Chiu-Yueh Hung

Overexpression of human erythropoietin in tobacco does not affect plant fertility or morphology.

Human erythropoietin (EPO) is a leading product in the biopharmaceutical market, but functional EPO has only been produced in mammalian cells, which limits its application and drives up the production costs. Using plants to produce human proteins may be an alternative way to reduce the cost. However, a recent report demonstrated that overexpression of the human EPO gene (EPO) in tobacco or Arabidopsis rendered males sterile and retarded vegetative growth, which raises concern whether EPO might interfere with hormone levels in transgenic plants. In the present study, we demonstrated that overexpressing EPO with additional 5′-His tag and 3′ ER-retention peptides in tobacco did not cause any developmental defect compared to GUS plants. With our method, all 20 transgenic plants grew on selective medium and, further confirmed by PCR, were fertile. Most of them grew similarly compared to GUS plants. Only one transgenic plant (EPO2) was shorter in plant height but had twice the life span compared to other transgenic plants. When 11 randomly selected EPO plants, along with the abnormal plant EPO2, were subjected to RT-PCR analysis, all of them had detectable EPO transcripts. However, their protein levels varied considerably; seven of them had detectable EPO proteins analyzed by western blot. Our results indicate that overexpressing human EPO protein in plants does not have detrimental effects on growth and development. Our transformation systems allow us to further explore the possibility of glycoengineering tobacco plants for producing functional EPO and its derivatives.

N -Glycosylation engineering of tobacco plants to produce asialoerythropoietin

Erythropoietin (EPO) is a glycoprotein hormone that displays both hematopoietic and tissue-protective functions by binding to two distinct receptors. Recombinant human EPO (rhuEPO) is widely used for the treatment of anemia, but its use for tissue protection is limited because of potentially harmful increases in red blood cell mass when higher doses of rhuEPO are used. Recent studies have shown that asialoerythropoietin (asialo-rhuEPO), a desialylated form of rhuEPO, lacks hematopoietic activity, but retains cytoprotective activity. Currently, a small amount of asialo-rhuEPO is produced by enzymatic desialylation of rhuEPO. The prohibitive cost of rhuEPO, however, is a major limitation of this method. Plants have the ability to synthesize complex N-glycans, but lack enzymatic activities to add sialic acid and β1,4-galactose to N-glycan chains. Plants could be genetically engineered to produce asialo-rhuEPO by introducing human β1,4-galactosyltransferase. The penultimate β1,4-linked galactose residues are important for in vivo biological activity. In this proof of concept study, we show that tobacco plants co-expressing human β1,4-galactosyltransferase and EPO genes accumulated asialo-rhuEPO. Purified asialo-rhuEPO binds to an Erythrina cristagalli lectin column, indicating that its N-glycan chains bear terminal β1,4-galactose residues and that the co-expressed GalT is functionally active. Asialo-rhuEPO interacted with the EPO receptor (EPOR) with similar affinity as rhuEPO, implying that it was properly folded. The strategy described here provides a straightforward way to produce asialo-rhuEPO for research and therapeutic purposes.Key messageN-glycosylation pathway in tobacco plants could be genetically engineered to produce a tissue-protective cytokine, asialoerythropoietin (a desialylated form of human hormone erythropoietin).

Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction

Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se0), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation

Summary: The primary metabolic pathway gene NtMTHFR negatively regulates the secondary metabolism pathway nicotine demethylation gene to potentially recycle methyl groups from alkaloids. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine, forming methionine, which is then used for the synthesis of S-adenosyl-methionine, a universal methyl donor for numerous methylation reactions, to produce primary and secondary metabolites. Here, we demonstrate that manipulating tobacco (Nicotiana tabacum) MTHFR gene (NtMTHFR1) expression dramatically alters the alkaloid profile in transgenic tobacco plants by negatively regulating the expression of a secondary metabolic pathway nicotine N-demethylase gene, CYP82E4. Quantitative real-time polymerase chain reaction and alkaloid analyses revealed that reducing NtMTHFR expression by RNA interference dramatically induced CYP82E4 expression, resulting in higher nicotine-to-nornicotine conversion rates. Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower nicotine-to-nornicotine conversion rates. However, the reduced expression of NtMTHFR did not affect the methionine and S-adenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine N-demethylation and suggests that the negative regulation of CYP82E4 expression may serve to recruit methyl groups from nicotine into the C1 pool under C1-deficient conditions.

Gibberellin deficiency is responsible for shy-flowering nature of Epipremnum aureum.

Epipremnum aureum is an extremely popular houseplant belonging to the Araceae family of angiosperms, but it does not flower either in the wild or under cultivation. We uncovered the potential causes of its shy-flowering nature by building the transcriptome using next-generation sequencing and identifying floral-related genes that are differentially expressed between vertical growth (VG, adult) and horizontal growth (HG, juvenile) plants. Transcripts of the gibberellin (GA) biosynthetic gene EaGA3ox1 and GA-responsive floral meristem identity gene EaLFY were absent in both VG and HG plants, suggesting that a deficiency of bioactive GAs may be responsible for its shy-flowering nature. This hypothesis is supported by undetectable or low levels of three bioactive GAs, and exogenous GA3 triggered flowering in both plants. Our study resolves the mystery why E. aureum fails to flower, and reveals the positive role of GAs in floral transition in perennials.

Two-step purification procedure for recombinant human asialoerythropoietin expressed in transgenic plants

Asialoerythropoietin (asialo-EPO) is a desialylated form of human glycoprotein hormone erythropoietin (EPO), which has been reported to be neuro-, cardio-, and renoprotective in animal models of organ injuries. Since the current method of production of asialo-EPO from mammalian cell-made recombinant human EPO (rhuEPO(M)) by enzymatic desialylation is not commercially viable, we and others used plant-based expression systems to produce recombinant human asialo-EPO (asialo-rhuEPO(P)). Despite achieving high expression levels in plants, its purification from plant extracts has remained a greater challenge, which has prevented studying its tissue-protective effects and translating it into clinical practice. In this study, a procedure was developed to purify asialo-rhuEPO(P) from transgenic tobacco leaf tissues in two steps: ion-exchange chromatography based on its high pI (8.75) to separate it from acidic plant proteins, and immunoaffinity chromatography to obtain pure asialo-rhuEPO(P). Using this process, up to 31% of the asialo-rhuEPO(P) could be recovered to near homogeneity from plant extracts. This work demonstrates that asialo-rhuEPO(P) expressed in tobacco plants could be purified in high yield and purity using minimal steps, which might be suitable for scale-up. Furthermore, the ion-exchange chromatography step together with the use of protein-specific antibody column could be used to purify a wide variety of basic recombinant proteins from transgenic leaf tissues.

Plant-Produced Asialo-Erythropoietin Restores Pancreatic Beta-Cell Function by Suppressing Mammalian Sterile-20-like Kinase (MST1) and Caspase-3 Activation

Pancreatic beta-cell death adversely contributes to the progression of both type I and II diabetes by undermining beta-cell mass and subsequently diminishing endogenous insulin production. Therapeutics to impede or even reverse the apoptosis and dysfunction of beta-cells are urgently needed. Asialo-rhuEPO, an enzymatically desialylated form of recombinant human erythropoietin (rhuEPO), has been shown to have cardioprotective and neuroprotective functions but with no adverse effects like that of sialylated rhuEPO. Heretofore, the anti-apoptotic effect of asialo-rhuEPO on pancreatic beta-cells has not been reported. In the current study, we investigated the cytoprotective properties of plant-produced asialo-rhuEPO (asialo-rhuEPOP) against staurosporine-induced cell death in the pancreatic beta-cell line RIN-m5F. Our results showed that 60 IU/ml asialo-rhuEPOP provided 41% cytoprotection while 60 IU/ml rhuEPO yielded no effect. Western blotting results showed that asialo-rhuEPOP treatment inhibited both MST1 and caspase-3 activation with the retention of PDX1 and insulin levels close to untreated control cells. Our study provides the first evidence indicating that asialo-rhuEPOP-mediated protection involves the reduction of MST1 activation, which is considered a key mediator of apoptotic signaling in beta-cells. Considering the many advantages its plant-based expression, asialo-rhuEPOP could be potentially developed as a novel and inexpensive agent to treat or prevent diabetes after further performing studies in cell-based and animal models of diabetes.

C-Terminally fused affinity Strep-tag II is removed by proteolysis from recombinant human erythropoietin expressed in transgenic tobacco plants

Key messageC-terminally fusedStrep-tag II is removed from rhuEPO expressed in tobacco plants. The finding suggests that direct fusion of purification tags at theC-terminus of rhuEPO should be avoided.AbstractAsialo-erythropoietin (asialo-EPO), a desialylated form of EPO, is a potent tissue-protective agent. Recently, we and others have exploited a low-cost plant-based expression system to produce recombinant human asialo-EPO (asialo-rhuEPOP). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPOP was efficiently expressed in transgenic tobacco plants, affinity purification based on Strep-tag II did not result in the recovery of the protein. In this study, we investigated the stability of Strep-tag II tagged asialo-rhuEPOP expressed in tobacco plants to understand whether this fused tag is cleaved or inaccessible. Sequencing RT-PCR products confirmed that fused DNA sequences encoding Strep-tag II were properly transcribed, and three-dimensional protein structure model revealed that the tag must be fully accessible. However, Western blot analysis of leaf extracts and purified asialo-rhuEPOP revealed that the Strep-tag II was absent on the protein. Additionally, no peptide fragment containing Strep-tag II was identified in the LC–MS/MS analysis of purified protein further supporting that the affinity tag was absent on asialo-rhuEPOP. However, Strep-tag II was detected on asialo-rhuEPOP that was retained in the endoplasmic reticulum, suggesting that the Strep-tag II is removed during protein secretion or extraction. These findings together with recent reports that C-terminally fused Strep-tag II or IgG Fc domain are also removed from EPO in tobacco plants, suggest that its C-terminus may be highly susceptible to proteolysis in tobacco plants. Therefore, direct fusion of purification tags at the C-terminus of EPO should be avoided while expressing it in tobacco plants.

Accumulation of high OPDA level correlates with reduced ROS and elevated GSH benefiting white cell survival in variegated leaves

Variegated ‘Marble Queen’ (Epipremnum aureum) plant has white (VMW) and green (VMG) sectors within the same leaf. The white sector cells containing undifferentiated chloroplasts are viable, but the underlying mechanism for their survival and whether these white cells would use any metabolites as signal molecules to communicate with the nucleus for maintaining their viability remain unclear. We analyzed and compared phytohormone levels with their precursors produced in chloroplasts between VMW and VMG, and further compared their transcriptomes to understand the consequences related to the observed elevated 12-oxo phytodienoic acid (OPDA), which was 9-fold higher in VMW than VMG. Transcriptomic study showed that a large group of OPDA-responsive genes (ORGs) were differentially expressed in VMW, including stress-related transcription factors and genes for reactive oxygen species (ROS) scavengers, DNA replication and repair, and protein chaperones. Induced expression of these ORGs could be verified in OPDA-treated green plants. Reduced level of ROS and higher levels of glutathione in VMW were further confirmed. Our results suggest that elevated OPDA or its related compounds are recruited by white cells as a signaling molecule(s) to up-regulate stress and scavenging activity related genes that leads to reduced ROS levels and provides survival advantages to the white cells.

Differential expression of a novel gene EaF82a in green and yellow sectors of variegated Epipremnum aureum leaves is related to uneven distribution of auxin

EaF82, a gene identified in previous studies of the variegated plant Epipremnum aureum, exhibited a unique expression pattern with greater transcript abundance in yellow sectors than green sectors of variegated leaves, but lower abundance in regenerated pale yellow plants than in green plants derived from leaf tissue culture. Studies of its full-length cDNA and promoter region revealed two members with only the EaF82a expressed. Immunoblotting confirmed that EaF82a encodes a 12 kDa protein and its accumulation consistent with its gene expression patterns in different color tissues. Transient expression of EaF82a-sGFP fusion proteins in protoplasts showed that EaF82a seems to be present in the cytosol as unidentified spots. Sequence motif search reveals a potential auxin responsive element in promoter region. Using transgenic Arabidopsis seedlings carrying EaF82a promoter driving the bacterial uidA (GUS) gene, an increased GUS activity was observed when IAA (indole-3-acetic acid) concentration was elevated. In E. aureum, EaF82a is more abundant at the site where axillary buds emerge and at the lower side of bending nodes where more IAA accumulates relative to the upper side. The measurement of endogenous IAA levels in different color tissues revealed the same pattern of IAA distribution as that of EaF82a expression, further supporting that EaF82a is an IAA responsive gene. EaF82a expression in etiolated transgenic Arabidopsis seedlings responded to IAA under the influence of light suggesting a microenvironment of uneven light condition affects the EaF82a transcript levels and protein accumulation in variegated leaves.