Fresh Leaf Material Research Articles

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans.

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana-based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLIGnGn and AFLISia, respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLIGnGn and AFLISia displayed similar binding potency to VEGF165, with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario.

Analysis of tea quality of large-leaf black tea with different harvesting tenderness based on metabolomics

The harvesting tenderness of tea fresh leaves has a significant effect on the flavor and aroma quality of black tea (BT), which is one of the important standards for BT grading. In this study, sensory evaluation, electronic tongue technique and metabolomics were performed to evaluate the flavor and aroma qualities of three different tenderness of large-leaf BTs that were manufactured from the fresh leaves of single-bud, one-bud and one-bud two-leaf teas, respectively. The results of the electronic tongue and sensory evaluation indicated that the increase in the tenderness of the fresh leaf material enhanced the sweetness, freshness and fruity flavor of the tea and reduced the bitterness of the tea. A total of 519 differential flavor components and 84 differential aroma components were identified by metabolomics. Among them, elevated levels of maltitol, cyclic leucine and l-methionine and l-aspartic acid led to elevated sweetness and freshness in more tender tea leaves. Reduced levels of 4-aminosalicylic acid, tyrosol, uracil, inosine, benzamide, and l-tyrosine led to reduced bitterness in BT. Moreover, the content of volatiles such as (Z)-2-Decenal, Phenylacetic acid propyl ester, Acetophenone, and 2-phenylethyl ester were increased, which contributed to the fruity aroma in the high-tenderness tea. This study provides a theoretical basis for grading the flavor quality of BT with different tenderness.

Investigating the phytotoxic potential of Verbesina encelioides: effect on growth and performance of co-occurring weed species.

Allelopathy has been proposed as an efficient mechanism of invasion by plant species via growth inhibition and suppression of the resident plant community. Verbesina encelioides (Cav.) Benth. & Hook. f. ex A. Gray (golden crownbeard; Asteraceae), a native of south-western USA and Mexican Plateau, is an emerging troublesome invasive weed species of north-western states of India. We investigated the allelopathic potential of the aqueous extracts prepared from the fresh foliage and leaf litter of V. encelioides on its co-occurring species, Amaranthus viridis and Senna occidentalis. Phytotoxicity bioassay showed concentration-dependent (control < 0.5% < 1% < 2% < 4% extract) inhibition of growth and photosynthetic parameters in the test plants. Both the extracts induced ~ 50% inhibition of germination compared to control at 4% concentration. The maximum synthesis effect (collective effect on seedling length and dry weight) was observed to be - 0.69 and - 0.62 in A. viridis and - 0.68 and - 0.57 in S. occidentalis for the fresh leaf and leaf litter extracts, respectively, at 4% concentration. Also, an antagonistic concentration-dependent impact was observed on the photosynthetic pigments (total chlorophyll and chlorophyll a content) and photosynthetic efficiency. The liquid chromatography-mass spectrometry assay of leaf extracts revealed the presence of 15 allelochemicals including phenolic acids, flavonoids, phytosterols, phytophenols, dicarboxylic acid, guanidine, and triterpenes. Of these, 14 compounds were present in both fresh and leaf litter materials. However, a guanidine derivative, galegine, was only found in the fresh leaf material of the plant. The findings support the novel weapon hypothesis and suggest that V. encelioides competitively excludes its neighboring plants by virtue of allelopathic interference.

From Plants to Ants: Fungal Modification of Leaf Lipids for Nutrition and Communication in the Leaf-Cutter Ant Fungal Garden Ecosystem.

ABSTRACTLipids are essential to all living organisms, as an energy source, as an important cellular structural component, and as a communication tool. In this study, we used global lipidomic methods to evaluate the lipids in leaf-cutter ant fungal gardens. Leaf-cutter ants and their coevolved fungal cultivar, Leucoagaricus gongylophorus, are a model mutualistic system. The fungus enzymatically digests fresh plant material that the ants cut and deliver, converting energy and nutrients from plants and providing them to the ants through specialized hyphal swellings called gongylidia. Using combined liquid chromatography, ion mobility spectrometry, and tandem mass spectrometry, we evaluated differences between the molecular species of lipids in the leaf-cutter ant fungal garden ecosystem. This lipidomic study characterized leaves that are fed to the gardens, gongylidia that are produced by the fungus to feed the ants, and spatially resolved regions of the fungal garden through stages of leaf degradation. Lipids containing alpha-linolenic acid (18:3) were enriched in leaves and the top of the gardens but not dominant in the middle or bottom regions. Gongylidia were dominated by lipids containing linoleic acid (18:2). To evaluate the communicative potential of the lipids in fungal gardens, we conducted a behavioral experiment that showed Atta leaf-cutter ants responded differently to 18:3 and 18:2 fatty acids, with aggression toward 18:3 and attraction for 18:2. This work demonstrates the role of lipids in both the transfer of energy and as an interkingdom communication tool in leaf-cutter ant fungal gardens.IMPORTANCE In this work, we examined the role of lipids in the mutualism between leaf-cutter ants and fungus. These ants cut fresh leaf material, which they provide to their fungal cultivar, that converts energy and nutrients from the plants and provides it to the ants in specialized hyphal swellings called gongylidia. This work constitutes the first example of a global lipidomics study of a symbiotic system and provides insights as to how the fungus modifies plant lipids into a usable source for the ants. Through a behavioral experiment, this work also demonstrates how lipids can be used as an interkingdom communication tool, in this case, as an attractant rather than as a repellant, which is more often seen.Author Video: An author video summary of this article is available.

DNA Profiling of Biotypes of Trichosanthes Tricuspidata; an Important Ethno medicinal Cucurbit Using RAPD, ISSR and Scot Molecular Marke

Trichosanthes tricuspidata Lour., is an important ethnomedicinal plant belonging to the Cucurbitaceae family. It is known by various vernacular names like Red ball snake gourd in English, Lal Indrayan in Hindi, Kalayar in Malayalam, and Avaduta in Telugu. There are several studies about the medicinal applications of Trichosanthes tricuspidata, but there is scanty information about molecular level identification of different biotypes of Trichosanthes tricuspidata. Hence, the present study was undertaken to develop a protocol for genetic analysis of three biotypes of Trichosanthes tricuspidata collected from wild areas of Warangal, Karimnagar and Adilabad districts of Telangana State, India by using Randomly Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR) and Start Codon Targeted (SCoT) primers-based PCR method. Genomic DNA was isolated from 1-gram fresh leaf material of three biotypes of T. tricuspidata. The primers OPA-2 of RAPD, S-5 of ISSR and S-8 of SCoT has shown good amplification with generation of monomorphic DNA bands ranging from 250 bp to 1000 bp by OPA-2 of RAPD primer; 500 bp to 1500 bp by S-5 of ISSR primer and 1450 bp by S-8 of SCoT primer. In conclusion, no genetic variation was detected in three biotypes, as the generated DNA bands from OPA-2 of RAPD, S-5 of ISSR and S-8 of SCoT primer-based amplifications were monomorphic. The developed protocol of genetic analysis of three biotypes of T. tricuspidata by RAPD, ISSR and SCoT primers could be used in near future for molecular level studies.

Using acetone for rapid PCR-amplifiable DNA extraction from recalcitrant woody plant taxa.

PremiseQuick and effective DNA extraction from plants for subsequent PCR amplification is sometimes challenging when working across diverse plant taxa that may contain a variety of inhibitory compounds. Time‐consuming methods may be needed to overcome these inhibitory effects as well as the effects of various preservation and collection methods to extract DNA from leaf samples. Our objective was to develop a rapid DNA extraction protocol that could be used with diverse plant taxa to produce high‐quality DNA suitable for downstream PCR applications.Methods and ResultsWe tested the efficacy of acetone in extracting DNA from fresh, frozen, oven‐dried, acetone‐fixed, and herbarium leaf material of 22 species from 16 woody and herbaceous plant families. An improved simplified DNA extraction protocol was developed using acetone‐fixed leaf material. The addition of 1% sodium dodecyl sulfate solution resulted in the optimal extraction from all tissue samples. The DNA resulting from the extraction protocol was readily amplified using real‐time PCR assays.ConclusionsThe protocol described here resulted in the extraction of DNA from recalcitrant plant species that was of sufficient quality and quantity for PCR amplification, as indicated by the low threshold cycle values from real‐time assays. This method is simple, fast, and cost‐effective, and is a reliable tool for extracting high‐quality DNA from plant material containing PCR inhibitors.

Selenium Biofortification Differentially Affects Sulfur Metabolism and Accumulation of Phytochemicals in Two Rocket Species (Eruca Sativa Mill. and Diplotaxis Tenuifolia) Grown in Hydroponics.

Biofortification can be exploited to enrich plants in selenium (Se), an essential micronutrient for humans. Selenium as selenate was supplied to two rocket species, Eruca sativa Mill. (salad rocket) and Diplotaxis tenuifolia (wild rocket), at 0–40 μM in hydroponics and its effects on the content and profile of sulphur (S)-compounds and other phytochemicals was evaluated. D. tenuifolia accumulated more total Se and selenocysteine than E. sativa, concentrating up to ~300 mg Se kg−1 dry weight from 10–40 μM Se. To ensure a safe and adequate Se intake, 30 and 4 g fresh leaf material from E. sativa grown with 5 and 10–20 μM Se, respectively or 4 g from D. tenuifolia supplied with 5 μM Se was estimated to be optimal for consumption. Selenium supplementation at or above 10 μM differentially affected S metabolism in the two species in terms of the transcription of genes involved in S assimilation and S-compound accumulation. Also, amino acid content decreased with Se in E. sativa but increased in D. tenuifolia and the amount of phenolics was more reduced in D. tenuifolia. In conclusion, selenate application in hydroponics allowed Se enrichment of rocket. Furthermore, Se at low concentration (5 μM) did not significantly affect accumulation of phytochemicals and plant defence S-metabolites.

Characterization and novel Est-SSR marker development of an important Chinese medicinal plant, Morinda officinalis How (Rubiaceae)

Morinda officinalis How (Rubiaceae) is well known as one of the four most popular medicinal plants in the southern region of China. In order to assess its genetic diversity and assist the development of its molecular assisted-selection breeding in China, we constructed the transcriptome database of M. officinalis using fresh leaf material and developed novel expressed sequence tag–simple sequence repeat (EST-SSR) markers for this important medicinal plant species. Our study identified 8064 potential EST-SSRs from 6668 unigenes. For perfect repeat motifs, the mononucleotide repeats were most abundant (4221; 52.34%), followed by the trinucleotide repeats (1588; 19.69%), and the dinucleotide ones (1040; 12.90%). The seven most commonly occurring repeat motifs were A/T (4661), AG/CT (588), AAG/CTT (408), AC/GT (353), ATC/ATG (338), AT/AT (296) and AGG/CCT (277). Of a total of 5605 SSR primer sets that were successfully designed de novo, 100 were selected for validation analysis. There, 24 primer sets were polymorphic across the 96 M. officinalis individuals collected from four different populations in Guangdong province. Based on cross-species transferability test, 19 and 22 primer sets produced successful amplification in M. umbellata subsp. obovata and M. parvifolia, respectively. These novel EST-SSR markers with high cross-species transferability between M. officinalis and its closely-related species could serve as aids to future studies in genetic diversity, conservation, assisted-selection breeding and genes associated with the medical uses of the genus.

Interactive touch-screen monitors facilitate collaborative learning of microscopy skills in an introductory-level plant biology lab

Microscopy skills are critical for many aspects of biology, yet they are notoriously difficult to teach, especially to large classes. We trialled the use of widescreen monitors with touch-screen capability connected to compound microscopes in an introductory-level plant biology lab. Working in teams, students used the screens to interpret, annotate and measure images of their sections taken through fresh leaf material. The lab progressed more rapidly than in previous years in which it was run as a traditional microscopy exercise; it was also far noisier and more congested. Students reported that they valued the size and quality of the images, and the opportunity to work collaboratively to identify the leaf’s structural features. All teams expressed a strong appreciation for the group discussions facilitated by the monitors, which presented a safe environment to bounce ideas off one another, and assisted their comprehension. The monitors were seen as an effective and time-efficient tool for collaborative learning of fundamental microscopy skills.

Novel expression of immunogenic foot-and-mouth disease virus-like particles in Nicotiana benthamiana

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals and is endemic in Africa, parts of South America and southern Asia. The causative agent, FMD virus (FMDV) is a member of the genus Aphthovirus, family Picornaviridae. Vaccines currently used against FMDV are chemically inactivated virus strains which are produced under high-level biocontainment facilities, thus raising their cost. The development of recombinant FMDV vaccines has focused predominantly on FMDV virus-like particle (VLP) subunit vaccines for which promising results have been achieved. These VLPs are attractive candidates because they avoid the use of live virus in production facilities, but conserve the complete repertoire of conformational epitopes of the virus. Recombinant FMDV VLPs are formed by the expression and assembly of the three structural proteins VP0, VP1 and VP3. This can be attained by co-expression of the three individual structural capsid proteins or by co-expression of the viral capsid precursor P1-2A together with the viral protease 3C. The latter proteolytically cleaves P1-2A into the respective structural proteins. These VLPS are produced in mammalian or insect cell culture systems, which are expensive and can be easily contaminated. Plants, such as Nicotiana benthamiana, potentially provide a more cost-effective and very highly scalable platform for recombinant protein and VLP production. In this study, P1-2A was transiently expressed in N. benthamiana alone, without the 3C protease. Surprisingly, there was efficient processing of the P1-2A polyprotein into its component structural proteins, and subsequent assembly into VLPs. The yield was ∼0.030μg per gram of fresh leaf material. Partially purified VLPs were preliminarily tested for immunogenicity in mice and shown to stimulate the production of FMDV-specific antibodies. This study, has important implications for simplifying the production and expression of potential vaccine candidates against FMDV in plants, in the absence of 3C expression.

Black Sparrowhawk Accipiter melanoleucus breeding behaviour and reproductive success in KwaZulu-Natal, South Africa

Black Sparrowhawk Accipiter melanoleucus breeding behaviour and nest success was determined using camera traps and field observations in KwaZulu-Natal, South Africa. Nest preparation was done predominantly by the male with incubation and brooding largely performed by the female. Brooding duration decreased with chick age, while feeding frequency and duration increased. Similarly, delivery of fresh leaf material to the nest site increased with chick age. Over the 2011 and 2012 breeding seasons respectively 74% and 41% of nests successfully fledged chicks, with the majority of these producing two offspring.

Nitrate analysis ofDiplotaxis tenuifolia: fresh versus dry material for meeting international standards and regulations

Nitrate fertilizers are the most commonly used nitrogen sources for growing vegetables. The use of nitrate fertilizers in vegetable production may result in its accumulation in the plants if uptake exceeds assimilation. High nitrate concentrations in leafy vegetables represent a possible threat to human health. Leafy vegetables are the highest contributor to total food nitrate intake as a result of their high specific concentration and the large amount consumed. Consequently, European Union regulations were established for nitrate concentrations in leafy vegetables such as lettuce, spinach and rucola ( Diplotaxis tenuifolia ). Therefore, the need for a reliable, easy, fast and universal method to meet these demands is essential. We reassessed nitrate determination by comparing fresh and dry leaf material extracts of rucola. Our results indicate that the leaf nitrate concentration with both measurement methods is similar. Consequently, the use of the dry leaf extracts method is feasible and worth recommending to the European Union as an official method for determining nitrate concentrations in leafy vegetables.

Feed protein value of acidic precipitates obtained from press juices of three types of green forage leaves

The present study investigated the potential of three types of green forage leaves for the extraction of a protein rich precipitate. Samples were taken during calendar weeks 19, 21 and 23 of 2012 from three grass rich pastures (GRP), two legume rich pastures (LRP) and one Trifolium pratense (TP) monoculture around the City of Freising (Germany) (n=9, n=6 and n=3 samples of GRP, LRP and TP). Trifolium pratense and LRP yielded the highest average amounts of precipitate per kg fresh leaf material. The highest amounts of crude protein (CP) within the precipitate were obtained from TP and GRP. Contents of essential and non-essential amino acids (AA) were highest within the precipitates of TP. The extraction efficiency as expressed by total precipitate yield, precipitate CP as well as the amounts of essential and non-essential AA declined in the course of plant development. The amounts of essential AA relative to total AA within the obtained precipitates were in most cases comparable or even higher than average amounts in soybean meal (480g CP/kg fresh matter). Taken together, we were able to extract a CP rich precipitate from green forage leafs. Products of that kind represent a valuable protein source and may replace soybean meal to some extent in practical livestock diets.

Isolation of Arabidopsis Leaf Peroxisomes and the Peroxisomal Membrane.

To date, less than 150 proteins have been located to plant peroxisomes, indicating that unbiased large-scale approaches such as experimental proteome research are required to uncover the remaining yet unknown metabolic functions of this organelle as well as its regulatory mechanisms and membrane proteins. For experimental proteome research, Arabidopsis thaliana is the model plant of choice and an isolation methodology that obtains peroxisomes of sufficient yield and high purity is vital for research on this organelle. However, organelle enrichment is more difficult from Arabidopsis when compared to other plant species and especially challenging for peroxisomes. Leaf peroxisomes from Arabidopsis are very fragile in aqueous solution and show pronounced physical interactions with chloroplasts and mitochondria in vivo that persist in vitro and decrease peroxisome purity. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes using two different types of density gradients (Percoll and sucrose) sequentially that yields approximately 120 μg of peroxisome proteins from 60 g of fresh leaf material. A method is also provided to assess the relative purity of the isolated peroxisomes by immunoblotting to allow selection of the purest peroxisome isolates. To enable the analysis of peroxisomal membrane proteins, an enrichment strategy using sodium carbonate treatment of isolated peroxisome membranes has been adapted to suit isolated leaf peroxisomes and is described here.

Responses of the two-spotted oak buprestid, Agrilus biguttatus (Coleoptera: Buprestidae), to host tree volatiles.

BACKGROUND Agrilus bigutattus (Fabricius) is a forest pest of increasing importance in the United Kingdom. The larvae damage weakened native oaks and are thought to contribute to premature tree death. Suspected links with acute oak decline (AOD) are not yet confirmed, but AOD‐predisposed trees appear to become more susceptible to A. biguttatus attack. Thus, management may be necessary for control of this insect. To explore the possibility of monitoring beetle populations by baited traps, the host tree volatiles regulating A. biguttatus–oak interactions were studied.RESULTSBiologically active volatile organic compounds in dynamic headspace extracts of oak foliage and bark were identified initially by coupled gas chromatography–electroantennography (GC‐EAG) and GC–mass spectrometry (GC‐MS), and the structures were confirmed by GC coinjection with authentic compounds. Of two synthetic blends of these compounds comprising the active leaf volatiles, the simpler one containing three components evoked strongly positive behavioural responses in four‐arm olfactometer tests with virgin females and males, although fresh leaf material was more efficient than the blend. The other blend, comprising a five‐component mixture made up of bark volatiles, proved to be as behaviourally active for gravid females as bark tissue.CONCLUSIONSThese initial results on A. biguttatus chemical ecology reveal aspects of the role of attractive tree volatiles in the host‐finding of beetles and underpin the development of semiochemically based surveillance strategies for this forest insect. © 2015 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector.

Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE(®) tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapatite resins. This procedure yields on average 400 mg of low-endotoxin CTBp from 1 kg of fresh leaf material.

Detritusphere effects on P availability and C mineralization in soil

SummaryThis paper investigates two possible processes in the detritusphere: the effect of various fresh plant materials on extractable phosphorus in soil and the effect of decomposing litter on mineralization of soil microbial residues. The objective was to quantify these two poorly‐studied processes. The work was done in a soil at two different fertility levels following 30 years of different fertilizer practice, as we have previously observed effects of fertilizer history on soil carbon dynamics. We incubated fresh leaf material from Lupinus, Raphanus and Rumex separated from the soil by a fine mesh. At 4, 7, 14 and 28 days after incubation, extractable phosphate was measured in 1‐mm soil layers at increasing distance from the litter. In order to establish a labelled microbial residue pool, the soil had been labelled with 14C‐glucose 3 weeks prior to incubation, and respiration was measured throughout the incubation. Extractable soil phosphorus (P) increased up to 3 mm from the litter layer, most in Rumex and least in Lupinus. Extractable soil P increased throughout the sampling period, corresponding to a maximum of 20% of the P lost from the plant litter layer. Measurement of residue‐derived organic acids in the soil showed no specific pattern. The study did not indicate any solubilization of soil P as the increase in soluble P can be fully accounted for by leakage from the litter. Mineralization of soil 14C in the form of microbial biomass and microbial residues was significantly accelerated by the addition of litter. The magnitude was correlated with decomposition of the plant litter, and corresponded to a maximum of 5% of the soil 14C. Because of the localized impact of litter on the detritusphere soil layer, the impact on soil 14C in this soil fraction was potentially even greater. In conclusion, the decomposition of litter had significant effects on soil carbon mineralization, although we could not demonstrate an effect of plant‐derived compounds on soil P availability, beyond the direct contribution of litter P.

Chemical Composition of Syzygium benthamianum (Wt. ex Duthie) Gamble Essential oil - An endemic and Vulnerable Tree Species

Fresh leaf material of Syzygium benthamianum a vulnerable tree species endemic to India, was steam distillation and analyzed by GC-MS for its chemical composition. The essential oil contained a total of 63 compounds. The major compounds obtained were sitosteryl acetate (11.83 %), stigmastan-3,5,22trien (7.0 %), 2,6-dimethyl-2-octene (6.99 %), estra-1,3,5(10)-trien-17.beta.-ol (6.3 %), ergosta-4,7,22-trien3.beta-ol (5.19 %), 1-methylcholest-1,3,5(10)-trien-3-ol (5.06 %) and a number of other minor compounds. This is the first report of essential oil chemical composition from leaf materials of S. benthamianum from India.

Rapid and Scalable Plant-based Production of a Cholera Toxin B Subunit Variant to Aid in Mass Vaccination against Cholera Outbreaks

IntroductionCholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants.Methodology/Principal FindingsIn a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB) was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro.Conclusions/SignificanceTaken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major biological activities of the original protein. This rapid and easily scalable system may enable the implementation of pCTB to mass vaccination against outbreaks, thereby providing better protection of high-risk populations in developing countries.

Expression of functionally active sialylated human erythropoietin in plants

Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO. cDNA constructs were delivered to Nicotiana benthamiana (N. benthamiana) and transiently expressed by a viral based expression system. Expression levels up to 85 mg rhEPO/kg fresh leaf material were achieved. Moreover, co-expression of rhEPO with six mammalian genes required for in planta protein sialylation resulted in the synthesis of rhEPO decorated mainly with bisialylated N-glycans (NaNa), the most abundant glycoform of circulating hEPO in patients with anemia. A newly established peptide tag (ELDKWA) fused to hEPO was particularly well-suited for purification of the recombinant hormone based on immunoaffinity. Subsequent lectin chromatography allowed enrichment of exclusively sialylated rhEPO. All plant-derived glycoforms exhibited high biological activity as determined by a cell-based receptor-binding assay. The generation of rhEPO carrying largely homogeneous glycosylation profiles (GnGnXF, GnGn, and NaNa) will facilitate further investigation of functionalities with potential implications for medical applications.