The pyrenoidal linker protein EPYC1 phase separates with hybrid Arabidopsis-Chlamydomonas Rubisco through interactions with the algal Rubisco small subunit

Photosynthetic efficiencies in plants are restricted by the CO2-fixing enzyme Rubisco but could be enhanced by introducing a CO2-concentrating mechanism (CCM) from green algae, such as Chlamydomonas reinhardtii (hereafter Chlamydomonas). A key feature of the algal CCM is aggregation of Rubisco in the pyrenoid, a liquid-like organelle in the chloroplast. Here we have used a yeast two-hybrid system and higher plants to investigate the protein-protein interaction between Rubisco and essential pyrenoid component 1 (EPYC1), a linker protein required for Rubisco aggregation. We showed that EPYC1 interacts with the small subunit of Rubisco (SSU) from Chlamydomonas and that EPYC1 has at least five SSU interaction sites. Interaction is crucially dependent on the two surface-exposed α-helices of the Chlamydomonas SSU. EPYC1 could be localized to the chloroplast in higher plants and was not detrimental to growth when expressed stably in Arabidopsis with or without a Chlamydomonas SSU. Although EPYC1 interacted with Rubisco in planta, EPYC1 was a target for proteolytic degradation. Plants expressing EPYC1 did not show obvious evidence of Rubisco aggregation. Nevertheless, hybrid Arabidopsis Rubisco containing the Chlamydomonas SSU could phase separate into liquid droplets with purified EPYC1 in vitro, providing the first evidence of pyrenoidlike aggregation for Rubisco derived from a higher plant.This work was funded by the UK BBSRC (BB/M006468/1) and Leverhulme Trust (RPG-2017-402). TEM was carried out with the support of the Wellcome Trust Multi User Equipment Grant (WT104915MA)

Transcriptome-wide analysis of pseudouridylation of mRNA and non-coding RNAs in Arabidopsis

Pseudouridine (Ψ) is widely distributed in mRNA and various non-coding RNAs in yeast and mammals, and the specificity of its distribution has been determined. However, knowledge about Ψs in the RNAs of plants, particularly in mRNA, is lacking. In this study, we performed genome-wide pseudouridine-sequencing in Arabidopsis and for the first time identified hundreds of Ψ sites in mRNA and multiple Ψ sites in non-coding RNAs. Many predicted and novel Ψ sites in rRNA and tRNA were detected. mRNA was extensively pseudouridylated, but with Ψs being under-represented in 3′-untranslated regions and enriched at position 1 of triple codons. The phenylalanine codon UUC was the most frequently pseudouridylated site. Some Ψs present in chloroplast 23S, 16S, and 4.5S rRNAs in wild-type Col-0 were absent in plants with a mutation of SVR1 (Suppressor of variegation 1), a chloroplast pseudouridine synthase gene. Many plastid ribosomal proteins and photosynthesis-related proteins were significantly reduced in svr1 relative to the wild-type, indicating the roles of SVR1 in chloroplast protein biosynthesis in Arabidopsis. Our results provide new insights into the occurrence of pseudouridine in Arabidopsis RNAs and the biological functions of SVR1, and will pave the way for further exploiting the mechanisms underlying Ψ modifications in controlling gene expression and protein biosynthesis in plants.This work was supported by the Ministry of Agriculture of China (2016ZX08009-003) and National Program on Key Basic Research Project (973 Program, 2012CB1143001)

Shade compromises the photosynthetic efficiency of NADP-ME less than that of PEP-CK and NAD-ME C4 grasses

The high energy cost and apparently low plasticity of C4 photosynthesis compared with C3 photosynthesis may limit the productivity and distribution of C4 plants in low light (LL) environments. C4 photosynthesis evolved numerous times, but it remains unclear how different biochemical subtypes perform under LL. We grew eight C4 grasses belonging to three biochemical subtypes [NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK)] under shade (16% sunlight) or control (full sunlight) conditions and measured their photosynthetic characteristics at both low and high light. We show for the first time that LL (during measurement or growth) compromised the CO2-concentrating mechanism (CCM) to a greater extent in NAD-ME than in PEP-CK or NADP-ME C4 grasses by virtue of a greater increase in carbon isotope discrimination (∆P) and bundle sheath CO2 leakiness (ϕ), and a greater reduction in photosynthetic quantum yield (Φmax). These responses were partly explained by changes in the ratios of phosphoenolpyruvate carboxylase (PEPC)/initial Rubisco activity and dark respiration/photosynthesis (Rd/A). Shade induced a greater photosynthetic acclimation in NAD-ME than in NADP-ME and PEP-CK species due to a greater Rubisco deactivation. Shade also reduced plant dry mass to a greater extent in NAD-ME and PEP-CK relative to NADP-ME grasses. In conclusion, LL compromised the co-ordination of the C4 and C3 cycles and, hence, the efficiency of the CCM to a greater extent in NAD-ME than in PEP-CK species, while CCM efficiency was less impacted by LL in NADP-ME species. Consequently, NADP-ME species are more efficient at LL, which could explain their agronomic and ecological dominance relative to other C4 grasses.This research was funded by the Australian Research Council: DP120101603 (OG and SMW), DE130101760 (RES), and CE140100015 (OG and SMW)

Effects of leaf age during drought and recovery on photosynthesis, mesophyll conductance and leaf anatomy in wheat leaves

Summary statement: Mesophyll conductance (gm) was negatively correlated with wheat leaf age but was positively correlated with the surface area of chloroplasts exposed to intercellular airspaces (Sc). The rate of decline in photosynthetic rate and gm as leaves aged was slower for water-stressed than well-watered plants. Upon rewatering, the degree of recovery from water-stress depended on the age of the leaves, with the strongest recovery for mature leaves, rather than young or old leaves. Diffusion of CO2 from the intercellular airspaces to the site of Rubisco within C3 plant chloroplasts (gm) governs photosynthetic CO2 assimilation (A). However, variation in gm in response to environmental stress during leaf development remains poorly understood. Age-dependent changes in leaf ultrastructure and potential impacts on gm, A, and stomatal conductance to CO2 (gsc) were investigated for wheat (Triticum aestivum L.) in well-watered and water-stressed plants, and after recovery by re-watering of droughted plants. Significant reductions in A and gm were found as leaves aged. The oldest plants (15 days and 22 days) in water-stressed conditions showed higher A and gm compared to irrigated plants. The rate of decline in A and gm as leaves aged was slower for water-stressed compared to well-watered plants. When droughted plants were rewatered, the degree of recovery depended on the age of the leaves, but only for gm. The surface area of chloroplasts exposed to intercellular airspaces (Sc) and the size of individual chloroplasts declined as leaves aged, resulting in a positive correlation between gm and Sc. Leaf age significantly affected cell wall thickness (tcw), which was higher in old leaves compared to mature/young leaves. Greater knowledge of leaf anatomical traits associated with gm partially explained changes in physiology with leaf age and plant water status, which in turn should create more possibilities for improving photosynthesis using breeding/biotechnological strategies

The rubisco chaperone BSD2 may regulate chloroplast coverage in maize bundle sheath cells

In maize (Zea mays), Bundle Sheath Defective2 (BSD2) plays an essential role in Rubisco biogenesis and is required for correct bundle sheath (BS) cell differentiation. Yet, BSD2 RNA and protein levels are similar in mesophyll (M) and BS chloroplasts, although Rubisco accumulates only in BS chloroplasts. This raises the possibility of additional BSD2 roles in cell development. To test this hypothesis, transgenic lines were created that overexpress and underexpress BSD2 in both BS and M cells, driven by the cell type-specific Rubisco Small Subunit (RBCS) or Phosphoenolpyruvate Carboxylase (PEPC) promoters or the ubiquitin promoter. Genetic crosses showed that each of the transgenes could complement Rubisco deficiency and seedling lethality conferred by the bsd2 mutation. This was unexpected, as RBCS-BSD2 lines lacked BSD2 in M chloroplasts and PEPC-BSD2 lines expressed half the wild-type BSD2 level in BS chloroplasts.We conclude that BSD2 does not play a vital role inM cells and that BS BSD2 is in excess of requirements for Rubisco accumulation. BSD2 levels did affect chloroplast coverage in BS cells. In PEPC-BSD2 lines, chloroplast coverage decreased 30% to 50%, whereas lines with increased BSD2 content exhibited a 25% increase. This suggests that BSD2 has an ancillary role in BS cells related to chloroplast size. Gas exchange showed decreased photosynthetic rates in PEPC-BSD2 lines despite restored Rubisco function, correlating with reduced chloroplast coverage and pointing to CO2 diffusion changes. Conversely, increased chloroplast coverage did not result in increased Rubisco abundance or photosynthetic rates. This suggests another limitation beyond chloroplast volume, most likely Rubisco biogenesis and/or turnover rates

Synthetic biology and opportunities within agricultural crops

Conventional breeding techniques have been integral to the development of many agronomically important traits in numerous crops. The adoption of modern biotechnology approaches further advanced and refined trait development and introduction beyond the scope possible through conventional breeding. However, crop yields continue to be challenged by abiotic and biotic factors that require the development of traits that are more genetically complex than can be addressed through conventional breeding or traditional genetic engineering. Therefore, more advanced trait development approaches are required to maintain and improve yields and production efficiency, especially as climate change accelerates the incidence of biotic and abiotic challenges to food and fibre crops. Synthetic biology (SynBio) encompasses approaches that design and construct new biological elements (e.g., enzymes, genetic circuits, cells) or redesign existing biological systems to build new and improved functions. SynBio ‘upgrades’ the potential of genetic engineering, which involves the transfer of single genes from one organism to another. This technology can enable the introduction of multiple genes in a single transgenic event, either derived from a foreign organism or synthetically generated. It can also enable the assembly of novel genomes from the ground up from a set of standardised genetic parts, which can then be transferred into the target cell or organism. New opportunities to advance breeding applications through exploiting SynBio technology include the introduction of new genes of known function, artificially creating genetic variation, topical applications of small RNAs as pesticides and potentially speeding up the production of new cultivars with elite traits. This review will draw upon case studies to demonstrate the potential application of SynBio to improve crop productivity and resistance to various challenges. Here, we outline specific solutions to challenges including fungal diseases, insect pests, heat and drought stress and nutrient acquisition in a range of important crops using the SynBio toolkit

Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms

While marine phytoplankton rival plants in their contribution to global primary productivity, our understanding of their photosynthesis remains rudimentary. In particular, the kinetic diversity of the CO₂-fixing enzyme, Rubisco, in phytoplankton remains unknown. Here we quantify the maximum rates of carboxylation (Kcatᶜ), oxygenation (Kcatᵒ), Michaelis constants (K m) for CO₂ (K C) and O₂ (K O), and specificity for CO₂ over O₂ (SC/O) for Form I Rubisco from 11 diatom species. Diatom Rubisco shows greater variation in KC (23-68 μM), SC/O (57-116mol mol⁻¹), and KO (413-2032 μM) relative to plant and algal Rubisco. The broad range of KC values mostly exceed those of C₄ plant Rubisco, suggesting that the strength of the carbon-concentrating mechanism (CCM) in diatoms is more diverse, and more effective than previously predicted. The measured k cat c for each diatom Rubisco showed less variation (2.1-3.7s⁻¹), thus averting the canonical trade-off typically observed between KC and kcatᶜ for plant Form I Rubisco. Uniquely, a negative relationship between KC and cellular Rubisco content was found, suggesting variation among diatom species in how they allocate their limited cellular resources between Rubisco synthesis and their CCM. The activation status of Rubisco in each diatom was low, indicating a requirement for Rubisco activase. This work highlights the need to better understand the correlative natural diversity between the Rubisco kinetics and CCM of diatoms and the underpinning mechanistic differences in catalytic chemistry among the Form I Rubisco superfamily

Photons to food: genetic improvement of cereal crop photosynthesis

Photosynthesis has become a major trait of interest for cereal yield improvement as breeders appear to have reached the theoretical genetic limit for harvest index, the mass of grain as a proportion of crop biomass. Yield improvements afforded by the adoption of green revolution dwarfing genes to wheat and rice are becoming exhausted, and improvements in biomass and radiation use efficiency are now sought in these crops. Exploring genetic diversity in photosynthesis is now possible using high-throughput techniques, and low-cost genotyping facilitates discovery of the genetic architecture underlying this variation. Photosynthetic traits have been shown to be highly heritable, and significant variation is present for these traits in available germplasm. This offers hope that breeding for improved photosynthesis and radiation use efficiency in cereal crops is tractable and a useful shorter term adjunct to genetic and genome engineering to boost yield potential.the New South Wales Environmental Trust (2016/RD/0006), the Cotton Research and Development Corporation (CRDC), and the Grains Research and Development Corporation (GRDC

Linking photosynthesis and leaf N allocation under future elevated CO2 and climate warming in Eucalyptus globulus

Leaf-level photosynthetic processes and their environmental dependencies are critical for estimating CO2 uptake from the atmosphere. These estimates use biochemical-based models of photosynthesis that require accurate Rubisco kinetics. We investigated the effects of canopy position, elevated atmospheric CO2 [eC; ambient CO2 (aC)+240 ppm] and elevated air temperature (eT; ambient temperature (aT)+3 °C) on Rubisco content and activity together with the relationship between leaf N and Vcmax (maximal Rubisco carboxylation rate) of 7 m tall, soil-grown Eucalyptus globulus trees. The kinetics of E. globulus and tobacco Rubisco at 25 °C were similar. In vitro estimates of Vcmax derived from measures of E. globulus Rubisco content and kinetics were consistent, although slightly lower, than the in vivo rates extrapolated from gas exchange. In E. globulus, the fraction of N invested in Rubisco was substantially lower than for crop species and varied with treatments. Photosynthetic acclimation of E. globulus leaves to eC was underpinned by reduced leaf N and Rubisco contents; the opposite occurred in response to eT coinciding with growth resumption in spring. Our findings highlight the adaptive capacity of this key forest species to allocate leaf N flexibly to Rubisco and other photosynthetic proteins across differing canopy positions in response to future, warmer and elevated [CO2] climates.This is a contribution from the Hawkesbury Forest Experiment. We thank Burhan Amiji and Dr Craig Barton for their assistance in undertaking gas exchange. This research was supported by funding from ARC grant DP160102452, the Forest Industries Climate Change Research Fund from the Australian Department of Agriculture, and the Commonwealth Government through the Education Investment Fund

Short-term thermal photosynthetic responses of C4 grasses are independent of the biochemical subtype

C4 photosynthesis evolved independently many times, resulting in multiple biochemical pathways; however, little is known about how these different pathways respond to temperature. We investigated the photosynthetic responses of eight C4 grasses belonging to three biochemical subtypes (NADP-ME, PEP-CK, and NAD-ME) to four leaf temperatures (18, 25, 32, and 40 °C). We also explored whether the biochemical subtype influences the thermal responses of (i) in vitro PEPC (Vpmax) and Rubisco (Vcmax) maximal activities, (ii) initial slope (IS) and CO2-saturated rate (CSR) derived from the A-Ci curves, and (iii) CO2 leakage out of the bundle sheath estimated from carbon isotope discrimination. We focussed on leakiness and the two carboxylases because they determine the coordination of the CO2-concentrating mechanism and are important for parameterizing the semi-mechanistic C4 photosynthesis model. We found that the thermal responses of Vpmax and Vcmax, IS, CSR, and leakiness varied among the C4 species independently of the biochemical subtype. No correlation was observed between Vpmax and IS or between Vcmax and CSR; while the ratios Vpmax/Vcmax and IS/CSR did not correlate with leakiness among the C4 grasses. Determining mesophyll and bundle sheath conductances in diverse C4 grasses is required to further elucidate how C4 photosynthesis responds to temperature.BVS was supported by a postgraduate research award funded by the Australian Research Council and the Hawkesbury Institute for the Environment at Western Sydney University. This research was funded by the following grants from the Australian Research Council: DP120101603 (OG, SMW), DE130101760 (RES), and CE140100015 (OG, SvC, SMW)