Evaluating the Antioxidant Properties of the Ancient-Crop Tef (Eragrostis tef) Grain Extracts in THP-1 Monocytes.

Tef (Eragrostis tef) is an orphan crop that is widely grown in East Africa, primarily in Ethiopia as a staple crop. It is becoming popular in the Western world owing to its nutritious and gluten-free grains and the forage quality of its biomass. Tef is also considered to have a high antioxidant capacity based on cell-free studies. However, the antioxidant activity of tef has never been validated using a physiologically relevant cell model. The purpose of this study was to investigate the antioxidant capacity of tef grain extracts using a mammalian cell model. We hypothesized that the tef grain extracts are capable of modulating the cellular antioxidant response via the modulation of glutathione (GSH) biosynthetic pathways. Therefore, we evaluated the antioxidant activity of purified tef grain extracts in the human acute monocytic leukemia (THP-1) cell line. Our findings revealed that the organic fraction of grain extracts increased the cellular GSH level, which was more evident for brown-colored tef than the ivory variety. Moreover, a brown-tef fraction increased the expressions of GSH-pathway genes, including γ-glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits and glutathione reductase (GR), an enzyme that plays a key role in GSH biosynthesis, suggesting that tef extracts may modulate GSH metabolism. Several compounds were uniquely identified via mass spectrometry (MS) in GSH-modulating brown-tef samples, including 4-oxo-ß-apo-13-carotenone, γ-linolenic acid (methyl ester), 4,4'-(2,3-dimethyl-1,4-butanediyl)bis-phenol (also referred to as 8,8'-lignan-4,4'-diol), and (3ß)-3-[[2-[4-(Acetylamino)phenoxy]acetyl]oxy]olean-12-en-28-oic acid. Tef possesses antioxidant activity due to the presence of phytochemicals that can act as direct antioxidants, as well as modulators of antioxidant-response genes, indicating its potential role in alleviating diseases triggered by oxidative stresses. To the best of our knowledge, this is the first report revealing the antioxidant ability of tef extracts in a physiologically relevant human cell model.

Polyvalent guide RNAs for CRISPR antivirals.

CRISPR effector Cas13 recognizes and degrades RNA molecules that are complementary to its guide RNA (gRNA) and possesses potential as an antiviral biotechnology because it can degrade viral mRNA and RNA genomes. Because multiplexed targeting is a critical strategy to improve viral suppression, we developed a strategy to design of gRNAs where individual gRNAs have maximized activity at multiple viral targets, simultaneously, by exploiting the molecular biophysics of promiscuous target recognition by Cas13. These "polyvalent" gRNA sequences ("pgRNAs") provide superior antiviral elimination across tissue/organ scales in a higher organism (Nicotiana benthamiana) compared to conventionally-designed gRNAs-reducing detectable viral RNA by >30-fold, despite lacking perfect complementarity with either of their targets and, when multiplexed, reducing viral RNA by >99.5%. Pairs of pgRNA-targetable sequences are abundant in the genomes of RNA viruses, and this work highlights the need for specific approaches to the challenges of targeting viruses in eukaryotes using CRISPR.

Seasonal Variation in Transcriptomic Profiling of Tetrastigma hemsleyanum Fully Developed Tuberous Roots Enriches Candidate Genes in Essential Metabolic Pathways and Phytohormone Signaling.

Tetrastigma hemsleyanum Diels et Gilg (Sanyeqing, SYQ) is a perennial climbing liana and an endemic plant to southern China. Its tuberous roots (TRs) are used in traditional Chinese medicine for treating some diseases such as high fever, pneumonia, asthma, hepatitis, and cancers. However, the mechanisms underlying the development of TR and the content of flavonoids and phenylpropanoids (FPs) are not well-understood. In this study, we performed a transcriptomic analysis of 12 fully developed TR (FD-TR) samples harvested in four seasons [spring (Sp), summer (Su), autumn (Au), and winter (Wi)] using the RNA-Sequencing (RNA-Seq). We obtained a total of 78.54 Gb raw data and 65,578 unigenes. Then, the unigenes were annotated by using six databases such as non-redundant protein database (NR), Pfam, eggNOG, SWISSProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene ontology (GO). The transcriptomic profiling showed closer relationships between the samples obtained in Su and Au than those obtained in Sp and Wi based on the results of both total unigenes and differentially expressed genes (DEGs). Three pathways, including the biosynthesis of FPs, metabolism of starch and sucrose, and signaling of phytohormones, were highly enriched, suggesting a gene-level seasonal variation. Based on the numbers of DEGs, brassinosteroid (BR) signal transduction factors appeared to play a key role in modulating the development of TRs while most of the auxin signaling genes were mainly activated in Wi and Sp FD-TRs. Most genes in the biosynthesis and biodegradation of starch and biodegradation of cellulose were activated in Wi FD-TRs. As determined by the high performance liquid chromatography (HPLC) and aluminum nitrate colorimetric method, the contents of total flavonoids and most detected FP components increased from Sp to Au but decreased in Wi. Enhanced expression levels of some genes in the biosynthetic pathways of FPs were detected in Su and Au samples, which corroborated well with metabolite content. Our findings provide the first transcriptomic and biochemical data on a seasonal variation in the composition of medically important metabolites in SYQ FD-TRs.

The Maize Corngrass1 miRNA-Regulated Developmental Alterations Are Restored by a Bacterial ADP-Glucose Pyrophosphorylase in Transgenic Tobacco.

Crop-based bioethanol has raised concerns about competition with food and feed supplies, and technologies for second- and third-generation biofuels are still under development. Alternative feedstocks could fill this gap if they can be converted to biofuels using current sugar- or starch-to-ethanol technologies. The aim of this study was to enhance carbohydrate accumulation in transgenic Nicotiana benthamiana by simultaneously expressing the maize Corngrass1 miRNA (Cg1) and E. coli ADP-glucose pyrophosphorylase (glgC), both of which have been reported to enhance carbohydrate accumulation in planta. Our findings revealed that expression of Cg1 alone increased shoot branching, delayed flowering, reduced flower organ size, and induced loss of fertility. These changes were fully restored by coexpressing Escherichia coli glgC. The transcript level of miRNA156 target SQUAMOSA promoter binding-like (SPL) transcription factors was suppressed severely in Cg1-expressing lines as compared to the wild type. Expression of glgC alone or in combination with Cg1 enhanced biomass yield and total sugar content per plant, suggesting the potential of these genes in improving economically important biofuel feedstocks. A possible mechanism of the Cg1 phenotype is discussed. However, a more detailed study including genome-wide transcriptome and metabolic analysis is needed to determine the underlying genetic elements and pathways regulating the observed developmental and metabolic changes.

Reducing biomass recalcitrance by heterologous expression of a bacterial peroxidase in tobacco (Nicotiana benthamiana).

Commercial scale production of biofuels from lignocellulosic feed stocks has been hampered by the resistance of plant cell walls to enzymatic conversion, primarily owing to lignin. This study investigated whether DypB, the lignin-degrading peroxidase from Rodococcus jostii, depolymerizes lignin and reduces recalcitrance in transgenic tobacco (Nicotiana benthamiana). The protein was targeted to the cytosol or the ER using ER-targeting and retention signal peptides. For each construct, five independent transgenic lines were characterized phenotypically and genotypically. Our findings reveal that expression of DypB in the cytosol and ER does not affect plant development. ER-targeting increased protein accumulation, and extracts from transgenic leaves showed higher activity on classic peroxidase substrates than the control. Intriguingly, in situ DypB activation and subsequent saccharification released nearly 200% more fermentable sugars from transgenic lines than controls, which were not explained by variation in initial structural and non-structural carbohydrates and lignin content. Pyrolysis-GC-MS analysis showed more reduction in the level of lignin associated pyrolysates in the transgenic lines than the control primarily when the enzyme is activated prior to pyrolysis, consistent with increased lignin degradation and improved saccharification. The findings reveal for the first time that accumulation and in situ activation of a peroxidase improves biomass digestibility.

Loss-of-function mutation of the calcium sensor CBL1 increases aluminum sensitivity in Arabidopsis.

Despite the physiological importance of aluminum (Al) phytotoxicity for plants, it remained unknown if, and how, calcineurin B-like calcium sensors (CBLs) and CBL-interacting protein kinases (CIPKs) are involved in Al resistance. We performed a comparative physiological and whole transcriptome investigation of an Arabidopsis CBL1 mutant (cbl1) and the wild-type (WT). cbl1 plants exudated less Al-chelating malate, accumulated more Al, and displayed a severe root growth reduction in response to Al. Genes involved in metabolism, transport, cell wall modification, transcription and oxidative stress were differentially regulated between the two lines, under both control and Al stress treatments. Exposure to Al resulted in up-regulation of a large set of genes only in WT and not cbl1 shoots, while a different set of genes were down-regulated in cbl1 but not in WT roots. These differences allowed us, for the first time, to define a calcium-regulated/dependent transcriptomic network for Al stress responses. Our analyses reveal not only the fundamental role of CBL1 in the adjustment of central transcriptomic networks involved in maintaining adequate physiological homeostasis processes, but also that a high shoot-root dynamics is required for the proper deployment of Al resistance responses in the root.