Multigene Editing Via CRISPR/Cas9 Guided By a Single-sgRNA Seed in Arabidopsis

We report that a single-sgRNA seed is capable of guiding CRISPR/Cas9 to simultaneously edit multiple genes AtRPL10A, AtRPL10B and AtRPL10C in Arabidopsis. Our results also demonstrate that it is possible to use CRISPR/Cas9 technology to create AtRPL10 triple mutants which otherwise cannot be generated by conventional genetic crossing. Compared to other conventional multiplex CRISPR/Cas systems, a single sgRNA seed has the advantage of reducing off-target gene-editing. Such a single sgRNA seed-induced gene editing system might be also applicable to modify other homologous genes or even less-homologous sequences for multiple gene-editing in plants and other organisms. [Abstract copyright: This article is protected by copyright. All rights reserved.

Flavonols protect Arabidopsis plants against UV-B deleterious effects

Flavonols are synthesized by flavonol synthase (FLS) enzymes (Martens et al., 2010). These compounds absorb UV-B light in the 280?320 nm region, and their concentration increases in plants exposed to environmental abiotic and biotic stresses, including UV-B; consequently, flavonols are thought to act as UV-B filters (Agati et al., 2011). It has been also suggested that these metabolites function as reactive oxygen species (ROS) scavengers, as they contain an OH- group in the 3-position of the flavonoid skeleton, which allows them to chelate metals, inhibiting the formation of free radicals and ROS accumulation, once formed (Agati et al., 2009). For these reasons, it has been suggested that flavonols play uncharacterized roles in UV responses (Verdan et al., 2011). Nevertheless, despite the fact that the role of flavonols in UV-B protection has been inferred; the protection conferred by flavonols on the target sites of UV-B-damage has not been directly proven in planta. Recently, we demonstrated that maize FLS1 (ZmFLS1) complements the flavonol deficiency of the Arabidopsis fls1 mutant and decreases its high level of anthocyanins, characteristic of this mutant plant (Falcone Ferreyra et al., 2010). In order to demonstrate that flavonols protect plants against UV-B damage, we generated Arabidopsis transgenic plants overexpressing the maize FLS1 cDNA (35S:ZmFLS1), and evaluated different responses of these transgenic plants against UV-B damage.Fil: Emiliani, Julia.Fil: Grotewold, Erich.Fil: Falcone Ferreyra, María Lorena.Fil: Casati, Paula

The Effect of Different Interfaces and Confinement on the Structure of the Ionic Liquid 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide Entrapped in Cationic and Anionic Reverse Micelles.

The behavior of the ionic liquid (IL) 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([bmim][Tf 2N]) entrapped in two reverse micelles (RMs) formed in an aromatic solvent as dispersant pseudophase: [bmim][Tf 2N]/benzyl-n-hexadecyldimethylammonium chloride (BHDC)/chlorobenzene and [bmim][Tf 2N]/sodium 1,4-bis-2- ethylhexylsulfosuccinate (AOT)/chlorobenzene, was investigated using dynamic light scattering (DLS), FT-IR and 1H NMR spectroscopies. DLS results reveal the formation of RMs containing [bmim][Tf 2N] as a polar component since the droplet size values increase as the W s (W s = [[bmim][Tf 2N]]/[surfactant]) increases. Furthermore, it shows that the RMs consist of discrete spherical and non-interacting droplets of [bmim][Tf 2N] stabilized by the surfactants. Important differences in the structure of [bmim][Tf 2N] entrapped inside BHDC RMs, in comparison with the neat IL, are observed from the FT-IR and 1H NMR measurements. The electrostatic interactions between anions and cations from [bmim][Tf 2N] and BHDC determine the solvent structure encapsulated inside the nano-droplets. It seems that the IL structure is disrupted due to the electrostatic interaction between the [Tf 2N] - and the cationic BHDC polar head (BHD +) giving a high ion pair degree between BHD + and [Tf 2N] - at a low IL content. On the other hand, for the AOT RMs there is no evidence of strong IL-surfactant interaction. The electrostatic interaction between the SO 3 - group and the Na + counterion in AOT seems to be stronger than the possible [bmim] +-SO 3 - interaction at the interface. Thus, the structure of [bmim][Tf 2N] encapsulated is not particularly disrupted by the anionic surfactant at all W s studied, in contrast to the BHDC RM results. Nevertheless, there is evidence of confinement in the AOT RMs because the [bmim] +-[Tf 2N] - interaction is stronger than in bulk solution. Thus, the IL is more associated upon confinement. Our results reveal that the [bmim][Tf 2N] structure can be modified in a different manner inside RMs by varying the kind of surfactant used to create the RMs and the IL content (W s). These facts can be very important if these media are used as nanoreactors because unique microenvironments can be easily created by simply changing the RM components and W s.Fil: Ferreyra, Darío David. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Correa, Nestor Mariano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Silber, Juana J.. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; ArgentinaFil: Falcone, Ruben Dario. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentin

Identification of a Bifunctional Maize C- and O-Glucosyltransferase

Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.Fil: Falcone Ferreyra, María Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Rodriguez, Eduardo Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Casas, María Isabel. The Ohio State University. Plant Biotechnology Center. Department Plant Cell Molecular Biology; Estados UnidosFil: Labadie, Guillermo Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; ArgentinaFil: Grotewold, Erich. The Ohio State University. Plant Biotechnology Center; Estados UnidosFil: Casati, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentin

Evolution and Expression of Tandem Duplicated Maize Flavonol Synthase Genes

Flavonoids are specialized compounds widely distributed and with diverse functions throughout the plant kingdom and with several benefits for human health. In particular, flavonols, synthesized by flavonol synthase (FLS), protect plants against UV-B radiation and are essential for male fertility in maize and other plants. We have recently characterized a UV-B inducible ZmFLS1, corresponding to the first to be described in monocot plants. Interestingly, the new assembly of the B73 maize genome revealed the presence of a second putative FLS gene (ZmFLS2), with very high identity with ZmFLS1. ZmFLSs expression was analyzed in different maize tissues, and by combining electrophoretic mobility shift assays and transient expression experiments, we show that both genes are direct targets of anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. ZmFLS expression analyses show higher levels of both transcripts in high altitude landraces than inbred lines, and both genes are regulated by UV-B radiation in all lines analyzed. Moreover, the high sequence conservation of the ZmFLS promoters between maize lines suggests that the differences observed in ZmFLS expression are due to allelic variations in the transcription factors that regulate their activities. Finally, we generated pFLS1::FLS1-RFP transgenic plants and analyzed ZmFLS1 expression in different maize tissues; we found that this enzyme is localized in the ER and the perinuclear region

A role for β,β-xanthophylls in Arabidopsis UV-B photoprotection

Plastidial isoprenoids, such as carotenoids and tocopherols, are important anti-oxidant metabolites synthesized in plastids from precursors generated by the methylerythritol 4-phosphate (MEP) pathway. In this study, we found that irradiation of Arabidopsis thaliana plants with UV-B caused a strong increase in the accumulation of the photoprotective xanthophyll zeaxanthin but also resulted in slightly higher levels of γ-tocopherol. Plants deficient in the MEP enzymes 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase showed a general reduction in both carotenoids and tocopherols and this was associated with increased DNA damage and decreased photosynthesis after exposure to UV-B. Genetic blockage of tocopherol biosynthesis did not affect DNA damage accumulation. In contrast, lut2 mutants that accumulate β,β-xanthophylls showed decreased DNA damage when irradiated with UV-B. Analysis of aba2 mutants showed that UV-B protection was not mediated by ABA (a hormone derived from β,β-xanthophylls). Plants accumulating β,β-xanthophylls also showed decreased oxidative damage and increased expression of DNA-repair enzymes, suggesting that this may be a mechanism for these plants to decrease DNA damage. In addition, in vitro experiments also provided evidence that β,β-xanthophylls can directly protect against DNA damage by absorbing radiation. Together, our results suggest that xanthophyll-cycle carotenoids that protect against excess illumination may also contribute to protection against UV-B

Circadian oscillation and development-dependent expression of glycine-rich RNA binding proteins in tomato fruits

Glycine-rich RNA-binding proteins (GRPs) are involved in the modulation of the post-transcriptional processing of transcripts and participate as an output signal of the circadian clock. However, neither GRPs nor the circadian rhythmic have been studied in detail in fleshy fruits as yet. In the present work, the GRP1 gene family was analysed in Micro-Tom tomato (Solanum lycopersicum L.) fruit. Three highly homologous LeGRP1 genes (LeGRP1a-c) were identified. For each gene, three products were found, corresponding to the unspliced pre RNA, the mature mRNA and the alternatively spliced mRNA (preLeGRP1a-c, mLeGRP1a-c and asLeGRP1a-c, respectively). Tomato GRPs (LeGRPs) show the classic RNA recognition motif and glycine-rich region, and were found in the nucleus and in the cytosol of tomato fruit. By using different Escherichia coli mutants, it was found that LeGRP1s contained in vivo RNA-melting abilities and were able to complement the cold-sensitive phenotype of BX04 cells. Particular circadian profiles of expression, dependent on the fruits-developmental stage, were found for each LeGRP1 form. During ripening off the vine of fruits harvested at the mature green stage, the levels of all LeGRP1a-c forms drastically increased; however, incubation at 4°C prevented such increases. Analysis of the expression of all LeGRP1a-c forms suggests a positive regulation of expression in tomato fruit. Overall, the results obtained in this work reveal a complex pattern of expression of GRPs in tomato fruit, suggesting they might be involved in post-transcriptional modulation of circadian processes of this fleshy fruit.Fil: Müller, Gabriela Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Triassi, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Alvarez, Clarisa Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Falcone Ferreyra, María Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Andreo, Carlos Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Lara, Maria Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Drincovich, Maria Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentin

Transcriptomic, proteomic and metabolomic analysis of UV-B signaling in maize

<p>Abstract</p> <p>Background</p> <p>Under normal solar fluence, UV-B damages macromolecules, but it also elicits physiological acclimation and developmental changes in plants. Excess UV-B decreases crop yield. Using a treatment twice solar fluence, we focus on discovering signals produced in UV-B-irradiated maize leaves that translate to systemic changes in shielded leaves and immature ears.</p> <p>Results</p> <p>Using transcriptome and proteomic profiling, we tracked the kinetics of transcript and protein alterations in exposed and shielded organs over 6 h. In parallel, metabolic profiling identified candidate signaling molecules based on rapid increase in irradiated leaves and increased levels in shielded organs; pathways associated with the synthesis, sequestration, or degradation of some of these potential signal molecules were UV-B-responsive. Exposure of just the top leaf substantially alters the transcriptomes of both irradiated and shielded organs, with greater changes as additional leaves are irradiated. Some phenylpropanoid pathway genes are expressed only in irradiated leaves, reflected in accumulation of pathway sunscreen molecules. Most protein changes detected occur quickly: approximately 92% of the proteins in leaves and 73% in immature ears changed after 4 h UV-B were altered by a 1 h UV-B treatment.</p> <p>Conclusions</p> <p>There were significant transcriptome, proteomic, and metabolomic changes under all conditions studied in both shielded and irradiated organs. A dramatic decrease in transcript diversity in irradiated and shielded leaves occurs between 0 h and 1 h, demonstrating the susceptibility of plants to short term UV-B spikes as during ozone depletion. Immature maize ears are highly responsive to canopy leaf exposure to UV-B.</p

Light-controlled flavonoid biosynthesis in fruits

Light is one of the most important environmental factors affecting flavonoid biosynthesis in plants. The absolute dependency of light to the plant development has driven evolvement of sophisticated mechanisms to sense and transduce multiple aspects of the light signal. Light effects can be categorized in photoperiod (duration), intensity (quantity), direction and quality (wavelength) including UV-light. Recently, new information has been achieved on the regulation of light-controlled flavonoid biosynthesis in fruits, in which flavonoids have a major contribution on quality. This review focuses on the effects of the different light conditions on the control of flavonoid biosynthesis in fruit producing plants. An overview of the currently known mechanisms of the light-controlled flavonoid accumulation is provided. R2R3 MYB transcription factors are known to regulate by differential expression the biosynthesis of distinct flavonoids in response to specific light wavelengths. Despite recent advances, many gaps remain to be understood in the mechanisms of the transduction pathway of light-controlled flavonoid biosynthesis. A better knowledge on these regulatory mechanisms is likely to be useful for breeding programs aiming to modify fruit flavonoid pattern