The price of pure cellulase enzyme for the recovery of fermentable cellulosic sugars is one of the major challenges that limit the commercialization of second-generation biofuels and bio-based products. This work shows a means to greatly reduce the cost of cellulases. The abundant capacity of plants to synthesize and hyperaccumulate transgenic proteins in their plastids has been demonstrated for two gene constructs using either NPTII or TetC to regulate the expression of bacterial cellulase Cel6A. Previously, the expression of Cel6A to 20-35% of total soluble leaf protein in tobacco was shown in two consecutive field trials. Moving forward, the key challenge was the extraction of active enzymes from the transgenic tobacco leaves hyperaccumulating bacterial cellulases in a cost-effective manner. The study showed that unpurified crude extracts of NPTII and TetC transgenic tobacco leaves recovered similar to 23% and similar to 29% w/w cellulosic sugars from energycane bagasse, respectively. A supplementation of as low as 25% of purified commercial cellulase improved the glucose recovery by 2.9 times (similar to 85% w/w) compared to 100% crude extract, which is comparable to the glucose recovery obtained by commercial cellulases, thereby suggesting a reduced requirement of commercial cellulases leading to a cost reduction of 75% for biorefineries. Assuming a stable 40% cellulase yield in total soluble protein under field conditions and multiple harvests (one to three) a year, the study estimates that the potential cost for saccharification of 1 t of lignocellulosic biomass can be reduced to 67-200 USD by using crude leaf extracts of transgenic tobacco. (c) 2026 The Author(s). Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd.

Abstract Accelerating photoprotective regulation to improve carbon assimilation is a promising strategy to increase crop productivity. Although rapid non-photochemical quenching (NPQ) relaxation has been validated as a target through metabolic engineering, it remains unclear whether conventional breeding has improved this trait. Here, we investigated whether more than a century of soybean breeding enhanced NPQ relaxation alongside light-saturated carbon assimilation and seed traits. We evaluated a historical panel of 24 soybean genotypes across vegetative and reproductive developmental stages by integrating NPQ relaxation, gas exchange parameters, xanthophyll-cycle pigment profiles, expression of key photoprotective genes ( VDE , PsbS , and ZEP ), seed number and seed weight. NPQ relaxation parameters were not consistently associated with genotype release year, seed number, or seed weight at either developmental stage. The only exception was the amplitude of the rapidly relaxing NPQ component (AqE), which was negatively correlated with all three variables during the reproductive stage. In contrast, genotype release year was positively associated with maximum net CO₂ assimilation rate ( A max ), maximum carboxylation rate of Rubisco ( V cmax ), maximum electron transport rate ( J max ), seed number, and seed weight, while A max and V cmax were positively correlated with seed number and seed weight. These findings indicate that the greater photosynthetic capacity of modern genotypes was not accompanied by faster photoprotective response. Thus, photoprotective regulation has not kept pace with gains in photosynthetic capacity under field conditions. We conclude that rapid NPQ relaxation remains an important target for synchronizing photoprotection with the high photosynthetic capacity of modern soybean lines.

ABSTRACT Miscanthus × giganteus (M×g) is a high‐yielding perennial C4 bioenergy crop, but genetic improvement by breeding is constrained by triploid sterility and clonal propagation. Improving genetic transformation methods for M×g would provide opportunities for advantageous trait introgression. Use of an easy to phenotype reporter gene is a promising strategy to improve transformation processes and efficiency. This study presents an efficient novel method for biolistic transformation of inflorescence‐derived callus in M×g and demonstrates its efficacy using RUBY, a betalain‐based noninvasive reporter that is visible throughout the transformation process. RUBY expression (Zea mays codon optimized) was visible from callus stage through plantlet development into maturity. RUBY expressing independently transformed plants were confirmed by hygromycin phosphotransferase ELISA and by genomic PCR demonstrating that the RUBY phenotype is sufficient for screening transformants. The Zea mays codon optimized hygromycin selection marker was driven by previously established promoters for Miscanthus, ZmUBI and 2×35S, while the RUBY gene expression was controlled by a known Zea mays C4 promoter, Brachypodium UBI10, newly employed in Miscanthus. The construct containing the 2×35S promoter for hygromycin had a 15.1% transformation efficiency while the ZmUBI promoter had a 20.5% transformation efficiency. This study provides a novel, highly efficient protocol for successful biolistic transformation of M×g for stable expression. This study also demonstrates that RUBY expression can be used as a convenient and powerful monitor of transformation in ongoing and future work to engineer M×g into an improved bioproduct feedstock.

Upstream open reading frames (uORFs) are regulatory elements present in the 5' leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome-wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild-type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost-efficient alternative to investigate transcript leaders by co-expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO2 assimilation during sun-shade transitions.

Continued greenhouse gas emissions will accelerate global warming and intensity of heat waves, which already harm crop productivity. From the stability of key enzymes to canopy processes, photosynthesis is affected by temperature. All crops suffer declines in photosynthetic rate when temperatures cross critical thresholds, with irreversible losses typically occurring above 40° to 45°C. Protective measures within plants can be induced by growth at elevated temperatures but not from the sudden temperature elevation of heat waves. Strategies to improve the heat resilience of photosynthesis include modifying surface energy balance, optimizing canopy architecture, improving enzymatic heat tolerance, and (re)engineering key metabolic pathways for greater efficiency or to remove bottlenecks. This Review summarizes present knowledge on the major mechanisms that underlie high-temperature inhibition of photosynthesis and explores opportunities for breeding and biotechnological interventions to overcome them.

Arctic environments are changing rapidly. To assess climate change impacts and guide conservation, there is a need to effectively monitor areas of high biodiversity that are difficult to access, such as the deep sea. Greenland (Kalaallit Nunaat), like many remote countries with large deep-sea exclusive economic zones (EEZs), lacks consistent access to the funding and logistics required to maintain advanced and expensive technologies for seafloor exploration. To fill this need, video and camera imaging technologies have been adapted to suit the unique requirements of Arctic environments and the social and economic needs of Greenland. Since 2015, a benthic monitoring program carried out by the Greenland Institute of Natural Resources (GINR) has provided the only large-scale, comprehensive survey in this region, including collection and analysis of photos and GoPro video footage recorded as deep as 1,600 m (Blicher, and Arboe, 2021). In line with the “collect once, use many times” principle, GINR is exploring the versatility of these data, which were originally designated for monitoring and evidence-based management. A potential research avenue for these data is polar blue carbon—the carbon stored and sequestered in ocean habitats—including benthic communities that either live on the seafloor (such as corals and sponges) or are transported there by ocean currents (such as algal detritus). This paper outlines Greenland’s affordable deep-sea technology, based on a towed camera system (Yesson, 2023), and its potential application to rapid, standardized artificial intelligence (AI)-based analysis.

Plant architecture influences the microenvironment throughout the canopy layer. Plants with a more erect leaf architecture allow for an increase in planting densities and allow more light to reach lower canopy leaves. This is predicted to increase crop carbon assimilation. Frictional resistance to wind reduces air movement in the lower canopy, resulting in higher humidity. By increasing the proportion of canopy photosynthesis in the more humid lower canopy, gains in the efficiency of water use might be expected, although this may be slightly offset by the more open erectophile form canopy. An anatomical feature in members of the Poaceae family that impacts leaf angle is the articulated junction of the sheath and blade, which also bares the ligule and auricles. Mutants, which lack ligules and auricles, show no articulation at this junction, resulting in leaves that are near vertical. In maize, these phenotypes termed liguleless result from null mutations of genes: ZmLG1 (Zm00001eb67740) and ZmLG2 (Zm00001eb147220). In sorghum, SbiRTx430.06G264300 (SbLG1) and SbiRTx430.03G392300 (SbLG2) are annotated as the respective maize homologues. A hair-pin element designed to down-regulate both SbLG1 and SbLG2 was introduced into the grain sorghum genotype RTx430. Derived transgenic events harbouring the hair-pin failed to develop ligules and displayed reduced leaf angles to the vertical, but less vertical than in null mutations. Under field settings, plots sown with these sorghum events having an erect architecture phenotype displayed an increase in photosynthesis in lower canopy levels, which led to increases in above-ground biomass and seed yield, without an increase in water use.

Crop leaves absorb approximately 90% of visible photons (400 - 700 nm) but transmit or reflect most far-red (FR) photons (700 - 800 nm). However, some cyanobacteria use FR photons up to 800 nm by incorporating chlorophyll (Chl) d or/and f into their photosystems. Here, we use a 3D canopy model to evaluate whether introducing these pigments could improve photosynthetic performance of field grown soybean. We simulate photon absorption and CO2 assimilation show that the introduction of Chl d and f would significantly enhance photosynthesis, especially in the lower canopy, where visible light is depleted and FR light is abundant. When FR absorption is regulated by phytochrome-sensed FR/red (FR/R) photon ratio, the benefit is further increased, with canopy CO2 assimilation improving up to 26%, without increasing the risk of photodamage. These results show that introducing FR-absorbing Chls into crops could have great value in boosting productivity.

Meta-analyses and theory show that with rising atmospheric [CO2], Rubisco has become the greatest limitation to light-saturated leaf CO2 assimilation rates (Asat) in C4 crops. So would transgenically increasing Rubisco increase Asat and result in increased productivity in the field? Here, we successfully overexpressed the Rubisco small subunit (RbcS) with Rubisco accumulation factor 1 (Raf1) in both sorghum and sugarcane, resulting in significant increases in Rubisco content of 13 to 25% and up to 90% respectively. Asat increased 12 to 15% and Rubisco enzyme activity ~40% in three independent transgenic events of both species. Sorghum plants also showed increased speeds of photosynthetic induction and decreased bundle sheath leakiness. These improvements translated into average increases of 15.5% in biomass in field-grown sorghum and a 37 to 81% increase in greenhouse-grown sugarcane. This suggests a potential opportunity to achieve substantial increases in productivity of this key economically important clade of C4 crops, future proofing their value under global atmospheric change.

Predicted changes in atmospheric composition and climate affecting crop productivity are reviewed. These include changes in both average conditions and extreme events, with respect to temperature, drought, flooding and surface ozone, coupled with rising atmospheric [CO2]. Impacts on, and means to adapt, crops to these changes are reviewed and outlined. Particular emphasis is given to (i) the results from open air field manipulations of surface atmosphere, temperature and soil water to understand impacts and adaptation and (ii) demonstrated genetic manipulations of photosynthesis and water use that could support future food supply under current and future conditions. Finally, attention is given to means by which crop systems could serve as CO2 collectors and carbon storage systems. Here, apparent opportunities are outlined for (i) manipulations of crops to enhance carbon storage and (ii) use of high-productivity sustainable perennial C4 grasses coupled with carbon capture and storage.This article is part of the theme issue 'Crops under stress: can we mitigate the impacts of climate change on agriculture and launch the 'Resilience Revolution'?'.

Light detection and ranging (lidar) scanning tools are available that can make rapid digital estimations of biomass. Voxelization and convex hull are two algorithms used to calculate the volume of the scanned plant canopy, which is correlated with biomass, often the primary trait of interest. Voxelization splits the scans into regular-sized cubes, or voxels, whereas the convex hull algorithm creates a polygon mesh around the outermost points of the point cloud and calculates the volume within that mesh. In this study, digital estimates of biomass were correlated against hand-harvested biomass for field-grown corn, broom corn, and energy sorghum. Voxelization (r = 0.92) and convex hull (r = 0.95) both correlated well with plant dry biomass. Lidar data were also collected in a large breeding trial with nearly 900 genotypes of energy sorghum. In contrast to the manual harvest studies, digital biomass estimations correlated poorly with yield collected from a forage harvester for both voxel count (r = 0.32) and convex hull volume (r = 0.39). However, further analysis showed that the coefficient of variation (CV, a measure of variability) for harvester-based estimates of biomass was greater than the CV of the voxel and convex-hull-based biomass estimates, indicating that poor correlation was due to harvester imprecision, not digital estimations. Overall, results indicate that the lidar-based digital biomass estimates presented here are comparable or more precise than current approaches.

Soybean (Glycine max) is the single most important global source of vegetable protein. Yield improvements per unit land area are needed to avoid further expansion onto natural systems. Mesophyll conductance (gm) quantifies the ease with which CO2 can diffuse from the sub-stomatal cavity to Rubisco. Increasing gm is attractive since it increases photosynthesis without increasing water use. Most measurements of gm have been made during steady-state light saturated photosynthesis. In field crop canopies, light fluctuations are frequent and the speed with which gm can increase following shade to sun transitions affects crop carbon gain. Is there variability in gm within soybean germplasm? If so, indirect selection may have indirectly increased gm during domestication and subsequent breeding for sustainability and yield. A modern elite cultivar (LD11) was compared with four ancestor accessions of Glycine soja from the assumed area of domestication by concurrent measurements of gas exchange and carbon isotope discrimination (∆13C). gm was a significant limitation to soybean photosynthesis both at steady state and through light induction but was twice the value of the ancestors in LD11. This corresponded to a substantial increase in leaf photosynthetic CO2 uptake and water use efficiency.

Bioenergy with carbon capture and geological storage (BECCS) is considered one of the top options for both offsetting CO2 emissions and removing atmospheric CO2. BECCS requires using limited land resources efficiently while ensuring minimal adverse impacts on the delicate food-energy-water nexus. Perennial C4 biomass crops are productive on marginal land under low-input conditions avoiding conflict with food and feed crops. The eastern half of the contiguous U.S. contains a large amount of marginal land, which is not economically viable for food production and liable to wind and water erosion under annual cultivation. However, this land is suitable for geological CO2 storage and perennial crop growth. Given the climate variation across the region, three perennials are major contenders for planting. The yield potential and stability of Miscanthus, switchgrass, and energycane across the region were compared to select which would perform best under the recent (2000-2014) and future (2036-2050) climates. Miscanthus performed best in the Midwest, switchgrass in the Northeast and energycane in the Southeast. On average, Miscanthus yield decreased from present 19.1 t/ha to future 16.8 t/ha; switchgrass yield from 3.5 to 2.4 t/ha; and energycane yield increased from 14 to 15 t/ha. Future yield stability decreased in the region with higher predicted drought stress. Combined, these crops could produce 0.6-0.62 billion tonnes biomass per year for the present and future. Using the biomass for power generation with CCS would capture 703-726 million tonnes of atmospheric CO(2 )per year, which would offset about 11% of current total U.S. emission. Further, this biomass approximates the net primary CO(2 )productivity of two times the current baseline productivity of existing vegetation, suggesting a huge potential for BECCS. Beyond BECCS, C4 perennial grasses could also increase soil carbon and provide biomass for emerging industries developing replacements for non-renewable products including plastics and building materials.

Small-scale fisheries are a cornerstone of coastal livelihoods throughout Madagascar, providing income, nutrition, and a way of life to over half a million people. Due to limited state capacity, community-based management of marine resources has proliferated, with locally-managed marine areas (LMMAs) becoming a major avenue for coastal resource governance in Madagascar. Though case studies of LMMAs exist, little research has tracked their effectiveness over time. In 2016, the Marine Protected Areas Governance (MPAG) framework was used to critically assess the governance of the Ankobohobo wetland’s mud crab fishery: the first analysis of a fishery produced using the framework. This study revisits Ankobohobo using the same methodology three years later, representing the first longitudinal application of the framework. Overall, participants throughout the fishery revealed a lack of progress towards management goals and emphasized the vulnerability of the governance model to external drivers of change. Despite substantial efforts from fishers and fishing associations to secure sustainable crab fishing, progress is hindered by an absence of support from the state or NGOs. Persistent challenges include intensified mangrove logging, declining crab stocks, increased fishing effort, and entrenched poverty. These are driven by factors beyond the communities’ control: global markets, population growth, migration, and a lack of viable, income-generating activities. These findings emphasise that a purely bottom-up, community-led approach cannot address external drivers. Effective governance requires a diversity of actors and incentives combining bottom-up and top-down approaches. The absence of this in Ankobohobo explains the lack of progress in the three years between these assessments.

The gap between genomics and phenomics is narrowing. The rate at which it is narrowing, however, is being slowed by improper statistical comparison of methods. Quantification using Pearson’s correlation coefficient (r) is commonly used to assess method quality, but it is an often misleading statistic for this purpose as it is unable to provide information about the relative quality of two methods. Using r can both erroneously discount methods that are inherently more precise and validate methods that are less accurate. These errors occur because of logical flaws inherent in the use of r when comparing methods, not as a problem of limited sample size or the unavoidable possibility of a type I error. A popular alternative to using r is to measure the limits of agreement (LOA). However both r and LOA fail to identify which instrument is more or less variable than the other and can lead to incorrect conclusions about method quality. An alternative approach, comparing variances of methods, requires repeated measurements of the same subject, but avoids incorrect conclusions. Variance comparison is arguably the most important component of method validation and, thus, when repeated measurements are possible, variance comparison provides considerable value to these studies. Statistical tests to compare variances presented here are well established, easy to interpret and ubiquitously available. The widespread use of r has potentially led to numerous incorrect conclusions about method quality, hampering development, and the approach described here would be useful to advance high throughput phenotyping methods but can also extend into any branch of science. The adoption of the statistical techniques outlined in this paper will help speed the adoption of new high throughput phenotyping techniques by indicating when one should reject a new method, outright replace an old method or conditionally use a new method.

As the leading global grain crop, maize significantly impacts agricultural water usage. Presently, photosynthesis ( A net ${A}_{\text{net}}$ ) in leaves of modern maize crops is saturated with CO 2 ${\text{CO}}_{2}$ , implying that reducing stomatal conductance ( g s ${g}_{{\rm{s}}}$ ) would not affect A net ${A}_{\text{net}}$ but reduce transpiration ( τ $\tau $ ), thereby increasing water use efficiency (WUE). While g s ${g}_{{\rm{s}}}$ reduction benefits upper canopy leaves under optimal conditions, the tradeoffs in low light and nitrogen-deficient leaves under nonoptimal microenvironments remain unexplored. Moreover, g s ${g}_{{\rm{s}}}$ reduction increases leaf temperature ( T leaf ${T}_{\text{leaf}}$ ) and water vapor pressure deficit, partially counteracting transpiratory water savings. Therefore, the overall impact of g s ${g}_{{\rm{s}}}$ reduction on water savings remains unclear. Here, we use a process-based leaf model to investigate the benefits of reduced g s ${g}_{{\rm{s}}}$ in maize leaves under different microenvironments. Our findings show that increases in T leaf ${T}_{\text{leaf}}$ due to g s ${g}_{{\rm{s}}}$ reduction can diminish WUE gains by up to 20%. However, g s ${g}_{{\rm{s}}}$ reduction still results in beneficial WUE tradeoffs, where a 29% decrease in g s ${g}_{{\rm{s}}}$ in upper canopy leaves results in a 28% WUE gain without loss in A net ${A}_{\text{net}}$ . Lower canopy leaves exhibit superior tradeoffs in g s ${g}_{{\rm{s}}}$ reduction with 178% gains in WUE without loss in A net ${A}_{\text{net}}$ . Our simulations show that these WUE benefits are resilient to climate change.

Mesophyll conductance (g(m)) describes the ease with which CO2 passes from the sub-stomatal cavities of the leaf to the primary carboxylase of photosynthesis, Rubisco. Increasing g(m) is suggested as a means to engineer increases in photosynthesis by increasing [CO2] at Rubisco, inhibiting oxygenation and accelerating carboxylation. Here, tobacco was transgenically up-regulated with Arabidopsis Cotton Golgi-related 3 (CGR3), a gene controlling methylesterification of pectin, as a strategy to increase CO2 diffusion across the cell wall and thereby increase g(m). Across three independent events in tobacco strongly expressing AtCGR3, mesophyll cell wall thickness was decreased by 7%-13%, wall porosity increased by 75% and g(m) measured by carbon isotope discrimination increased by 28%. Importantly, field-grown plants showed an average 8% increase in leaf photosynthetic CO2 uptake. Up-regulating CGR3 provides a new strategy for increasing g(m) in dicotyledonous crops, leading to higher CO2 assimilation and a potential means to sustainable crop yield improvement.

Development of sustainable and scalable technologies to convert lignocellulosic biomass to biofuels is critical to achieving carbon neutrality. The potential of transgenic bioenergy crops as a renewable source of sugars and lipids has been demonstrated at bench-scale. However, scaling up these processes is important for holistic analysis. Here proof-of-concept for chemical-free hydrothermal pretreatment of transgenic energycane-oilcane line L13 at an industrially relevant scale to recover vegetative lipids along with cellulosic sugars is presented. Pilot-scale processing of 97 kg of transgenic energycane-oilcane L13 stems and high solids pretreatment of bagasse enhanced the recovery of cellulosic glucose and xylose by 5-fold as compared to untreated bagasse and helped in the enrichment of vegetative lipids in the biomass residues which allowed its recovery at the end of the bioprocess. Palmitic and oleic acids were the predominant fatty acids (FAs) extracted from stems and leaves. The processing did not affect lipid composition. The efficiency of lipid recovery from untreated biomass was 75.9% which improved to 88.7% upon pretreatment. The vegetative tissues of transgenic energycane-oilcane L13 contained 0.42 metric tons/hectare of lipids. Processing vegetative tissues yielded 0.38 metric tons/hectare of lipids. This approaches an oil yield similar to soybean (global average 0.44 metric tons/hectare) and is almost twice as high as the oil yield from sugarcane engineered to hyperaccumulate lipids (0.20 metric tons/hectare). The study suggests that further optimization by state-of-the-art metabolic engineering and biomass processing can establish transgenic bioenergy crops for commercial drop-in fuel production.

Orthodox scholarship claims the CIA-MI6 covert actions in Albania and Ukraine in the early Cold War exemplified a singular effort to rollback Soviet power in Europe. Using recently declassified files, this article presents a case study of Operation BGFiend/Valuable and Aerodynamic to challenge the existence of an aggressive rollback programme. These were limited, diverse and strategically dynamic projects that did not seek regime change in either territory. The west subverted Hoxha's Albanian regime, focusing on intelligence collection and propaganda. In Ukraine, emphasis was on gathering intelligence, particularly to provide early warning of an anticipated Soviet invasion of Western Europe.