Biology subjects

Smith, A. G.

Publications and source records attributed to Smith, A. G..

At least 19 recordsLinked to original sources

bioRxiv2026

Cellular basis of B12 uptake and remodelling in microalgae revealed using a novel bioassay

O_LIVitamin B12, an essential micronutrient for many microalgae and humans, is synthesised only by certain prokaryotes. B12 is a complex tetrapyrrole that can exist in many forms (vitamers), some more bioavailable than others. Some microalgae are able to interconvert, or remodel, different B12 vitamers. As microalgae are important primary producers, it is crucial to understand how diverse microalgae acquire, utilise, and remodel this micronutrient. C_LIO_LIThrough the development of a novel algal bioassay for B12 quantification that distinguishes between B12 vitamers with different lower axial ligands, and the generation of targeted knock-out lines, we characterised the role of proteins involved in algal B12 uptake and remodelling. C_LIO_LIWe found that the previously characterised protein CoBalamin-Acquisition protein 1 (CBA1) is also necessary for the acquisition of pseudocobalamin, a less bioavailable form of B12. In addition, we provide the first experimental evidence that COBT is required for Chlamydomonas reinhardtii to remodel B12. C_LIO_LIWe apply the algal B12 bioassay to show that the edible alga Chlorella vulgaris can accumulate pseudocobalamin but is unable to remodel it, highlighting the need for thorough investigation of the metabolic requirements and capabilities of microalgae, especially given the growing interest in microalgae-based food additives. C_LI

microbiology↗

bioRxiv2026

A Root Foundation Model for Zero-Shot Segmentation

Foundation models pre-trained on massive datasets have demonstrated impressive performance, but in some specialised domains have been found to have lower accuracy. Domain-specific foundation models target a particular domain such as retinal or plant images. These domain-specific models have shown inconsistent results and the benefit to root segmentation is unknown. We train and evaluate the first domainspecific foundation model for root segmentation. Evaluation uses a leave-one-dataset-out design across nine diverse root datasets with two architectures. Applied zero-shot to unseen datasets, the root foundation model achieves 92% of fine-tuned Dice on average (0.636 versus 0.698), with 5 of 9 datasets above 90%. With 10 patches of few-shot fine-tuning, the root foundation model recovers 95% of its full-data Dice on average, versus 69% for a general pre-trained model. At low patch counts the general pre-trained model often failed to converge, with 5 of 9 datasets giving Dice below 0.05 at 3 patches, while the root foundation model produced Dice above 0.47 on every dataset and patch count. With full target-data fine-tuning, the two perform comparably, with mean improvements of +0.011 Dice for MobileSAM and +0.022 for M2F Swin-S, neither significant (Wilcoxon p = 0.150 and 0.064). We release our pre-trained MobileSAM root foundation model for use with RootPainter, enabling fully automatic root segmentation on new datasets with an ordinary laptop or desktop computer, with no need for annotation or training.

plant biology↗

bioRxiv2026

Transformers Outperform ConvNets for Root Segmentation: A Systematic Comparison Across Nine Datasets

Root segmentation is a fundamental yet challenging task in image-based plant phenotyping. We present the first systematic comparison of Transformer and Convolutional Neural Network (ConvNet) architectures for root segmentation, evaluating 21 architectures across nine diverse datasets and comparing pre-trained models to training from scratch. Transformer-based models significantly outperform ConvNets for segmentation accuracy and root-diameter agreement. Pre-training significantly improves mean Dice from 0.623 to 0.666 (p = 3.3 x 10-10). We also find that Transformers benefit more from pre-training than ConvNets, with Dice improvements of +0.072 versus +0.022 (p = 3.7 x 10-4), supporting the hypothesis that fine-tuned Transformers transfer more effectively across large domain gaps. Among evaluated models, MobileSAM achieved the highest Dice score while maintaining computational efficiency. Dataset choice explained far more performance variance (70.9%) than model architecture (6.7%), suggesting that data curation matters more than model selection.

plant biology↗

bioRxiv2025

MultiSpecies Canopy Segmentation: Interactive Machine-Learning and Pseudo-Labelling are key

This study investigates the challenge of creating datasets for training multiclass deep-learning segmentation models, specifically for segmenting multi-species canopy images. Creating training sets for deep-learning based segmentation of multispecies canopies is currently too labor-intensive and time-consuming to be viable. To address this challenge, we propose a novel pipeline that uses fully convolutional neural networks (FCNNs) to transition from single-species images to segmented multi-species images. This paper demonstrates that FCNNs can effectively generalize learning from single-species canopy images to multispecies canopy images, achieving accurate pixel classification in mixed species canopies even when the network was trained only on images of single-species canopies. Additionally, we introduce Interactive Machine Learning and pseudo labeling as a method for generating a single-species canopy training set in a matter of minutes. We also present two software packages to implement our approach and extensively evaluate them against several baselines. Our findings demonstrate that our approach can significantly reduce the human time load required for semantic segmentation of multispecies canopy images, achieving over 90% accuracy in less than 10 minutes. This new method has the potential to greatly facilitate the study of multispecies canopies.

bioengineering↗

bioRxiv2025

Inhibition of BRN2 in Melanoma Reverses Anoikis Resistance and Sensitizes Cells to Killing by Vemurafenib

Anoikis is an apoptotic cell death program triggered upon detachment from surrounding extracellular structures. However, the ability to evade cell death by anoikis in the presence of apoptosis-inducing stimuli is necessary for the formation of malignant tumors and progression to metastasis. Our findings indicate that the BRN2 (POU3F2) transcription factor is associated with anoikis resistance in melanoma cells. However, the BRN2 signaling cascade driving anoikis resistance remains unknown. Herein, we employed genome-wide CRISPR screens to validate BRN2 as a driver of anoikis resistance. Small molecule inhibition of BRN2 in melanoma cell lines with acquired anoikis resistance resensitized to death by anoikis in ultra-low attachment conditions. Our quantitative mass spectrometry analysis revealed that BRN2 functionally impacts oxidative phosphorylation and mitochondrial activity, whereby probes designed to inhibit BRN2 induced apoptosis and mitochondrial fragmentation through the MAPK and NF-{kappa}B signaling pathways and reduction in PPAR{gamma} expression. Our study suggests that inhibition of BRN2 might allow the targeting of metastatic cells in circulation, and sensitizes cells to BRAF-targeted therapy, improving the prognosis for melanoma patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/667908v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@12ffd89org.highwire.dtl.DTLVardef@1ae6c8eorg.highwire.dtl.DTLVardef@58e49forg.highwire.dtl.DTLVardef@9b83e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO Role of BRN2 in driving anoikis resistance in melanoma. Upon detachment from the extra-cellular matrix (ECM) melanoma cells must evade cell death by anoikis to seed distant metastases. This study expanded the understanding of the role of the BRN2 transcription factor as a driver of resistance to anoikis in melanoma. The use of small molecule inhibitors targeting BRN2 revealed that the transcription factor drives anoikis resistance via the MAPK and NF-{kappa}B signaling pathways, resulting in PPAR{gamma} dysregulation and subsequently driving mitochondrial dysfunction. Green boxes = previously published drivers of anoikis resistance in melanoma. Blue box = changes to mitochondrial function following inhibition of BRN2 as determined by proteomics analysis. C_FIG

cancer biology↗

bioRxiv2025

Cultivating Future Materials: Artificial Symbiosis for Bulk Production of Bacterial Cellulose Composites

Symbiotic relationships between micro-organisms are the basis of all global ecosystems. Here we extrapolate this concept for novel material fabrication by creating artificial symbiotic relationships between species that are usually not grown synergistically in nature. Specifically, we combine the cellulose-producing bacterium Komagataeibacter hansenii and the green microalga Chlamydomonas reinhardtii to obtain bulk growth of bacterial cellulose. Usually, bacterial cellulose is produced as floating pellicles at the air-liquid interface of the growing media, because free oxygen, together with the nutrients in the culture medium, is required for the bacteria to synthesise the cellulose fibres. In the co-culture, bacterial cellulose production can be achieved in bulk beyond the restriction of the liquid/air interface as the motile microalgae act as oxygen-generating sites within the culture medium. In exchange, the highly porous and mechanically robust scaffold provided by the cellulose allows the algal community to form a healthy biofilm, which can reach several centimetres thick. We demonstrate that our growing platform allows the simultaneous production of bulk bacterial cellulose in static incubation conditions, taking up an arbitrary and yet tunable 3D shape, dependent on the geometry of the culture vessel and with a relatively high biomass output.

microbiology↗

bioRxiv2025

Five seasons with DeepRootLab: A unique facility for easier deep root research in the field

Deep-rooted crops accessing water and nutrients from deep soil layers enhance the resource base for crop production. However, studying these roots in field conditions is labor-intensive, limiting research scope. We established a field root research facility with 48 plots for replicated experiments. The facility includes 144 six-meter-long minirhizotron tubes and an AI-based pipeline for rapid root trait analysis. We also attempted to install access-tubes and customized ingrowth-core production for less-invasive root activity determination. Our study revealed significant differences in deep root density among species, particularly at depths of 2.5 to 4.5 meters, over five years. The less invasive studies using ingrowth-cores reached depths of 4.2 meters. Nutrient tracer 15N analysis showed marked differences in deep root activity among crop species. TDR sensors indicated varying water depletion in deeper soil layers, influenced by crop species and root growth patterns. We established a field facility for studying deep root growth and function, demonstrating its effectiveness in analyzing diverse deep-rooted plant species. This facility provides an ideal platform for conducting meaningful research in deep soil layers, yielding statistically and biologically significant results for agricultural applications.

plant biology↗

bioRxiv2025

Chlamydomonas chloroplast genes tolerate compression of the genetic code to just 51 codons

Genome scale engineering has enabled codon compression of the universal genetic code of up to three codons in E. coli, providing the means for genetic code expansion. To go much beyond this number, smaller and simpler genetic systems are needed to avoid significant technical challenges. Chloroplast genomes offer multiple advantages for codon compression and reassignment. Here we report a recoding scheme for the Chlamydomonas reinhardtii chloroplast genome, in which two stop codons and one or more of the codons for arginine, glycine, isoleucine, leucine and serine, all of which have two cognate tRNAs, are absent, compressing the genetic code to 51 codons. Firstly, several recoding strategies were tested on the essential rpoA gene, encoding a subunit of the chloroplast RNA polymerase. A defined compression scheme, which relied on swapping the target codons with the permitted frequent codons, could replace the native protein coding sequence without affecting chloroplast protein expression levels or the strain fitness. The same strategy was successfully used for codon compression of ycf1, encoding a subunit of the chloroplast translocon, psaA and psbA, intron-containing highly expressed genes encoding reaction centres subunits of both photosystems, and an 8.5 kb operon encoding essential and non-essential genes. Finally, we tested degeneracy of the 51-codon genetic code by exploring the combinatorial design for the large subunit of RuBisCO, relying on restoration of photosynthesis in an rbcL mutant strain. More than 70 functional sequences with diverse codon adaptation indices were recovered. In all codon-compressed genes there was no observable penalty on photosynthetic growth. Significance StatementWe demonstrate the use of a chloroplast genome as a synthetic biology platform to test modifications of the universal genetic code. Elimination of 13 of the 64 possible triplet codes (codons) was possible for several essential and/or highly expressed genes without affecting chloroplast function. The technology is mature, paving the way for codon compression of the entire genome and thus enabling further radical rearrangement or expansion of the genetic code.

synthetic biology↗

bioRxiv2024

Automated Seminal Root Angle Measurement with Corrective Annotation

Measuring seminal root angle is an important aspect of root phenotyping, yet automated methods are lacking. We introduce SeminalRootAngle, a novel open-source automated method that measures seminal root angles from images. To ensure our method is flexible and user-friendly we build on an established corrective annotation training method for image segmentation. We tested SeminalRootAngle on a heterogeneous dataset of 662 spring barley rhizobox images, which presented challenges in terms of image clarity and root obstruction. Validation of our new automated pipeline against manual measurements yielded a Pearson correlation coefficient of 0.71. We also measure inter-annotator agreement, obtaining a Pearson correlation coefficient of 0.68, indicating that our new pipeline provides similar root angle measurement accuracy to manual approaches. We use our new SeminalRootAngle tool to identify SNPs significantly associated with angle and length, shedding light on the genetic basis of root architecture.

plant biology↗

bioRxiv2023

New Interactive Machine Learning Tool for Marine Image Analysis

Due to advances in imaging technologies, the rate of marine video and image data collection is drastically increasing. Often these datasets are not analysed to their full potential as extracting information for multiple species, such as their presence and surface area, is incredibly time-consuming. This study demonstrates the potential of a new open-source interactive machine learning tool, RootPainter, to analyse large marine image datasets quickly and accurately. The tool was initially developed to measure plant roots, but here was tested on its ability to measure the presence and surface area of the cold-water coral reef associate sponge species, Mycale lingua, in two types of underwater image data: 18,346 time-lapse images and 1,420 remotely operated vehicle video frames. New corrective annotation metrics integrated with RootPainter, such as dice score and species area error, allow for the objective assessment of when to stop model training and reduce the need for manual model validation. Three highly accurate Mycale lingua models were created using RootPainter, as indicated by their average dice score of 0.94 {+/-} 0.06. Model transfer and optimisation aided in the production of two of these models, increasing analysis efficiency from 6 to 16 times faster than manual annotation in Photoshop, for underwater observatory images. Sponge and surface area measurements were extracted from both datasets allowing future investigation of sponge behaviours and distributions. This study demonstrates that interactive machine learning tools and model sharing have the potential to dramatically increase image analysis speeds, collaborative research, and our collective knowledge on spatiotemporal patterns in biodiversity.

ecology↗

bioRxiv2023

Light management by algal aggregates in living photosynthetic hydrogels.

Rapid progress in algal biotechnology has triggered a growing interest in hydrogel-encapsulated microalgal cultivation, especially for the engineering of functional photosynthetic materials and biomass production. An overlooked characteristic of gel-encapsulated cultures is the emergence of cell aggregates, which are the result of the mechanical confinement of the cells. Such aggregates have a dramatic effect on the light management of gel-encapsulated photobioreactors and hence strongly affect the photosynthetic outcome. In order to evaluate such an effect, we experimentally studied the optical response of hydrogels containing algal aggregates and developed optical simulations to study the resultant light intensity profiles. The simulations are validated experimentally via transmittance measurements using an integrating sphere and aggregate volume analysis with confocal microscopy. Specifically, the heterogeneous distribution of cell aggregates in a gel matrix can increase light penetration while alleviating photoinhibition compared to a flat biofilm. Finally, we demonstrate that light harvesting efficiency can be further enhanced with the introduction of scattering particles within the hydrogel matrix, leading to a four-fold increase in biomass growth. Our study, therefore, highlights a new strategy for the design of spatially efficient photosynthetic living materials that have important implications for the engineering of future algal cultivation systems. Significance StatementThe ability to cultivate microalgae at scale efficiently would allow more sustainable production of food and food additives. However, efficient growth of microalgae requires optimised light conditions, which are usually challenging to obtain using biofilm cultivations mode: as the outer layer of cells are necessarily more exposed to incoming light than the inner layer, posing the problem of photoinhibition on the outer cells receiving too much light, and shading the ones below. Here we study both experimentally and numerically, how microalgae aggregates growing in the confinement of a hydrogel can provide an improved light distribution and therefore biomass growth is maximised. This study proposes new strategies on how to engineer future photobioreactors.

plant biology↗

bioRxiv2023

Presence of vitamin B12 metabolism in the last common ancestor of land plants

Vitamin B12, or cobalamin, (hereinafter B12) is an essential organic micronutrient, required by humans as a cofactor for methionine synthase (METH) and for methylmalonyl CoA mutase (MCM), involved in the propionate shunt. B12 is a complex corrinoid molecule made only by a subset of bacteria. Plants and fungi have an alternative methionine synthase (METE) that does not need a B12 cofactor, so these organisms are typically considered to neither synthesise nor utilise B12. In contrast many algal species utilise B12 if it is available, because they encode both METE and METH. Moreover, a large proportion of algal species encode METH only, and so are like animals in being dependent on an external source of the vitamin. Here, we performed a detailed phylogenetic analysis of the distribution of METE, METH and eleven further proteins implicated in B12 metabolism in eukaryotic cells across an exhaustive library of over 1,500 plant and algal genomes and transcriptomes. The results reveal the hitherto undetected existence of B12-associated metabolism deep into the streptophytes. The B12-dependent synthase METH, and the accessory proteins MTRR, CblB, CblC, CblD and CblJ were detected in the basally divergent plant lineage of hornworts, and CblB and CblJ were further identified in liverworts. Using phylogenetic and PFAM analysis we demonstrate this is due to retention of ancestral B12-metabolism pathways in the last common ancestor of land plants, followed by at least two independent complete losses in mosses and vascular plants. We further show more limited distributions of genes encoding B12-related proteins across the algal tree of life, including MCM and type II ribonucleotide reductase, alongside an obligate B12-dependency across several major marine algal orders. Finally, by considering the functional biology of early-diverging land plants, together with the collection sites of ten further algal species inferred to have lost B12-dependent metabolism, we propose freshwater-to-land transitions and symbiotic associations to have been major constraining factors in B12 availability in early plant evolution.

plant biology↗

bioRxiv2023

The conserved protein CBA1 is required for vitamin B12 uptake in different algal lineages

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling, but despite their phototrophic lifestyle, over half of algal species depend on a supply of the corrinoid vitamin B12 (cobalamin) for growth. This essential organic micronutrient is produced only by a subset of prokaryotic organisms, which implies that for algal species to use this compound, they must first acquire it from external sources. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about how it is taken up in algae. Here, we demonstrate the essential role of a protein, CBA1 (for cobalamin acquisition protein 1), in B12 uptake in Phaeodactylum tricornutum, using CRISPR-Cas9 to generate targeted knockouts, and in Chlamydomonas reinhardtii, by insertional mutagenesis. In both cases, CBA1 knockout lines are no longer able to take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wildtype CBA1 gene restores B12 uptake, and regulation of CBA1 expression via a riboswitch element can be used to control the phenotype. When visualised by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 shows association with membranes. A bioinformatics analysis found that CBA1-like sequences are present in all the major eukaryotic phyla. Its presence is correlated with B12-dependent enzymes in many, although not all, taxa, suggesting CBA1 has a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities. One sentence summaryKnockout mutants and physiological studies demonstrate that the CBA1 protein is essential for uptake of vitamin B12 in both Chlamydomonas reinhardtii and the unrelated Phaeodactylum tricornutum.

biochemistry↗

bioRxiv2022

Overexpression of PSR1 in Chlamydomonas reinhardtii induces luxury phosphorus uptake

Remediation using micro-algae offers an attractive solution to environmental phosphate (Pi) pollution. However, for maximum efficiency, pre-conditioning of algae to induce luxury phosphorus (P) uptake is needed. Here we show that natural pre-conditioning can be mimicked through over-expression of a single gene, the global regulator PSR1 (Myb transcription factor: Phosphate Starvation Response 1), raising P levels to 8% dry cell weight from 2% in control. Complete removal of Pi occurred in log phase, unlike the control. This was associated with increases in PolyP granule size and uptake of Mg2+, the principal counterion. Hyper-accumulation of P depended on a feed-forward mechanism, where a small set of Class I genes were activated despite abundant external Pi levels. This drove a reduction in external Pi levels, permitting more genes to be expressed (Class II), leading to more Pi-uptake. These discoveries enable a bio-circular approach of recycling nutrients from wastewater back to agriculture. TeaserManipulating a single gene drove uptake of P and a Mg2+ counter-ion for increased PolyP accumulation.

bioengineering↗

bioRxiv2022

Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

Cobalamin (vitamin B12), is a cofactor for crucial metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonise the algal phycosphere, forming stable communities that gain preferential access to exudates and in return provide compounds, such as B12. Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12-independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesise that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12-dependent algae. Originality-Significance statementMicroalgae are fundamental to the global carbon cycle, and yet despite being photosynthetic they often rely on other organisms for micronutrients. One of these micronutrients is vitamin B12 (cobalamin), which they receive from bacteria. Many environmental studies support the widespread role of B12 in algal-bacterial mutualisms, so here we wished to investigate how these mutualisms may arise evolutionarily by using an experimentally evolved B12-dependent alga and various B12-producers. A B12-producing rhizobium, Mesorhizobium loti, could stably support the B12-dependent Chlamydomonas reinhardtii metE7 strain, and vice versa, but nutrient supplementation increased growth of both species further. metE7 could also be supported by E. coli strains engineered to produce B12, and engineering a strain to release a higher proportion of B12 led to better algal growth, which increased bacterial growth in turn. We suggest that as B12-based mutualisms develop, increased B12 release may be selected for and therefore lead to more productive symbioses.

microbiology↗

bioRxiv2022

Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches

O_LIThiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for approximately 20% of global primary production. They have been predicted to contain TPP aptamers in the 3UTR of some thiamine metabolism-related genes, but little is known about their function and regulation. C_LIO_LIWe used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. C_LIO_LIWe found that thiamine-related genes with putative TPP aptamers are not responsive to thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and the targeted mutation of the TPP aptamer in the HMP-P synthase (THIC) does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtSSSP is necessary for thiamine uptake and that PtTHIC is essential for thiamine biosynthesis. C_LIO_LIOur results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance. C_LI

molecular biology↗

bioRxiv2021

Dynamics of deep water and N uptake under varied N and water supply

AimsEnhanced nitrogen (N) and water uptake from deep soil layers may increase resource use efficiency whilst maintaining yield under stressed conditions. Winter oilseed rape (Brassica napus L.) can develop deep roots and access deep-stored resources such as N and water, while this potential has large uncertainties in variable environments. In this study, we aimed to evaluate the effects of reduced N and water supply on deep N and water uptake. MethodsOilseed rape plants grown in outdoor rhizotrons were supplied with 240 and 80 kg N ha-1 respectively in 2019 whereas a well-watered and a water-deficit treatment were established in 2020. To track deep water and N uptake, a mixture of 2H2O and Ca(15NO3)2 was injected into the soil column at 0.5 and 1.7 m depths. {delta}2H in transpiration water and {delta}15N in leaves were measured after injection. {delta}15N in biomass samples were also measured. ResultsDifferences in N or water supply had little effect on root growth. The low N treatment reduced water uptake throughout the soil profile, but caused a non-significant increment in 15N uptake efficiency at both 0.5 and 1.7 m. Water deficit in the upper soil layers led to compensatory deep water, while N uptake was not altered by soil water status. ConclusionOur findings demonstrate that for winter oilseed rape, high N application and water deficiency in shallow layers increases deep water uptake, and that the efficiency of deep N uptake is mainly sensitive to N supply rather than water supply.

plant biology↗

bioRxiv2021

Exploring the impact of terminators on transgene expression in Chlamydomonas reinhardtii with a synthetic biology approach

Chlamydomonas reinhardtii has many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in the C. reinhardtii genome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design.

synthetic biology↗