A Rapid Alkalinization Factor-like Peptide EaF82 Impairs Tapetum Degeneration during Pollen Development through Induced ATP Deficiency.

In plants, the timely degeneration of tapetal cells is essential for providing nutrients and other substances to support pollen development. Rapid alkalinization factors (RALFs) are small, cysteine-rich peptides known to be involved in various aspects of plant development and growth, as well as defense against biotic and abiotic stresses. However, the functions of most of them remain unknown, while no RALF has been reported to involve tapetum degeneration. In this study, we demonstrated that a novel cysteine-rich peptide, EaF82, isolated from shy-flowering 'Golden Pothos' (Epipremnum aureum) plants, is a RALF-like peptide and displays alkalinizing activity. Its heterologous expression in Arabidopsis delayed tapetum degeneration and reduced pollen production and seed yields. RNAseq, RT-qPCR, and biochemical analyses showed that overexpression of EaF82 downregulated a group of genes involved in pH changes, cell wall modifications, tapetum degeneration, and pollen maturation, as well as seven endogenous Arabidopsis RALF genes, and decreased proteasome activity and ATP levels. Yeast two-hybrid screening identified AKIN10, a subunit of energy-sensing SnRK1 kinase, as its interacting partner. Our study reveals a possible regulatory role for RALF peptide in tapetum degeneration and suggests that EaF82 action may be mediated through AKIN10 leading to the alteration of transcriptome and energy metabolism, thereby causing ATP deficiency and impairing pollen development.

Asialo-rhuEPO as a Potential Neuroprotectant for Ischemic Stroke Treatment.

Neuroprotective drugs to protect the brain against cerebral ischemia and reperfusion (I/R) injury are urgently needed. Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been demonstrated to have excellent neuroprotective functions in preclinical studies, but its neuroprotective properties could not be consistently translated in clinical trials. The clinical failure of rhuEPOM was thought to be mainly due to its erythropoietic activity-associated side effects. To exploit its tissue-protective property, various EPO derivatives with tissue-protective function only have been developed. Among them, asialo-rhuEPO, lacking terminal sialic acid residues, was shown to be neuroprotective but non-erythropoietic. Asialo-rhuEPO can be prepared by enzymatic removal of sialic acid residues from rhuEPOM (asialo-rhuEPOE) or by expressing human EPO gene in glycoengineered transgenic plants (asialo-rhuEPOP). Both types of asialo-rhuEPO, like rhuEPOM, displayed excellent neuroprotective effects by regulating multiple cellular pathways in cerebral I/R animal models. In this review, we describe the structure and properties of EPO and asialo-rhuEPO, summarize the progress on neuroprotective studies of asialo-rhuEPO and rhuEPOM, discuss potential reasons for the clinical failure of rhuEPOM with acute ischemic stroke patients, and advocate future studies needed to develop asialo-rhuEPO as a multimodal neuroprotectant for ischemic stroke treatment.

A plant-based mutant huntingtin model-driven discovery of impaired expression of GTPCH and DHFR.

Pathophysiology associated with Huntington's disease (HD) has been studied extensively in various cell and animal models since the 1993 discovery of the mutant huntingtin (mHtt) with abnormally expanded polyglutamine (polyQ) tracts as the causative factor. However, the sequence of early pathophysiological events leading to HD still remains elusive. To gain new insights into the early polyQ-induced pathogenic events, we expressed Htt exon1 (Httex1) with a normal (21), or an extended (42 or 63) number of polyQ in tobacco plants. Here, we show that transgenic plants accumulated Httex1 proteins with corresponding polyQ tracts, and mHttex1 induced protein aggregation and affected plant growth, especially root and root hair development, in a polyQ length-dependent manner. Quantitative proteomic analysis of young roots from severely affected Httex1Q63 and unaffected Httex1Q21 plants showed that the most reduced protein by polyQ63 is a GTP cyclohydrolase I (GTPCH) along with many of its related one-carbon (C1) metabolic pathway enzymes. GTPCH is a key enzyme involved in folate biosynthesis in plants and tetrahydrobiopterin (BH4) biosynthesis in mammals. Validating studies in 4-week-old R6/2 HD mice expressing a mHttex1 showed reduced levels of GTPCH and dihydrofolate reductase (DHFR, a key folate utilization/alternate BH4 biosynthesis enzyme), and impaired C1 and BH4 metabolism. Our findings from mHttex1 plants and mice reveal impaired expressions of GTPCH and DHFR and may contribute to a better understanding of mHtt-altered C1 and BH4 metabolism, and their roles in the pathogenesis of HD.

A Novel Plant-Produced Asialo-rhuEPO Protects Brain from Ischemic Damage Without Erythropoietic Action.

Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been shown to be a multimodal neuroprotectant targeting an array of key pathological mechanisms in experimental stroke models. However, the rhuEPOM clinical trials were terminated due to increased risk of thrombosis, largely ascribed to its erythropoietic function. We recently took advantage of a plant-based expression system lacking sialylation capacity to produce asialo-rhuEPOP, a rhuEPO derivative without sialic acid residues. In the present study, we proved that asialo-rhuEPOP is non-erythropoietic by repeated intravenous injection (44 µg/kg bw) in mice showing no increase in hemoglobin levels and red blood cell counts, and confirmed that it is non-immunogenic by measuring humoral response after immunizing the mice. We demonstrate that it is neuroprotective in a cerebral ischemia and reperfusion (I/R) mouse model, exhibiting ~ 50% reduction in cerebral infarct volume and edema, and significant improvement in neurological deficits and histopathological outcome. Our studies further revealed that asialo-rhuEPOP, like rhuEPOM, displays pleiotropic neuroprotective effects, including restoring I/R-interrupted mitochondrial fission and fusion proteins, preventing I/R injury-induced increase in mitophagy and autophagy markers, and inhibiting apoptosis to benefit nerve cell survival. Most importantly, asialo-rhuEPOP lacking erythropoietic activity and immunogenicity holds great translational potential as a multimodal neuroprotectant for stroke treatment.

Rethinking the necessity of low glucose intervention for cerebral ischemia/reperfusion injury.

Glucose is the essential and almost exclusive metabolic fuel for the brain. Ischemic stroke caused by a blockage in one or more cerebral arteries quickly leads to a lack of regional cerebral blood supply resulting in severe glucose deprivation with subsequent induction of cellular homeostasis disturbance and eventual neuronal death. To make up ischemia-mediated adenosine 5'-triphosphate depletion, glucose in the ischemic penumbra area rapidly enters anaerobic metabolism to produce glycolytic adenosine 5'-triphosphate for cell survival. It appears that an increase in glucose in the ischemic brain would exert favorable effects. This notion is supported by in vitro studies, but generally denied by most in vivo studies. Clinical studies to manage increased blood glucose levels after stroke also failed to show any benefits or even brought out harmful effects while elevated admission blood glucose concentrations frequently correlated with poor outcomes. Surprisingly, strict glycaemic control in clinical practice also failed to yield any beneficial outcome. These controversial results from glucose management studies during the past three decades remain a challenging question of whether glucose intervention is needed for ischemic stroke care. This review provides a brief overview of the roles of cerebral glucose under normal and ischemic conditions and the results of managing glucose levels in non-diabetic patients. Moreover, the relationship between blood glucose and cerebral glucose during the ischemia/reperfusion processes and the potential benefits of low glucose supplements for non-diabetic patients are discussed.

Transformation of Long-Lived Albino Epipremnum aureum 'Golden Pothos' and Restoring Chloroplast Development.

Chloroplasts are organelles responsible for chlorophyll biosynthesis, photosynthesis, and biosynthesis of many metabolites, which are one of key targets for crop improvement. Elucidating and engineering genes involved in chloroplast development are important approaches for studying chloroplast functions as well as developing new crops. In this study, we report a long-lived albino mutant derived from a popular ornamental plant Epipremnum aureum 'Golden Pothos' which could be used as a model for analyzing the function of genes involved in chloroplast development and generating colorful plants. Albino mutant plants were isolated from regenerated populations of variegated 'Golden Pothos' whose albino phenotype was previously found to be due to impaired expression of EaZIP, encoding Mg-protoporphyrin IX monomethyl ester cyclase. Using petioles of the mutant plants as explants with a traceable sGFP gene, an efficient transformation system was developed. Expressing Arabidopsis CHL27 (a homolog of EaZIP) but not EaZIP in albino plants restored green color and chloroplast development. Interestingly, in addition to the occurrence of plants with solid green color, plants with variegated leaves and pale-yellow leaves were also obtained in the regenerated populations. Nevertheless, our study shows that these long-lived albino plants along with the established efficient transformation system could be used for creating colorful ornamental plants. This system could also potentially be used for investigating physiological processes associated with chlorophyll levels and chloroplast development as well as certain biological activities, which are difficult to achieve using green plants.

Quantitative Proteomics Reveals the Beneficial Effects of Low Glucose on Neuronal Cell Survival in an in vitro Ischemic Penumbral Model.

Understanding proteomic changes in the ischemic penumbra are crucial to rescue those salvageable cells and reduce the damage of an ischemic stroke. Since the penumbra region is dynamic with heterogeneous cells/tissues, tissue sampling from animal models of stroke for the molecular study is a challenge. In this study, cultured hippocampal HT22 cells under hypoxia treatment for 17.5 h with 0.69 mM low glucose (H+LG) could mimic ischemic penumbral cells since they had much higher cell viability and viable cell number compared to hypoxia without glucose (H-G) treatment. To validate established cell-based ischemic penumbral model and understand the beneficial effects of low glucose (LG), quantitative proteomics analysis was performed on H+LG, H-G, and normoxia with normal 22 mM glucose (N+G) treated cells. We identified 427 differentially abundant proteins (DAPs) between H-G and N+G and further identified 105 DAPs between H+LG and H-G. Analysis of 105 DAPs revealed that LG promotes cell survival by activating HIF1α to enhance glycolysis; preventing the dysregulations of extracellular matrix remodeling, cell cycle and division, and antioxidant and detoxification; as well as attenuating inflammatory reaction response, protein synthesis and neurotransmission activity. Our results demonstrated that this established cell-based system could mimic penumbral conditions and can be used for molecular studies.

Glycoengineering tobacco plants to stably express recombinant human erythropoietin with different N-glycan profiles.

Plant-based expression system has many potential advantages to produce biopharmaceuticals, but plants cannot be directly used to express human glycoproteins because of their differences in glycosylation abilities from mammals. To exploit plant-based expression system for producing recombinant human erythropoietin (rhuEPO), we glycoengineered tobacco plants by stably introducing seven to eight mammalian genes including a target human EPO into tobacco in order to generate capacities for ß1,4-galactosylation, bisecting N-acetylglucosamine (GlcNAc) and sialylation. Wild type human ß1,4-galactosyltransferase gene (GalT) or a chimeric GalT gene (ST/GalT) was co-expressed to produce rhuEPO bearing ß1,4-galactose-extended N-glycan chains as well as compare their ß1,4-galactosylation efficiencies. Five mammalian genes encoding enzymes/transporter for sialic acid biosynthesis, transport and transfer were co-expressed to build sialylation capacity in plants. The human MGAT3 was co-expressed to produce N-glycan chains with bisecting GlcNAc. Our results demonstrated that the above transgenes were incorporated into tobacco genome and transcribed. ST/GalT was found to be more efficient than GalT for ß1,4-galactosylation. Furthermore, co-expressing MGAT3 generated N-glycans likely bearing bisected GlcNAc. However, our current efforts did not result in generating sialylation capacity. Created transgenic plants expressing EPO and ST/GalT could be used to produce rhuEPO with high proportion of ß1,4-galactose-extended N-glycan chains for tissue protective purposes.

Recombinant asialoerythropoetin protects HL-1 cardiomyocytes from injury via suppression of Mst1 activation.

BACKGROUND: Recombinant human erythropoietin (rhuEPO) and asialoerythropoietin (asialo-rhuEPO) are cardioprotective. However, the protective effects of rhuEPO could not be translated into clinical practice because of its hematopoiesis-associated side effects while non-erythropoietic asialo-rhuEPO is unavailable in large quantities for clinical studies. This study was designed to investigate the cardiomyocyte protective potential of plant-produced asialo-rhuEPO (asialo-rhuEPOP) against staurosporine (STS)-induced injury in HL-1 murine cardiomyocytes and identify cellular pathway(s) responsible for its cardioprotection. METHODS: HL-1 cardiomyocytes were simultaneously treated with STS and asialo-rhuEPOP. Cellular injury, apoptosis, and cell viabilities were measured by LDH assay, Hoechst staining and trypan blue exclusion method, respectively while western blotting was used to study its effects on apoptosis and autophagy hallmarks. RESULTS: Our results showed that 20 IU/ml asialo-rhuEPOP provided 39% protection to cardiomyocytes compared to STS-treated cells, which is 2-fold better than that of mammalian cell-produce rhuEPO (rhuEPOM). Asialo-rhuEPOP was found to suppress activation of proapoptotic kinase Mst1 (mammalian Sterile-20-like kinase 1) and FOXO3, leading to inhibition of apoptotic pathway and restoration of autophagy as indicated by the reduction of fragmented/condensed nuclei, altered ratios of Bax/Bcl2, p-Bad/Bad, cytosol/mitochondrial cyt c and caspase-3 activation, and the restored levels of autophagy markers Beclin1, p62 and LC3B-II. Additionally, Akt was found to be activated and FOXO3 was phosphorylated on Ser253, suggesting inhibition of FOXO3 transcriptional function. CONCLUSIONS: Asialo-rhuEPOP-mediated cardioprotection occurs through activation of PI3K/Akt pathway leading to suppression of Mst1 activation and promoting cardiomyocyte survival. GENERAL SIGNIFICANCE: Asialo-rhuEPOP could be used to modulate Mst1 activity elevated under numerous pathological states.

An Effective Way of Producing Fully Assembled Antibody in Transgenic Tobacco Plants by Linking Heavy and Light Chains via a Self-Cleaving 2A Peptide.

Therapeutic monoclonal antibodies (mAbs) have evolved into an important class of effective medicine in treatment of various diseases. Since the antibody molecule consists of two identical heavy chains (HC) and two light chains (LC), each chain encoded by two different genes, their expressions at similar levels are critical for efficient assembly of functional recombinant mAbs. Although the plant-based expression system has been tested to produce fully assembled recombinant mAbs, coordinately expressing HC and LC at similar levels in a transgenic plant remains a challenge. A sequence coding for a foot-and-mouth disease virus (FMDV) 2A peptide has been successfully used to link two or more genes, which enable the translated polyprotein to be "self-cleaved" into individual protein in various genetically modified organisms. In the present study, we exploited the usage of F2A in Ebola virus monoclonal antibody (EBOV mAb) production. We found that transgenic tobacco plants carrying a transcription unit containing HC and LC linked by 2A not only produced similar levels of HC and LC but also rendered a higher yield of fully assembled EBOV mAb compared to those expressing HC and LC in two independent transcription units. Purified EBOV mAb bound to an Ebola epitope peptide with apparent Kd -values of 90.13-149.2 nM, indicating its proper assembly and high affinity binding to Ebola epitope peptide. To our knowledge, this is the first report showing mAb production by overexpressing a single transcription unit consisting of HC, LC and 2A in stable transformed tobacco plants.

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.

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.

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.

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

KEY MESSAGE: C -terminally fused Strep -tag II is removed from rhuEPO expressed in tobacco plants. The finding suggests that direct fusion of purification tags at the C -terminus of rhuEPO should be avoided. Asialo-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-rhuEPO(P)). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPO(P) 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-rhuEPO(P) 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-rhuEPO(P) 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-rhuEPO(P). However, Strep-tag II was detected on asialo-rhuEPO(P) 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.

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 (Se(0)), 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.

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.

Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M)) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and ß1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P)) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P) bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P) (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M) (21%). The cytoprotective effect of the asialo-rhuEPO(P) was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

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

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.