land plants, is enriched in the mesophyll cells of grasses that perform the NADP-ME subtype of C4 photosynthesis. In contrast, the grass-specific paralog PGRL1{beta} is enriched in bundle sheath cells. To investigate the functional significance of this cell-specific expression, we generated gene-edited lines of the NADP-ME C4 grass Setaria viridis lacking either PGRL1 paralog. We found that PGRL1{beta} in bundle sheath cells was required for rapid photoprotection, enabling Photosystem I oxidation five seconds faster during transitions from darkness to high light. In contrast, PGRL1 in mesophyll cells was essential for maintaining Photosystem I oxidation under steady-state high-light conditions. We propose that these complementary functions arise from structural differences within the lumen-facing regions of the two paralogs, providing a mechanistic basis for their distinct roles in regulating photoprotection. The conservation of this dual PGRL1 system across grasses suggests that functional specialisation of the paralogs expands the dynamic range of protective responses, enhancing photosynthetic performance under fluctuating and high-light stress conditions.

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↗

bioRxiv2023

Systems analysis of long-term heat stress responses in the C4 grass Setaria viridis

A substantial number of C4 plants are utilized as food and fodder crops and often display improved resource use efficiency compared to C3 counterparts. However, their response to future extreme climates such as heatwaves is less understood. Setaria viridis, an emerging C4 model grass closely related to important C4 crops, was grown under high temperature for two weeks (42{degrees}C as compared to 28{degrees}C). High temperature resulted in stunted growth, but surprisingly had little impact on leaf area based photosynthetic rates. Rates of dark respiration significantly increased and there were major alterations in carbon and nitrogen metabolism in the heat-stressed plants, including reduced starch levels, accumulation of soluble sugars and an increase in leaf nitrogen content. Measurements of major phytohormones revealed a dramatic increase in abscisic acid in the heat-stressed plants. Leaf transcriptomics, proteomics and metabolomics analyses were carried out and mapped onto metabolic pathways of photosynthesis, respiration, carbon/nitrogen metabolism and hormone synthesis and signaling. Overall, upregulation of a number of stress-signaling pathways was observed, consistent with multiple potent signals leading to reduced plant growth. A systems model of plant response is presented based on oxidative stress, hormone and sugar signaling pathways.

plant biology↗

bioRxiv2023

NDH complex-mediated cyclic electron flow in bundle sheath cells enables C4 photosynthesis

The superior productivity of C4 plants is achieved via a metabolic C4 cycle which acts as a CO2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. Chloroplast NADH dehydrogenase-like complex (NDH) increases ATP production in C3 plants by operating cyclic electron flow (CEF) around Photosystem I (PSI), and its importance for C4 photosynthesis has been proposed from evolutionary and reverse genetics studies. We used the gene-edited C4 species Setaria viridis with null ndhO alleles lacking NDH to study a contribution of the complex to the cell-level electron transport. Our results indicate that NDH is the primary PSI electron acceptor mediating the majority of CEF in BS cells whilst the contribution of the complex to CEF in mesophyll cells is minimal. Moreover, the reduced leaf CO2 assimilation rate and growth of plants lacking the complex cannot be rescued by supplying additional CO2, indicating that NDH is essential for generating ATP required for CO2 fixation by the C3 cycle. Hereby we resolve a cell-level mechanism for the contribution of NDH to supporting high CO2 assimilation rates in C4 photosynthesis.

plant biology↗

bioRxiv2023

Mesophyll-specific overexpressing of SvHXK6 gene improves water use efficiency without yield loss in C4 Setaria viridis

Hexokinases (HXK) were the first sugar signalling proteins identified in plants and are well known for their feedback regulation of photosynthetic gene expression. In some C3 plants, HXKs have been found to regulate stomatal function. However, the role of HXK in C4 photosynthesis, which is inherently more water use efficient than C3 metabolism, remains poorly understood. Here, we report on the first tissue-specific modification of HXK in a C4 plant. SvHXK6 was expressed in the model C4 grass Setaria viridis under the control of the ZmPEPC promoter (ZmPEPCpro), which directs expression in the leaf mesophyll tissue. Three S. viridis transgenic lines with increased abundance of SvHXK6 transcripts in the leaf tissue showed significant reduction in stomatal conductance with minimal effects on leaf CO2 assimilation rate. Consequently, the transgenic lines had higher leaf-level water use efficiency relative to the control (wild-type and null) plants. Overexpression of SvHXK6 had no effect on shoot biomass or seed yield of the S. viridis plants. Our study shows conserved function of HXK in regulating stomatal conductance in a C4 grass, demonstrating possible widespread utility in improving water use efficiency in C4 as well as C3 species.

plant biology↗

bioRxiv2022

Faster responses of photosynthesis to light transitions increase biomass and grain yield in transgenic Sorghum bicolor overexpressing Rieske FeS

Sorghum is one of the most important crops providing food and feed in many of the worlds harsher environments. Sorghum utilises the C4 pathway of photosynthesis in which a biochemical carbon concentrating mechanism results in high CO2 assimilation rates. Overexpressing the Rieske subunit of the Cytochrome b6f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO2 assimilation in the model C4 plant Setaria viridis. To test whether productivity of C4 crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor plants with increased Rieske content. The transgenic plants showed no marked changes in abundance of other photosynthetic proteins or chlorophyll content. Increases in yield of Photosystem II and CO2 assimilation rate as well as faster responses of non-photochemical quenching during transient photosynthetic responses were observed as a result of an elevated in vivo Cytochrome b6f activity in plants overexpressing Rieske. The steady-state rates of electron transport and CO2 assimilation did not differ between transgenic and control plants, suggesting that Cytochrome b6f is not the only factor limiting electron transport in sorghum at high light and high CO2. Nevertheless, more agile responses of photosynthesis to light transitions led to increases in biomass and grain yield in plants overexpressing Rieske. Our results indicate that increasing Rieske content could boost productivity of C4 crops by improving the efficiency of light utilisation and conversion to biomass.

plant biology↗

bioRxiv2022

A cross-scale analysis to understand and quantify effects of photosynthetic enhancement on crop growth and yield

Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying effectively how the seasonal growth and yield dynamics of target crops might be affected over a wide range of environments is limited. Using a state-of-the-art cross-scale model we predicted crop-level impacts of a broad list of promising photosynthesis manipulation strategies for C3 wheat and C4 sorghum. The manipulation targets have varying effects on the enzyme-limited (Ac) and electron transport-limited (Aj) rates of photosynthesis. In the top decile of seasonal outcomes, yield gains with the list of manipulations were predicted to be modest, ranging between 0 and 8%, depending on the crop type and manipulation. To achieve the higher yield gains, large increases in both Ac and Aj are needed. This could likely be achieved by stacking Rubisco function and electron transport chain enhancements or installing a full CO2 concentrating system. However, photosynthetic enhancement influences the timing and severity of water and nitrogen stress on the crop, confounding yield outcomes. Strategies enhancing Ac alone offers more consistent but smaller yield gains across environments, Aj enhancement alone offers higher gains but is undesirable in less favourable environments. Understanding and quantifying complex cross-scale interactions between photosynthesis and crop yield will challenge and stimulate photosynthesis and crop research. Summary StatementLeaf-canopy-crop prediction using a state-of-the-art cross-scale model improves understanding of how photosynthetic manipulation alters wheat and sorghum growth and yield dynamics. This generates novel insights for quantifying impacts of photosynthetic enhancement on crop yield across environments.

plant biology↗

bioRxiv2022

Increased sedoheptulose-1,7-bisphosphatase content in the C4 species Setaria viridis does not affect photosynthesis

Sedoheptulose-1,7-bisphosphatase (SBPase) is one of the rate-limiting enzymes of the Calvin cycle, and, in C3 plants, increasing the abundance of SBPase is known to provide higher photosynthetic rates and stimulate biomass and yield. C4 plants usually have higher photosynthetic rates because they operate a biochemical CO2 concentrating mechanism between mesophyll and bundle sheath cells. In the C4 system, SBPase and other enzymes of Calvin cycle are localised to the bundle sheath cells. Here we tested what effect increasing abundance of SBPase would have on C4 photosynthesis. Using Setaria viridis, a model C4 plant of NADP-ME subtype, we created transgenic plants with 1.5 to 3.2-times higher SBPase content, compared to wild type plants. Transcripts of the transgene were found predominantly in the bundle sheaths suggesting the correct cellular localisation of the protein. Abundance of RBCL, the large subunit of Rubisco, was not affected in transgenic plants overexpressing SBPase, and neither was relative chlorophyll content or photosynthetic electron transport parameters. We found no correlation between SBPase content in S. viridis and saturating rates of CO2 assimilation. Moreover, detailed analysis of CO2 assimilation rates at different CO2 partial pressure, irradiance and leaf temperature, showed no improvement of photosynthesis in plants overexpressing SBPase. We discuss potential implications of these results for understanding the regulation of C4 photosynthesis.

plant biology↗

bioRxiv2021

Expression of a CO2-permeable aquaporin enhances mesophyll conductance in the C4 species Setaria viridis

A fundamental limitation of photosynthetic carbon fixation is the availability of CO4. In C4 plants, primary carboxylation occurs in mesophyll cytosol, and little is known about the role of CO2 diffusion in facilitating C4 photosynthesis. We have examined the expression, localization, and functional role of selected plasma membrane intrinsic aquaporins (PIPs) from Setaria italica (foxtail millet) and discovered that SiPIP2;7 is CO2-permeable. When ectopically expressed in mesophyll cells of S. viridis (green foxtail), SiPIP2;7 was localized to the plasma membrane and caused no marked changes in leaf biochemistry. Gas-exchange and C18O16O discrimination measurements revealed that targeted expression of SiPIP2;7 enhanced the conductance to CO2 diffusion from the intercellular airspace to the mesophyll cytosol. Our results demonstrate that mesophyll conductance limits C4 photosynthesis at low pCO2 and that SiPIP2;7 is a functional CO2 permeable aquaporin that can improve CO2 diffusion at the airspace/mesophyll interface and enhance C4 photosynthesis.

plant biology↗

bioRxiv2021

Explainable machine learning models of major crop traits from satellite-monitored continent-wide field trial data

Four species of grass generate half of all human-consumed calories1. However, abundant biological data on species that produce our food remains largely inaccessible, imposing direct barriers to understanding crop yield and fitness traits. Here, we assemble and analyse a continent-wide database of field experiments spanning ten years and hundreds of thousands of machine-phenotyped populations of ten major crop species. Training an ensemble of machine learning models, using thousands of variables capturing weather, ground-sensor, soil, chemical and fertiliser dosage, management, and satellite data, produces robust cross-continent yield models exceeding R2 = 0.8 prediction accuracy. In contrast to black box analytics, detailed interrogation of these models reveals fundamental drivers of crop behaviour and complex interactions predicting yield and agronomic traits. These results demonstrate the capacity of machine learning models to build unified, interpretable, and explainable models of crop behaviour, and highlight the powerful role of data in the future of food.

plant biology↗

bioRxiv2021

Up-regulation of bundle sheath electron transport capacity under limiting light in C4 Setaria viridis

C4 photosynthesis is a biochemical pathway that operates across mesophyll and bundle sheath (BS) cells to increase CO2 concentration at the site of CO2 fixation. C4 plants benefit from high irradiance but their efficiency decreases under shade causing a loss of productivity in crop canopies. We investigated shade acclimation responses of a model NADP-ME monocot Setaria viridis focussing on cell-specific electron transport capacity. Plants grown under low light (LL) maintained CO2 assimilation rates similar to high light plants but had an increased chlorophyll and light-harvesting-protein content, predominantly in BS cells. Photosystem II (PSII) protein abundance, oxygen-evolving activity and the PSII/PSI ratio all increased in LL BS cells indicating a higher capacity for linear electron flow. PSI, ATP synthase, Cytochrome b6f and the chloroplastic NAD(P) dehydrogenase complex, which constitute the BS cyclic electron flow machinery, were all upregulated in LL plants. A decline in PEP carboxylase activity in mesophyll cells and a consequent shortage of reducing power in BS chloroplasts was associated with the more oxidised redox state of the plastoquinone pool in LL plants and the formation of PSII - light-harvesting complex II supercomplexes with an increased oxygen evolution rate. Our results provide evidence of a redox regulation of the supramolecular composition of Photosystem II in BS cells in response to shading. This newly identified link contributes to understanding the regulation of PSII activity in C4 plants and will support strategies for crop improvement including the engineering of C4 photosynthesis into C3 plants. Significance statementThe efficiency of C4 photosynthesis decreases under low irradiance causing a loss of productivity in crop canopies. We investigate shade acclimation of a model NADP-ME monocot, analysing cell-specific protein expression and electron transport capacity. We propose a regulatory pathway controlling abundance and activity of Photosystem II in bundle sheath cells in response to irradiance.

plant biology↗

bioRxiv2020

Targeted knockdown of ribulose-1, 5-bisphosphate carboxylase-oxygenase in rice mesophyll cells impact on photosynthesis and growth

We generated antisense constructs targeting two of the five Rubisco small subunit genes (OsRBCS2 and 4) which account for between 30-40% of the RBCS transcript abundance in leaf blades. The constructs were driven by a maize phosphoenolpyruvate carboxylase (PEPC) promoter known to have enriched expression in mesophyll cells (MCs). In the resulting lines leaf Rubisco protein content was reduced by between 30-50% and CO2 assimilation rate was limited under photorespiratory and non-photorespiratory conditions. A relationship between Rubisco protein content and CO2 assimilation rate was found. This was associated with a significant reduction in dry biomass accumulation and grain yield of between 37 to 70%. In addition to serving as a resource for reducing Rubisco accumulation in a cell-preferential manner, these lines allow us to characterize gene function and isoform specific suppression on photosynthesis and growth. Our results suggest that the knockdown of multiple genes is required to completely reduce Rubisco accumulation in MCs.

plant biology↗

bioRxiv2020

Uncovering candidate genes involved in photosynthetic capacity using unexplored genetic variation in Spring Wheat

To feed an ever-increasing population we must leverage advances in genomics and phenotyping to harness the variation in wheat breeding populations for traits like photosynthetic capacity which remains unoptimized. Here we survey a diverse set of wheat germplasm containing elite, introgression and synthetic derivative lines uncovering previously uncharacterised variation. We demonstrate how strategic integration of exotic material alleviates the D genome genetic bottleneck in wheat, increasing SNP rate by 62% largely due to Ae. tauschii synthetic wheat donors. Across the panel, 67% of the Ae. tauschii donor genome is represented as introgressions in elite backgrounds. We show how observed genetic variation together with hyperspectral reflectance data can be used to identify candidate genes for traits relating to photosynthetic capacity using association analysis. This demonstrates the value of genomic methods in uncovering hidden variation in wheat and how that variation can assist breeding efforts and increase our understanding of complex traits.

plant biology↗

bioRxiv2020

A partial C4 photosynthetic biochemical pathway in rice

Introduction of a C4 photosynthetic pathway into C3 rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously expresses four of the core C4 photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME) and pyruvate phosphate dikinase (ZmPPDK), in a cell-specific manner. This led to enhanced enzyme activity but was largely neutral in its effects on photosynthetic rate and growth. Measurements of the flux of 13CO2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13C into malate, consistent with increased fixation of 13CO2 via PEP carboxylase in lines expressing the maize PEPC enzyme. We also showed 13C labelling of aspartate indicating additional 13CO2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. However, there were no significant differences in labelling of 3-phosphoglycerate (3PGA) or phosphoenolpyruvate (PEP) indicating limited carbon flux through C4 enzymes into the Calvin-Benson cycle. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reduced OsGDCH line and higher labelling of malate, aspartate and citrate. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C4 cycle, these results demonstrate for the first-time flux through the carboxylation phase of C4 metabolism in transgenic rice containing the key metabolic steps in the C4 pathway.

plant biology↗