Comprehensive in silico analysis of the underutilized crop tef (Eragrostis tef (Zucc.) Trotter) genome reveals drought tolerance signatures.

BACKGROUND: Tef (Eragrostis tef) is a C4 plant known for its tiny, nutritious, and gluten-free grains. It contains higher levels of protein, vitamins, and essential minerals like calcium (Ca), iron (Fe), copper (Cu), and zinc (Zn) than common cereals. Tef is cultivated in diverse ecological zones under diverse climatic conditions. Studies have shown that tef has great diversity in withstanding environmental challenges such as drought. Drought is a major abiotic stress severely affecting crop productivity and becoming a bottleneck to global food security. Here, we used in silico-based functional genomic analysis to identify drought-responsive genes in tef and validated their expression using quantitative RT-PCR. RESULTS: We identified about 729 drought-responsive genes so far reported in six crop plants, including rice, wheat, maize, barley, sorghum, pearl millet, and the model plant Arabidopsis, and reported 20 genes having high-level of GO terms related to drought, and significantly enriched in several biological and molecular function categories. These genes were found to play diverse roles, including water and fluid transport, resistance to high salt, cold, and drought stress, abscisic acid (ABA) signaling, de novo DNA methylation, and transcriptional regulation in tef and other crops. Our analysis revealed substantial differences in the conserved domains of some tef genes from well-studied rice orthologs. We further analyzed the expression of sixteen tef orthologs using quantitative RT-PCR in response to PEG-induced osmotic stress. CONCLUSIONS: The findings showed differential regulation of some drought-responsive genes in shoots, roots, or both tissues. Hence, the genes identified in this study may be promising candidates for trait improvement in crops via transgenic or gene-editing technologies.

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.

Micro(nano)plastic pollution in terrestrial ecosystem: emphasis on impacts of polystyrene on soil biota, plants, animals, and humans.

Pollution with emerging microscopic contaminants such as microplastics (MPs) and nanoplastics (NPs) including polystyrene (PS) in aquatic and terrestrial environments is increasingly recognized. PS is largely used in packaging materials and is dumped directly into the ecosystem. PS micro-nano-plastics (MNPs) can be potentially bioaccumulated in the food chain and can cause human health concerns through food consumption. Earlier MP research has focused on the aquatic environments, but recent researches show significant MP and NP contamination in the terrestrial environments especially agricultural fields. Though PS is the hotspot of MPs research, however, to our knowledge, this systematic review represents the first of its kind that specifically focused on PS contamination in agricultural soils, covering sources, effects, and ways of PS mitigation. The paper also provides updated information on the effects of PS on soil organisms, its uptake by plants, and effects on higher animals as well as human beings. Directions for future research are also proposed to increase our understanding of the environmental contamination of PS in terrestrial environments.

Genome-wide transcriptome analysis of the orphan crop tef (Eragrostis tef (Zucc.) Trotter) under long-term low calcium stress.

Calcium (Ca2+) is one of the essential mineral nutrients for plant growth and development. However, the effects of long-term Ca2+ deficiency in orphan crops such as Tef [(Eragrostis tef) (Zucc.) Trotter], which accumulate high levels of Ca in the grains, remained unknown. Tef is a staple crop for nearly 70 million people in East Africa, particularly in Ethiopia and Eritrea. It is one of the most nutrient-dense grains, and is also more resistant to marginal soils and climatic conditions than main cereals like corn, wheat, and rice. In this study, tef plants were grown in a hydroponic solution containing optimum (1 mM) or low (0.01 mM) Ca2+, and plant growth parameters and whole-genome transcriptome were analyzed. Ca+2-deficient plants exhibited leaf necrosis, leaf curling, and growth stunting symptoms. Ca2+ deficiency significantly decreased root and shoot Ca, potassium (K), and copper content in both root and shoots. At the same time, it greatly increased root iron (Fe) content, suggesting the role of Ca2+ in the uptake and/or translocation of these minerals. Transcriptomic analysis using RNA-seq revealed that members of Ca2+ channels, including the cyclic nucleotide-gated channels and glutamate receptor-like channels, Ca2+-transporters, Ca2+-binding proteins and Ca2+-dependent protein kinases were differentially regulated by Ca+2 treatment. Moreover, several Fe/metal transporters, including members of vacuolar Fe transporters, yellow stripe-like, natural resistance-associated macrophage protein, and oligo-peptide transporters, were differentially regulated between shoot and root in response to Ca2+ treatment. Taken together, our findings suggest that Ca2+ deficiency affects plant growth and mineral accumulation by regulating the transcriptomes of several transporters and signaling genes.

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.

Analysis of miRNAs responsive to long-term calcium deficiency in tef (Eragrostis tef (Zucc.) Trotter).

MicroRNAs (miRNAs) play an important role in growth, development, stress resilience, and epigenetic modifications of plants. However, the effect of calcium (Ca2+) deficiency on miRNA expression in the orphan crop tef (Eragrostis tef) remains unknown. In this study, we analyzed expression of miRNAs in roots and shoots of tef in response to Ca2+ treatment. miRNA-seq followed by bioinformatic analysis allowed us to identify a large number of small RNAs (sRNAs) ranging from 17 to 35 nt in length. A total of 1380 miRNAs were identified in tef experiencing long-term Ca2+ deficiency while 1495 miRNAs were detected in control plants. Among the miRNAs identified in this study, 161 miRNAs were similar with those previously characterized in other plant species and 348 miRNAs were novel, while the remaining miRNAs were uncharacterized. Putative target genes and their functions were predicted for all the known and novel miRNAs that we identified. Based on gene ontology (GO) analysis, the predicted target genes are known to have various biological and molecular functions including calcium uptake and transport. Pairwise comparison of differentially expressed miRNAs revealed that some miRNAs were specifically enriched in roots or shoots of low Ca2+-treated plants. Further characterization of the miRNAs and their targets identified in this study may help in understanding Ca2+ deficiency responses in tef and related orphan crops.

Microplastics interaction with terrestrial plants and their impacts on agriculture.

Microplastics (MPs) are widespread in natural ecosystems and have attracted considerable attention from scientists all over the world because they are believed to threaten every life form. In addition to their potential physical and chemical effects on organisms, MPs may act as a carrier for many micropollutants, including antibiotics, heavy metals, and others. Over the last 10-15 yr, extensive research has been carried out on MPs in marine environments regarding their sources, fate, and toxicity. However, studies concerning their accumulation in the soil ecosystem, uptake, internalization, and impacts on photosynthetic components of the terrestrial ecosystem and risk assessments have been scanty. Thus, there is a large knowledge gap on the extent to which terrestrial environments, especially agroecosystems, are affected by MPs and their subsequent risks to human health. This review summarizes up-to-date findings about MPs on terrestrial environments and provides guidelines for future studies regarding the phytotoxic effects of MPs on plants; the mechanism of uptake and translocation in plant tissues; detection tools for MPs in plants; impacts on plant growth, plant development, and agricultural productivity; and, most important, the future prospects of MPs interaction and accumulation in plants.

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.

Alternative Strategies for Multi-Stress Tolerance and Yield Improvement in Millets.

Millets are important cereal crops cultivated in arid and semiarid regions of the world, particularly Africa and southeast Asia. Climate change has triggered multiple abiotic stresses in plants that are the main causes of crop loss worldwide, reducing average yield for most crops by more than 50%. Although millets are tolerant to most abiotic stresses including drought and high temperatures, further improvement is needed to make them more resilient to unprecedented effects of climate change and associated environmental stresses. Incorporation of stress tolerance traits in millets will improve their productivity in marginal environments and will help in overcoming future food shortage due to climate change. Recently, approaches such as application of plant growth-promoting rhizobacteria (PGPRs) have been used to improve growth and development, as well as stress tolerance of crops. Moreover, with the advance of next-generation sequencing technology, genome editing, using the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system are increasingly used to develop stress tolerant varieties in different crops. In this paper, the innate ability of millets to tolerate abiotic stresses and alternative approaches to boost stress resistance were thoroughly reviewed. Moreover, several stress-resistant genes were identified in related monocots such as rice (Oryza sativa), wheat (Triticum aestivum), and maize (Zea mays), and other related species for which orthologs in millets could be manipulated by CRISPR/Cas9 and related genome-editing techniques to improve stress resilience and productivity. These cutting-edge alternative strategies are expected to bring this group of orphan crops at the forefront of scientific research for their potential contribution to global food security.

From Traditional Breeding to Genome Editing for Boosting Productivity of the Ancient Grain Tef [Eragrostis tef (Zucc.) Trotter].

Tef (Eragrostis tef (Zucc.) Trotter) is a staple food crop for 70% of the Ethiopian population and is currently cultivated in several countries for grain and forage production. It is one of the most nutritious grains, and is also more resilient to marginal soil and climate conditions than major cereals such as maize, wheat and rice. However, tef is an extremely low-yielding crop, mainly due to lodging, which is when stalks fall on the ground irreversibly, and prolonged drought during the growing season. Climate change is triggering several biotic and abiotic stresses which are expected to cause severe food shortages in the foreseeable future. This has necessitated an alternative and robust approach in order to improve resilience to diverse types of stresses and increase crop yields. Traditional breeding has been extensively implemented to develop crop varieties with traits of interest, although the technique has several limitations. Currently, genome editing technologies are receiving increased interest among plant biologists as a means of improving key agronomic traits. In this review, the potential application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) technology in improving stress resilience in tef is discussed. Several putative abiotic stress-resilient genes of the related monocot plant species have been discussed and proposed as target genes for editing in tef through the CRISPR-Cas system. This is expected to improve stress resilience and boost productivity, thereby ensuring food and nutrition security in the region where it is needed the most.

Silicon Enhances Biomass and Grain Yield in an Ancient Crop Tef [Eragrostis tef (Zucc.) Trotter].

Silicon (Si) is one of the beneficial plant mineral nutrients which is known to improve biotic and abiotic stress resilience and productivity in several crops. However, its beneficial role in underutilized or "orphan" crop such as tef [Eragrostis tef (Zucc.) Trotter] has never been studied before. In this study, we investigated the effect of Si application on tef plant performance. Plants were grown in soil with or without exogenous application of Na2SiO3 (0, 1.0, 2.0, 3.0, 4.0, and 5.0 mM), and biomass and grain yield, mineral content, chlorophyll content, plant height, and expression patterns of putative Si transporter genes were studied. Silicon application significantly increased grain yield (100%) at 3.0 mM Si, and aboveground biomass yield by 45% at 5.0 mM Si, while it had no effect on plant height. The observed increase in grain yield appears to be due to enhanced stress resilience and increased total chlorophyll content. Increasing the level of Si increased shoot Si and Na content while it significantly decreased the content of other minerals including K, Ca, Mg, P, S, Fe, and Mn in the shoot, which is likely due to the use of Na containing Si amendment. A slight decrease in grain Ca, P, S, and Mn was also observed with increasing Si treatment. The increase in Si content with increasing Si levels prompted us to analyze the expression of Si transporter genes. The tef genome contains seven putative Si transporters which showed high homology with influx and efflux Lsi transporters reported in various plant species including rice. The tef Lsi homologs were deferentially expressed between tissues (roots, leaves, nodes, and inflorescences) and in response to Si, suggesting that they may play a role in Si uptake and/or translocation. Taken together, these results show that Si application improves stress resilience and yield and regulates the expression of putative Si transporter genes. However, further study is needed to determine the physiological function of the putative Si transporters, and to study the effect of field application of Si on tef productivity.

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.

Novel Bioengineered Cassava Expressing an Archaeal Starch Degradation System and a Bacterial ADP-Glucose Pyrophosphorylase for Starch Self-Digestibility and Yield Increase.

To address national and global low-carbon fuel targets, there is great interest in alternative plant species such as cassava (Manihot esculenta), which are high-yielding, resilient, and are easily converted to fuels using the existing technology. In this study the genes encoding hyperthermophilic archaeal starch-hydrolyzing enzymes, α-amylase and amylopullulanase from Pyrococcus furiosus and glucoamylase from Sulfolobus solfataricus, together with the gene encoding a modified ADP-glucose pyrophosphorylase (glgC) from Escherichia coli, were simultaneously expressed in cassava roots to enhance starch accumulation and its subsequent hydrolysis to sugar. A total of 13 multigene expressing transgenic lines were generated and characterized phenotypically and genotypically. Gene expression analysis using quantitative RT-PCR showed that the microbial genes are expressed in the transgenic roots. Multigene-expressing transgenic lines produced up to 60% more storage root yield than the non-transgenic control, likely due to glgC expression. Total protein extracted from the transgenic roots showed up to 10-fold higher starch-degrading activity in vitro than the protein extracted from the non-transgenic control. Interestingly, transgenic tubers released threefold more glucose than the non-transgenic control when incubated at 85°C for 21-h without exogenous application of thermostable enzymes, suggesting that the archaeal enzymes produced in planta maintain their activity and thermostability.

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.

Characterization and Expression Analysis of Common Bean Histone Deacetylase 6 during Development and Cold Stress Response.

Histone deacetylases (HDACs) are important regulators of gene transcription thus controlling multiple cellular processes. Despite its essential role in plants, HDA6 is yet to be validated in common bean. In this study, we show that HDA6 is involved in plant development and stress response. Differential expression of HDA6 was determined in various tissues and the expression was seen to be upregulated with plant age (seedling < flowering < maturity). Higher expression was observed in flowers and pods than in stem, leaf, and root. Upregulation of HDA6 gene during cold stress implies its prominent role in abiotic stress. Furthermore, the HDA6 gene was isolated from three common bean genotypes and sequence analyses revealed homology with functionally characterized homologs in model species. The 53 kDa translated product was detected using an HDA6 specific antibody and recombinant protein overexpressed in Escherichia coli showed HDAC activity in vitro. To our knowledge, this is the first report in the agriculturally important crop common bean describing the functional characterization and biological role of HDA6.