BY-2 Cells Research Articles

Production and characterization of novel Anti-HIV Fc-fusion proteins in plant-based systems: Nicotiana benthamiana & tobacco BY-2 cell suspension

Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

Cold tolerance of woodland strawberry (Fragaria vesca) is linked to Cold Box Factor 4 and the dehydrin Xero2.

Domesticated strawberry is susceptible to sudden frost episodes, limiting the productivity of this cash crop in regions where they are grown during early spring. In contrast, the ancestral woodland strawberry (Fragaria vesca) has successfully colonized many habitats of the Northern Hemisphere. Thus, this species seems to harbour genetic factors promoting cold tolerance. Screening a germplasm established in the frame of the German Gene Bank for Crop Wild Relatives, we identified, among 70 wild accessions, a pair with contrasting cold tolerance. By following the physiological, biochemical, molecular, and metabolic responses of this contrasting pair, we identified the transcription factor Cold Box Factor 4 and the dehydrin Xero2 as molecular markers associated with superior tolerance to cold stress. Overexpression of green fluorescent protein fusions with Xero2 in tobacco BY-2 cells conferred cold tolerance to these recipient cells. A detailed analysis of the metabolome for the two contrasting genotypes allows the definition of metabolic signatures correlated with cold tolerance versus cold stress. This work provides a proof-of-concept for the value of crop wild relatives as genetic resources to identify genetic factors suitable to increase the stress resilience of crop plants.

Toxin Homology Domain in Plant Type 2 Prolyl 4-Hydroxylases Acts as a Golgi Localization Domain.

Prolyl 4-hydroxylase (P4H) generates hydroxyproline residues in proteins. Two classes of P4H have been found in plants. Type 1 P4H has a signal anchor at the N-terminus, while type 2 P4H has both an N-terminal signal peptide and a C-terminal toxin homology domain (Tox1 domain) with six conserved cysteine residues. We analyzed the localization of tobacco type 2 P4H (NtP4H2.2) in tobacco BY-2 cells. Cell fractionation studies, immunostaining of cells, and GFP fusion study indicated that NtP4H2.2 localizes predominantly to the Golgi apparatus and is a peripheral membrane protein associated with the luminal side of organelles. Expression of the GFP-Tox1 domains of NtP4H2.2 and another tobacco type 2 P4H NtP4H2.1 in BY-2 cells and Arabidopsis epidermal cells indicated that these proteins were targeted to the Golgi. The Tox1 domains from Arabidopsis and rice type 2 P4Hs also directed GFP to the Golgi in tobacco BY-2 cells. The Tox1 domain of NtP4H2.2 increased the membrane association of GFP, and mutation of the cysteine residues in this domain abolished Golgi localization. Furthermore, the catalytic domain of NtP4H2.2 also directed GFP to the Golgi. Thus, the Tox1 domains of plant P4Hs are the Golgi localization domains, and tobacco P4H2.2 localizes to the Golgi by the action of both this domain and the catalytic domain.

Spearmint targets microtubules by (-)-carvone.

Allelopathy can provide sustainable alternatives to herbicides because it is based on specific signals rather than generic toxicity. We show that the allelopathic activity of Spearmint and Watermint is linked with their main compounds, (-)-carvone and (+)-menthofuran, both deriving from (-)-limonene. Germination of Poppy and Cress, and root growth of Arabidopsis thaliana are inhibited by very low concentrations of (-)-carvone, acting even through the gas phase. (+)-Menthofuran is active as well, but at lower efficacy. Using fluorescently tagged marker lines in tobacco BY-2 cells and Arabidopsis roots, we demonstrate a rapid degradation of microtubules and a remodeling of actin filaments in response to (-)-carvone and, to a milder extent, to (+)-menthofuran. This cytoskeletal response is followed by cell death. By means of a Root Chip system, we can follow the tissue dependent response of the cytoskeleton and show a cell-type dependent gradient of sensitivity between meristem and distal elongation zone, accompanied by programmed cell death.

The cAMP-dependent phosphorylation footprint in response to heat stress

Key messagecAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress.In plants, 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP–dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular “sponge” that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

Advancing Engineered Plant Living Materials through Tobacco BY-2 Cell Growth and Transfection within Tailored Granular Hydrogel Scaffolds.

In this study, an innovative approach is presented in the field of engineered plant living materials (EPLMs), leveraging a sophisticated interplay between synthetic biology and engineering. We detail a 3D bioprinting technique for the precise spatial patterning and genetic transformation of the tobacco BY-2 cell line within custom-engineered granular hydrogel scaffolds. Our methodology involves the integration of biocompatible hydrogel microparticles (HMPs) primed for 3D bioprinting with Agrobacterium tumefaciens capable of plant cell transfection, serving as the backbone for the simultaneous growth and transformation of tobacco BY-2 cells. This system facilitates the concurrent growth and genetic modification of tobacco BY-2 cells within our specially designed scaffolds. These scaffolds enable the cells to develop into predefined patterns while remaining conducive to the uptake of exogenous DNA. We showcase the versatility of this technology by fabricating EPLMs with unique structural and functional properties, exemplified by EPLMs exhibiting distinct pigmentation patterns. These patterns are achieved through the integration of the betalain biosynthetic pathway into tobacco BY-2 cells. Overall, our study represents a groundbreaking shift in the convergence of materials science and plant synthetic biology, offering promising avenues for the evolution of sustainable, adaptive, and responsive living material systems.

On Interactions of Sulfamerazine with Cyclodextrins from Coupled Diffusometry and Toxicity Tests.

This scientific study employs the Taylor dispersion technique for diffusion measurements to investigate the interaction between sulfamerazine (NaSMR) and macromolecular cyclodextrins (β-CD and HP-β-CD). The results reveal that the presence of β-CD influences the diffusion of the solution component, NaSMR, indicating a counterflow of this drug due to solute interaction. However, diffusion data indicate no inclusion of NaSMR within the sterically hindered HP-β-CD cavity. Additionally, toxicity tests were conducted, including pollen germination (Actinidia deliciosa) and growth curve assays in BY-2 cells. The pollen germination tests demonstrate a reduction in sulfamerazine toxicity, suggesting potential applications for this antimicrobial agent with diminished adverse effects. This comprehensive investigation contributes to a deeper understanding of sulfamerazine-cyclodextrin interactions and their implications for pharmaceutical and biological systems.

Neopetromin, a Cyclic Tripeptide with a C-N Cross-Link, from the Marine Sponge Neopetrosia sp., That Causes Vacuole Fragmentation in Tobacco BY-2 Cells.

HPLC-MS analysis revealed the presence of an unreported peptide in the extract of the marine sponge Neopetrosia sp. Its structure was determined as a tripeptide, named neopetromin (1), composed of two tyrosine and one tryptophan residues with a heteroaromatic C-N cross-link between side chains. The absolute configuration of amino acids was determined using Marfey's method after ozonolysis and hydrolysis of 1. Compound 1 promoted vacuole fragmentation in an actin-independent manner in tobacco BY-2 cells.

Assessment of cytotoxic and genotoxic effects of Colletotrichum gloeosporioides and C. capsici toxins on tobacco BY-2 cells

Assessment of cytotoxic and genotoxic effects of Colletotrichum gloeosporioides and C. capsici toxins on tobacco BY-2 cells

MYB3R-mediated and cell cycle-dependent transcriptional regulation of a tobacco ortholog of SCARECROW-LIKE28 in synchronized cultures of BY-2 cells.

Although it is well known that hierarchical transcriptional networks are essential for various aspects of plant development and environmental response, little has been investigated about whether and how they also regulate the plant cell cycle. Recent studies on cell cycle regulation in Arabidopsis thaliana identified SCARECROW-LIKE28 (SCL28), a GRAS-type transcription factor, that constitutes a hierarchical transcriptional pathway comprised of MYB3R, SCL28 and SIAMESE-RELATED (SMR). In this pathway, MYB3R family proteins regulate the G2/M-specific transcription of the SCL28 gene, of which products, in turn, positively regulate the transcription of SMR genes encoding a group of plant-specific inhibitor proteins of cyclin-dependent kinases. However, this pathway with a role in cell cycle inhibition is solely demonstrated in A. thaliana, thus leaving open the question of whether and to what extent this pathway is evolutionarily conserved in plants. In this study, we conducted differential display RT-PCR on synchronized Nicotiana tabacum (tobacco) BY-2 cells and identified several M-phase-specific cDNA clones, one of which turned out to be a tobacco ortholog of SCL28 and was designated NtSCL28. We showed that NtSCL28 is expressed specifically during G2/M and early G1 in the synchronized cultures of BY-2 cells. NtSCL28 contains MYB3R-binding promoter elements, so-called mitosis-specific activator elements, and is upregulated by a hyperactive form of NtmybA2, one of the MYB3R proteins from tobacco. Our study indicated that a part of the hierarchical pathway identified in A. thaliana is equally operating in tobacco cells, suggesting the conservation of this pathway across different families in evolution of angiosperm.

Caffeine inhibits cell-wall regeneration in protoplasts isolated from tobacco BY-2 cells

Caffeine inhibits cell-wall regeneration in protoplasts isolated from tobacco BY-2 cells

Carbon nanoparticles alleviate oxidative stress on BY-2 cells via promoting potassium accumulation

Carbon nanomaterials have been widely applied in promoting plant growth, but the effects on plants should require careful investigation. In our study, BY-2 cells were exposed to carbon nanoparticles (CNPs) with concentration of 0–125 μg/mL, for the purpose of better investigating the relationship between oxidative stress and potassium accumulation in BY-2 cells induced by CNPs. The results showed that CNPs significantly increased in the content of K+ and reduced Na+ content compared to control group, while oxidative stress was observed with no significantly changes, including H2O2 and malondialdehyde (MDA) content, antioxidant activities. These results might be associated with the content of potassium in cells, which had negative correlation with H2O2 content, as well as CAT antioxidant enzymes activity. Furthermore, IBR value is all less than 2 under the experiment concentration, indicating BY-2 cells are insensitive to CNPs exposure. The most important metabolites were clustered into three groups according to the concentration of CNPs (control, low concentration (0, 6.25, 31.25) and high concentration (62.50 and 125.00). Our findings indicate that CNPs can alleviate oxidative stress through increasing in potassium content in BY-2 cells, and also shed light on understanding the underlying mechanism of toxicity of carbon nanomaterials in plant.

Issue when expressing a recombinant protein under the control of p35S in Nicotiana tabacum BY-2 cells.

Several recombinant proteins have been successfully produced in plants. This usually requires Agrobacterium-mediated cell transformation to deliver the T-DNA into the nucleus of plant cells. However, some genetic instability may threaten the integrity of the expression cassette during its delivery via A. tumefaciens, especially when the protein of interest is toxic to the bacteria. In particular, we found that a Tn3 transposon can be transferred from the pAL4404 Ti plasmid of A. tumefaciens LBA4404 into the expression cassette when using the widely adopted 35S promoter, thereby damaging T-DNA and preventing correct expression of the gene of interest in Nicotiana tabacum BY-2 suspension cells.

The seven rice vacuolar sorting receptors localize to prevacuolar compartments

Vacuolar sorting is critically important in plants as it regulates the mobilization of proteins and plays a major role in important agricultural traits like yield and seed protein content. Vacuolar sorting receptors (VSRs) are integral membrane proteins that mediate protein trafficking from the Golgi apparatus to the vacuole via the intermediate membrane-bound prevacuolar compartment (PVC)/multivesicular body (MVB). VSR proteins, such as an 80 kD (BP-80) from pea, also serve as markers for PVC/MVB. Dissecting VSR-mediated protein trafficking pathways may provide ways to enhance agronomic traits and crop yield. Green fluorescence protein (GFP) fusions with the seven Arabidopsis (Arabidopsis thaliana) VSRs were previously shown to localize to PVCs in transgenic tobacco BY-2 cells. The Rice (Oryza sativa) genome contains seven VSRs (OsVSR1-7), but little is known about their subcellular localizations. Here we studied the subcellular localization of OsVSR1-7 b y using a reporter approach, in which GFP-OsVSR1-7 fusions containing the transmembrane domain (TMD) and cytoplasmic tail (CT) of individual OsVSR were expressed in the protoplasts of rice, transgenic tobacco BY-2 cells and transgenic rice plants. Immunofluorescent labelling studies and confocal laser scanning microscope observation demonstrated that the seven OsVSRs are localized to PVCs and form ring-like structures upon wortmannin treatment. Therefore, we have verified the subcellular localization of OsVSR1-7 in this study. The OsVSRs tagged with GFP can serve as PVCs/MVBs markers in rice for the future studies.

A new method to measure aquaporin-facilitated membrane diffusion of hydrogen peroxide and cations in plant suspension cells.

Plant aquaporins (AQPs) facilitate the membrane diffusion of water and small solutes, including hydrogen peroxide (H2 O2 ) and, possibly, cations, essential signalling molecules in many physiological processes. While the determination of the channel activity generally depends on heterologous expression of AQPs in Xenopus oocytes or yeast cells, we established a genetic tool to determine whether they facilitate the diffusion of H2 O2 through the plasma membrane in living plant cells. We designed genetic constructs to co-express the fluorescent H2 O2 sensor HyPer and AQPs, with expression controlled by a heat shock-inducible promoter in Nicotiana tabacum BY-2 suspension cells. After induction of ZmPIP2;5 AQP expression, a HyPer signal was recorded when the cells were incubated with H2 O2 , suggesting that ZmPIP2;5 facilitates H2 O2 transmembrane diffusion; in contrast, the ZmPIP2;5W85A mutated protein was inactive as a water or H2 O2 channel. ZmPIP2;1, ZmPIP2;4and AtPIP2;1 also facilitated H2 O2 diffusion. Incubation with abscisic acid and the elicitor flg22 peptide induced the intracellular H2 O2 accumulation in BY-2 cells expressing ZmPIP2;5. We also monitored cation channel activity of ZmPIP2;5 using a novel fluorescent photo-switchable Li+ sensor in BY-2 cells. BY-2 suspension cells engineered for inducible expression of AQPs as well as HyPer expression and the use of Li+ sensors constitute a powerful toolkit for evaluating the transport activity and the molecular determinants of PIPs in living plant cells.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis

BackgroundUDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides.ResultsIn this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark.ConclusionOur findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

A Glycine soja S1 group bZIP transcription factor GsbZIP43 is a positive regulator of sodium bicarbonate stress tolerance

Soil salinization-alkalization has long restricted the yield and quality of crops. As a key factor in the regulation of gene expression, transcription factor (TF) controls the growth and development of crops. The basic leucine zipper (bZIP) family is one of the largest TF families in plants and has been widely reported regulating plant growth and development and stress responses. Here, in this study, we identified a sodium bicarbonate tolerance gene, GsbZIP43, by screening the cDNA library generated from the wild soybean G07256 with sodium bicarbonate tolerance. Sequence analysis revealed that GsbZIP43 is a member of S1 bZIP subfamily. The subcellular localization of GsbZIP43-RFP (red fluorescent protein) fusion protein in tobacco leaf cells suggested that GsbZIP43 localizes in cell nucleus. Further transactivation assay uncovered that GsbZIP43 acts as a transcriptional activator and the C-terminal region is vital for transcriptional activation. Overexpression of GsbZIP43 in BY-2 cells, Arabidopsis, and soybean hairy roots enhanced tolerance to sodium bicarbonate stress. Consistently, the loss of its close Arabidopsis homologous gene AtbZIP44 decreased tolerance to sodium bicarbonate stress. Taken together, the above results illustrate the positive role of GsbZIP43 in responding to sodium bicarbonate stress.

The Carbon Flow Shifts from Primary to Secondary Metabolism during Xylem Vessel Cell Differentiation inArabidopsis thaliana.

Xylem vessel cell differentiation is characterized by the deposition of a secondary cell wall (SCW) containing cellulose, hemicellulose and lignin. VASCULAR-RELATED NAC-DOMAIN7 (VND7), a plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor, is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). Previous metabolome analysis using the VND7-inducible system in tobacco BY-2 cells successfully revealed significant quantitative changes in primary metabolites during xylem vessel cell differentiation. However, the flow of primary metabolites is not yet well understood. Here, we performed a metabolomic analysis of VND7-inducible Arabidopsis T87 suspension cells. Capillary electrophoresis-time-of-flight mass spectrometry quantified 57 metabolites, and subsequent data analysis highlighted active changes in the levels of UDP-glucose and phenylalanine, which are building blocks of cellulose and lignin, respectively. In a metabolic flow analysis using stable carbon 13 (13C) isotope, the 13C-labeling ratio specifically increased in 3-phosphoglycerate after 12 h of VND7 induction, followed by an increase in shikimate after 24 h of induction, while the inflow of 13C into lactate from pyruvate was significantly inhibited, indicating an active shift of carbon flow from glycolysis to the shikimate pathway during xylem vessel cell differentiation. In support of this notion, most glycolytic genes involved in the downstream of glyceraldehyde 3-phosphate were downregulated following the induction of xylem vessel cell differentiation, whereas genes for the shikimate pathway and phenylalanine biosynthesis were upregulated. These findings provide evidence for the active shift of carbon flow from primary metabolic pathways to the SCW polymer biosynthetic pathway at specific points during xylem vessel cell differentiation.

Effects of lipoprotein nanoparticles' composition and size on their internalization in plant and mammalian cells.

The internalization of engineered high-density lipoprotein nanoparticles (engineered lipoproteins [eLPs]) with different lipid and protein compositions, zeta potentials, and/or sizes were analyzed in representative plant and mammalian cells. The impact of the addition of a cell-penetrating peptide to eLPs on the internalization was very small in Bright Yellow (BY)-2 protoplasts compared with HeLa cells. When eLPs were prepared with one of the abundant lipids in BY-2 cells, digalactosyldiacylglycerol (DGDG) (eLP4), its internalization was dramatically increased only in HeLa cells. Such an increase in HeLa cells was also obtained for liposomes containing DGDG in a DGDG content-dependent manner. Increasing the size and zeta potential of eLPs improved their internalization in both HeLa cells and in BY-2 protoplasts but to quite varying degrees. Although eLPs tended to stay at the plasma membrane (PM) in BY-2 protoplasts with much less internalization, the PM-bound eLPs somehow promoted the internalization of coexisting nanobeads in cell culture media. These results provide fundamental insight into the future design of lipid nanoparticles for drug delivery in mammalian and plant cells.

Hidden history of the tobacco BY-2 cell line.

For almost 50 years, tobacco (Nicotiana tabacum) BY-2 cells have been widely recognized as an important cell line for plant biology. The cell line grows rapidly, can be synchronized to a high degree, and is excellent for imaging; over the years, these features have led to many high-impact discoveries. However, certain other uses of this cell line are virtually unknown. In the early days, I was involved in distributing the cells to laboratories around the world. Many of these scientists wanted to study the cell cycle; however, I also distributed the cells to scientists who were elucidating the mechanism of plant transformation by Agrobacterium tumefaciens. In fact, BY-2 cells played an essential role in the identification and analysis of Vir genes on the Ti plasmid; likewise, the cells were important for discovering the factor that induces the expression of Vir genes. Thus, BY-2 cells were crucial for the development of modern plant biotechnology. Here, I recount the story of how this came to pass and explain why the use of BY-2 cells in this work was never recognized.