Response of plasmodesmata formation in leaves of C₄ grasses to growth irradiance

Rapid metabolite diffusion across the mesophyll (M) and bundle sheath (BS) cellinterface in C4leaves is a key requirement for C4photosynthesis and occurs viaplasmodesmata (PD). Here, we investigated how growth irradiance affects PDdensity between M and BS cells and between M cells in two C4species using ourPD quantification method, which combines three‐dimensional laser confocal fluores-cence microscopy and scanning electron microscopy. The response of leaf anatomyand physiology of NADP‐ME species,Setaria viridisandZea maysto growth under dif-ferent irradiances, low light (100μmol m−2s−1), and high light (1,000μmol m−2s−1),was observed both at seedling and established growth stages. We found that theeffect of growth irradiance on C4leaf PD density depended on plant age and species.The high light treatment resulted in two to four‐fold greater PD density per unit leafarea than at low light, due to greater area of PD clusters and greater PD size in highlight plants. These results along with our finding that the effect of light on M‐BS PDdensity was not tightly linked to photosynthetic capacity suggest a complex mecha-nism underlying the dynamic response of C4leaf PD formation to growth irradiance.This research was funded by the Australian Government through the Australian Research Council Centre of Excellence for Translational Photosynthesis (CE1401000015). F. R. D is also financially supported by the Lee Rice Foundation scholarship through the International Rice Research Institute, Philippines

Multiple mechanisms for enhanced plasmodesmata density in disparate subtypes of C4 grasses

Proliferation of plasmodesmata (PD) connections between bundle sheath (BS) and mesophyll (M) cells has been proposed as a key step in the evolution of two-cell C4 photosynthesis; However, a lack of quantitative data has hampered further exploration and validation of this hypothesis. In this study, we quantified leaf anatomical traits associated with metabolite transport in 18 species of BEP and PACMAD grasses encompassing four origins of C4 photosynthesis and all three C4 subtypes (NADP-ME, NAD-ME, and PCK). We demonstrate that C4 leaves have greater PD density between M and BS cells than C3 leaves. We show that this greater PD density is achieved by increasing either the pit field (cluster of PD) area or the number of PD per pit field area. NAD-ME species had greater pit field area per M–BS interface than NADP-ME or PCK species. In contrast, NADP-ME and PCK species had lower pit field area with increased number of PD per pit field area than NAD-ME species. Overall, PD density per M–BS cell interface was greatest in NAD-ME species while PD density in PCK species exhibited the largest variability. Finally, the only other anatomical characteristic that clearly distinguished C4 from C3 species was their greater Sb value, the BS surface area to subtending leaf area ratio. In contrast, BS cell volume was comparable between the C3 and C4 grass species examined.FRD is supported by scholarship awards from the Lee Foundation (IRRI) and the Australian Research Council Centre of Excellence for Translational Photosynthesis (CE140100015). SK is a Royal Society University Research Fellow. Work in SK’s lab is supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement number 637765

On the road to C-4 rice: advances and perspectives

The international C4 rice consortium aims to introduce into rice a high capacity photosynthetic mechanism, the C4 pathway, to increase yield. The C4 pathway is characterised by a complex combination of biochemical and anatomical specialisation that ensures high CO2 partial pressure at RuBisCO sites in bundle sheath (BS) cells. Here we report an update of the progress of the C4 rice project. Since its inception in 2008 there has been an exponential growth in synthetic biology and molecular tools. Golden Gate cloning and synthetic promoter systems have facilitated gene building block approaches allowing multiple enzymes and metabolite transporters to be assembled and expressed from single gene constructs. Photosynthetic functionalisation of the BS in rice remains an important step and there has been some success overexpressing transcription factors in the cytokinin signalling network which influence chloroplast volume. The C4 rice project has rejuvenated the research interest in C4 photosynthesis. Comparative anatomical studies now point to critical features essential for the design. So far little attention has been paid to the energetics. C4 photosynthesis has a greater ATP requirement, which is met by increased cyclic electron transport in BS cells. We hypothesise that changes in energy statues may drive this increased capacity for cyclic electron flow without the need for further modification. Although increasing vein density will ultimately be necessary for high efficiency C4 rice, our modelling shows that small amounts of C4 photosynthesis introduced around existing veins could already provide benefits of increased photosynthesis on the road to C4 rice.The Research was funded by a C4 rice projectgrant from The Bill & Melinda Gates Foundation to the Universityof Oxford (2015–2019; OPP1129902) and Australian Research Council Centre of Excellence for Translational Photosynthesis(CE1401000015

Installation of C4 photosynthetic pathway enzymes in rice using a single construct

Introduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13CO2 labelling demonstrated a 10-fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP-malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.This work was funded by a C4 Rice Project grant from Bill & Melinda Gates Foundation to the University of Oxford (2015– 2019; OPP1129902), Max Planck Society (SA, HI, RF, MG, JEL, MS) and Australian Research Council (DP150101037 to ML, JEL, MS and CE140100015). Work in the SK laboratory was supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement number 637765; SK is a Royal Society University Research Fello

Niraparib in patients with metastatic castration-resistant prostate cancer and DNA repair gene defects (GALAHAD): a multicentre, open-label, phase 2 trial

Background Metastatic castration-resistant prostate cancers are enriched for DNA repair gene defects (DRDs) that can be susceptible to synthetic lethality through inhibition of PARP proteins. We evaluated the anti-tumour activity and safety of the PARP inhibitor niraparib in patients with metastatic castration-resistant prostate cancers and DRDs who progressed on previous treatment with an androgen signalling inhibitor and a taxane. Methods In this multicentre, open-label, single-arm, phase 2 study, patients aged at least 18 years with histologically confirmed metastatic castration-resistant prostate cancer (mixed histology accepted, with the exception of the small cell pure phenotype) and DRDs (assessed in blood, tumour tissue, or saliva), with progression on a previous next-generation androgen signalling inhibitor and a taxane per Response Evaluation Criteria in Solid Tumors 1.1 or Prostate Cancer Working Group 3 criteria and an Eastern Cooperative Oncology Group performance status of 0–2, were eligible. Enrolled patients received niraparib 300 mg orally once daily until treatment discontinuation, death, or study termination. For the final study analysis, all patients who received at least one dose of study drug were included in the safety analysis population; patients with germline pathogenic or somatic biallelic pathogenic alterations in BRCA1 or BRCA2 (BRCA cohort) or biallelic alterations in other prespecified DRDs (non-BRCA cohort) were included in the efficacy analysis population. The primary endpoint was objective response rate in patients with BRCA alterations and measurable disease (measurable BRCA cohort). This study is registered with ClinicalTrials.gov, NCT02854436. Findings Between Sept 28, 2016, and June 26, 2020, 289 patients were enrolled, of whom 182 (63%) had received three or more systemic therapies for prostate cancer. 223 (77%) of 289 patients were included in the overall efficacy analysis population, which included BRCA (n=142) and non-BRCA (n=81) cohorts. At final analysis, with a median follow-up of 10·0 months (IQR 6·6–13·3), the objective response rate in the measurable BRCA cohort (n=76) was 34·2% (95% CI 23·7–46·0). In the safety analysis population, the most common treatment-emergent adverse events of any grade were nausea (169 [58%] of 289), anaemia (156 [54%]), and vomiting (111 [38%]); the most common grade 3 or worse events were haematological (anaemia in 95 [33%] of 289; thrombocytopenia in 47 [16%]; and neutropenia in 28 [10%]). Of 134 (46%) of 289 patients with at least one serious treatment-emergent adverse event, the most common were also haematological (thrombocytopenia in 17 [6%] and anaemia in 13 [4%]). Two adverse events with fatal outcome (one patient with urosepsis in the BRCA cohort and one patient with sepsis in the non-BRCA cohort) were deemed possibly related to niraparib treatment. Interpretation Niraparib is tolerable and shows anti-tumour activity in heavily pretreated patients with metastatic castration-resistant prostate cancer and DRDs, particularly in those with BRCA alterations

The role of leaf plasmodesmata in C4 photosynthesis

In a C4 leaf, numerous plasmodesmata in the wall interface between the mesophyll and bundle sheath cells provide symplastic transport for exchange of C4 acids and metabolites during photosynthesis. Despite their importance in understanding the function and efficiency of C4 photosynthesis, quantification studies of the plasmodesmata that exist between these two photosynthetic cells are very few. The main reason for this is the difficulty involved in capturing the plasmodesmata that occur as clusters or pit field units particularly in leaves. In this work, this problem was overcame through the development of a new and more robust method to accurately quantify plasmodesmata in leaves without resorting to serial sectioning (Chapter 3). This was achieved by combining scanning electron microscopy and three-dimensional laser confocal fluorescence microscopy (e.i. PEA-CLARITY). This new method allowed empirical quantification of plasmodesmata per cell interface area, and consequently, estimation of plasmodesmata flux when these values are combined with photosynthetic measurements (Chapter 3). The new plasmodesmata quantification method provided the opportunity to examine a diversity of grass species within the non-C4 BEP clade and PACMAD clade including representative species from C3, C3-C4 intermediate, and all the C4 biochemical subtypes, nicotinamide adenine dinucleotide phosphate - malic enzyme (NADP-ME), nicotinamide adenine dinucleotide - malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PCK) (Chapter 4). Results showed that C3 species, regardless of clade, have fewer plasmodesmata than C4 species. Panicum milioides, a C3-C4 intermediate species, had plasmodesmata density within the C4 species range. Within the C4 species, there was a substantial variation of plasmodesmata densities at the mesophyll-bundle sheath cell interfaces among the subtypes, where NAD-ME subtype had the greatest value, NADP-ME had the least, and PCK had the largest variability. The greater plasmodesmata density in C4 leaves was a result of having larger pit fields and an increased number of plasmodesmata per pit field area. Conversely, plasmodesmata densities at the mesophyll-mesophyll cell interfaces were similar in C3 and C4 species. Other C4 evolution-related anatomical traits such as bundle sheath cell size, interveinal distance, and bundle sheath surface area per leaf area (Sb) were also measured. Results revealed that Sb and, to a lesser extent, interveinal distance, but not bundle sheath cell size, were statistically different between C3 species and C4 species. In addition to plasmodesmata density survey, the new method developed here was also applied to investigate the response of plasmodesmata formation in C4 grasses to changes in photosynthetic capacity. Results showed that when C4 plants were subjected to different growth light environments (Chapter 5) or when the initial carbon fixation enzyme, phosphoenolpyruvate carboxylase (PEPC) was downregulated reducing photosynthetic capacity in these plants (Chapter 6), plasmodesmata connection between the mesophyll and bundle sheath cells were altered. Low growth irradiance caused fewer plasmodesmata connection to form between mesophyll and bundle sheath cells in leaves of both Setaria viridis and Zea mays compared to the high light-grown plants, albeit manifested at different plant ages (Chapter 5). On the other hand, an increased number of pit fields in the mesophyll-bundle sheath cell interface of the leaf resulted from impaired C4-associated PEPC gene expression in S. viridis (Chapter 6). From these results it can be concluded that increased plasmodesmata density between the mesophyll and bundle sheath cells is an essential anatomical feature of C4 photosynthesis and is responsive to changes in photosynthetic capacity in C4 plants

CO₂ diffusion in tobacco: A link between mesophyll conductance and leaf anatomy

The partial pressure of CO 2 at the sites of carboxylation within chloroplasts depends on the conductance to CO 2 diffusion from intercellular airspace to the sites of carboxylation, termed mesophyll conductance (g m). We investigated how g m varies with leaf age and through a tobacco (Nicotiana tabacum) canopy by combining gas exchange and carbon isotope measurements using tunable diode laser spectroscopy. We combined these measurements with the anatomical characterization of leaves. CO 2 assimilation rate, A, and g m decreased as leaves aged and moved lower in the canopy and were linearly correlated. This was accompanied by large anatomical changes including an increase in leaf thickness. Chloroplast surface area exposed to the intercellular airspace per unit leaf area (S c) also decreased lower in the canopy. Older leaves had thicker mesophyll cell walls and g m was inversely proportional to cell wall thickness. We conclude that reduced g m of older leaves lower in the canopy was associated with a reduction in S c and a thickening of mesophyll cell walls.This research was supported by a sub-award from the Univer-sity of Illinois as part of the Realizing Increased PhotosyntheticEfficiency (RIPE) project, funded by the Bill & Melinda GatesFoundation to V.C.C. and S.v.C. and the Australian ResearchCouncil Centre of Excellence for Translational Photosynthesis(CE1401000015) for S.v.C. and F.R.D

Peeking at a plant through the holes in the wall - exploring the roles of plasmodesmata

Plasmodesmata (PD) are membrane-line pores that connect neighbouring plant cells and allow molecular exchange via the symplast. Past studies have revealed the basic structure of PD, some of the transport mechanisms for molecules through PD, and a variety of physiological processes in which they function. Recently, with the help of newly developed technologies, several exciting new features of PD have been revealed. New PD structures were observed during early formation of PD and between phloem sieve elements and phloem pole pericycle cells in roots. Both observations challenge our current understanding of PD structure and function. Research into novel physiological responses, which are regulated by PD, indicates that we have not yet fully explored the potential contribution of PD to overall plant function. In this Viewpoint article, we summarize some of the recent advances in understanding the structure and function of PD and propose the challenges ahead for the community

The Metabolite Pathway between Bundle Sheath and Mesophyll: Quantification of Plasmodesmata in Leaves of C-3 and C-4 Monocots

C4 photosynthesis is characterized by a CO2-concentrating mechanism between mesophyll (M) and bundle sheath (BS) cells of leaves. This generates high metabolic fluxes between these cells, through interconnecting plasmodesmata (PD). Quantification of these symplastic fluxes for modeling studies requires accurate quantification of PD, which has proven difficult using transmission electron microscopy. Our new quantitative technique combines scanning electron microscopy and 3D immunolocalization in intact leaf tissues to compare PD density on cell interfaces in leaves of C3 (rice [Oryza sativa] and wheat [Triticum aestivum]) and C4 (maize [Zea mays] and Setaria viridis) monocot species. Scanning electron microscopy quantification of PD density revealed that C4 species had approximately twice the number of PD per pitfield area compared with their C3 counterparts. 3D immunolocalization of callose at pitfields using confocal microscopy showed that pitfield area per M-BS interface area was 5 times greater in C4 species. Thus, the two C4 species had up to nine times more PD per M-BS interface area (S. viridis, 9.3 PD µm−2; maize, 7.5 PD µm−2; rice 1.0 PD µm−2; wheat, 2.6 PD µm−2). Using these anatomical data and measured photosynthetic rates in these C4 species, we have now calculated symplastic C4 acid flux per PD across the M-BS interface. These quantitative data are essential for modeling studies and gene discovery strategies needed to introduce aspects of C4 photosynthesis to C3 crops.F.R.D. is supported by scholarship awards from Lee Foundation (IRRI) and Centre of Excellence for Translational Photosynthesis (ANU)