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Recent advancements in molecular tools for plant genetic engineering, particularly CRISPR-based technologies, have created new opportunities for targeted genome editing. However, applying these tools remains challenging in crop species such as sunflower (Helianthus annuus) that lack established and effective transformation pipelines, including transient reagent delivery methods for functional screening and validation of genetic engineering tools. To address this gap, three major reagent delivery platforms, namely protoplast transfection, leaf infiltration, and Agrobacterium-mediated tissue co-culture, were systematically adapted and assessed for use in sunflower seedlings. While each method enabled successful reagent delivery, they differed in their levels of scalability and efficiency. With these platforms, delivery by different Agrobacterium strains and the effectiveness of various reporter gene expression cassettes were compared to define the most experimentally suitable components for different applications in sunflowers. Together, these results establish a foundational toolkit for transient functional testing in sunflower and pave the way for more sophisticated genetic engineering approaches in this agriculturally important oilseed, confectionary seed, and horticultural crop. 

Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible. 

Abstract U2 auxiliary factor 1 (U2AF1) functions in 3′-splice site selection during pre-mRNA processing. Alternative usage of duplicated tandem exons in U2AF1 produces two isoforms, U2AF1a and U2AF1b, but their functional differences are unappreciated due to their homology. Through integrative approaches of genome editing, customized-transcriptome profiling and crosslinking-mediated interactome analyses, we discovered that the expression of U2AF1 isoforms is controlled by mTOR and they exhibit a distinctive molecular profile for the splice site and protein interactomes. Mechanistic dissection of mutually exclusive alternative splicing events revealed that U2AF1 isoforms’ inherent differential preferences of nucleotide sequences and their stoichiometry determine the 3′-splice site. Importantly, U2AF1a-driven transcriptomes feature alternative splicing events in the 5′-untranslated region (5′-UTR) that are favorable for translation. These findings unveil distinct roles of duplicated tandem exon-derived U2AF1 isoforms in the regulation of the transcriptome and suggest U2AF1a-driven 5′-UTR alternative splicing as a molecular mechanism of mTOR-regulated translational control.