Similar results were observed in LUX ARRHYTHMO mutants in diploid wheat, another component of the evening complex . These results suggest that in the temperate cereals, the evening complex of the circadian clock acts as a transcriptional repressor of PPD1 . Interestingly, two LUX-binding sites are present in the PPD1 promoter, including one in the region deleted in the Ppd-A1a allele. In barley, changes in photoperiod have been shown to have rapid effects on the expression of circadian clock genes . However, we did not observe significant changes in the expression profiles ofany of the core circadian clock genes after 21 NBs, suggesting that changes in the clock played a limited role in the induction of flowering by NBs . Moreover, the strong delay in heading time observed in the Kronos and Paragon ppd1-null mutants under NBmax demonstrated that PPD1 is the major driver of the acceleration of heading time by NBs. This does not rule out the possibility that the circadian clock may play an important role in the regulation of the intermediate steps between PPD1 and FT1 induction or in the PPD1-independent photoperiod pathway in the temperate grasses.In this study, we show that while a single NB as short as 15 min in duration is sufficient to induce PPD1, the peak of expression is not observed until 3 h after the NB . This timeline of events suggests that additional molecular steps may be involved in the transcriptional activation of PPD1 following the initial short exposure to white light. NB responses have previously been shown to be rapid, hydroponic equipment and red-light NBs of 2 min were shown to be sufficient to accelerate flowering . The short length of the light pulse required to trigger the NB response is consistent with a role of the phytochromes in the initial steps of the NB response. In Arabidopsis, conversion of phytochromes from Pfr to Pr forms occurs within 5 min of exposure to high radiance R light, and 2 min of R light treatment is sufficient to initiate the phosphorylation of PIFs, which are direct targets of activated phytochromes .
Phosphorylated PIFs are targeted for degradation by the 26S proteasome, triggering downstream transcriptional responses within 15 min of the light signals . The time lag between the light application and the up-regulation of PPD1 transcript levels suggests the existence of intermediate molecular steps. Based on the involvement of wheat PHYB and PHYC in the light activation of PPD1 transcription and the known interactions between phytochromes and PIFs in Arabidopsis, we hypothesize that the degradation of one or more PIFs acting as PPD1 transcriptional repressors may be involved in the light activation of this gene. A putative PIF binding site is present within the region of the PPD1 promoter that is deleted in the Ppd-A1a allele . According to this hypothesis, the application of FR after NB reduces Pfr levels and limits the degradation of this putative PIF, thereby maintaining some transcriptional repression of PPD1 . Although NBs do not perfectly mimic the LD response, there are several similarities between the two processes, particularly in the PPD1-dependent photoperiodic response. Both processes are dependent on the PHYB/PHYC-mediated light activation of PPD1, both processes require multiple inductive cycles to accelerate flowering, and in both NBs and in plants carrying the Ppd-A1a allele, expression of PPD1 during the night is associated with accelerated flowering. Based on these similarities and on previous studies, we propose a tentative working model for the PPD1-dependent photoperiodic regulation of flowering in wheat . According to this model, flowering is accelerated only when the light-induced expression of PPD1 coincides with the expression and/or activity of one or more circadian-regulated factor required for the induction of FT1. Under LD, but not under SD, PPD1 expression coincides with the putative additional factor, inducing FT1 expression .
When NBs are applied in the middle of the night, light-induced PPD1 expression coincides with a peak of the putative additional factor, resulting in maximal activation of FT1 and early flowering . Although NBs applied earlier or later than this point still result in the induction of PPD1, these NBs no longer coincide with a peak of the putative circadian-regulated factor required for the activation of FT1. In Arabidopsis, the sensitivity of the flowering response to the induction of FT expression is most effective when FT is artificially induced during the evening and early night , suggesting that the timing of FT induction can also carry information relevant to the acceleration of flowering. Other studies support the hypothesis that the timing of PPD1 induction is critical for flowering. In wheat plants carrying the Ppd-A1a allele conferring reduced sensitivity to photoperiod, PPD1 is expressed during darkness . Therefore, even in non-inductive SD photoperiods, PPD1 expression coincides with the peak activity of the putative circadian-regulated factor required for the activation of FT1 and the induction of flowering . This last result suggests that no light stimuli are required to induce FT1 and flowering when PPD1 is mis-expressed during the night. However, in both the phyB-null and phyC-null mutants, the relatively high transcript levels of PPD-A1a were insufficient to induce FT1. A possible explanation for this observation is that PHYB and PHYC are important for some of the intermediate molecular steps required for the FT1 up-regulation by PPD1.The putative additional factor required for FT1 induction is likely to be regulated by the circadian clock, with its expression or activity peaking between 6 and 10 h after dusk under a SD photoperiod of 16 h of darkness. This putative factor could function to stabilize or activate the PPD1 protein or be an additional factor that acts either in a complex with PPD1 or downstream of PPD1 to activate FT1. Alternatively, PPD1 may activate a protein that degrades a repressor of FT1 or induce epigenetic changes in FT1 or other intermediate genes.
The identification of this clock-regulated putative factor involved in the PPD1 activation of FT1 is an outstanding question of the PPD1-mediated photoperiodic response in wheat.Plant-based pharmaceutical production is appealing given its inexpensive facility and production cost, linear scale-up, the absence of animal pathogens, and capability to produce complex proteins and perform post-translational modifications, which overcomes one or more drawbacks of traditional recombinant protein expression systems such as animal cell culture and bacterial fermentation.Much work has been carried out using stably transformed plants, but the significantly reduced development and production timeline makes transient expression of proteins in whole plants a particularly attractive option, cutting the time to bring critical medications to the market during a pandemic. Vacuum agroinfiltration is the most widely used method for uniformly introducing agrobacterium harboring an expression cassette containing a gene of interest into plant tissue given its natural ability to transfer T-DNA into plant cells, which is ideal for transient protein production in plants. Although a plant-based recombinant protein production system provides distinct advantages over traditional systems, the differences between N-glycosylation of proteins produced in plants and humans could limit the use of plant systems for the production of glycoprotein-based pharmaceuticals. In higher eukaryotes, the initial steps of N-glycosylation processing are well conserved between plants and human, resulting in oligomannose-type N-glycosylation. However, late N-glycosylation maturation in the Golgi apparatus is kingdom-specific, and thus results in different N-glycosylation on proteins produced in plants compared with human. These plant-specific glycans may lead to potential safety issues such as hypersensitivity or allergy, as plant-specific α-fucose and β-xylose are known to be important IgE binding determinants of plant allergens. Thus, if these plant-specific glycans are present in an injected pharmaceutical product, the glycoprotein could trigger immunological response, or at least, result in a short circulation half-life. N-glycans of proteins produced from mammals are often terminated in β-galactose and sialic acid; sialic acid is particularly important as it typically increases the circulation half-life of proteins. There are a number of ways to avoid incorporating plant-specific glycans in the product such as adding a signal sequence at the C-terminus of the target protein to retain it within ER, or RNAi-mediated knock-down of α-fucose and β-xylose. These methods require modification to either the protein sequence or to the expression system, which can potentially affect protein structure and require a long developmental time. As an alternative, the use of small molecule inhibitors of intracellular glycosidases is a highly flexible bio-processing approach for controlling protein N-glycosylation patterns in transient agroinfiltration processes, and it is the approach investigated in this study. Here, we report an easy and fast way to modify N-glycosylation of recombinant proteins produced transiently in N. benthamiana through the addition of kifunensine in the agrobacterium suspension prior to vacuum agroinfiltration,vertical grow table which avoids modification to protein sequence or expression system while producing recombinant protein with oligomannose-type N-glycans that are similar between plant and human. Oligomannose-type N-glycan is preferred for the HIV-1 viral vaccine development as a vast majority of broad and potent neutralizing antibody responses during HIV-1 infection target mannose-glycan-dependent epitopes. In addition, monoclonal antibodies with oligomannose N-glycans show increased ADCC activity and affinity for FcγRIIIA. Protein N-glycosylation starts in the endoplasmic reticulum , where N-glycan precursors Glc3Man9GlcNAc2 are first synthesized, followed by the removal of terminal Glc residues, resulting in Man9GlcNAc2 structures. Then, a single α1,2 linked mannose is removed by ER class I α-mannosidase, producing Man8GlcNAc2 structures. The trimming of α1-2 mannose residues continues with the action of Golgi class I α-mannosidases in cis-Golgi to give Man5 structures. Kifunensine is a highly selective inhibitor of class I α-mannosidases in both plants and animals, and it has been used in cell cultures to produce recombinant proteins with oligomannose-type N-glycans. Although the general effects of kifunensine and other alkaloid-like processing glycosidases inhibitors are well understood, for the most part this information comes from cell culture system studies. Meanwhile, the study of kifunensine on whole-plant transient protein expression through agroinfiltration is new.
There are only two published papers on whole-plant kifunensine treatment, where kifunensine was supplied hydroponically throughout the whole incubation period, which requires larger quantities of kifunensine, a more expensive hydroponic system and constant monitoring especially at large scale as compared to our method. In addition, it was also shown that hydroponic kifunensine treatment resulted in dramatic decrease of protein expression level which was not observed with our method. In this study, Fc-fused capillary morphogenesis gene-2 , an anthrax decoy protein, served as a model protein, which contains single N-glycosylation site within its Fc domain . CMG2-Fc is a potent anthrax decoy protein as shown previously, where the CMG2 domain binds to anthrax protective antigen and prevents the anthrax toxin from entering the cell. Meanwhile, the presence of Fc domain significantly increases the serum half-life, which prolongs therapeutic activity owing the slower renal clearance for larger sized molecules and interaction with the salvage neonatal Fc-receptor. CMG2-Fc thus can be used as potent anthrax therapeutic and prophylactic without frequent redosing. The expression levels of CMG2-Fc produced transiently in wild type N. benthamiana under kifunensine treated and untreated conditions were measured with a sandwich ELISA, and protein N-glycosylation profiles were evaluated with mass spectrometry for kifunensine treated and untreated conditions. The findings in this study can be applied for N-glycosylation modification of other plant recombinant proteins when oligomannose-type N-glycans lacking core fucose are preferred, without the need to modify protein sequence and/or subcellular targeting.The CMG2-Fc expression levels in crude leaf extract were quantified through a sandwich ELISA to confirm the expression of CMG2-Fc, and to evaluate the effect of kifunensine on protein expression. The ELISA relies on binding of CMG2-Fc through the Fc region to protein A coated on a 96-well plate. A secondary anti-Fc polyclonal antibody linked to a horseradish peroxidase enzyme binds to the CMG2-Fc allowing colorimetric detection. The potential interference of plant host cell proteins and nonspecific binding were determined to be negligible. Twenty wild-type 5–6-week old N. benthamiana plants were divided equally into experimental and control groups, agro-infiltrated and incubated for 6 days, then whole leaves were extracted under identical conditions to determine protein expression. Kifunensine at a concentration of 5.4 µM was included in the agrobacterium suspension in the Kifunensine group. This kifunensine concentration was chosen as a starting point by taking the average of concentrations used in a previous CHO cell culture study, as no reference concentration is available for vacuum infiltration of kifunensine.