Antisense suppression of NtAQP1 in tobacco lowered the level of expression of several PIP1 homologues and resulted in a significant decrease in protoplast membrane water permeability, reduced root hydraulic conductivity and decreased transpiration. The results of heterologous expression in Xenopus oocytes suggest that, in addition to functioning as a water channel, NtAQP1 is also a membrane CO2 pore that facilitates the transport of CO2 across membranes. The movement of CO2 between the substomatal cavities and the sites of carboxylation within chloroplasts, through plasma and chloroplast membranes, is generally termed leaf mesophyll conductance. The ability of NtAQP1 and its Arabidopsis homolog AtPIP1,2 to function as CO2 membrane transport facilitators has been demonstrated in in vivo experiments. Increased expression of NtAQP1 in tobacco plants enhanced CO2 incorporation and stomatal conductance; whereas antisense suppression of NtAQP1 had the opposite effect. In other studies, over expression of AtPIP1,2 or NtAQP1 in tobacco plants significantly enhanced the rates of growth, transpiration and photosynthesis; whereas antisense suppression of NtAQP1 in tobacco plants and T-DNA insertion Arabidopsis mutants in AtPIP1,2 reduced gm and led to lower rates of photosynthesis. Unlike NtAQP1, over expression of Arabidopsis hexokinase in Arabidopsis and tomato plants decreased photosynthesis, transpiration and growth. AtHXK1 is a sugar sensing enzyme that monitors glucose levels, hydroponics growing system most likely in mesophyll cells of photosynthetic tissues.
When glucose levels are sufficiently high, this enzyme inhibits the expression of photosynthetic genes, decreases chlorophyll levels and reduces the rate of photosynthesis. In addition, AtHXK1 also stimulates stomatal closure and decreases transpiration in response to increasing sugar levels. In light of the opposite effects of AtHXK1 and NtAQP1 on photosynthesis and growth, we examined the relationship between AtHXK1 and NtAQP1 using double transgenic plants that express AtHXK1 and NtAQP1 simultaneously. We found that NtAQP1 significantly compensated for the growth inhibition imposed by AtHXK1, primarily by enhancing mesophyll CO2 conductance and the rate of photosynthesis, while the hydraulic conductivity in those plants remained unchanged.The hydraulic conductance of the tomato root system was assessed using plants grown hydroponically and was determined by measuring the flow induced in response to 1 bar of applied pressure. De-topped root systems were fitted with a plastic tube filled with deionized water and connected to a beaker located on a balance . The root system was sealed in a chamber containing the hydroponic solution in which the plants had been grown. The pressure in the chamber was regulated using a needle valve, which was adjusted to allow a small leak into the chamber, so that the air used to pressurize the chamber also served to aerate the medium. Water flow through the root system was automatically recorded by a computer at 30 s intervals. At the end of each experiment, the roots were dried in an oven for 72 h at 90uC and the dry weight of the root system was then measured.Whole-plant transpiration rates and relative daily transpiration were determined using lysimeters, as described in detail by Sade et al.. WT, AQP1, HK4, AQP1xHK4 and grafted plants were planted in 3.9-L pots and grown under controlled conditions. Each pot was placed on a temperature-compensated load cell with digital output and was sealed to prevent evaporation from the surface of the growth medium. A wet, vertical wick made of 0.14 m2 cotton fibers partially submerged in a 1-L water tank was placed on a similar load cell and used as a reference for the temporal variations in the potential transpiration rate. The output of the load cells was monitored every 10 s and the average readings over 3-min intervals were logged in a data logger for further analysis.
The whole-plant transpiration rate was calculated as a numerical derivative of the load cell output following a data smoothing process.Over expression of NtAQP1 in tobacco plants enhanced leaf mesophyll CO2 conductance , hydraulic conductivity, stomatal conductance , transpiration and photosynthesis. Expression of NtAQP1 in tomato plants also enhanced photosynthesis, stomatal conductance and transpiration. However, in our study, NtAQP1 did not enhance photosynthesis, stomatal conductance or hydraulic conductivity relative to WT plants and enhanced transpiration only slightly . These differences may be due to the different tomato genotype used in our study or to different expression levels of NtAQP1. Nevertheless, photosynthesis, stomatal conductance and transpiration were elevated by NtAQP1 in the double-transgenic plants , as compared to the HK4 parental line. Yet, the hydraulic conductivity of AQP1xHK4 remained low as in the HK4 plants, implying that the increased transpiration that was observed is not directly related to hydraulic characteristics. Rather, the increased transpiration is most likely due to high gm values in the mesophyll, which opens stomata and increases the influx of CO2 to help maintain constant levels of Ci in the sub-stomatal cavity. High levels of AN, gs and gm, accompanied by constant Ci , were also reported in previous studies of tobacco plants over expressing NtAQP1. AtHXK1 is a sugar-sensing enzyme that inhibits the expression of photosynthetic genes, decreases chlorophyll levels and reduces the rate of photosynthesis in response to increasing sugar levels. As a result, tomato and Arabidopsis plants with high levels of AtHXK1 expression display severe growth inhibition directly correlated to AtHXK1 expression and activity levels. It is likely that part of the growth inhibition imposed by AtHXK1 is the result of insufficient photosynthesis, since the increased photosynthesis rate observed in AQP1xHK4 plants partially eliminated this growth inhibition. The increased rate of photosynthesis observed in AQP1xHK4 plants, despite the low level of expression of the photosynthetic gene CAB1 in those plants, can probably be attributed to NtAQP1, which accelerates CO2 mesophyll conductance.
The CO2 mesophyll conductance of HK4 plants is significantly lower than that of WT plants and is enhanced by simultaneous expression of NtAQP1, indicating that CO2 mesophyll conductance significantly affects growth. It appears that, in addition to its known sugar-sensing effect , AtHXK1 also reduces gm, perhaps by reducing the expression of TRAMP , the tomato homolog of NtAQP1. Indeed, lower gm levels have been observed in tobacco NtAQP1 antisense lines and Arabidopsis pip1;2 mutants . In those studies, the decrease in gm was accompanied by lower Cc. In agreement with the findings of those studies, the HK4 plants in our study exhibited lower Cc than the WT plants and the expression of NtAQP1in the double-transgenic plants led to full complementation of Cc . Interestingly, the HK4 plants had lower electron transport rates than the WT plants, while a clear recovery was observed in the AQP1xHK4 plants despite the low level of expression of the photosynthetic gene CAB1 in the AQP1xHK4 plants . It has previously been shown that expression level of NtAQP1 which affects gm levels also affects electron transport rates. Flexas et al. hypothesized that modified intercellular CO2 concentrations may trigger differences in the leaf photosynthetic capacity, so that the photosynthetic machinery can adjust to the change in mesophyll conductance. This would also explain why gm usually scales with photosynthetic capacity, as has been observed in broad comparisons of different species. The effect of AtHXK1 on gm suggests that HXK might coordinate photosynthesis with sugar levels by several mechanisms in different cell types. It inhibits expression of photosynthetic genes and reduces gm most likely in mesophyll photosynthetic cells. In guard cells HXK mediates stomatal closure in response to sugars and reduces stomatal conductance. These findings support the existence of a multilevel feedback-inhibition mechanism that is mediated by HXK in response to sugars. When sugar levels are high,hydroponic grow systems likely when the rate of photosynthesis exceeds the rate at which the sugar is loaded and carried by the phloem, the surplus of sugar is sensed by HXK in mesophyll and guard cells, which respond in concert to reduce both unnecessary investments in photosynthetic capacity and water loss. This response includes reducing the expression of photosynthetic genes, slowing chlorophyll production, diminishing mesophyll CO2 conductance and closing the stomata. In addition to these effects in shoots, HXK reduces the hydraulic conductivity of stem and roots via an as yet unknown mechanism. This reduction in hydraulic conductivity occurs independently of stomatal conductance, as it also happens in the double-transgenic plants that have WT levels of stomatal conductance . Nevertheless, grafting experiments indicate that neither over expression of AtHXK1 in roots nor expression of AtHXK1 in the stem has any visible physiological effects. Rather, over expression of AtHXK1 in shoots is necessary and sufficient to obtain a photosynthesis effect and growth inhibition. The dominant effect of AtHXK1, lowering hydraulic conductance in AQP1xHK4, might be the reason for the intermediate transpiration rate of AQP1xHK4 plants, which is lower than that of WT plants , despite the increase in stomatal conductance to levels similar to that of WT plants . It has been suggested that NtAQP1 might play independent roles in leaves and roots, a hydraulic role in roots and a membrane CO2 permeability role in shoots. The improved gm observed in the double-transgenic plants supports the notion that, in leaves, NtAQP1 functions as a CO2 transmembrane facilitator and that the complementation effect of NtAQP1 may be primarily attributed to its affect on CO2 conductance in leaf mesophyll. The roles of HXK and PIP1 in the regulation of photosynthesis, stomatal conductance and transpiration are well established. This study suggests that HXK and PIP1 together may influence these central properties of plant physiology and, eventually, plant growth.
Monoclonal antibodies represent the fastest growing class of therapeutics and have been especially beneficial in the treatment of cancer. Since the approval of the first anti-cancer monoclonal antibody in 1986, several innovations have improved the potency of monoclonal antibodies used in immunotherapies that offer increased drug efficiency and/or lower drug dosage for a specific treatment. Among them, glycan engineering of the oligosaccharides attached to Asn297 of the Fc region of the heavy chain has been shown to affect antibody-dependent cell-mediated cytotoxicity , complement dependent cytotoxicity , and binding to the neonatal Fc receptor, FcRn. specific oligosaccharides influence the affinity of the antibody Fc domain to Fc receptor present on effector cells resulting in altered biological functions. For example, the removal of terminal galactose residues on mammalian cell-derived antibodies lowered C1q binding, while ADCC activity is almost completely dependent on the presence or absence of fucose residues bound to the glycosylation core. Several approaches have been employed to manufacture a monoclonal antibody with a decreased or absent core fucosylation. One strategy is to use cell lines or organisms with modified glycosylation pathways. The alteration of the expression of key enzymes in the host glycosylation pathway such as the mammalian α1,6-fucosyltransferase, the plant α1,3-fucosyltransferase, the GDP -mannose 4,6-dehydratase, or the β1,4-N-Acetylglucosaminyltransferase III led to afucosylated antibodies with improved anti-tumor activity. This led to the approval of mogamulizumab and obinutuzumab in 2012 and 2013, respectively, both produced in glycoengineered mammalian cell lines. Another approach to alter the antibody glycosylation profile is to modify the culture conditions of the host cells by adjusting the growth environment or supplementing the media with inhibitors of enzymes in the glycosylation pathway such as N-butyldeoxynojirimycin , mannostatin A, swainsonine, or kifunensine. Kifunensine from the actinomycete Kitasatosporia kifunense 9482 inhibits class I α-mannosidases and blocks N-glycan synthesis at the Man8GlcNAc2 or Man9GlcNAc2 stage before the core fucose is added. In mammalian cell culture, kifunensine was successfully employed to produce protein with >90% high-mannose content. This effect was similar across many different proteins including antibodies, suggesting that this simple treatment could be applied broadly. Compared to other α-mannosidase I inhibitors, kifunensine is highly effective on mammalian cell culture without significantly affecting cell growth or protein yield, even at concentrations as low as 100 ng/mL culture. Similar to mammalian cell studies, kifunensine was used in conjunction with the Nicotiana benthamiana transient protein expression systems to produce proteins with >98% afucosylated high-mannose glycans. In plants, the non-human α1,3-fucose and β1,2-xylose residues are commonly added in the Golgi apparatus after mannose trimming by mannosidases in the endoplasmic reticulum. Upon kifunensine treatment, addition of α1,3-fucose and β1,2-xylose residues were not observed on the Man3 to Man9 structures. However, the amount of kifunensine used in these studies was at or above 1.16 µg/mL, which significantly increases production costs at the manufacturing scale. Kifunensine is currently being used to manufacture a recombinant glucocerebrosidase in HT1080 fibrosarcoma cells to treat type 1 Gaucher disease.