Transgenic Populations Research Articles

Abstract 1998: ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature

Abstract Uveal melanoma (UM) is the leading form of intraocular cancer in adults and the second most common form of melanoma. Although rare, UM has a median survival rate of ~1 year after metastasis occurs. Metastatic UM is largely refractory to treatment and there are no effective pharmacological therapies, resulting in poor overall survival. In >90% UM cases, the oncogenic initiator proteins are constitutively active mutant GNAQ or GNA11 proteins (GNAQ/11). FR900359 (FR) is a natural compound that inhibits GNAQ/11 and has anti-proliferative effects on UM cells. To identify key oncogenic mechanisms downstream of mutant GNAQ/11, we performed a Sleeping Beauty (SB) transposon forward genetic screen to identify drivers of FR resistance. The SB screen identified ATP-binding cassette sub-family B member 1 (ABCB1) as one of the top genes upregulated by SB in FR-resistant UM cell populations. We determined that although ABCB1 does not provide immediate resistance to FR, the overexpression of ABCB1 leads to emergence of resistant colonies at a much higher rate than spontaneous resistance, validating ABCB1 as contributors to FR resistance. Unexpectedly, immunofluorescence revealed that a relatively small fraction of UM cells strongly expresses the transgenes after stable transfection. Immunoblotting confirmed that ABCB1 expression was initially modest in ABCB1-transfected populations but increased in the cells that developed resistance to FR. To examine the kinetics of FR resistance, population doubling versus time (PDVT) assays were performed in UM cells expressing ABCB1 or empty vector control treated with FR or vehicle. FR potently suppressed the proliferation of vector control cells. Drug-naïve ABCB1-transfected cells developed resistance to FR over time but accomplished fewer population doublings than vehicle-treated cells in the same time interval. Once transgenic populations emerged as resistant populations in FR, they grew similarly in drug or vehicle. Ongoing RNA-sequencing analysis from PDVT samples has identified distinct gene expression signatures associated with FR treatment and ABCB1 expression. These findings are expected to illuminate tumor cell heterogeneity amongst UM cell populations that may be related to drug resistance and metastasis. Citation Format: Sarina Dion Murray, Jesse D. Riordan, Eric R. Anderson, Michael D. Onken, Kendall J. Blumer, Adam J. Dupuy, Christopher S. Stipp. ABCB1 promotes uveal melanoma cell resistance to FR900359 and identifies a tumor cell subpopulation with a distinct gene expression signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1998.

Developing efficient strategies for localizing the enhanced yellow fluorescent protein subcellularly in transgenic Eimeria parasites

Eimeria species serve as promising eukaryotic vaccine vectors. And that the location of heterologous antigens in the subcellular components of genetically modified Eimeria may determine the magnitude and type of immune responses. Therefore, our study aimed to target a heterologous fluorescent protein to the cell surface or microneme, two locations where are more effective in inducing protective immunity, of Eimeria tenella and E. acervulina sporozoites. We used an enhanced yellow fluorescent protein (EYFP) as a tagging biomarker, fusing variously with some localization or whole sequences of compartmental proteins for targeting. After acquiring stable transgenic Eimeria populations, we observed EYFP expressing in expected locations with certain strategies. That is, EYFP successfully localized to the surface when it was fused between signal peptides and mature products of surface antigen 1 (SAG1). Furthermore, EYFP was efficiently targeted to the apical end, an optimal location for secretory organelle known as the microneme, when fused to the C terminus of microneme protein 2. Unexpectedly, EYFP exhibited dominantly in the apical end with only weak expression on the surface of the transgenic sporozoites when the parasites were transfected with plasmid with EYFP fused between signal peptides and mature products of E. tenella SAG 13. These strategies worked in both E. tenella and E. acervulina, laying a solid foundation for studying E. tenella and E. acervulina-based live vaccines that can be further tailored to the inclusion of cargo immunogens from other pathogens.

CRISPR-Cas9-based method for isolating microgametes of Eimeria tenella

Eimeria tenella infections are known to cause severe caecal damage and death of the infected chicken. Gamogony is an essential stage in E. tenella life cycle and in the establishment of coccidiosis. Prior research had extensively explored isolation and separation of the parasite gametes - microgamete (male) and macrogamete (female). However, there is little information on the efficient, highly purified and distinctly separated male and female gametes. In this study, we generated a genome editing line expressing mCherry fluorescent protein fused with GCS1 protein in E. tenella by using Toxoplasma gondii CRISPR-Cas9 system, flow cytometry and fluorescence microscopy. This allowed precise separation of E. tenella male and female gametes in the transgenic parasite population. The separation of male and female gametes would not only build on our understanding of E. tenella transmission, but it would also facilitate development of gametocidal compounds as drug targets for E. tenella infection.

High-throughput light sheet imaging of adult and larval C. elegans Parkinson's disease model using a low-cost optofluidic device and a fluorescent microscope.

Recent advancements at the interface of microfluidics technology and light sheet fluorescence microscopy have opened the door for high-throughput and high-content investigation of C. elegans disease models. In this paper, we report on the development of a simple, miniaturized, and low-cost optofluidic platform that can be added to a conventional inverted fluorescent microscope for continuous light sheet imaging of transgenic worm populations with high lateral and axial resolutions of 1.1 µm and 2.4 µm, respectively. The optofluidic device is made entirely of PDMS with integrated optics for light sheet generation. Laser excitation is delivered to the device via a low-cost free space laser, and cross-sections of worm populations are imaged as they pass continuously through a channel. Results show the platform can image NW1229 whole worms with pan-neural fluorescent expression at a throughput of >20 worms per minute at L3 and young adult (YA) stages. As a benchmark test, we show that the low-cost device can quantify the reduced neuronal expressions of L3 and YA NW1229 worms when exposed to 500 µM 6-OHDA neurodegenerative agent. Following the benchmark validation, we utilized the platform in a novel application for imaging human alpha-synuclein reporter in populations of Parkinson's transgenic model (ERS100). Results show the ability of the low-cost platform to reliably detect and quantify the anomalous neural phenotypic changes in ERS100 populations at L3 and YA stages with high spatial resolution. The findings of this study show the potential of our low-cost optofluidic add-on platform to equip conventional fluorescent microscopes with light sheet capability for quantitative phenotypic studies of transgenic C. elegans at high resolution and throughput.

A versatile 2A peptide-based strategy for ectopic expression and endogenous gene tagging in Trypanosoma cruzi

Nearly all expression vectors currently available for Trypanosoma cruzi were conceived to produce a single primary transcript containing the genes of interest along with those that confer antibiotic resistance. However, since each messenger RNA (mRNA) matures separately, drug selection will only guarantee the expression of those derived from the selectable marker. Therefore, commonly a considerable fraction of the cells recovered after selection with these expression vectors, although resistant do not express the protein of interest. Consequently, in order to counteract this disadvantage, we developed vectors with an alternative arrangement in which the gene of interest and antibiotic resistance are fused sharing the same mRNA. To test this configuration, we included the coding sequence for the green fluorescent protein (mEGFP) linked to the one conferring neomycin resistance (Neo). Additionally, to allow for the production of two independent proteins the sequence for a Thosea asigna virus self-cleaving 2A peptide (T2A) was inserted in-between. Cells obtained with these vectors displayed higher mEGFP expression levels with more homogeneous transgenic parasite populations than those transfected with more conventional independent mRNA-based alternatives. Moreover, as determined by Western blot, 2A mediated fusion protein dissociation occurred with high efficiency in all parasite stages. In addition, these vectors could easily be transformed into endogenous tagging constructs that allowed the insertion, by ends-in homologous recombination, of a hemagglutinin tag (HA) fused to the actin gene. The use of 2A self-cleaving peptides in the context of single mRNA vectors represents an interesting strategy capable of improving ectopic transgene expression in T. cruzi as well as providing a simple alternative to more sophisticated methods, such as the one based on CRISPR/Cas9, for the endogenous labeling of genes.

Fitness and Hard Seededness of F2 and F3 Descendants of Hybridization between Herbicide-Resistant Glycine max and G. soja.

The commercial cultivation of herbicide-resistant (HR) transgenic soybeans (Glycine max L. Merr.) raises great concern that transgenes may introgress into wild soybeans (Glycine soja Sieb. et Zucc.) via pollen-mediated gene flow, which could increase the ecological risks of transgenic weed populations and threaten the genetic diversity of wild soybean. To assess the fitness of hybrids derived from transgenic HR soybean and wild soybean, the F2 and F3 descendants of crosses of the HR soybean line T14R1251-70 and two wild soybeans (LNTL and JLBC, which were collected from LiaoNing TieLing and JiLin BaiCheng, respectively), were planted along with their parents in wasteland or farmland soil, with or without weed competition. The fitness of F2 and F3 was significantly increased compared to the wild soybeans under all test conditions, and they also showed a greater competitive ability against weeds. Seeds produced by F2 and F3 were superficially similar to wild soybeans in having a hard seed coat; however, closer morphological examination revealed that the hard-seededness was lower due to the seed coat structure, specifically the presence of thicker hourglass cells in seed coat layers and lower Ca content in palisade epidermis. Hybrid descendants containing the cp4-epsps HR allele were able to complete their life cycle and produce a large number of seeds in the test conditions, which suggests that they would be able to survive in the soil beyond a single growing season, germinate, and grow under suitable conditions. Our findings indicate that the hybrid descendants of HR soybean and wild soybean may pose potential ecological risks in regions of soybean cultivation where wild soybean occurs.

In maize, co-expression of GAT and GR79-EPSPS provides high glyphosate resistance, along with low glyphosate residues

Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified (GM) crops. Herbicide-tolerant crops, especially glyphosate-resistant crops, offer great advantages for weed management; however, despite these benefits, glyphosate-resistant maize (Zea mays L.) has not yet been commercially deployed in China. To develop a new bio-breeding resource for glyphosate-resistant maize, we introduced a codon-optimized glyphosate N-acetyltransferase gene, gat, and the enolpyruvyl-shikimate-3-phosphate synthase gene, gr79-epsps, into the maize variety B104. We selected a genetically stable high glyphosate resistance (GR) transgenic event, designated GG2, from the transgenic maize population through screening with high doses of glyphosate. A molecular analysis demonstrated that single copy of gat and gr79-epsps were integrated into the maize genome, and these two genes were stably transcribed and translated. Field trials showed that the transgenic event GG2 could tolerate 9000 g acid equivalent (a.e.) glyphosate per ha with no effect on phenotype or yield. A gas chromatography-mass spectrometry (GC–MS) analysis revealed that, shortly after glyphosate application, the glyphosate (PMG) and aminomethylphosphonic acid (AMPA) residues in GG2 leaves decreased by more than 90% compared to their levels in HGK60 transgenic plants, which only harbored the epsps gene. Additionally, PMG and its metabolic residues (AMPA and N-acetyl-PMG) were not detected in the silage or seeds of GG2, even when far more than the recommended agricultural dose of glyphosate was applied. The co-expression of gat and gr79-epsps, therefore, confers GG2 with high GR and a low risk of herbicide residue accumulation, making this germplasm a valuable GR event in herbicide-tolerant maize breeding.

Identifying yield-related genes in maize based on ear trait plasticity

BackgroundPhenotypic plasticity is defined as the phenotypic variation of a trait when an organism is exposed to different environments, and it is closely related to genotype. Exploring the genetic basis behind the phenotypic plasticity of ear traits in maize is critical to achieve climate-stable yields, particularly given the unpredictable effects of climate change. Performing genetic field studies in maize requires development of a fast, reliable, and automated system for phenotyping large numbers of samples.ResultsHere, we develop MAIZTRO as an automated maize ear phenotyping platform for high-throughput measurements in the field. Using this platform, we analyze 15 common ear phenotypes and their phenotypic plasticity variation in 3819 transgenic maize inbred lines targeting 717 genes, along with the wild type lines of the same genetic background, in multiple field environments in two consecutive years. Kernel number is chosen as the primary target phenotype because it is a key trait for improving the grain yield and ensuring yield stability. We analyze the phenotypic plasticity of the transgenic lines in different environments and identify 34 candidate genes that may regulate the phenotypic plasticity of kernel number.ConclusionsOur results suggest that as an integrated and efficient phenotyping platform for measuring maize ear traits, MAIZTRO can help to explore new traits that are important for improving and stabilizing the yield. This study indicates that genes and alleles related with ear trait plasticity can be identified using transgenic maize inbred populations.

Control of Grain Weight and Size in Rice (Oryza sativa L.) by OsPUB3 Encoding a U-Box E3 Ubiquitin Ligase

Grain weight and size, mostly determined by grain length, width and thickness, are crucial traits affecting grain quality and yield in rice. A quantitative trait locus controlling grain length and width in rice, qGS1-35.2, was previously fine-mapped in a 57.7-kb region on the long arm of chromosome 1. In this study, OsPUB3, a gene encoding a U-box E3 ubiquitin ligase, was validated as the causal gene for qGS1-35.2. The effects were confirmed firstly by using CRISPR/Cas9-based mutagenesis and then through transgenic complementation of a Cas9-free knock-out (KO) mutant. Two homozygous KO lines were produced, each having a 1-bp insertion in OsPUB3 which caused frameshift mutation and premature termination. Compared with the recipient and a transgenic-negative control, both mutants showed significant decreases in grain weight and size. In transgenic complementation populations derived from four independent T0 plants, grain weight of transgenic-positive plants was significantly higher than transgenic-negative plants, coming with increased grain length and a less significant decrease in grain width. Based on data documented in RiceVarMap V2.0, eight haplotypes were classified according to six single-nucleotide polymorphisms (SNPs) found in the OsPUB3 coding region of 4695 rice accessions. Significant differences on grain size traits were detected between the three major haplotypes, Hap1, Hap2 and Hap3 that jointly occupy 98.6% of the accessions. Hap3 having the largest grain weight and grain length but intermediate grain width exhibits a potential for simultaneously improving grain yield and quality. In another set of 257 indica rice cultivars tested in our study, Hap1 and Hap2 remained to be the two largest groups. Their differences on grain weight and size were significant in the background of non-functional gse5, but non-significant in the background of functional GSE5, indicating a genetic interaction between OsPUB3 and GSE5. Cloning of OsPUB3 provides a new gene resource for investigating the regulation of grain weight and size.

Blind cavefish retain functional connectivity in the tectum despite loss of retinal input

Sensory systems display remarkable plasticity and are under strong evolutionary selection. The Mexican cavefish, Astyanax mexicanus, consists of eyed river-dwelling surface populations and multiple independent cave populations that have converged on eye loss, providing the opportunity to examine the evolution of sensory circuits in response to environmental perturbation. Functional analysis across multiple transgenic populations expressing GCaMP6s showed that functional connectivity of the optic tectum largely did not differ between populations, except for the selective loss of negatively correlated activity within the cavefish tectum, suggesting positively correlated neural activity is resistant to an evolved loss of input from the retina. Furthermore, analysis of surface-cave hybrid fish reveals that changes in the tectum are genetically distinct from those encoding eye loss. Together, these findings uncover the independent evolution of multiple components of the visual system and establish the use of functional imaging in A.mexicanus to study neural circuit evolution.

Control of Thousand-Grain Weight by OsMADS56 in Rice.

Grain weight and size are important traits determining grain yield and influencing grain quality in rice. In a previous study, a quantitative trait locus controlling thousand-grain weight (TGW) in rice, qTGW10-20.8, was mapped in a 70.7 kb region on chromosome 10. Validation of the candidate gene for qTGW10-20.8, OsMADS56 encoding a MADS-box transcription factor, was performed in this study. In a near-isogenic line (NIL) population segregated only at the OsMADS56 locus, NILs carrying the OsMADS56 allele of IRBB52 were 1.9% and 2.9% lower in TGW than NILs carrying the OsMADS56 allele of Teqing in 2018 and 2020, respectively. Using OsMADS56 knock-out mutants and overexpression transgenic plants, OsMADS56 was validated as the causal gene for qTGW10-20.8. Compared with the recipients, the TGW of the knock-out mutants was reduced by 6.0–15.0%. In these populations, decreased grain weight and size were associated with a reduction in the expression of OsMADS56. In transgenic populations of OsMADS56 driven by a strong constitutive promoter, grain weight and size of the positive plants were significantly higher than those of the negative plants. Haplotype analysis showed that the Teqing-type allele of OsMADS56 is the major type presented in cultivated rice and used in variety improvement. Cloning of OsMADS56 provides a new gene resource to improve grain weight and size through molecular design breeding.

Inheritance of NmDef02 synthetic defensin gene and hpt selectable marker in four successive generations of transgenic rice (Oryza sativa, cv. J-104)

The evaluation of the inheritance and stability of the transgenes is essential for the application of transgenic plants in agriculture. In this work, we studied the inheritance of two transgenes in T1, T2, T3 and T4 rice progenies. Transgenic rice plants (cv. J-104) was obtained by biolistic method using a synthetic defensin gene (NmDef02) and hpt as selectable marker gene, co-transformed in a 4:1 proportion, respectively. Regenerated plants were acclimated under natural conditions. The study started from the primary transformants that fulfilled the agronomic characters reported by the experts for the J-104 rice cultivar in the maturation stage. The integration and relative expression of NmDef02 in T1 plants was verified by Southern blot and qRT-PCR, respectively. The inheritance of transgenes over four generations was analyzed by PCR. The following transgene combinations were identified: NmDef02(+)hpt(+), NmDef02(+)hpt(-) and NmDef02(-)hpt(-). The most advantageous combination was NmDef02(+)hpt(-), which corresponded to the marker-free plants harboring the gene of interest. The inheritance of NmDef02 was confirmed in T1 and T2 progenies, but in some T3 and T4 lines the loss of this gene was verified. This inheritance analysis provided information concerning the transgenic population, but the mechanisms that destabilize the inheritance of the gene of interest will be the goal of future research.

Resistance of transgenic papaya trees to papaya ringspot in Brazil

Abstract: The objective of this work was to evaluate the resistance of transgenic papaya populations (PTPs) to Papaya ringspot virus-P (PRSV-P). 'Sunrise Solo' transgenic papaya plants were produced with the gene of the PRSV-P protein coat, and PRSV was mechanically inoculated in plants in greenhouse conditions. The presence of the CP/PRSV gene and homozygosis were evaluated by polymerase chain reaction (PCR). Selected plants and the 'Sunrise Solo' control were transplanted to the field for agronomic evaluations. The plants evaluated in greenhouse conditions showed resistance between 96.3 and 5.8%, without variation of symptoms. The PTPs 1/6, 1/7, 1/9, 1/10, 1/15, 2/38, 2/41, 2/56, 2/65, 3/27, 3/46, 3/48, 4/9, 4/27, 8/4, 8/23, 8/33, 18/3, 18/4, 18/8, 18/22, 18/27, 28/97, 28/104, and 28/110 showed no symptoms, they were ELISA negative, and most of them contained the CP and NPT II genes. PTPs 1/6 and 3/46 had the CP gene in homozygosis and in double insertion. PTPs 1/6/20, 1/6/59, 1/6/64, 1/6/90, 3/46/44, 3/46/52, and 18/27/97 had a well-formed fruit cluster, piriform fruit weighing approximately 500 grams, orange pulp, and less than 10% carpelloidy. PTPs 1/6/59 and 3/46/52 show resistance to PRSV, good agronomic characteristics, and the CP gene in homozygosis.

Engineered Maize Hybrids with Diverse Carotenoid Profiles and Potential Applications in Animal Feeding.

Multi-gene transformation methods need to be able to introduce multiple transgenes into plants in order to reconstitute a transgenic locus where the introduced genes express in a coordinated manner and do not segregate in subsequent generations. This simultaneous multiple gene transfer enables the study and modulation of the entire metabolic pathways and the elucidation of complex genetic control circuits and regulatory hierarchies. We used combinatorial nuclear transformation to produce multiplex-transgenic maize plants. In proof of principle experiments, we co-expressed five carotenogenic genes in maize endosperm. The resulting combinatorial transgenic maize plant population, equivalent to a "mutant series," allowed us to identify and complement rate-limiting steps in the extended endosperm carotenoid pathway and to recover corn plants with extraordinary levels of β-carotene and other nutritionally important carotenoids. We then introgressed the induced (transgenic) carotenoid pathway in a transgenic line accumulating high levels of nutritionally important carotenoids into a wild-type yellow-endosperm variety with a high β:ε ratio. Novel hybrids accumulated zeaxanthin at unprecedented amounts. We introgressed the same pathway into a different yellow corn line with a low β:ε ratio. The resulting hybrids, in this case, had a very different carotenoid profile. The role of genetic background in determining carotenoid profiles in corn was elucidated, and further rate-limiting steps in the pathway were identified and resolved in hybrids. Astaxanthin accumulation was engineered by overexpression of a β-carotene ketolase in maize endosperm. In early experiments, limited astaxanthin accumulation in transgenic maize plants was attributed to a bottleneck in the conversion of adonixanthin (4-ketozeaxanthin) to astaxanthin. More recent experiments showed that a synthetic β-carotene ketolase with a superior β-carotene/zeaxanthin ketolase activity is critical for the high-yield production of astaxanthin in maize endosperm. Engineered lines were used in animal feeding experiments which demonstrated not only the safety of the engineered lines but also their efficacy in a range of different animal production applications.

Reaction-diffusion Model Applied to the Population Dynamics of Wild and Transgenic Mosquitoes

Due to recent advances in genetic manipulation, transgenic mosquitoes can be a viable alternative to reduce some diseases. Viability conditions are obtained by the simulation and analysis of mathematical models that describe the behavior of wild and transgenic mosquitoes population living in the same geographic area. In this work, we present a reaction-diffusion model, where the term reaction is a nonlinear function that describes the interaction between wild and transgenic mosquitoes taking into account their zygosity and the diffusive term representing a uniform spatial spread characterized by a fixed diffusion parameter. The system of partial differential equations obtained is solved numerically by combining the implicit Runge-Kutta method and the finite element method through the sequential operator splitting technique. Several scenarios are analyzed simulating the spatial release of transgenic mosquitoes, demonstrating an intrinsic relationship between the transgenic and wild varieties for different initial conditions.

Rolling Circle Amplification (RCA)-Mediated Genome-Wide ihpRNAi Mutant Library Construction in Brassica napus.

With the successful completion of genomic sequencing for Brassica napus, identification of novel genes, determination of functions performed by genes, and exploring the molecular mechanisms underlying important agronomic traits were challenged. Mutagenesis-based functional genomics techniques including chemical, physical, and insertional mutagenesis have been used successfully in the functional characterization of genes. However, these techniques had their disadvantages and inherent limitations for allopolyploid Brassica napus, which contained a large number of homologous and redundant genes. Long intron-spliced hairpin RNA (ihpRNA) constructs which contained inverted repeats of the target gene separated by an intron, had been shown to be very effective in triggering RNAi in plants. In the present study, the genome-wide long ihpRNA library of B. napus was constructed with the rolling circle amplification (RCA)-mediated technology. Using the phytoene desaturase (PDS) gene as a target control, it was shown that the RCA-mediated long ihpRNA construct was significantly effective in triggering gene silence in B. napus. Subsequently, the resultant long ihpRNA library was transformed into B. napus to produce corresponding RNAi mutants. Among the obtained transgenic ihpRNA population of B. napus, five ihpRNA lines with observable mutant phenotypes were acquired including alterations in the floral model and the stamen development. The target genes could be quickly identified using specific primers. These results showed that the RCA-mediated ihpRNA construction method was effective for the genome-wide long ihpRNA library of B. napus, therefore providing a platform for study of functional genomics in allopolyploid B. napus.

Partitioning stable and unstable expression level variation in cell populations: A theoretical framework and its application to the T cell receptor.

Phenotypic variation in the copy number of gene products expressed by cells or tissues has been the focus of intense investigation. To what extent the observed differences in cellular expression levels are persistent or transient is an intriguing question. Here, we develop a quantitative framework that resolves the expression variation into stable and unstable components. The difference between the expression means in two cohorts isolated from any cell population is shown to converge to an asymptotic value, with a characteristic time, τT, that measures the timescale of the unstable dynamics. The asymptotic difference in the means, relative to the initial value, measures the stable proportion of the original population variance . Empowered by this insight, we analysed the T-cell receptor (TCR) expression variation in CD4 T cells. About 70% of TCR expression variance is stable in a diverse polyclonal population, while over 80% of the variance in an isogenic TCR transgenic population is volatile. In both populations the TCR levels fluctuate with a characteristic time of 32 hours. This systematic characterisation of the expression variation dynamics, relying on time series of cohorts’ means, can be combined with technologies that measure gene or protein expression in single cells or in bulk.

A Decoy Library Uncovers U-Box E3 Ubiquitin Ligases That Regulate Flowering Time in Arabidopsis.

Targeted degradation of proteins is mediated by E3 ubiquitin ligases and is important for the execution of many biological processes. Redundancy has prevented the genetic characterization of many E3 ubiquitin ligases in plants. Here, we performed a reverse genetic screen in Arabidopsis using a library of dominant-negative U-box-type E3 ubiquitin ligases to identify their roles in flowering time and reproductive development. We identified five U-box decoy transgenic populations that have defects in flowering time or the floral development program. We used additional genetic and biochemical studies to validate PLANT U-BOX 14 (PUB14), MOS4-ASSOCIATED COMPLEX 3A (MAC3A), and MAC3B as bona fide regulators of flowering time. This work demonstrates the widespread importance of E3 ubiquitin ligases in floral reproductive development. Furthermore, it reinforces the necessity of dominant-negative strategies for uncovering previously unidentified regulators of developmental transitions in an organism with widespread genetic redundancy, and provides a basis on which to model other similar studies.

Heterologous expression of a mutant Orange gene from Brassica oleracea increases carotenoids and induces phenotypic changes in the microalga Chlamydomonas reinhardtii

In plants, a mutation in the Orange (Or) gene is known to enhance the accumulation of carotenoids in comparison to the wild-type (WT) allele. We investigated the effect of a point mutation in the Or gene of Brassica oleracea on its protein structure via in silico analysis. Subsequently we studied the effect of the Or mutation on carotenoid regulation in Chlamydomonas reinhardtii. The Or mutation in Brassica oleracea—mBoOr (R88H)—results in a small β-sheet in place of the α-helix that is present in the WT protein, and was found to be associated with enhanced carotenoid content when screened in Escherichia coli. The expression of the equivalent mutant from C. reinhardtii—mCrOr (R106H)—did not result in any change in protein structure and exhibited lower levels of carotenoid in E. coli. An in silico analysis of microalgal genomes revealed the presence of Or homologues in several microalgae from the class Trebouxiphyceae, while C. reinhardtii was the only representative of the Chlorophyceae. The heterologous expression of mBoOr in C. reinhardtii resulted in a 1.7-fold increase in carotenoid content, as well as an increase in cell size and the appearance of multicellularity in C. reinhardtii, which was evidenced by the presence of both orange and green transgenic cells grouping together as a population. A palmelloid formation of the orange and green cells impaired the motility of the transgenic population, resulting in flocculation. This study establishes mBoOr expression as a promising strategy for enhancing carotenoid content in microalgae.

Genome-wide analyses reveal the role of noncoding variation in complex traits during rice domestication.

Genomes carry millions of noncoding variants, and identifying the tiny fraction with functional consequences is a major challenge for genomics. We assessed the role of selection on long noncoding RNAs (lncRNAs) for domestication-related changes in rice grains. Among 3363 lncRNA transcripts identified in early developing panicles, 95% of those with differential expression (329 lncRNAs) between Oryza sativa ssp. japonica and wild rice were significantly down-regulated in the domestication event. Joint genome and transcriptome analyses reveal that directional selection on lncRNAs altered the expression of energy metabolism genes during domestication. Transgenic experiments and population analyses with three focal lncRNAs illustrate that selection on these loci led to increased starch content and grain weight. Together, our findings indicate that genome-wide selection for lncRNA down-regulation was an important mechanism for the emergence of rice domestication traits.