Biology subjects

Searle, I. R.

Publications and source records attributed to Searle, I. R..

9 recordsLinked to original sources

bioRxiv2025

The Arabidopsis CYSTM α 5'UTR increases protein production from transgenes in plants and bacteria.

Achieving high levels of transgene expression is often crucial for the effective production of high value pharmaceutical proteins and bioactive compounds. Here, we show a 79-nucleotide sequence of the 5 UTR of the Arabidopsis thaliana cysteine-rich transmembrane module 1 (CYSTM1) significantly increases transient and stable transgene expression at the post-transcriptional level in multicellular plants, A. thaliana and Nicotiana benthamiana, in monocotyledon wheat germ extracts, and in the unicellular bacterium Escherichia coli by up to 7-fold. We also show the 79 nt CYSTM sequence has 50 % higher transgene expression than the broadly used Tobacco Mosaic Virus omega ({Omega}) sequence in both A. thaliana and N. benthamiana. Taken together, this study provides a short transgene expression enhancer for multicellular and unicellular organisms.

plant biology↗

bioRxiv2025

Mutations in HOMEOBOX DOMAIN-2 improve grain protein content in wheat without significantly affecting grain yield and senescence

MicroRNA-resistant alleles of HOMEOBOX DOMAIN-2 (HB-2) were recently reported to improve grain protein content (GPC) in wheat by modifying vascular development in stems to increase amino acid distribution to grain. However, it is not known if these alleles increase GPC at the expense of other physiologically important traits, such as senescence and yield-component traits. Here, we evaluated the effects of mutations in HB-2 on grain yield and nitrogen (N)-use efficiency as well as senescence and plant architecture in multi-environment glasshouse and field experiments. In glasshouse experiments, the mutations in HB-2 moderately improved GPC irrespective of N supply and without accelerating senescence or decreasing grain number or weight compared to the wildtype spring wheat cv. Cadenza. The trait analysis indicated mutant plants were more tolerant of N limitation in terms of above-ground N reduction compared to the wild type. The expression of all HB-2 homeologues increased in spikes at the lemma primordium stage in response to N limitation, indicating the later GPC increase could be a result of enhanced N sink strength determined during early development, rather than processes caused by direct changes in HB-2 expression in the grain. In field experiments, the introgression of HB-2 alleles into Australian elite spring wheat cultivars significantly improved GPC without compromising grain yield. The outcomes of this study support the introduction of miRNA-resistant HB-2 alleles, with a preference to HB-A2, into commercial wheat for improving grain quality without a yield penalty.

plant biology↗

bioRxiv2025

Two routes from the Fertile Crescent led to the introduction of common vetch into Europe.

Vicia sativa (common vetch, n=6) is an annual leguminous plant with high drought tolerance and high grain protein content. Historically and still today, vetches are important for nitrogen fixation to maintain cereal yields and feeding grazing livestock. In this study, we re-sequenced the genomes of 279 common vetch accessions from a wide geographic range covering western Eurasia and North Africa to construct a comprehensive nucleotide variation map. Population structure analyses indicate that the Middle Eastern region was the centre of origin of common vetch, which then entered Europe in two distinct waves. One wave propagated by humans through Turkey into the Carpathian Basin before spreading throughout Europe via "Danubian" and "Mediterranean" routes. Demographic inferences revealed a significant bottleneck for all common vetch groups initiated during the Last Glacial Maximum, followed by an expansion that coincides with the onset of the warm Holocene epoch and the Neolithic Revolution. Interestingly, we identified selective sweeps for the flowering time regulators VsSOC1 and VsFTb2 in northern latitude populations suggesting that both genes are important regulators of latitudinal adaptation. Overall, we provide valuable genomic resources for conservation and breeding programs to optimize food production in the face of population growth and reduced agricultural resources.

plant biology↗

bioRxiv2025

Insights from modern & historical genomes of Neorhizobium vitis, a new pathogen causing neoplastic growths on grapevine.

Grapevine crown gall is a globally significant disease typically attributed to Agrobacterium species carrying tumour-inducing (Ti) plasmids. In this study, we identify Neorhizobium vitis as a previously unrecognised crown gall-associated pathogen. Four modern isolates from symptomatic grapevines in South Australia and four historical strains collected in New South Wales in 1989 induced weak but consistent neoplastic growths on sunflower hypocotyls and carrot discs. Long-read genome sequencing revealed that all strains lack canonical Ti plasmids and the oncogenes iaaM and ipt, which are essential for classical auxin- and cytokinin-mediated tumorigenesis. Phylogenomic analyses reassigned all strains to the Neorhizobium clade, correcting the original classification of historical isolates as Agrobacterium vitis. The persistence of tumorigenic activity despite the absence of known virulence genes suggests the involvement of alternative, yet uncharacterized pathogenic mechanisms. These findings revise the non-pathogenic status of Neorhizobium, establish N. vitis as a novel tumorigenic lineage, and underscore the value of re-evaluating legacy isolates with modern genomic tools.

plant biology↗

bioRxiv2025

TMT-based quantitative proteomic assessment of Vicia sativa induced neurotoxicity by β-cyano-L-alanine and γ-glutamyl-β-cyano-L-alanine in SH-SY5Y cells.

{beta}-Cyano-L-alanine (BCA) and {gamma}-glutamyl-{beta}-cyano-L-alanine (GBCA) are the primary antinutritional compounds present within the seeds of the high protein and drought tolerant orphan legume Vicia sativa. Evidence of neurotoxicity is limited to symptom analysis from animal feed trials whilst the molecular mechanisms underlying suspected neurotoxicity in monogastric animals are largely unknown. In this study, we first optimised an in vitro cell-based assay for rapid testing of BCA and GBCA toxicity in the retinoic acid differentiated SH-SY5Y human neuroblastoma cells. Using this system, we then performed proteomics analyses to determine dysregulated expression of proteins in BCA or GBCA treated of differentiated SH-SY5Y cells. Our findings indicate that BCA affects expression of proteins involved in DNA damage and translation whilst GBCA treatment causes dysregulation of those involved in mitosis and cell cycle. Following BCA treatment, we identified changes in many proteins previously suggested to have close association with neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimers disease as well as cancers. Following GBCA treatment, dysregulation of proteins involved in apoptosis pathways was observed. Finally, the lack of common dysregulated proteins and pathways in BCA or GBCA treated cells indicated that they most likely cause neurotoxicity via distinct mechanisms. SignificanceAntinutritional compounds or toxins limit the use of many potential grain crops including V. sativa (Common Vetch), and BCA and GBCA are the two principal toxic compounds in Common Vetch grain. Vetch grain toxicity is associated with neurotoxic symptoms in animals. Characterizing the proteome of BCA- and GBCA-treated neural cells gives insights into the excitotoxicity mechanism and identification of biomarkers for screening higher quality grain. Quantitative tandem mass tag (TMT) mass spectrometry-based proteome profiling of BCA treated neural cells characterized 6,827 proteins, of which 26 were significantly up-regulated and 73 significantly downregulated. However, TMT proteome analysis of GBCA treated cells identified 76 significantly up regulated and 86 downregulated proteins. These results provide insight into the toxicity of BCA and GBCA and enable future identification of putative direct protein targets. We describe for the first time BCA playing a role in modulating the expression DNA damage proteins and translation, and GBCA playing a role in modulating the expression of mitosis and cell cycle proteins, elucidating the mechanisms of plant-derived toxins in mammalian cell neurotoxicity HighlightsO_LIEstablished in vitro cell-based toxicity assay for {beta}-cyano-L-alanine and {gamma}-glutamyl-{beta}-cyano-L-alanine. C_LIO_LI{beta}-Cyano-L-alanine treatment dysregulated many proteins associated with DNA damage in retinoic acid differentiated SH-SY5Y cells. C_LIO_LI{gamma}-Glutamyl-{beta}-cyano-L-alanine treatment dysregulated proteins associated with cell cycle. C_LI

pharmacology and toxicology↗

bioRxiv2021

Chromosome-level assembly of the common vetch reference genome (Vicia sativa)

BackgroundVicia sativa L. (Common Vetch, n = 6) is an annual, herbaceous, climbing legume that is distributed in tropical, sub-tropical and temperate climates. Originating in the Fertile Crescent of the Middle East, V. sativa is now widespread and grows in the Mediterranean basin, West, Central and Eastern Asia, North and South America. V. sativa is of economic importance as a forage legume in countries such as Australia, China, and the USA and contributes valuable nitrogen to agricultural rotation cropping systems. To accelerate precision genome breeding and genomics-based selection of this legume, we here present a chromosome-level reference genome sequence for V. sativa. ResultsWe applied a combination of long-read Oxford Nanopore sequencing, short-read Illumina sequencing, and high-throughput chromosome conformation data (CHiCAGO and Hi-C) analysis to construct a chromosome-level genome of V. sativa. The chromosome-level assembly of six pseudo-chromosomes has a total genome length of 1.9 gigabases (Gb) with a median contig length of 684 kb. Benchmarking Universal Single-Copy Orthologs (BUSCO) of the assembly demonstrated a very high completeness of 98 % of the dicotyledonous orthologs. RNA-seq analysis and gene modelling enabled the annotation of 58,415 protein-coding genes. ConclusionsThe high-quality chromosome-level genome assembly of V. sativa will provide novel insights into vetch genome evolution and be a valuable resource for genomic breeding, genetic diversity and for understanding adaption to diverse arid environments.

genomics↗

bioRxiv2020

An efficient root transformation system for recalcitrant Vicia sativa.

Common vetch (Vicia sativa) is a multi-purpose legume widely used in pasture and crop rotation systems. Vetch seeds have desirable nutritional characteristics and are often used to feed ruminant animals. Although transcriptomes are available for vetch, problems with genetic transformation and plant regeneration hinder functional gene studies in this legume species. Therefore, the aim of this study was to develop an efficient and rapid hairy root transformation system for common vetch to facilitate functional gene analysis. We infected the hypocotyls of five-day old in vitro or in vivo, soil grown, seedlings with Agrobacterium rhizogenes and produced transformed hairy roots 28 days later at 24% and 43% efficiency, respectively. Seventy-nine percent of the hairy roots from the in vitro plants showed stable expression of a co-transformed marker {beta}-glucuronidase (GUS). In summary, transgenic hairy roots were obtained within 28 days, and are sufficient to facilitate functional gene analysis in common vetch.

plant biology↗

bioRxiv2020

Common Vetch: a drought tolerant, high protein neglected leguminous crop with potential as a sustainable food source.

Global demand for protein is predicted to increase by 50% by 2050. To meet the increasing demand whilst ensuring sustainability, protein sources that generate low-greenhouse gas emissions are required and protein-rich legume seeds have the potential to make a significant contribution. Legumes like common vetch (Vicia sativa) that grow in marginal cropping zones and are drought tolerant and resilient to changeable annual weather patterns, will be in high demand as the climate changes. In common vetch, the inability to eliminate the {gamma}-glutamyl-{beta}-cyano-alanine (GBCA) toxin present in the seed has hindered its utility as a human and animal food for many decades, leaving this highly resilient species an "orphan" legume. However, the availability of the vetch genome and transcriptome data together with the application of CRISPR-Cas genome editing technologies lay the foundations to eliminate the GBCA toxin constraint. In the near future, we anticipate that a zero-toxin vetch variety will become a significant contributor to global protein demand.

plant biology↗

bioRxiv2018

Arabidopsis TRM5 encodes a nuclear-localised bifunctional tRNA guanine and inosine-N1-methyltransferase that is important for growth.

Modified nucleosides in tRNAs are critical for protein translation. N1-methylguanosine-37 and N1-methylinosine-37 in tRNAs, both located at the 3-adjacent to the anticodon, are formed by Trm5 and here we describe Arabidopsis thaliana AtTrm5 (At3g56120) as a Trm5 ortholog. We show that AtTrm5 complements the yeast trm5 mutant, and in vitro methylates tRNA guanosine-37 to produce N1-methylguanosine (m1G). We also show in vitro that AtTRM5 methylates tRNA inosine-37 to produce N1-methylinosine (m1I) and in Attrm5 mutant plants, we show a reduction of both N1-methylguanosine and N1-methylinosine. We also show that AtTRM5 is localized to the nucleus in plant cells. Attrm5 mutant plants have overall slower growth as observed by slower leaf initiation rate, delayed flowering and reduced primary root length. In Attrm5 mutants, mRNAs of flowering time genes are less abundant and correlated with delayed flowering. Finally, proteomics data show that photosynthetic protein abundance is affected in mutant plants. Our findings highlight the bifunctionality of AtTRM5 and the importance of the post-transcriptional tRNA modifications m1G and m1I at tRNA position 37 in general plant growth and development.

molecular biology↗