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Lipopolysaccharides (LPS) are highly toxic compounds, even at a trace amount. When recombinant proteins are produced in E . coli , it is inevitable that LPS contaminates. However, LPS removal is still technically challenging and costly due to the high degree of solubility in a wide range of solvents. In this study, we explored the possibility of using the N-terminal region containing cysteine-rich, EGF-like, and sushi1–3 domains (CES3) of Factor C from the horseshoe crab Carcinoscorpius rotundicauda to develop a platform to remove LPS from recombinant proteins. We expressed CES3 as part of a recombinant protein, BiP:NT:CBM3:SUMO:CES3:His:HDEL, in Nicotiana benthamiana and found that purified or microcrystalline cellulose (MCC) bead-immobilised CES3 showed strong binding to LPS-containing E . coli . To produce CES3:CBM3 in an LPS-free environment, we generated Arabidopsis transgenic plants harbouring a recombinant gene, BiP:NT:SUMO:CES3:CBM3:HDEL , and found that transgenic plants mainly produce CES3:CBM3:His:HDEL, a truncated version of BiP:NT:SUMO:CES3:CBM3:HDEL via endogenous protease-mediated proteolytic processing in vivo. CES3:CBM3:HDEL purified from Arabidopsis plant extracts and immobilised onto MCC beads removed LPS contamination from protein samples. We propose that the CES3:CBM3 fusion protein produced in plants and immobilised on MCC beads can be a robust and easy platform for LPS removal from recombinant proteins.

Legume plants produce one-third of the total yield of primary crops and are important food sources for both humans and animals worldwide. Frequent exposure to abiotic stresses, such as drought, salt, and cold, greatly limits the production of legume crops. Several morphological, physiological, and molecular studies have been conducted to characterize the response and adaptation mechanism to abiotic stresses. The tolerant mechanisms of the model legume plant Medicago truncatula to abiotic stresses have been extensively studied. Although many potential genes and integrated networks underlying the M. truncatula in responding to abiotic stresses have been identified and described, a comprehensive summary of the tolerant mechanism is lacking. In this review, we provide a comprehensive summary of the adaptive mechanism by which M. truncatula responds to drought, salt, and cold stress. We also discuss future research that need to be explored to improve the abiotic tolerance of legume plants.

Many strategies have been developed to produce high levels of biologically active recombinant proteins in plants for biopharmaceutical purposes. However, the production of an active form of human iduronate-2-sulfatase (hIDS) for the treatment of Hunter syndrome by enzyme replacement therapy (ERT) is challenging due to the requirement for cotranslational modification by a formylglycine-producing enzyme encoded by sulfatase modifying factor 1 (hSUMF1) at the Cys84 residue, which converts it to C(alpha)-formylglycine. In this study, we have shown that hIDS can be highly expressed in N. benthamiana by using different constructs. Among them, BiP-GB1-L-dCBD1-2L-8xHis-L-6xHis-3L-EK-hIDS-HDEL ( GB1-CBD1-hIDS ) showed a high expression level when transiently co-expressed with the turnip crinkle virus gene silencing suppressor P38 and GB1-fused human calreticulin ( GB1-CRT1 ) as a folding enhancer. The hSUMF1 was co-expressed with hIDS for cotranslational modification. The full-length recombinant proteins were purified using Ni 2+ -NTA affinity resin followed by enterokinase treatment to obtain tag-free hIDS. The N-terminal fragment was removed using microcrystalline cellulose (MCC) beads. The purified active form of hIDS can successfully cleave the sulfate group from an artificial substrate, 4-nitrocatechol sulfate, at a similar level to commercial hIDS expressed in animal cells. These results suggest that plants could be a promising platform for the production of recombinant hIDS.

During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.

Subcellular trafficking is required for a multitude of functions in eukaryotic cells. It involves regulation of cargo sorting, vesicle formation, trafficking and fusion processes at multiple levels. Adaptor protein （AP） complexes are key regulators of cargo sorting into vesicles in yeast and mammals but their existence and function in plants have not been demonstrated. Here we report the identification of the protein-affected trafficking 4 （pat4） mutant defective in the putative 6 subunit of the AP-3 complex, pat4 and pat2, a mutant isolated from the same GFP imaging-based for- ward genetic screen that lacks a functional putative AP-3 β, as well as dominant negative AP-3 μ transgenic lines dis- play undistinguishable phenotypes characterized by largely normal morphology and development, but strong intra- cellular accumulation of membrane proteins in aberrant vacuolar structures. All mutants are defective in morphol- ogy and function of lytic and protein storage vacuoles （PSVs） but show normal sorting of reserve proteins to PSVs. Immunoprecipitation experiments and genetic studies revealed tight functional and physical associations of putative AP-3 β and AP-3 δ subunits. Furthermore, both proteins are closely linked with putative AP-3μvand σ subunits and several components of the clathrin and dynamin machineries. Taken together, these results demonstrate that AP complexes, similar to those in other eukaryotes, exist in plants, and that AP-3 plays a specific role in the regulation of biogenesis and function of vacuoles in plant ceils.

Key message Plant-produced SazCA and its application to CO 2 capture. Technologies that rely on chemical absorption or physical adsorption have been developed to capture CO 2 from industrial flue gases and sequester it at storage sites. Carbonic anhydrases (CAs), metalloenzymes, that catalyze the reversible hydration of CO 2 have recently received attention as biocatalysts in the capture of CO 2 from flue gases, but their cost presents a major obstacle for use at an industrial scale. This cost, however, can be reduced either by producing a long-lasting enzyme suitable for CO 2 capture or by lowering production costs. High-level expression, easy purification, and immobilization of CAs from Sulfurihydrogenibium azorense (SazCA) were investigated in a plant system. Fusion of the 60-amino acid-long ectodomain (M-domain) of the human receptor-type tyrosine-protein phosphatase C increased the levels of SazCA accumulation. Fusion of the cellulose-binding module (CBM3) from Clostridium thermocellum resulted in tight binding of recombinant protein to microcrystalline cellulose beads, enabling easy purification. The chimeric fusion protein, BMC-SazCA, which consisted of SazCA with the M and CBM3 domains, was expressed in tobacco ( Nicotiana benthamiana ), giving a recombinant protein yield in leaf extracts of 350 mg/kg fresh weight. BMC-SazCA produced in planta was active in the presence of various chemicals used in CO 2 capture. Immobilization of BMC-SazCA on the surface of microcrystalline cellulose beads extended its heat stability, allowing its reuse in multiple rounds of the CO 2 hydration reaction. These results suggest that production of SazCA in plants has great potential for CA-based CO 2 sequestration and mineralization.

The phytohormone abscisic acid (ABA) is crucial for plant growth and adaptive responses to various stress conditions. Plants continuously adjust the ABA level to meet physiological needs, but how ABA homeostasis occurs is not fully understood. This study provides evidence that UGT71B6, an ABA uridine diphosphate glucosyltransferase (UGT), and its two closely related homologs, UGT71B7 and UGT71B8, play crucial roles in ABA homeostasis and in adaptation to dehydration, osmotic stress, and high-salinity stresses in Arabidopsis (Arabidopsis thaliana). UGT RNA interference plants that had low levels of these three UGT transcripts displayed hypersensitivity to exogenous ABA and high-salt conditions during germination and exhibited a defect in plant growth. However, the ectopie expression of UGT71B6 in the atbgl (for β-glucosidase) mutant background aggravated the ABA-defìcient phenotype of atbgl mutant plants. In addition, modulation of the expression of the three UGTs affects the expression of CYP707A1 to CYP707A4, which encode ABA 8'-hydroxylases; four CYP707As were expressed at higher levels in the UGT RNA interference plants but at lower levels in the UGT71B6:GFP-overexpressing plants. Based on these data, this study proposes that UGT71B6 and its two homologs play a critical role in ABA homeostasis by converting active ABA to an inactive form (abscisic acid-glucose ester) depending on intrinsic cellular and environmental conditions in plants.

The production of recombinant proteins in plant systems is receiving wider attention. Indeed, various plant-produced pharmaceuticals have been shown to be biologically active. However, the production of human growth factors and cytokines in heterologous systems is still challenging because they often act as complex forms, such as homo- or hetero-dimers, and their production is tightly regulated in vivo . In this study, we demonstrated that the mature form of human TGFβ1 produced and purified from Nicotiana benthamiana shows biological activity in animal cells. To produce the mature form of TGFβ1, various recombinant genes containing the mature form of TGFβ1 were generated and produced in N . benthamiana . Of these, a recombinant construct, BiP:M:CBM3:LAP[C33S]:EK:TGF β 1 , was expressed at a high level in N . benthamiana . Recombinant proteins were one-step purified using cellulose-binding module 3 (CBM3) as an affinity tag and microcrystalline cellulose (MCC) beads as a matrix. The TGFβ1 recombinant protein bound on MCC beads was proteolytically processed with enterokinase to separate mature TGFβ1. The mature TGFβ1 still associated with Latency Associated Protein, [LAP(C33S)] that had been immobilized on MCC beads was released by HCl treatment. Purified TGFβ1 activated TGFβ1-mediated signaling in the A549 cell line, thereby inducing phosphorylation of SMAD-2, the expression of ZEB-2 and SNAIL1 , and the formation of a filopodia-like structure. Based on these results, we propose that active mature TGFβ1, one of the most challenging growth factors to produce in heterologous systems, can be produced from plants at a high degree of purity via a few steps.

Infection with human papillomavirus (HPV) can cause cervical cancers in women, and vaccination against the virus is one of most effective ways to prevent these cancers. Two vaccines made of virus-like particles (VLPs) of HPV L1 proteins are currently commercially available. However, these HPV vaccines are highly expensive, and thus not affordable for women living in developing countries. Therefore, great demand exists to produce a cost-effective vaccine. Here, we investigate the production of self-assembled HPV16 VLPs in plants. We generated a chimeric protein composed of N-terminal 79 amino acid residues of RbcS as a long-transit peptide to target chloroplasts, the SUMO domain, and HPV16 L1 proteins. The chimeric gene was expressed in plants with chloroplast-targeted bdSENP1, a protein that specifically recognizes the SUMO domain and cleaves its cleavage site. This co-expression of bdSENP1 led to the release of HPV16 L1 from the chimeric proteins without any extra amino acid residues. HPV16 L1 purified by heparin chromatography formed VLPs that mimicked native virions. Moreover, the plant-produced HPV16 L1 VLPs elicited strong immune responses in mice without adjuvants. Thus, we demonstrated the cost-effective production of HPV16 VLPs in plants.

Plants have recently received much attention as a means of producing recombinant proteins because they are easy to grow at a low cost and at a large scale. Although many plant protein expression systems have been developed, there remains a need for improved systems that deliver high yields of recombinant proteins. Transcription of the recombinant gene is a key step in increasing the yield of recombinant proteins. However, revealed strong promoters, terminators, and transcription factors that have been identified do not necessarily lead to high level production of recombinant proteins. Thus, in this study, a robust expression system was designed to produce high levels of recombinant protein consisting of a novel hybrid promoter, FM′M-UD, coupled with an artificial terminator, 3PRt. FM′M-UD contained fragments from three viral promoters (the promoters of Mirabilis mosaic caulimovirus (MMV) full-length transcript, the MMV subgenomic transcript, and figwort mosaic virus subgenomic transcript) and two types of cis -acting elements (four GAL4 binding sites and two zinc finger binding sites). The artificial terminator, 3PRt, consisted of the PINII and 35S terminators plus RB7, a matrix attachment region. The FM′M-UD promoter increased protein levels of reporters GFP, RBD : SD1 (part of S protein from SARS-CoV-2), and human interleukin-6 (hIL6) by 4–6-fold, 2-fold, and 6-fold, respectively, relative to those of the same reporters driven by the CaMV 35S promoter. Furthermore, when the FM′M-UD/3PRt expression cassette was expressed together with GAL4/TAC3d2, an artificial transcription factor that bound the GAL4 binding sites in FM′M-UD, levels of hIL6 increased by 10.7-fold, relative to those obtained from the CaMV 35S promoter plus the RD29B terminator. Thus, this novel expression system led to the production of a large amount of recombinant protein in plants.