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El triaje busca identificar a aquellos pacientes que necesitan atención inmediata y a los que pueden esperar con seguridad durante un tiempo más prolongado. Se realizó un estudio observacional, descriptivo y prospectivo, con pacientes que acuden al servicio de emergencias del Hospital Infantil Dr. Robert Reid Cabral en los meses de septiembre, octubre y noviembre de 2018, para un total de 32 %, 21 %, y 47 %, respectivamente. El total de pacientes captados fue 660, 54 % masculinos y 46 % femeninos. Edad de mayor asistencia de 1-6 años, 311 para un 47 %. El 4to nivel fue el de mayor atención, 219 para un 33 %. Mayor flujo horario de 11:00 a.m. a 4:00 p.m., para un 5.8 % y 6.1 %. Tiempo de respuesta nivel 1: 50 seg; nivel 2: 22 min con 5 seg; nivel 3: 41 min con 12 seg; nivel 4: 49 min con 35 seg; y nivel 5: 48 min con 53 seg. Como conclusión, la respuesta a las atenciones se ve afectada por la falta de triaje estructurado, una respuesta más rápida y oportuna a pacientes con niveles 3 a 4, gracias al alto volumen de pacientes que acuden y no así a los de nivel 1 y 2.

Stomata regulate CO2 and water vapor exchange between leaves and the atmosphere. Stomata are a target for engineering to improve crop intrinsic water use efficiency (iWUE). One example is by expressing genes that lower stomatal density (SD) and reduce stomatal conductance (gsw). However, the quantitative relationship between reduced SD, gsw, and the mechanisms underlying it is poorly understood. We addressed this knowledge gap using low-SD sugarcane (Saccharum spp. hybrid) as a case study alongside a meta-analysis of data from 10 species. Transgenic expression of EPIDERMAL PATTERNING FACTOR 2 from Sorghum bicolor (SbEFP2) in sugarcane reduced SD by 26-38% but did not affect gsw compared to wildtype. Further, no changes occurred in stomatal complex size or proxies for photosynthetic capacity. Measurements of gas exchange at low CO2 concentrations that promote complete stomatal opening to normalize aperture size between genotypes were combined with modeling of maximum gsw from anatomical data. These data suggest that increased stomatal aperture is the only possible explanation for maintaining gsw when SD is reduced. Meta-analysis across C3 dicots, C3 monocots, and C4 monocots revealed engineered reductions in SD are strongly correlated with lower gsw (r2=0.60-0.98), but this response is damped relative to the change in anatomy., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Engineering lower stomatal density to reduce stomatal conductance (gs) has improved WUE in multiple C3 crop species. However, reducing gs in C3 species often reduces photosynthetic carbon gain. A different response is expected in C4 plants because they possess specialized anatomy and biochemistry which concentrates CO2 at the site of fixation. This modifies the photosynthesis (AN) relationship with intracellular CO2 concentration (ci) so that photosynthesis is CO2-saturated and reductions in gs are unlikely to limit AN. To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C4 crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum, led to reduced stomatal densities, reduced gs, reduced plant water use and avoidance of stress during a period of water deprivation. In addition, moderate reduction in stomatal density did not increase stomatal limitation to AN. However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues could provide a pathway to improved agronomic outcomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Maize grain is deficient in lysine. While the opaque2 mutation increases grain lysine, o2 is a transcription factor that regulates a wide network of genes beyond zeins, which leads to pleiotropic and often negative effects. Additionally, the drastic reduction in 19 kDa and 22 kDa alpha-zeins causes a floury kernel, unsuitable for agricultural use. Quality protein maize (QPM) overcame the undesirable kernel texture through the introgression of modifying alleles. However, QPM still lacks a functional o2 transcription factor, which has a penalty on non-lysine amino acids due to the o2 mutation. CRISPR/cas9 gives researchers the ability to directly target genes of interest. In this paper, gene editing was used to specifically target the 19 kDa alpha zein gene family. This allows for proteome rebalancing to occur without an o2 mutation and without a total alpha-zein knockout. The results showed that editing some, but not all, of the 19 kDa zeins resulted in up to 30% more lysine. An edited line displayed an increase of 30% over the wild type. While not quite the 55% lysine increase displayed by QPM, the line had little collateral impact on other amino acid levels compared to QPM. Additionally, the edited line containing a partially reduced 19 kDa showed an advantage in kernel texture that had a complete 19 kDa knockout. These results serve as proof of concept that editing the 19 kDa alpha-zein family alone can enhance lysine while retaining vitreous endosperm and a functional O2 transcription factor., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Microtubule-associated protein 65-1 (MAP65-1) protein plays an essential role in plant cellular dynamics through impacting stabilization of the cytoskeleton by serving as a crosslinker of microtubules. The role of MAP65-1 in plants has been associated with phenotypic outcomes in response to various environmental stresses. The Arabidopsis MAP65-1 ( At MAP65-1) is a known virulence target of plant bacterial pathogens and is thus a component of plant immunity. Soybean events were generated that carry transgenic alleles for both At MAP65-1 and Gm MAP65-1, the soybean At MAP65-1 homolog, under control of cauliflower mosaic virus 35S promoter. Both At MAP65-1 and Gm MAP65-1 transgenic soybeans are more resistant to challenges by the soybean bacterial pathogen Pseudomonas syringae pv. glycinea and the oomycete pathogen Phytophthora sojae , but not the soybean cyst nematode, Heterodera glycines. Soybean plants expressing At MAP65-1 and Gm MAP65-1 also display a tolerance to the herbicide oryzalin, which has a mode of action to destabilize microtubules. In addition, Gm MAP65-1-expressing soybean plants show reduced cytosol ion leakage under freezing conditions, hinting that ectopic expression of Gm MAP65-1 may enhance cold tolerance in soybean. Taken together, overexpression of At MAP65-1 and Gm MAP65-1 confers tolerance of soybean plants to various biotic and abiotic stresses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady-state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans. Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl-acyl carrier protein (ACPs) levels were quantified overdevelopment. Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5-2% of seed biomass (i.e. 2-9% change in oil). Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans., (© 2023 The Authors. New Phytologist © New Phytologist Foundation U.S. Government work - Not all authors are U.S. Government employees.)

Introduction: Sorghum is a resilient and widely cultivated grain crop used for feed and food. However, it's grain is deficient in lysine, an essential amino acid. This is due to the primary seed storage proteins, the alpha-kafirins, lacking lysine. It has been observed that reductions in alpha-kafirin protein results in rebalancing of the seed proteome and a corresponding increase in non-kafirin proteins which leads to an increased lysine content. However, the mechanisms underlying proteome rebalancing are unclear. This study characterizes a previously developed gene edited sorghum line, with deletions at the alpha kafirin locus., Methods: A single consensus guide RNA leads to tandem deletion of multiple members of the gene family in addition to the small target site mutations in remaining genes. RNA-seq and ATAC-seq were utilized to identify changes in gene expression and chromatin accessibility in developing kernels in the absence of most alpha-kafirin expression., Results: Several differentially accessible chromatin regions and differentially expressed genes were identified. Additionally, several genes upregulated in the edited sorghum line were common with their syntenic orthologues differentially expressed in maize prolamin mutants. ATAC-seq showed enrichment of the binding motif for ZmOPAQUE 11, perhaps indicating the transcription factor's involvement in the kernel response to reduced prolamins., Discussion: Overall, this study provides a resource of genes and chromosomal regions which may be involved in sorghum's response to reduced seed storage proteins and the process of proteome rebalancing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hurst, Yobi, Li, Sato, Clemente, Angelovici and Holding.)

Cas9-based genome editing is a powerful genetic tool for loci specifically targeted for genome modification. This chapter describes up-to-date protocols using Cas9-based genome editing technology, including vector construction with GoldenBraid assembly, Agrobacterium-mediated soybean transformation, and identification of editing in the genome., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariflorus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites., Results: We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariflorus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors., Conclusions: We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a good editing target in species other than Z. mays. The ability to target specific loci to enable endogenous gene editing presents a new avenue for genetic improvement of this important biomass crop., (© 2022. The Author(s).)

Cannabis sativa is a versatile crop that can be cultivated for fiber, seed, or phytochemicals. To take advantage of this versatility and the potential of Cannabis as a feedstock for the bioeconomy, genomics-enabled breeding programs must be strengthened and expanded. This work contributes to the foundation for such by investigating the phytochemistry and genomics of feral Cannabis populations collected from seventeen counties across the climate gradient of Nebraska. Flower tissue from male and female plants (28 total) was studied using (i) gas chromatography-mass spectrometry to assess cannabinoid profiles and (ii) RNA sequencing to determine transcript abundances. Both male and female flower tissues produced cannabinoids, and, though the compounds were more abundant in female flower tissue, the primary cannabinoid in both was usually cannabidiol. The expression of genes that mediate early steps on the cannabinoid biosynthetic pathway were upregulated in female relative to male flowers, suggesting that female versus male flower tissue cannabinoid abundance may be controlled at least in part at the transcriptional level. DNA sequencing was used to place feral Cannabis plants from Nebraska into a previously described genomic context, revealing that all the plants studied here are much more similar to previously characterized hemp-type Cannabis plants than to drug-type Cannabis plants, at least at the genetic level. This work provides foundational phytochemical knowledge and a large set of high-quality single nucleotide polymorphism markers for future studies of feral Nebraska Cannabis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Soybean oil is one of the most consumed vegetable oils worldwide. Genetic improvement of its concentration in seeds has been historically pursued due to its direct association with its market value. Engineering attempts aiming to increase soybean seed oil presented different degrees of success that varied with the genetic design and the specific variety considered. Understanding the embryo's responses to the genetic modifications introduced, is a critical step to successful approaches. In this work, the metabolic and transcriptional responses to AtWRI1 and AtDGAT1 expression in soybean seeds were evaluated. AtWRI1 is a master regulator of fatty acid (FA) biosynthesis, and AtDGAT1 encodes an enzyme catalysing the final and rate-limiting step of triacylglycerides biosynthesis. The events expressing these genes in the embryo did not show an increase in total FA content, but they responded with changes in the oil and carbohydrate composition. Transcriptomic studies revealed a down-regulation of genes putatively encoding for oil body packaging proteins, and a strong induction of genes annotated as lipases and FA biosynthesis inhibitors. Novel putative AtWRI1 targets, presenting an AW-box in the upstream region of the genes, were identified by comparison with an event that harbours only AtWRI1. Lastly, targeted metabolomics analysis showed that carbon from sugar phosphates could be used for FA competing pathways, such as starch and cell wall polysaccharides, contributing to the restriction in oil accumulation. These results allowed the identification of key cellular processes that need to be considered to break the embryo's natural restriction to uncontrolled seed lipid increase., (© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin-1 receptor (TIR) domain homologous to immunity-related TIR domains of plant nucleotide-binding leucine-rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD + ) and activate immunity. In vitro and in vivo assays were conducted to determine if HopAM1 hydrolyzes NAD + and if the activity is essential for HopAM1's suppression of plant immunity and contribution to virulence. HPLC and LC-MS were utilized to analyze metabolites produced from NAD + by HopAM1 in vitro and in both yeast and plants. Agrobacterium-mediated transient expression and in planta inoculation assays were performed to determine HopAM1's intrinsic enzymatic activity and virulence contribution. HopAM1 is catalytically active and hydrolyzes NAD + to produce nicotinamide and a novel cADPR variant (v2-cADPR). Expression of HopAM1 triggers cell death in yeast and plants dependent on the putative catalytic residue glutamic acid 191 (E191) within the TIR domain. Furthermore, HopAM1's E191 residue is required to suppress both pattern-triggered immunity and effector-triggered immunity and promote P. syringae virulence. HopAM1 manipulates endogenous NAD + to produce v2-cADPR and promote pathogenesis. This work suggests that HopAM1's TIR domain possesses different catalytic specificity than other TIR domain-containing NAD + hydrolases and that pathogens exploit this activity to sabotage NAD + metabolism for immune suppression and virulence., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20 . Transgenic soybean events carrying mPing -based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system., (© 2021 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd.)

Scope: Previous studies have suggested that diets rich in omega-3 and low in omega-6 long-chain polyunsaturated fatty acids (PUFAs) can limit the development of metabolic syndrome (MetS). Transgenic soybeans yielding oils enriched for omega-3 PUFAs represent a new and readily-available option for incorporating omega-3 PUFAs into diets to provide health benefits., Methods and Results: Transgenic soybean oils, enriched for either stearidonic acid (SDA) or eicosapentaenoic acid (EPA), are incorporated into diets to test their effects on limiting the development of MetS in a mouse model of diet-induced obesity. Supplementation with SDA- but not EPA-enriched oils improved features of MetS compared to feeding a control wild-type oil. Because previous studies have linked the gut microorganism Akkermansia muciniphila to the metabolic effects of feeding omega-3 PUFAs, the causal contribution of A. muciniphila to mediating the metabolic benefits provided by SDA-enriched diets is investigated. Although A. muciniphila is not required for SDA-induced metabolic improvements, this microorganism does modulate levels of saturated and mono-unsaturated fatty acids in host adipose tissues., Conclusion: Together, these findings support the utilization of SDA-enriched diets to modulate weight gain, glucose metabolism, and fatty acid profiles of liver and adipose tissue., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Drought stress is the major limiting factor in agriculture. Wheat, which is the most widely grown crop in the world, is predominantly cultivated in drought-prone rainfed environments. Since roots play a critical role in water uptake, root response to water limitations is an important component for enhancing wheat adaptation. In an effort to discover novel genetic sources for improving wheat adaptation, we characterized a wheat translocation line with a chromosomal segment from Agropyron elongatum, a wild relative of wheat, which unlike common wheat maintains root growth under limited-water conditions. By exploring the root transcriptome data, we found that reduced transcript level of LATERAL ROOT DENSITY (LRD) gene under limited water in the Agropyron translocation line confers it the ability to maintain root growth. The Agropyron allele of LRD is down-regulated in response to water limitation in contrast with the wheat LRD allele, which is up-regulated by water deficit stress. Suppression of LRD expression in wheat RNAi plants confers the ability to maintain root growth under water limitation. We show that exogenous gibberellic acid (GA) promotes lateral root growth and present evidence for the role of GA in mediating the differential regulation of LRD between the common wheat and the Agropyron alleles under water stress. Suppression of LRD also had a positive pleiotropic effect on grain size and number under optimal growth conditions. Collectively, our findings suggest that LRD can be potentially useful for improving wheat response to water stress and altering yield components., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Wheat streak mosaic virus (WSMV) and triticum mosaic virus (TriMV) are economically important viruses of wheat ( Triticum aestivum L.), causing significant yield losses in the Great Plains region of the United States. These two viruses are transmitted by wheat curl mites, which often leads to mixed infections with synergistic interaction in grower fields that exacerbates yield losses. Development of dual-resistant wheat lines would provide effective control of these two viruses. In this study, a genetic resistance strategy employing an RNA interference (RNAi) approach was implemented by assembling a hairpin element composed of a 202-bp (404-bp in total) stem sequence of the NIb (replicase) gene from each of WSMV and TriMV in tandem and of an intron sequence in the loop. The derived RNAi element was cloned into a binary vector and was used to transform spring wheat genotype CB037. Phenotyping of T 1 lineages across eight independent transgenic events for resistance revealed that i) two of the transgenic events provided resistance to WSMV and TriMV, ii) four events provided resistance to either WSMV or TriMV, and iii) no resistance was found in two other events. T 2 populations derived from the two events classified as dual-resistant were subsequently monitored for stability of the resistance phenotype through the T 4 generation. The resistance phenotype in these events was temperature-dependent, with a complete dual resistance at temperatures ≥25°C and an increasingly susceptible response at temperatures below 25°C. Northern blot hybridization of total RNA from transgenic wheat revealed that virus-specific small RNAs (vsRNAs) accumulated progressively with an increase in temperature, with no detectable levels of vsRNA accumulation at 20°C. Thus, the resistance phenotype of wheat harboring an RNAi element was correlated with accumulation of vsRNAs, and the generation of vsRNAs can be used as a molecular marker for the prediction of resistant phenotypes of transgenic plants at a specific temperature.

Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in δ- and γ-forms, which were shifted largely to α- and β-tocochromanols with γ-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4 Δ6,9,12,15 )-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3 Δ9,12,15 or GLA, 18:3 Δ6,9,12 ), from transgene segregation. Oil extracted from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

To increase phenylpropanoid constituents and energy content in the versatile C 4 grass sorghum ( Sorghum bicolor [L.] Moench), sorghum genes for proteins related to monolignol biosynthesis were overexpressed: SbMyb60 (transcriptional activator), SbPAL (phenylalanine ammonia lyase), SbCCoAOMT (caffeoyl coenzyme A [CoA] 3- O -methyltransferase), Bmr2 (4-coumarate:CoA ligase), and SbC3H (coumaroyl shikimate 3-hydroxylase). Overexpression lines were evaluated for responses to stalk pathogens under greenhouse and field conditions. Greenhouse-grown plants were inoculated with Fusarium thapsinum (Fusarium stalk rot) and Macrophomina phaseolina (charcoal rot), which cause yield-reducing diseases. F. thapsinum -inoculated overexpression plants had mean lesion lengths not significantly different than wild-type, except for significantly smaller lesions on two of three SbMyb60 and one of two SbCCoAOMT lines. M. phaseolina -inoculated overexpression lines had lesions not significantly different from wild-type except one SbPAL line (of two lines studied) with mean lesion lengths significantly larger. Field-grown SbMyb60 and SbCCoAOMT overexpression plants were inoculated with F. thapsinum . Mean lesions of SbMyb60 lines were similar to wild-type, but one SbCCoAOMT had larger lesions, whereas the other line was not significantly different than wild-type. Because overexpression of SbMyb60 , Bmr2 , or SbC3H may not render sorghum more susceptible to stalk rots, these lines may provide sources for development of sorghum with increased phenylpropanoid concentrations.

Solanaceae have played an important role in elucidating how flower color is specified by the flavonoid biosynthesis pathway (FBP), which produces anthocyanins and other secondary metabolites. With well-established reverse genetics tools and rich genomic resources, Solanaceae provide a robust framework to examine the diversification of this well-studied pathway over short evolutionary timescales and to evaluate the predictability of genetic perturbation on pathway flux. Genomes of eight Solanaceae species, nine related asterids, and four rosids were mined to evaluate variation in copy number of the suite of FBP enzymes involved in anthocyanin biosynthesis. Comparison of annotation sources indicated that the NCBI annotation pipeline generated more and longer FBP annotations on average than genome-specific annotation pipelines. The pattern of diversification of each enzyme among asterids was assessed by phylogenetic analysis, showing that the CHS superfamily encompasses a large paralogous family of ancient and recent duplicates, whereas other FBP enzymes have diversified via recent duplications in particular lineages. Heterologous expression of a pansy F3'5'H gene in tobacco changed flower color from pink to dark purple, demonstrating that anthocyanin production can be predictably modified using reverse genetics. These results suggest that the Solanaceae FBP could be an ideal system to model genotype-to-phenotype interactions for secondary metabolism.

Soybean (Glycine max [L.] Merr.) is a commodity crop highly valued for its protein and oil content. The high percentage of polyunsaturated fatty acids in soybean oil results in low oxidative stability, which is a key parameter for usage in baking, high temperature frying applications, and affects shelf life of packaged products containing soybean oil. Introduction of a seed-specific expression cassette carrying the Arabidopsis transcription factor WRINKLED1 (AtWRI1) into soybean, led to seed oil with levels of palmitate up to approximately 20%. Stacking of the AtWRI1 transgenic allele with a transgenic locus harbouring the mangosteen steroyl-ACP thioesterase (GmFatA) resulted in oil with total saturates up to 30%. The creation of a triple stack in soybean, wherein the AtWRI1 and GmFatA alleles were combined with a FAD2-1 silencing allele led to the synthesis of an oil with 28% saturates and approximately 60% oleate. Constructs were then assembled that carry a dual FAD2-1 silencing element/GmFatA expression cassette, alone or combined with an AtWRI1 cassette. These plasmids are designated pPTN1289 and pPTN1301, respectively. Transgenic events carrying the T-DNA of pPTN1289 displayed an oil with stearate levels between 18% and 25%, and oleate in the upper 60%, with reduced palmitate (<5%). While soybean events harboring transgenic alleles of pPTN1301 had similar levels of stearic and oleate levels as that of the pPTRN1289 events, but with levels of palmitate closer to wild type. The modified fatty acid composition results in an oil with higher oxidative stability, and functionality attributes for end use in baking applications., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Triterpenes are thirty-carbon compounds derived from the universal five-carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large-scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T 0 mature plants) were obtained using the cytosolic-targeting strategy. Plastid-targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid-targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Sorghum (Sorghum bicolor) is a drought tolerant crop, which is being developed as a bioenergy feedstock. The monolignol biosynthesis pathway is a major focus for altering the abundance and composition of lignin. Caffeoyl coenzyme-A O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase that methylates caffeoyl-CoA to generate feruloyl-CoA, an intermediate required for the biosynthesis of both G- and S-lignin. SbCCoAOMT was overexpressed to assess the impact of increasing the amount of this enzyme on biomass composition. SbCCoAOMT overexpression increased both soluble and cell wall-bound (esterified) ferulic and sinapic acids, however lignin concentration and its composition (S/G ratio) remained unaffected. This increased deposition of hydroxycinnamic acids in these lines led to an increase in total energy content of the stover. In stalk and leaf midribs, the increased histochemical staining and autofluorescence in the cell walls of the SbCCoAOMT overexpression lines also indicate increased phenolic deposition within cell walls, which is consistent with the chemical analyses of soluble and wall-bound hydroxycinnamic acids. The growth and development of overexpression lines were similar to wild-type plants. Likewise, RNA-seq and metabolite profiling showed that global gene expression and metabolite levels in overexpression lines were also relatively similar to wild-type plants. Our results demonstrate that SbCCoAOMT overexpression significantly altered cell wall composition through increases in cell wall associated hydroxycinnamic acids without altering lignin concentration or affecting plant growth and development., Competing Interests: The authors have declared that no competing interests exist.

Kafirins are the major storage proteins in sorghum ( Sorghum bicolor ) grains and form protein bodies with poor digestibility. Since kafirins are devoid of the essential amino acid lysine, they also impart poor protein quality to the kernel. The α-kafirins, which make up most of the total kafirins, are largely encoded by the k1C family of highly similar genes. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to target the k1C genes to create variants with reduced kafirin levels and improved protein quality and digestibility. A single guide RNA was designed to introduce mutations in a conserved region encoding the endoplasmic reticulum signal peptide of α-kafirins. Sequencing of kafirin PCR products revealed extensive edits in 25 of 26 events in one or multiple k1C family members. T1 and T2 seeds showed reduced α-kafirin levels, and selected T2 events showed significantly increased grain protein digestibility and lysine content. Thus, a single consensus single guide RNA carrying target sequence mismatches is sufficient for extensive editing of all k1C genes. The resulting quality improvements can be deployed rapidly for breeding and the generation of transgene-free, improved cultivars of sorghum, a major crop worldwide., (© 2018 American Society of Plant Biologists. All rights reserved.)

Losses of floral pigmentation represent one of the most common evolutionary transitions in flower color, yet the genetic basis for these changes has been elucidated in only a handful of cases. Here we used crossing studies, bulk-segregant RNA sequencing, phylogenetic analyses and functional tests to identify the gene(s) responsible for the transition to white flowers in Iochroma loxense. Crosses between I. loxense and its blue-flowered sister species, I. cyaneum, suggested that a single locus controls the flower color difference and that the white allele causes a nearly complete loss of pigmentation. Examining sequence variation across phenotypic pools from the crosses, we found that alleles at a novel R3 MYB transcription factor were tightly associated with flower color variation. This gene, which we term MYBL1, falls into a class of MYB transcriptional repressors and, accordingly, higher expression of this gene is associated with downregulation of multiple anthocyanin pigment pathway genes. We confirmed the repressive function of MYBL1 through stable transformation of Nicotiana. The mechanism underlying the evolution of white flowers in I. loxense differs from that uncovered in previous studies, pointing to multiple mechanisms for achieving fixed transitions in flower color intensity., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Few transcription factors have been identified in C 4 grasses that either positively or negatively regulate monolignol biosynthesis. Previously, the overexpression of SbMyb60 in sorghum (Sorghum bicolor) has been shown to induce monolignol biosynthesis, which leads to elevated lignin deposition and altered cell wall composition. To determine how SbMyb60 overexpression impacts other metabolic pathways, RNA-Seq and metabolite profiling were performed on stalks and leaves. 35S::SbMyb60 was associated with the transcriptional activation of genes involved in aromatic amino acid, S-adenosyl methionine (SAM) and folate biosynthetic pathways. The high coexpression values between SbMyb60 and genes assigned to these pathways indicate that SbMyb60 may directly induce their expression. In addition, 35S::SbMyb60 altered the expression of genes involved in nitrogen (N) assimilation and carbon (C) metabolism, which may redirect C and N towards monolignol biosynthesis. Genes linked to UDP-sugar biosynthesis and cellulose synthesis were also induced, which is consistent with the observed increase in cellulose deposition in the internodes of 35S::SbMyb60 plants. However, SbMyb60 showed low coexpression values with these genes and is not likely to be a direct regulator of cell wall polysaccharide biosynthesis. These findings indicate that SbMyb60 can activate pathways beyond monolignol biosynthesis, including those that synthesize the substrates and cofactors required for lignin biosynthesis., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Background: Quantitative trait loci (QTLs) detected in one mapping population may not be detected in other mapping populations at all the time. Therefore, before being used for marker assisted breeding, QTLs need to be validated in different environments and/or genetic backgrounds to rule out statistical anomalies. In this regard, we mapped the QTLs controlling various agronomic traits in a recombinant inbred line (RIL) population in response to Nitrogen (N) stress and validated these with the reported QTLs in our earlier study to find the stable and consistent QTLs across populations. Also, with Illumina RNA-sequencing we checked the differential expression of gene (DEG) transcripts between parents and pools of RILs with high and low nitrogen use efficiency (NUE) and overlaid these DEGs on to the common validated QTLs to find candidate genes associated with N-stress tolerance in sorghum., Results: An F 7 RIL population derived from a cross between CK60 (N-stress sensitive) and San Chi San (N-stress tolerant) inbred sorghum lines was used to map QTLs for 11 agronomic traits tested under different N-levels. Composite interval mapping analysis detected a total of 32 QTLs for 11 agronomic traits. Validation of these QTLs revealed that of the detected, nine QTLs from this population were consistent with the reported QTLs in earlier study using CK60/China17 RIL population. The validated QTLs were located on chromosomes 1, 6, 7, 8, and 9. In addition, root transcriptomic profiling detected 55 and 20 differentially expressed gene (DEG) transcripts between parents and pools of RILs with high and low NUE respectively. Also, overlay of these DEG transcripts on to the validated QTLs found candidate genes transcripts for NUE and also showed the expected differential expression. For example, DEG transcripts encoding Lysine histidine transporter 1 (LHT1) had abundant expression in San Chi San and the tolerant RIL pool, whereas DEG transcripts encoding seed storage albumin, transcription factor IIIC (TFIIIC) and dwarfing gene (DW2) encoding multidrug resistance-associated protein-9 homolog showed abundant expression in CK60 parent, similar to earlier study., Conclusions: The validated QTLs among different mapping populations would be the most reliable and stable QTLs across germplasm. The DEG transcripts found in the validated QTL regions will serve as future candidate genes for enhancing NUE in sorghum using molecular approaches.

Soybean C 3 photosynthesis can suffer a severe loss in efficiency due to photorespiration and the lack of a carbon concentrating mechanism (CCM) such as those present in other plant species or cyanobacteria. Transgenic soybean (Glycine max cv. Thorne) plants constitutively expressing cyanobacterial ictB (inorganic carbon transporter B) gene were generated using Agrobacterium-mediated transformation. Although more recent data suggest that ictB does not actively transport HCO3-/CO 2 , there is nevertheless mounting evidence that transformation with this gene can increase higher plant photosynthesis. The hypothesis that expression of the ictB gene would improve photosynthesis, biomass production and seed yield in soybean was tested, in two independent replicated greenhouse and field trials. Results showed significant increases in photosynthetic CO 2 uptake (A net ) and dry mass in transgenic relative to wild type (WT) control plants in both the greenhouse and field trials. Transgenic plants also showed increased photosynthetic rates and biomass production during a drought mimic study. The findings presented herein demonstrate that ictB, as a single-gene, contributes to enhancement in various yield parameters in a major commodity crop and point to the significant role that biotechnological approaches to increasing photosynthetic efficiency can play in helping to meet increased global demands for food., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Nitrogen is essential for plant growth and development. Improving the ability of plants to acquire and assimilate nitrogen more efficiently is a key agronomic parameter that will augment sustainability in agriculture. A transcription factor approach was pursued to address improvement of nitrogen use efficiency in two major commodity crops. To this end, the Zea mays Dof1 ( ZmDof1 ) transcription factor was expressed in both wheat ( Triticum aestivum ) and sorghum ( Sorghum bicolor ) either constitutively, UBI4 promoter from sugarcane, or in a tissue specific fashion via the maize rbcS1 promoter. The primary transcription activation target of ZmDof1 , phospho enol pyruvate carboxylase (PEPC), is observed in transgenic wheat events. Expression ZmDof1 under control of the rbcs1 promoter translates to increase in biomass and yield components in wheat. However, constitutive expression of ZmDof1 led to the down-regulation of genes involved in photosynthesis and the functional apparatus of chloroplasts, and an outcome that negatively impacts photosynthesis, height, and biomass in wheat. Similar patterns were also observed in sorghum transgenic events harboring the constitutive expression cassette of ZmDof1 . These results indicate that transcription factor strategies to boost agronomic phenotypic outcomes in crops need to consider expression patterns of the genetic elements to be introduced.

Soya bean (Glycine max (L.) Merr.) is sought after for both its oil and protein components. Genetic approaches to add value to either component are ongoing efforts in soya bean breeding and molecular biology programmes. The former is the primary vegetable oil consumed in the world. Hence, its primary usage is in direct human consumption. As a means to increase its utility in feed applications, thereby expanding the market of soya bean coproducts, we investigated the simultaneous displacement of marine ingredients in aquafeeds with soya bean-based protein and a high Omega-3 fatty acid soya bean oil, enriched with alpha-linolenic and stearidonic acids, in both steelhead trout (Oncorhynchus mykiss) and Kampachi (Seriola rivoliana). Communicated herein are aquafeed formulations with major reduction in marine ingredients that translates to more total Omega-3 fatty acids in harvested flesh. Building off of these findings, subsequent efforts were directed towards a genetic strategy that would translate to a prototype design of an optimal identity-preserved soya bean-based feedstock for aquaculture, whereby a multigene stack approach for the targeted synthesis of two value-added output traits, eicosapentaenoic acid and the ketocarotenoid, astaxanthin, were introduced into the crop. To this end, the systematic introduction of seven transgenic cassettes into soya bean, and the molecular and phenotypic evaluation of the derived novel events are described., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided., (© 2015 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Background: Sorghum is an important C4 crop which relies on applied Nitrogen fertilizers (N) for optimal yields, of which substantial amounts are lost into the atmosphere. Understanding the genetic variation of sorghum in response to limited nitrogen supply is important for elucidating the underlying genetic mechanisms of nitrogen utilization., Results: A bi-parental mapping population consisting of 131 recombinant inbred lines (RILs) was used to map quantitative trait loci (QTLs) influencing different agronomic traits evaluated under normal N (100 kg.ha(-1) fertilizer) and low N (0 kg.ha(-1) fertilizer) conditions. A linkage map spanning 1614 cM was developed using 642 polymorphic single nucleotide polymorphisms (SNPs) detected in the population using Genotyping-By-Sequencing (GBS) technology. Composite interval mapping detected a total of 38 QTLs for 11 agronomic traits tested under different nitrogen levels. The phenotypic variation explained by individual QTL ranged from 6.2 to 50.8%. Illumina RNA sequencing data generated on seedling root tissues revealed 726 differentially expressed gene (DEG) transcripts between parents, of which 108 were mapped close to the QTL regions., Conclusions: Co-localized regions affecting multiple traits were detected on chromosomes 1, 5, 6, 7 and 9. These potentially pleiotropic regions were coincident with the genomic regions of cloned QTLs, including genes associated with flowering time, Ma3 on chromosome 1 and Ma1 on chromosome 6, gene associated with plant height, Dw2 on chromosome 6. In these regions, RNA sequencing data showed differential expression of transcripts related to nitrogen metabolism (Ferredoxin-nitrate reductase), glycolysis (Phosphofructo-2-kinase), seed storage proteins, plant hormone metabolism and membrane transport. The differentially expressed transcripts underlying the pleiotropic QTL regions could be potential targets for improving sorghum performance under limited N fertilizer through marker assisted selection.

Agrobacterium-mediated transformation of sorghum (Sorghum bicolor L. Moench) targeting immature embryo explants is a route to introduce transgenic alleles into the crop. The protocol requires maintenance of quality stock plants under greenhouse conditions for a constant supply of immature embryo explants. This is typically carried out by a regular sowing of seeds, minimal use of pesticides, and monitoring of plants to document pollen dispersal and bagging of heads. The time frame from explant inoculation to establishment of a primary transgenic event in the greenhouse typically ranges from 4 to 6 months. Seed set in the primary transformants is comparable to greenhouse-grown stock plants, with the majority of the transgenic alleles being inherited as a single functional locus.

Seed oils enriched in omega-7 monounsaturated fatty acids, including palmitoleic acid (16:1∆9) and cis-vaccenic acid (18:1∆11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega-7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ∆9 desaturation of stearoyl (18:0)-acyl carrier protein (ACP) to ∆9 desaturation of palmitoyl (16:0)-acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed-specific co-expression of a mutant ∆9-acyl-ACP and an acyl-CoA desaturase with high specificity for 16:0-ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega-7 monounsaturated fatty acids were obtained. Further increases in omega-7 fatty acid accumulation to 60-65% of the total fatty acids in camelina seeds were achieved by inclusion of seed-specific suppression of 3-keto-acyl-ACP synthase II and the FatB 16:0-ACP thioesterase genes to increase substrate pool sizes of 16:0-ACP for the ∆9-acyl-ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Analysis of fatty acid methyl esters (FAMEs) by gas chromatography (GC) is a common technique for the quantitative and qualitative analysis of acyl lipids. Methods for FAME preparation are typically time-consuming and labor-intensive and require multiple transfers of reagents and products between reaction tubes and autosampler vials. In order to increase throughput and lower the time and materials costs required for FAME preparation prior to GC analysis, we have developed a method in which 10-to-20-mg samples of microbial biomass are transferred to standard GC autosampler vials, transesterified using an emulsion of methanolic trimethylsulfonium hydroxide and hexane, and analyzed directly by GC without further sample handling. This method gives results that are essentially identical to those obtained by the more labor- and material-intensive FAME preparation methods, such as transmethylation with methanolic HCl. We applied this method to the screening of laboratory and environmental isolates of the green alga Chlamydomonas for variations in fatty acid composition. This screening method facilitated two novel discoveries. First, we identified a common laboratory strain of C. reinhardtii, CC-620, completely lacking all ω-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ω-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make Δ6-unsaturated polyunsaturated fatty acids (PUFA) rather than the Δ5 species normally made by the previously characterized laboratory strains of Chlamydomonas, suggesting that there is species-specific variation in the regiospecificity and substrate selectivity of front-end desaturases in this algal genus., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Soybean (Glycine max (L.) Merr) is valued for both its protein and oil, whose seed is composed of 40% and 20% of each component, respectively. Given its high percentage of polyunsaturated fatty acids, linoleic acid and linolenic acid, soybean oil oxidative stability is relatively poor. Historically food processors have employed a partial hydrogenation process to soybean oil as a means to improve both the oxidative stability and functionality in end-use applications. However, the hydrogenation process leads to the formation of trans-fats, which are associated with negative cardiovascular health. As a means to circumvent the need for the hydrogenation process, genetic approaches are being pursued to improve oil quality in oilseeds. In this regard, we report here on the introduction of the mangosteen (Garcinia mangostana) stearoyl-ACP thioesterase into soybean and the subsequent stacking with an event that is dual-silenced in palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase expression in a seed-specific fashion. Phenotypic analyses on transgenic soybean expressing the mangosteen stearoyl-ACP thioesterase revealed increases in seed stearic acid levels up to 17%. The subsequent stacked with a soybean event silenced in both palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase activity, resulted in a seed lipid phenotype of approximately 11%-19% stearate and approximately 70% oleate. The oil profile created by the stack was maintained for four generations under greenhouse conditions and a fifth generation under a field environment. However, in generation six and seven under field conditions, the oleate levels decreased to 30%-40%, while the stearic level remained elevated., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Background and Aims: During seed fill in cereals, nutrients are symplasmically unloaded to vascular parenchyma in ovules, but thereafter nutrient transport is less certain. In Zea mays, two mechanisms of nutrient passage through the chalaza and nucellus have been hypothesized, apoplasmic and symplasmic. In a recent study, nutrients first passed non-selectively to the chalazal apoplasm and were then selectively absorbed by the nucellus before being released to the endosperm apoplasm. This study reports that the promoter of OUTER CELL LAYER3 (PSbOCL3) from Sorghum bicolor (sorghum) directs gene expression to chalazal cells where the apoplasmic barrier is thought to form. The aims were to elucidate PSbOCL3 expression patterns in sorghum and relate them to processes of nutrient pathway development in kernels and to recognized functions of the homeodomain-leucine zipper (HD-Zip) IV transcription factor family to which the promoter belongs., Methods: PSbOCL3 was cloned and transformed into sorghum as a promoter-GUS (β-glucuronidase) construct. Plant tissues from control and transformed plants were then stained for GUS, and kernels were cleared and characterized using differential interference contrast microscopy., Key Results: A symplasmic disconnect between the chalaza and nucellus during seed fill is inferred by the combination of two phenomena: differentiation of a distinct nucellar epidermis adjacent to the chalaza, and lysis of GUS-stained chalazal cells immediately proximal to the nucellar epidermis. Compression of the GUS-stained chalazal cells during kernel maturation produced the kernel abscission zone (closing layer)., Conclusions: The results suggest that the HD-Zip IV transcription factor SbOCL3 regulates kernel nutrition and abscission. The latter is consistent with evidence that members of this transcription factor group regulate silique abscission and dehiscence in Arabidopsis thaliana. Collectively, the findings suggest that processes of floral organ abscission are conserved among angiosperms and may in some respects differ from processes of leaf abscission., (© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Main Conclusions: A Chlorovirus aquaglyceroporin expressed in tobacco is localized to the plastid and plasma membranes. Transgenic events display improved response to water deficit. Necrosis in adult stage plants is observed. Aquaglyceroporins are a subclass of the water channel aquaporin proteins (AQPs) that transport glycerol along with other small molecules transcellular in addition to water. In the studies communicated herein, we analyzed the expression of the aquaglyceroporin gene designated, aqpv1, from Chlorovirus MT325, in tobacco (Nicotiana tabacum), along with phenotypic changes induced by aqpv1 expression in planta. Interestingly, aqpv1 expression under control of either a constitutive or a root-preferred promoter, triggered local lesion formation in older leaves, which progressed significantly after induction of flowering. Fusion of aqpv1 with GFP suggests that the protein localized to the plasmalemma, and potentially with plastid and endoplasmic reticulum membranes. Physiological characterizations of transgenic plants during juvenile stage growth were monitored for potential mitigation to water dry-down (i.e., drought) and recovery. Phenotypic analyses on drought mimic/recovery of juvenile transgenic plants that expressed a functional aqpv1 transgene had higher photosynthetic rates, stomatal conductance, and water use efficiency, along with maximum carboxylation and electron transport rates when compared to control plants. These physiological attributes permitted the juvenile aqpv1 transgenic plants to perform better under drought-mimicked conditions and hastened recovery following re-watering. This drought mitigation effect is linked to the ability of the transgenic plants to maintain cell turgor.

Similar to Arabidopsis thaliana, the wild soybeans (Glycine soja) and many cultivars exhibit indeterminate stem growth specified by the shoot identity gene Dt1, the functional counterpart of Arabidopsis TERMINAL FLOWER1 (TFL1). Mutations in TFL1 and Dt1 both result in the shoot apical meristem (SAM) switching from vegetative to reproductive state to initiate terminal flowering and thus produce determinate stems. A second soybean gene (Dt2) regulating stem growth was identified, which, in the presence of Dt1, produces semideterminate plants with terminal racemes similar to those observed in determinate plants. Here, we report positional cloning and characterization of Dt2, a dominant MADS domain factor gene classified into the APETALA1/SQUAMOSA (AP1/SQUA) subfamily that includes floral meristem (FM) identity genes AP1, FUL, and CAL in Arabidopsis. Unlike AP1, whose expression is limited to FMs in which the expression of TFL1 is repressed, Dt2 appears to repress the expression of Dt1 in the SAMs to promote early conversion of the SAMs into reproductive inflorescences. Given that Dt2 is not the gene most closely related to AP1 and that semideterminacy is rarely seen in wild soybeans, Dt2 appears to be a recent gain-of-function mutation, which has modified the genetic pathways determining the stem growth habit in soybean., (© 2014 American Society of Plant Biologists. All rights reserved.)

Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene's expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg, and Mo), pod walls (Fe, K, P, Cu, and Ni), leaves (Fe, P, Cu, Ca, Ni, and Mg) and seeds (Fe, Zn, Cu, and Ni). Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves, and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and distribution.

Suppression of seed shattering was a key step during crop domestication that we have previously suggested to be convergent among independent cereal lineages. Positional, association, expression, and mutant complementation data all implicate a WRKY transcription factor, SpWRKY, in conferring shattering to a wild sorghum relative, Sorghum propinquum. We hypothesize that SpWRKY functions in a manner analogous to Medicago and Arabidopsis homologs that regulate cell wall biosynthesis genes, with low expression toward the end of floral development derepressing downstream cell wall biosynthesis genes to allow deposition of lignin that initiates the abscission zone in the seed-pedicel junction. The recent discovery of a YABBY locus that confers shattering within Sorghum bicolor and other cereals validated our prior hypothesis that some parallel domestication may have been convergent. Ironically, however, the shattering allele of SpWRKY appears to be recently evolved in S. propinquum and illustrates a case in which the genetic control of a trait in a wild relative fails to extrapolate even to closely related crops. Remarkably, the SpWRKY and YABBY loci lie only 300 kb apart and may have appeared to be a single genetic locus in some sorghum populations.

Zeins, the maize (Zea mays) prolamin storage proteins, accumulate at very high levels in developing endosperm in endoplasmic reticulum membrane-bound protein bodies. Products of the multigene α-zein families and the single-gene γ-zein family are arranged in the central hydrophobic core and the cross-linked protein body periphery, respectively, but little is known of the specific roles of family members in protein body formation. Here, we used RNA interference suppression of different zein subclasses to abolish vitreous endosperm formation through a variety of effects on protein body density, size, and morphology. We showed that the 27-kilodalton (kD) γ-zein controls protein body initiation but is not involved in protein body filling. Conversely, other γ-zein family members function more in protein body expansion and not in protein body initiation. Reduction in both 19- and 22-kD α-zein subfamilies severely restricted protein body expansion but did not induce morphological abnormalities, which result from reduction of only the 22-kD α-zein class. Concomitant reduction of all zein classes resulted in severe reduction in protein body number but normal protein body size and morphology.

The constitutive and drought-induced activities of the Arabidopsis thaliana RD29A and RD29B promoters were monitored in soybean (Glycine max (L.) Merr.] via fusions with the visual marker gene β-glucuronidase (GUS). Physiological responses of soybean plants were monitored over 9 days of water deprivation under greenhouse conditions. Data were used to select appropriate time points to monitor the activities of the respective promoter elements. Qualitative and quantitative assays for GUS expression were conducted in root and leaf tissues, from plants under well-watered and dry-down conditions. Both RD29A and RD29B promoters were significantly activated in soybean plants subjected to dry-down conditions. However, a low level of constitutive promoter activity was also observed in both root and leaves of plants under well-watered conditions. GUS expression was notably higher in roots than in leaves. These observations suggest that the respective drought-responsive regulatory elements present in the RD29X promoters may be useful in controlling targeted transgenes to mitigate abiotic stress in soybean, provided the transgene under control of these promoters does not invoke agronomic penalties with leaky expression when no abiotic stress is imposed.

The rhg1-b allele of soybean is widely used for resistance against soybean cyst nematode (SCN), the most economically damaging pathogen of soybeans in the United States. Gene silencing showed that genes in a 31-kilobase segment at rhg1-b, encoding an amino acid transporter, an α-SNAP protein, and a WI12 (wound-inducible domain) protein, each contribute to resistance. There is one copy of the 31-kilobase segment per haploid genome in susceptible varieties, but 10 tandem copies are present in an rhg1-b haplotype. Overexpression of the individual genes in roots was ineffective, but overexpression of the genes together conferred enhanced SCN resistance. Hence, SCN resistance mediated by the soybean quantitative trait locus Rhg1 is conferred by copy number variation that increases the expression of a set of dissimilar genes in a repeated multigene segment.

Cytological studies of fluorescent proteins are rapidly yielding insights into chromatin structure and dynamics. Here we describe the production and cytological characterization of new transgenic maize lines expressing a fluorescent histone fusion protein, H2B-mCherry. The transgene is expressed under the control of the maize ubiquitin1 promoter, including its first exon and intron. Polymerase chain reaction-based genotyping and root-tip microscopy showed that most of the lines carrying the transgene also expressed it, producing bright uniform staining of nuclei. Further, plants showing expression in root tips at the seedling stage also showed expression during meiosis, late in the life cycle. Detailed high-resolution three-dimensional imaging of cells and nuclei from various somatic and meiotic cell types showed that H2B-mCherry produced remarkably clear images of chromatin and chromosome fiber morphology, as seen in somatic, male meiotic prophase, and early microgametophyte cells. H2B-mCherry also yielded distinct nucleolus staining and was shown to be compatible with fluorescence in situ hybridization. We found several instances where H2B-mCherry was superior to DAPI as a generalized chromatin stain. Our study establishes these histone H2B-mCherry lines as new biological reagents for visualizing chromatin structure, chromosome morphology, and nuclear dynamics in fixed and living cells in a model plant genetic system.

Multicellular eukaryotes demonstrate nongenetic, heritable phenotypic versatility in their adaptation to environmental changes. This inclusive inheritance is composed of interacting epigenetic, maternal, and environmental factors. Yet-unidentified maternal effects can have a pronounced influence on plant phenotypic adaptation to changing environmental conditions. To explore the control of phenotypy in higher plants, we examined the effect of a single plant nuclear gene on the expression and transmission of phenotypic variability in Arabidopsis (Arabidopsis thaliana). MutS HOMOLOG1 (MSH1) is a plant-specific nuclear gene product that functions in both mitochondria and plastids to maintain genome stability. RNA interference suppression of the gene elicits strikingly similar programmed changes in plant growth pattern in six different plant species, changes subsequently heritable independent of the RNA interference transgene. The altered phenotypes reflect multiple pathways that are known to participate in adaptation, including altered phytohormone effects for dwarfed growth and reduced internode elongation, enhanced branching, reduced stomatal density, altered leaf morphology, delayed flowering, and extended juvenility, with conversion to perennial growth pattern in short days. Some of these effects are partially reversed with the application of gibberellic acid. Genetic hemicomplementation experiments show that this phenotypic plasticity derives from changes in chloroplast state. Our results suggest that suppression of MSH1, which occurs under several forms of abiotic stress, triggers a plastidial response process that involves nongenetic inheritance.

A key step during crop domestication is the loss of seed shattering. Here, we show that seed shattering in sorghum is controlled by a single gene, Shattering1 (Sh1), which encodes a YABBY transcription factor. Domesticated sorghums harbor three different mutations at the Sh1 locus. Variants at regulatory sites in the promoter and intronic regions lead to a low level of expression, a 2.2-kb deletion causes a truncated transcript that lacks exons 2 and 3, and a GT-to-GG splice-site variant in the intron 4 results in removal of the exon 4. The distributions of these non-shattering haplotypes among sorghum landraces suggest three independent origins. The function of the rice ortholog (OsSh1) was subsequently validated with a shattering-resistant mutant, and two maize orthologs (ZmSh1-1 and ZmSh1-5.1+ZmSh1-5.2) were verified with a large mapping population. Our results indicate that Sh1 genes for seed shattering were under parallel selection during sorghum, rice and maize domestication.

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.