A prion reduction filter does not completely remove endogenous prion infectivity from sheep blood.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy affecting humans, acquired initially through infection with bovine spongiform encephalopathy (BSE). A small number of vCJD cases have been acquired through the transfusion of blood from asymptomatic donors who subsequently developed vCJD. Filter devices that selectively bind the infectious agent associated with prion disease have been developed for removal of infection from blood. This study independently assessed one such filter, the P-CAPT filter, for efficacy in removing infectivity associated with the BSE agent in sheep blood. The sheep BSE model has previously been used to evaluate the distribution of infectivity in clinically relevant blood components. This is the first study to assess the ability of the P-CAPT filter to remove endogenous infectivity associated with blood components prepared from a large animal model. STUDY DESIGN AND METHODS: Paired units of leukoreduced red blood cells (LR-RBCs) were prepared from donors at the clinical stage of infection and confirmed as having BSE. One cohort of recipients was transfused with LR-RBCs alone, whereas a parallel cohort received LR and P-CAPT-filtered RBCs (LR-RBCs-P-CAPT). RESULTS: Of 14 recipients, two have been confirmed as having BSE. These sheep had received LR-RBCs and LR-RBCs-P-CAPT from the same donor. CONCLUSIONS: The results indicate that, after leukoreduction and P-CAPT filtration, there can still be sufficient residual infectivity in sheep RBCs to transmit infection when transfused into a susceptible recipient.

Expressing banana transcription factor MaERFVII3 in Arabidopsis confers enhanced waterlogging tolerance and root growth.

Background: Waterlogging poses a significant threat to plant growth and yield worldwide. Identifying the genes responsible for mitigating waterlogging stress is crucial. Ethylene-responsive factors (ERFs) are transcriptional regulators that respond to various biotic and abiotic stresses in plants. However, their roles and involvement in responding to waterlogging stress remain largely unexplored. Hence, this study aimed to elucidate the role of ERFs in enhancing banana plant resilience to waterlogging. Methods: We hypothesized that introducing a group VII ERF transcription factor in Arabidopsis could enhance waterlogging stress tolerance. To test this hypothesis, we isolated MaERFVII3 from banana roots, where it exhibited a significant induction in response to waterlogging stress. The isolated MaERFVII3 was introduced into Arabidopsis plants for functional gene studies. Results: Compared with wild-type plants, the MaERFVII3-expressing Arabidopsis showed increased survival and biomass under waterlogging stress. Furthermore, the abundance of transcripts related to waterlogging and hypoxia response showed an elevation in transgenic plants but a decrease in wild-type and empty vector plants when exposed to waterlogging stress. Our results demonstrate the significant contribution of MaERFVII3 to waterlogging tolerance in Arabidopsis, providing baseline data for further exploration and potentially contributing to crop improvement programs.

Unearth of small open reading frames (sORFs) in drought stress transcriptome of Oryza sativa subsp. indica.

Drought is a major abiotic stress that influences rice production. Although the transcriptomic data of rice against drought is widely available, the regulation of small open reading frames (sORFs) in response to drought stress in rice is yet to be investigated. Different levels of drought stress have different regulatory mechanisms in plants. In this study, drought stress was imposed on four-leaf stage rice, divided into two treatments, 40% and 30% soil moisture content (SMC). The RNAs of the samples were extracted, followed by the RNA sequencing analysis on their sORF expression changes under 40%_SMC and 30%_SMC, and lastly, the expression was validated through NanoString. A total of 122 and 143 sORFs were differentially expressed (DE) in 40%_SMC and 30%_SMC, respectively. In 40%_SMC, 69 sORFs out of 696 (9%) DEGs were found to be upregulated. On the other hand, 69 sORFs out of 449 DEGs (11%) were significantly downregulated. The trend seemed to be higher in 30%_SMC, where 112 (12%) sORFs were found to be upregulated from 928 significantly upregulated DEGs. However, only 8% (31 sORFs out of 385 DEGs) sORFs were downregulated in 30%_SMC. Among the identified sORFs, 110 sORFs with high similarity to rice proteome in the PsORF database were detected in 40%_SMC, while 126 were detected in 30%_SMC. The Gene Ontology (GO) enrichment analysis of DE sORFs revealed their involvement in defense-related biological processes, such as defense response, response to biotic stimulus, and cellular homeostasis, whereas enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways indicated that DE sORFs were associated with tryptophan and phenylalanine metabolisms. Several DE sORFs were identified, including the top five sORFs (OsisORF_3394, OsisORF_0050, OsisORF_3007, OsisORF_6407, and OsisORF_7805), which have yet to be characterised. Since these sORFs were responsive to drought stress, they might hold significant potential as targets for future climate-resilient rice development.

Foliar Application of Oil Palm Wood Vinegar Enhances Pandanus amaryllifolius Tolerance under Drought Stress.

Drought stress severely threatens plant growth, yield and survivability. Wood vinegar, formed by the condensation of smoke produced during biochar production, has been shown to promote plant growth and enhance stress tolerance. They have now been recognized as a sustainable alternative and are frequently used exogenously to support plants coping with environmental stress. This study aimed to evaluate the efficacy of oil palm wood vinegar (OPWV) in mitigating the adverse effects of drought stress on Pandanus amaryllifolius. The optimal concentrations and frequencies of OPWV application were determined before the drought treatment. The results showed that the imposed drought stress negatively affected the plant growth parameters but applying OPWV at 1:500 dilution at 3-day intervals for 12 days increased its tolerance. These include increased leaf relative water content, root-to-shoot ratio, relative stem circumference, chlorophyll pigments and antioxidant enzyme activities. In contrast, the drought-stressed plants treated with OPWV showed decreased relative electrolyte leakage, hydrogen peroxide, proline, malondialdehyde, and enhanced drought-responsive gene expressions, such as HSP70, GAPDH, and Thau, while ENO and ß-Fruc were reduced. These biostimulatory effects of OPWV might be due to several antioxidant compounds, such as anthranilic acid, tetrasiloxane, syringol, guaiacol, and catechol. Altogether, our results showed the effectiveness of OPWV in alleviating the adverse effects of drought stress, and as such, OPWV could be potentially applied in agriculture.

Significant differences in incubation times in sheep infected with bovine spongiform encephalopathy result from variation at codon 141 in the PRNP gene.

The susceptibility of sheep to prion infection is linked to variation in the PRNP gene, which encodes the prion protein. Common polymorphisms occur at codons 136, 154 and 171. Sheep which are homozygous for the A(136)R(154)Q(171) allele are the most susceptible to bovine spongiform encephalopathy (BSE). The effect of other polymorphisms on BSE susceptibility is unknown. We orally infected ARQ/ARQ Cheviot sheep with equal amounts of BSE brain homogenate and a range of incubation periods was observed. When we segregated sheep according to the amino acid (L or F) encoded at codon 141 of the PRNP gene, the shortest incubation period was observed in LL(141) sheep, whilst incubation periods in FF(141) and LF(141) sheep were significantly longer. No statistically significant differences existed in the expression of total prion protein or the disease-associated isoform in BSE-infected sheep within each genotype subgroup. This suggested that the amino acid encoded at codon 141 probably affects incubation times through direct effects on protein misfolding rates.

Green Revolution to Gene Revolution: Technological Advances in Agriculture to Feed the World.

Technological applications in agriculture have evolved substantially to increase crop yields and quality to meet global food demand. Conventional techniques, such as seed saving, selective breeding, and mutation breeding (variation breeding), have dramatically increased crop production, especially during the 'Green Revolution' in the 1990s. However, newer issues, such as limited arable lands, climate change, and ever-increasing food demand, pose challenges to agricultural production and threaten food security. In the following 'Gene Revolution' era, rapid innovations in the biotechnology field provide alternative strategies to further improve crop yield, quality, and resilience towards biotic and abiotic stresses. These innovations include the introduction of DNA recombinant technology and applications of genome editing techniques, such as transcription activator-like effector (TALEN), zinc-finger nucleases (ZFN), and clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR/Cas) systems. However, the acceptance and future of these modern tools rely on the regulatory frameworks governing their development and production in various countries. Herein, we examine the evolution of technological applications in agriculture, focusing on the motivations for their introduction, technical challenges, possible benefits and concerns, and regulatory frameworks governing genetically engineered product development and production.

Drought Stress Induces Morpho-Physiological and Proteome Changes of Pandanus amaryllifolius.

Drought is one of the significant threats to the agricultural sector. However, there is limited knowledge on plant response to drought stress and post-drought recovery. Pandanus amaryllifolius, a moderate drought-tolerant plant, is well-known for its ability to survive in low-level soil moisture conditions. Understanding the molecular regulation of drought stress signaling in this plant could help guide the rational design of crop plants to counter this environmental challenge. This study aimed to determine the morpho-physiological, biochemical, and protein changes of P. amaryllifolius in response to drought stress and during recovery. Drought significantly reduced the leaf relative water content and chlorophyll content of P. amaryllifolius. In contrast, relative electrolyte leakage, proline and malondialdehyde contents, and the activities of antioxidant enzymes in the drought-treated and recovered samples were relatively higher than the well-watered sample. The protein changes between drought-stressed, well-watered, and recovered plants were evaluated using tandem mass tags (TMT)-based quantitative proteomics. Of the 1415 differentially abundant proteins, 74 were significantly altered. The majority of proteins differing between them were related to carbon metabolism, photosynthesis, stress response, and antioxidant activity. This is the first study that reports the protein changes in response to drought stress in Pandanus. The data generated provide an insight into the drought-responsive mechanisms in P. amaryllifolius.

Small open reading frames in plant research: from prediction to functional characterization.

Gene prediction is a laborious and time-consuming task. The advancement of sequencing technologies and bioinformatics tools, coupled with accelerated rate of ribosome profiling and mass spectrometry development, have made identification of small open reading frames (sORFs) (< 100 codons) in various plant genomes possible. The past 50 years have seen sORFs being isolated from many organisms. However, to date, a comprehensive sORF annotation pipeline is as yet unavailable, hence, addressed in our review. Here, we also provide current information on classification and functions of plant sORFs and their potential applications in crop improvement programs.

CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in Nicotiana tabacum.

Flavonoids are an important class of natural compounds present in plants. However, despite various known biological activities and therapeutic potential, the low abundance of flavonoids in nature limits their development for industrial applications. In this study, we aimed to enhance flavonoid production by silencing cinnamate-4-hydroxylase (C4H), an enzyme involved in the branch point of the flavonoid biosynthetic pathway, using the clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach. We designed three sgRNAs targeting the promoter region of NtC4H and cloned them into a CRISPRi construct. After being introduced into Nicotiana tabacum cell suspension culture, the transformed cells were sampled for qPCR and liquid chromatography-mass spectrometry analyses. Sixteen of 21 cell lines showed PCR-positive, confirming the presence of the CRISPRi transgene. The NtC4H transcript in the transgenic cells was 0.44-fold lower than in the wild-type. In contrast, the flavonoid-related genes in the other branching pathways, such as Nt4CL and NtCHS, in the C4H-silenced cells showed higher expression than wild-type. The upregulation of these genes increased their respective products, including pinostrobin, naringenin, and chlorogenic acid. This study provides valuable insight into the future development of CRISPRi-based metabolic engineering to suppress target genes in plants.

Genome Editing for Sustainable Crop Improvement and Mitigation of Biotic and Abiotic Stresses.

Climate change poses a serious threat to global agricultural activity and food production. Plant genome editing technologies have been widely used to develop crop varieties with superior qualities or can tolerate adverse environmental conditions. Unlike conventional breeding techniques (e.g., selective breeding and mutation breeding), modern genome editing tools offer more targeted and specific alterations of the plant genome and could significantly speed up the progress of developing crops with desired traits, such as higher yield and/or stronger resilience to the changing environment. In this review, we discuss the current development and future applications of genome editing technologies in mitigating the impacts of biotic and abiotic stresses on agriculture. We focus specifically on the CRISPR/Cas system, which has been the center of attention in the last few years as a revolutionary genome-editing tool in various species. We also conducted a bibliographic analysis on CRISPR-related papers published from 2012 to 2021 (10 years) to identify trends and potential in the CRISPR/Cas-related plant research. In addition, this review article outlines the current shortcomings and challenges of employing genome editing technologies in agriculture with notes on future prospective. We believe combining conventional and more innovative technologies in agriculture would be the key to optimizing crop improvement beyond the limitations of traditional agricultural practices.

Waterlogging Stress Induces Antioxidant Defense Responses, Aerenchyma Formation and Alters Metabolisms of Banana Plants.

Flooding caused or exacerbated by climate change has threatened plant growth and food production worldwide. The lack of knowledge on how crops respond and adapt to flooding stress imposes a major barrier to enhancing their productivity. Hence, understanding the flooding-responsive mechanisms of crops is indispensable for developing new flooding-tolerant varieties. Here, we examined the banana (Musa acuminata cv. Berangan) responses to soil waterlogging for 1, 3, 5, 7, 14, and 24 days. After waterlogging stress, banana root samples were analyzed for their molecular and biochemical changes. We found that waterlogging treatment induced the formation of adventitious roots and aerenchyma with conspicuous gas spaces. In addition, the antioxidant activities, hydrogen peroxide, and malondialdehyde contents of the waterlogged bananas increased in response to waterlogging stress. To assess the initial response of bananas toward waterlogging stress, we analyzed the transcriptome changes of banana roots. A total of 3508 unigenes were differentially expressed under 1-day waterlogging conditions. These unigenes comprise abiotic stress-related transcription factors, such as ethylene response factors, basic helix-loop-helix, myeloblastosis, plant signal transduction, and carbohydrate metabolisms. The findings of the study provide insight into the complex molecular events of bananas in response to waterlogging stress, which could later help develop waterlogging resilient crops for the future climate.

CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences.

RNA-guided genomic transcriptional regulation tools, namely clustered regularly interspaced short palindromic repeats interference (CRISPRi) and CRISPR-mediated gene activation (CRISPRa), are a powerful technology for gene functional studies. Deriving from the CRISPR/Cas9 system, both systems consist of a catalytically dead Cas9 (dCas9), a transcriptional effector and a single guide RNA (sgRNA). This type of dCas9 is incapable to cleave DNA but retains its ability to specifically bind to DNA. The binding of the dCas9/sgRNA complex to a target gene results in transcriptional interference. The CRISPR/dCas9 system has been explored as a tool for transcriptional modulation and genome imaging. Despite its potential applications and benefits, the challenges and limitations faced by the CRISPR/dCas9 system include the off-target effects, protospacer adjacent motif (PAM) sequence requirements, efficient delivery methods and the CRISPR/dCas9-interfered crops being labeled as genetically modified organisms in several countries. This review highlights the progression of CRISPR/dCas9 technology as well as its applications and potential challenges in crop improvement.

Plant Nitric Oxide Signaling under Drought Stress.

Water deficit caused by drought is a significant threat to crop growth and production. Nitric oxide (NO), a water- and lipid-soluble free radical, plays an important role in cytoprotection. Apart from a few studies supporting the role of NO in drought responses, little is known about this pivotal molecular amendment in the regulation of abiotic stress signaling. In this review, we highlight the knowledge gaps in NO roles under drought stress and the technical challenges underlying NO detection and measurements, and we provide recommendations regarding potential avenues for future investigation. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool with which plant fitness can be improved under adverse growth conditions.

Cell-Free Expression of a Plant Membrane Protein BrPT2 From Boesenbergia Rotunda.

Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins.

Osmotic stress in banana is relieved by exogenous nitric oxide.

Drought is one of the severe environmental stresses threatening agriculture around the globe. Nitric oxide plays diverse roles in plant growth and defensive responses. Despite a few studies supporting the role of nitric oxide in plants under drought responses, little is known about its pivotal molecular amendment in the regulation of stress signaling. In this study, a label-free nano-liquid chromatography-mass spectrometry approach was used to determine the effects of sodium nitroprusside (SNP) on polyethylene glycol (PEG)-induced osmotic stress in banana roots. Plant treatment with SNP improved plant growth and reduced the percentage of yellow leaves. A total of 30 and 90 proteins were differentially identified in PEG+SNP against PEG and PEG+SNP against the control, respectively. The majority of proteins differing between them were related to carbohydrate and energy metabolisms. Antioxidant enzyme activities, such as superoxide dismutase and ascorbate peroxidase, decreased in SNP-treated banana roots compared to PEG-treated banana. These results suggest that the nitric oxide-induced osmotic stress tolerance could be associated with improved carbohydrate and energy metabolism capability in higher plants.

A New Plant Expression System for Producing Pharmaceutical Proteins.

In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.

Enhancing flavonoid production by promiscuous activity of prenyltransferase, BrPT2 from Boesenbergia rotunda.

Flavonoids and prenylated flavonoids are active components in medicinal plant extracts which exhibit beneficial effects on human health. Prenylated flavonoids consist of a flavonoid core with a prenyl group attached to it. This prenylation process is catalyzed by prenyltranferases (PTs). At present, only a few flavonoid-related PT genes have been identified. In this study, we aimed to investigate the roles of PT in flavonoid production. We isolated a putative PT gene (designated as BrPT2) from a medicinal ginger, Boesenbergia rotunda. The deduced protein sequence shared highest gene sequence homology (81%) with the predicted homogentisate phytyltransferase 2 chloroplastic isoform X1 from Musa acuminata subsp. Malaccensis. We then cloned the BrPT2 into pRI vector and expressed in B. rotunda cell suspension cultures via Agrobacterium-mediated transformation. The BrPT2-expressing cells were fed with substrate, pinostrobin chalcone, and their products were analyzed by liquid chromatography mass spectrometry. We found that the amount of flavonoids, namely alpinetin, pinostrobin, naringenin and pinocembrin, in BrPT2-expressing cells was higher than those obtained from the wild type cells. However, we were unable to detect any targeted prenylated flavonoids. Further in-vitro assay revealed that the reaction containing the BrPT2 protein produced the highest accumulation of pinostrobin from the substrate pinostrobin chalcone compared to the reaction without BrPT2 protein, suggesting that BrPT2 was able to accelerate the enzymatic reaction. The finding of this study implied that the isolated BrPT2 may not be involved in the prenylation of pinostrobin chalcone but resulted in high yield and production of other flavonoids, which is likely related to enzyme promiscuous activities.

Proteomics in commercial crops: An overview.

Proteomics is a rapidly growing area of biological research that is positively affecting plant science. Recent advances in proteomic technology, such as mass spectrometry, can now identify a broad range of proteins and monitor their modulation during plant growth and development, as well as during responses to abiotic and biotic stresses. In this review, we highlight recent proteomic studies of commercial crops and discuss the advances in understanding of the proteomes of these crops. We anticipate that proteomic-based research will continue to expand and contribute to crop improvement. SIGNIFICANCE: Plant proteomics study is a rapidly growing area of biological research that is positively impacting plant science. With the recent advances in new technologies, proteomics not only allows us to comprehensively analyses crop proteins, but also help us to understand the functions of the genes. In this review, we highlighted recent proteomic studies in commercial crops and updated the advances in our understanding of the proteomes of these crops. We believe that proteomic-based research will continue to grow and contribute to the improvement of crops.

Effects of Selected Physicochemical Parameters on Zerumbone Production of Zingiber zerumbet Smith Cell Suspension Culture.

Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement.