Ellagic acid (EA), which is widely distributed in many foods, has been found to possess inhibitory activity against xanthine oxidase (XO). However, there is ongoing debate about the difference in XO inhibitory activity between EA and allopurinol. Additionally, the inhibitory kinetics and mechanism of EA on XO are still unclear. Herein, the authors systematically studied the inhibitory effects of EA on XO. The authors’ findings showed that EA is a reversible inhibitor with mixed‐type inhibition, and its inhibitory activity is weaker than allopurinol. Fluorescence quenching experiments suggested that the generation of EA‐XO complex was exothermic and spontaneous. In silico analysis further confirmed that EA entered the XO catalytic centre. Furthermore, the authors verified the anti‐hyperuricemia effect of EA in vivo. This study elucidates the inhibition kinetics and mechanism of EA on XO, and lays a theoretical foundation for the further development of drugs and functional foods containing EA for the treatment of hyperuricemia. Inhibitory kinetics and mechanism of ellagic acid on xanthine oxidase were evaluated by using UV spectra, fluorescence spectra, molecular docking and the hyperuricemia mice model. With fo50 values reported. One study reported that EA had an IC50 (concentration required to inhibit 50% enzyme activity) value of 3.1 μmol/L, indicating weaker inhibitory activity than allopurinol (0.17 μmol/L)  is the concentration of EA. The curves , respectively. These values were all significantly larger than the maximum scattering collision quenching constant [2.0 × 1010 L/(mol·s)], indicating that a static quenching mechanism was involved in the interaction between EA and XO  and temperature (K) are represented by the symbols T and R, respectively. The calculated values of △H, △S and △G are summarised in Table G suggested that the binding process of EA and XO was spontaneous. Furthermore, the negative value of △H and the positive value of △S indicate that hydrogen bonding and hydrophobic interaction were the main driving forces in the formation of EA‐XO complex [Here, K complex .3.6The binding mode between EA and XO was analysed in silico, and the results are shown in Figure d = 3.0 Å), SER‐1082 (d = 1.9 Å), THR‐1083 (d = 2.1 Å), SER‐1080 (d = 2.2 Å), ALA‐1078 (d = 3.1 Å), and ALA‐1079 (d = 2.7 Å), as shown in Figure + analysis are shown in Figure Specifically, EA was inserted into the catalytic site by forming 6 hydrogen bonds (indicated by yellow dashed line) with some residues, including GLN‐1194 (3.7P < 0.001). Compared with MG, AG serum uric acid  decreased significantly (P < 0.001) to 2.01 ± 0.34 mg/dl, but there was no significant difference with CG (P > 0.05). These results demonstrated that the hyperuricemic mouse model was successfully established. After intragastric administration of EA , serum uric acid levels in mice decreased to 5.09 ± 0.57, 4.19 ± 0.53 and 2.87 ± 0.41 mg/dl, respectively, with significant differences compared to MG , indicating that EA does have the effect of lowering serum uric acid levels. Moreover, there were significant differences among different doses of EAGs (P < 0.05), indicating that the uric acid lowering effect of EA was dose‐dependent.The anti‐hyperuricemia effect of EA on mice was evaluated using the PO‐induced hyperuricemia mouse model. As shown in Figure 50, 22.97 ± 0.12) was much weaker than allopurinol . The results of in vivo experiments further clarified the confusion of XO inhibitory activity between EA and allopurinol. In addition, another reason may be the very poor bioavailability of EA due to its low solubility, limited permeability, and first pass effect [Compared to AG, the anti‐hyperuricemia effect of EA (even at 480 mg/kg) was significantly weaker than that of allopurinol (20 mg/kg). The main reason may be that EA's XO inhibitory activity (ICs effect . Howevers effect , which w450 of 22.97 ± 0.12 μmol/L, which is weaker than that of allopurinol. The mechanism of EA inhibition was explored through in vitro and silico studies, showing that EA formed a static complex with XO through hydrogen‐bond and hydrophobic interactions, hindering substrate entry and ultimately inhibiting enzyme catalytic activity. The animal study demonstrated that EA had an anti‐hyperuricemia effect in mice, although it was weaker than allopurinol, possibly due to its higher IC50 value and lower bioavailability. Nevertheless, EA's good drug safety profile suggests its potential as an active compound for treating hyperuricemia and gout. These findings may be useful for developing EA‐rich food nutrition and functional foods for lowering serum uric acid levels.This study demonstrated that EA is a reversible mixed XO inhibitor with an ICJianmin Chen designed the experiments and wrote the paper. Zemin He, Sijin Yu and Yanhua Lin conducted the experiments. Xiaozhen Cai and Danhong Zhu analysed the data. All authors read and approved the final manuscript.The authors declare that they have no conflict of interest.None.


Cryopreservation is a unique and practical method to facilitate extended access to biological materials. Because of this, cryopreservation of cells, tissues, and organs is essential to modern medical science, including cancer cell therapy, tissue engineering, transplantation, reproductive technologies, and bio-banking. Among diverse cryopreservation methods, significant focus has been placed on vitrification due to low cost and reduced protocol time. However, several factors, including the intracellular ice formation that is suppressed in the conventional cryopreservation method, restrict the achievement of this method. To enhance the viability and functionality of biological samples after storage, a large number of cryoprotocols and cryodevices have been developed and studied. Recently, new technologies have been investigated by considering the physical and thermodynamic aspects of cryopreservation in heat and mass transfer. In this review, we first present an overview of the physiochemical aspects of freezing in cryopreservation. Secondly, we present and catalog classical and novel approaches that seek to capitalize on these physicochemical effects. We conclude with the perspective that interdisciplinary studies provide pieces of the cryopreservation puzzle to achieve sustainability in the biospecimen supply chain. Cryopreservation is one of the approaches to preserve cells and tissues over long time periods. Because of this, cryopreservation of cells and tissues is essential to modern medical science, including cancer cell therapy, tissue engineering, transplantation, reproductive technologies, and bio-banking. For example, one such application, liver cell transplantation (LCT), treats diseases involving liver metabolism ,2,3,4. ICryopreservation often results in significantly reduced cell and tissue viability due to ice crystal formation. This reduced viability is attributed to several classes of damage, including osmotically induced volume changes , damaginChemical and biological materials, called cryoprotectant agents (CPAs), have been crucial in decreasing damage due to ice formation and excessive ion concentrations. However, a high concentration of CPAs harms cells and tissues because it leads to cellular osmotic and mechanical stress as the result of volume changes (shrinking and swelling) during the addition and removal of CPA. In addition, biophysical and biochemical interactions between CPA and cytoplasmic constituents cause toxic effects in a biological sample ,21,22,238 °C/min [One approach to avoid damage due to ice formation is to use ice-free cryopreservation, known as vitrification, to reduce the potential toxicity of high solute concentrations below 0 °C and avoid ice crystallization and growth. There is a non-crystalline state at extremely low temperatures in which cells and tissues are considered safe. This state is called a glassy or vitreous state, and the material forming in this state is called glass. A sufficiently concentrated solution can vitrify at any cooling rate , but the8 °C/min , not ach8 °C/min . However8 °C/min . Hence, 8 °C/min . TherefoTo achieve rapid cooling rates there have been two primary methods to reach vitrification: moving a cryogen over the sample (convection method) and using solid surface cooling (conduction method) . The conEach approach is associated with significant hurdles, including contamination due to direct contact between biospecimen and liquid nitrogen  and the In this review, we discuss the physical chemistry of cryopreservation and, specifically, vitrification. Further, we explore the effect of hydrostatic pressure in vitrification and on biological samples. Finally, a number of methods, technologies, and devices used in vitrification are presented, and results are summarized.Tm), it first passes into a region called the supercooled liquid state, as shown in As pure liquid water is cooled below its melting temperature  with a highly different probability. Homogeneous nucleation temperature (Th) is where homogeneous nucleation becomes increasingly probable [Tg), at which a glassy or vitreous state of non-crystalline material can form [Although liquid water can be prolonged in the supercooled state, homogeneous nucleation may occur at any temperature below melting temperature , the medium viscosity increases , facilitcroscope . On the croscope . To sum Tg) based on a sudden change in heat capacity using a differential scanning calorimeter (DSC) [In glasses, the values of thermodynamic response variables, such as the specific heat and coefficient of expansion, are closer to those of the crystalline form than to the liquid state  Figure . In conter (DSC) .Due to the influence of high hydrostatic pressure on reducing the melting temperature and increasing the glass transition temperature, the application of high hydrostatic pressure towards preserving biological systems commenced in the 1970s to reduce ice damage during freezing . This teUnder ambient pressure, water freezes in a hexagonal form known as Ice I . As showTg) increased linearly with pressure, but the slope decreased as solution concentration decreased and would be zero for pure water.To establish a theoretical foundation for further experimental designs, Miyata et al. investigated the vitrification of aqueous glycerol solutions under high pressures at low cooling rates (3 °C/min) . Miyata At atmospheric pressure, water is less viscous. Therefore, as the ice grows in the medium, additional water molecules are recruited to make larger ice crystals. However, under high pressure, water becomes much more viscous. Consequently, the growth rate of ice crystals is reduced by high pressure. At atmospheric pressure, freezing causes ice crystals with rough surfaces, resulting in mechanical injury to the membrane of cells and tissues . On the Tg due to the metastability of the solution with respect to ice formation. This nucleation forms a base for extensive ice crystal growth at higher solution temperatures. For example, the peak of the ice nucleation rate occurs around Tg, and ice nucleation even happens at temperatures lower than Tg. This is because in order to form ice nuclei, local replacement of molecules is all that is needed [Recrystallization is defined as the growth of ice crystals in the warming process . Ice nucs needed . Howevers needed  because s needed . Tiny ics needed ,64,65,66s needed ,64. For s needed  cryopress needed  reporteds needed  show thaTr) [Tr is recorded at higher temperatures. This Tr has practical implications: Bank [In some references, the temperature related to crystallization in the warming process is called recrystallization temperature (Tr) ,42,70. Ins: Bank  rapidly Tr is −50 °C for mouse embryos. Gilkey and Staehelin [Tr is reduced. For example, a CPA-free suspension of red blood cells has Tr of −30 °C, while Tr is −80 °C for this suspension with 30% glycerol [Rall et al.  mentionetaehelin  did not glycerol .Tg of water/CPA systems is around −140 °C, this is considered the maximal safe temperature for long-term storage of cryopreserved cells and tissues [Cryopreservation facilitates the nearly indefinite storage of cells and tissues in a state of biological arrest . The tem tissues ,76,77,78Generally, a cell and tissue “bank” is a collection of cryopreserved cells and tissues  that canDifferent approaches have improved the vitrification of precision-cut liver slices (PCLS). For example, de Kanter and Koster  showed tAnother approach for improving rapid freezing in PCLS was using a high thermal conductivity material such as aluminum plates in the freezing and thawing process . In thisThe cytoplasm tends to vitrify more readily than the extracellular medium because of the high concentration of intracellular proteins ,93. FurtSeveral sample-containing devices, or cryocarriers, designed to facilitate vitrification have been developed, including cryoloop, straw (and many variations thereof), Cryotip, Cryopette, VitriSafe, S3 μS-VTF, S3 system, Rapid-i ,97,98,99Pressure, similar to temperature, is a thermodynamic parameter that can affect any thermodynamic system, such as biomolecular media. In nature, the range of pressure that can be tolerated by living species ranges from 0.1 MPa (~1 atm) as an atmospheric pressure to 110 MPa (~1100 atm) at the deepest point of Mariana Trench at a depth of 11 km in the western Pacific Ocean .−10 Pa−1 [According to the principle of Le Chatelier, if an equilibrated system is exposed to an alteration, the equilibrium is adjusted to object to the alteration . For exa−10 Pa−1 . TherefoThe use of pressure as an additional control variable for vitrification is not without its own hazards. In the literature, the effects of high hydrostatic pressure on biological samples are divided into two categories: “physiological high hydrostatic pressure” (pHHP), which is between 1 and 100 MPa, and “non-physiological high hydrostatic pressure” (HHP), which is above 100 MPa ,149. MarThe induction of an external shock, such as heat and pressure, was suggested as a mechanism for initiating stress induction . When ceWhen pressure greater than 100 MPa is applied, changes in protein structure and even protein denaturation ,171,172,Moving to higher pressures, Frey et al.  found thCryoprotective agents (CPAs) are defined as chemicals that mitigate or eliminate ice damage and reduce high ionic concentrations during cryopreservation . As evidHowever, concentration-dependent CPA toxicity has been identified as a limitation in cryopreservation . EspeciaCPA toxicity has been frequently reported in the field of cryobiology. One of the toxicities related to CPAs is cell membrane toxicity. At the outer and inner surfaces of cell membrane bilayers, there are hydrophilic polar head groups, while in the middle of the cell membrane, there are hydrophobic fatty acid chains. Some factors can increase the permeability of CPAs, such as lipophilicity. On the other hand, increasing molecular size and hydrogen bond formation can reduce a CPA’s permeability . For exaWarner et al.  investigTo sum up, many factors play a role in the CPA toxicity of biospecimens, requiring specific cryoprotocol to reduce cytotoxicity based on cryopreserved biospecimen, CPA type, and freezing method.The growth of ice crystals can cause severe damage to cells and tissues. Thus, to address this issue, the use of ultra-rapid cooling is needed when low CPA concentrations are used. Ultra-rapid freezing is a cryopreservation technique much faster than slow freezing and even conventional vitrification methods in cryobiology. Slow freezing methods often require expensive programmable freezers with complicated protocols to cryopreserve each type of cell and tissue. On the other hand, the success of any fast-freezing approach is greatly dependent on the CPA concentration, which is generally associated with toxicity in cells and tissues. In short, vitrification approaches will trade the challenge of managing subzero ice formation and water transport via cooling rates in slow or equilibrium protocols for the challenge of managing CPA toxicity and mechanical stresses at above-zero temperatures in vitrification protocols.Cooling rates are generally limited by conductive heat transfer in tissues greater than 1 mm thick. A number of methods and devices have been presented to maximize heat transfer, and these methods fall into three categories used for rapid freezing: conventional rapid freezing, ultra-rapid freezing, and high-pressure freezing ,36.In the conventional rapid freezing method, a biological sample is immersed directly in a cryogen, such as liquid nitrogen, with a boiling point of −196 °C. In this method, the heat loss from the sample creates an insulating film of evaporated nitrogen around the sample, called the Leidenfrost effect. Therefore, heat loss and cooling rate are reduced [Concerning ultra-rapid freezing technology, there are four methods: plunging, spraying, jetting, and metal block .Plunge freezing commenced in the 1950′s in the application of electron microscopy . Among tDue to a number of improvements, this approach has a higher cooling rate than conventional rapid freezing; nonetheless, it faces significant challenges in freezing thick biospecimens, such as 1 mm thick tissue.−12 L), they achieved a maximum cooling rate of 2.2 × 106 °C/min, and they confirmed the vitrification of droplets by spectroscopy and a high-speed camera. Although a high cooling rate is achievable with this method for CPA-free cryopreservation, this system is limited in scope to cells in suspension, and as such cannot be used for tissue slices or other small tissues.The spray freezing method is the same as plunging, but a sample is reduced to small droplets in order to increase the surface-to-volume ratio of contact area . In this5 °C/min for a plastic cryovial containing 4 mL 15% glycerol. However, this cooling rate was not measured directly and, as such, may be an overestimation of the cooling rate. This approach may have other problems, such as not eliminating the Leidenfrost effect, completely.In the jetting method, liquid cryogen is shot toward a sample at a high velocity ,243,244.6 °C/min, but only for the surface . HoweveThere have been many designs, modifications, developments, and alterations based on the idea of a cold metal block ,271,272.In high-pressure freezing technology, in one method, a sample is preserved by high-pressure jetting liquid nitrogen ,276,277.A summary of the freezing techniques, samples tested, corresponding advantages, disadvantages, and achievements is given in This review investigates the physiochemical concepts involved in cryobiology, various cryopreserved biospecimens, and devices designed to improve biospecimens’ viability after thawing. Up to now, a few manuscripts have had an interdisciplinary viewpoint of all factors playing a role in cryopreservation. Here we study several significant parameters influencing the vitrification technology from different aspects.Recently, vitrification has been considered widely in stem cells, cell therapy, reproductive technology, and transplantation. Many protocols, methods, devices, and technologies have been used and tested to improve vitrification. However, there are impediments to vitrification technology, which can be solved by considering all disciplines involved in cryopreservation, such as physical chemistry, biomathematics, engineering, and biology.CPA toxicity is a significant concern that should be tackled through an interdisciplinary approach, including physical chemistry techniques such as differential scanning calorimetry (DSC) and mechanical and mathematical modeling of transport and toxicity ,210. It The idea of “non-harmful” ice crystals in cryobiology can enhance cell and tissue cryopreservation . This idThe warming process is another concern in cryobiology. Cryosolutions with high CPA concentrations, due to the high amount of vitrification in the solution during cooling, ice recrystallization is a significant obstacle to cell and tissue viability in the warming process. Under these circumstances, a high warming rate is required to suppress devitrification and large ice crystal formation . In factTo sum up, there are several limitations for the vitrification method in practical applications, such as low biospecimen volume, contamination, small tissue thickness, complicated techniques, and devices that need to be addressed through interdisciplinary investigations. There is no doubt that cryopreserved specimens will be highly demanded in the future, particularly in cancer cell therapy for cancer and on-shelf organ cryopreservation for transplantation .In this review, we presented some basic and interdisciplinary notions applicable to cryopreservation and a diverse cryopreserved biospecimen with different methods and devices, showing various results due to the type of cells and tissues with different cooling rates and cryoprotocols. Hence, it is essential to pay more attention to fundamental interdisciplinary concepts of cryobiology to build a bridge between all current gaps in the literature and achieve sustainability in this realm of technology.


Microbial communities and nutrient dynamics in glaciers and ice sheets continuously change as the hydrological conditions within and on the ice change. Glaciers and ice sheets can be considered bioreactors as microbiomes transform nutrients that enter these icy systems and alter the meltwater chemistry. Global warming is increasing meltwater discharge, affecting nutrient and cell export, and altering proglacial systems. In this review, we integrate the current understanding of glacial hydrology, microbial activity, and nutrient and carbon dynamics to highlight their interdependence and variability on daily and seasonal time scales, as well as their impact on proglacial environments. Although the movement of glaciers and ice sheets profoundly affects the landscape, the most immediate protagonist of glacial influences is glacial meltwater . Meltwat−1 [22 cells y−1 from Greenland alone [22 cells y−1 are expected from its surface under a medium emission scenario in the next 80 years [Ice sheets and glaciers occupy 10% of the Earth’s surface and represent about 68% of Earth’s freshwater . Their r−1 . Meltwat−1 ,28. Came80 years . This ex80 years ,31,32,33Although several recent reviews of the microbiomics of glacial systems are available ,34,35, tGlacial hydrological systems consist of interconnected micro- and macro-pathways, their size and location being influenced by the glacial thermal regime  and wateBox 1 Warm atmospheric temperatures and high precipitation rates lead to temperate ice , where snow deposition can isolate surface ice from constant subzero temperatures. On the contrary, surface cold ice  is the result of permanent cold temperatures . Whereas Ice temperature influences glacier and ice sheet functioning, where small changes in ice temperature can deeply change the glacier dynamics ,43. For  Contrary to temperate glaciers, water only flows through crevasses, moulins, englacial channels, and conduits (secondary permeability) in cold glaciers ,46. WherThe presence of water in glaciers is highly seasonal, as the generation of glacial water depends on the energy available to melt snow and ice. Water input also comes from rain events. For most glaciers in the temperate and subpolar regions, mass accumulates during the accumulation season , which is characterized by colder temperatures and snow accumulation . By compIn spring, the snowpack temperature increases through heat conduction from warm air and via refreezing of meltwater that percolates into the cold snow . Once thThe main pathways from the glacier surface and firn aquifers to the glacial interior are moulins and crevasses. Moulins constitute a direct connection to an englacial system of conduits, whereas crevasses drain water via a network of fractures thought to connect to englacial conduits ,70,71. TMost subglacial water originates from the glacier surface, although a small flux may originate englacially and subglacially due to frictional dissipation of heat generated by flowing water, ice deformation, or from geothermal heat at the bottom of the glacier ,73,74. TDuring summers with plenty of meltwater, surface streams flow over the ablation zone, and the weathering crust, including cryoconite holes, is full of water  Figure . SurfaceAt summer’s end and the beginning of winter’s accumulation season, much less water is present in the glacial system due to the low air temperatures and reduced solar radiation, which largely eliminates surface melt, and while precipitation falls mostly as snow . Some waThe residence time of water in various parts of the glacial hydrological system varies  and inflThe residence times of water in a glacier depend on the glacier’s size, where the bigger the glacier, the longer the water takes to flow through the system because of longer routing pathways . MeltwatBelow the photic zone, within the firn aquifer of the ablation zone, water residence times can range from hours to days ,104; perAs with meltwater discharge, water residence times change seasonally, with the shortest residence time being in the summer . In wintDry and wet deposition of atmospheric aerosols transports diverse chemistry and biology to the glacier surface ,110,111.Bioaerosols (particulates containing microorganisms) may inoculate glacier surfaces, and subsequent microbial community development depends on the surface environment and microbial adaptations ,118,119.In the supraglacial environment, organic and inorganic nutrients and carbon are often available in dissolved forms released by the biochemical weathering of deposited particles ,126. Wit3+ reduction in ice-hosted sediment particles [2 [2+ and Fe3+, are soluble only within certain pH and dO2 conditions, affecting their capacity to associate with other ions  or to adsorb onto ice crystal surfaces [In the photic layer of the weathering crust, UV- and visible-light-driven chemical transformations lead to the dissolution of iron oxide and silicates in mineral particles and Fearticles ,55,127. articles . Cryoconarticles ,129,130 icles [2 ,131. Howicles [2 . The oxiAncylonema nordenskiöldii, and Mesotaenium berggrenii on the surface of the ice/weathering crust) [2 and N2 when nitrogen-fixing cyanobacteria, such as Nostoc and Anabaena, are present [3− and NH4+) flush from the snowpack and are utilized by microbial communities, while later in the season (after the inorganic flush and when concentrations are low) microbial activity switches to dinitrogen fixation [With the onset of the ablation season, during which the primary nutrient input is from snowmelt, the exposed component of the supraglacial environment  is dominated by prokaryotic photoautotrophs  and ice algae  ,133. The present ,135. Micfixation ,137,138.fixation ,128,139.fixation . Microbifixation ,142 withfixation ,49,142. fixation . Most offixation ,35. Withfixation ,143,144.Box 2 Although the subject of this review is how glacial hydrology influences nutrient and microbial distribution, the contrary is also true. The distribution of nutrients and microbial cells influences supraglacial water flux. Albedo  is lower for darker surfaces compared to lighter surfaces. Therefore, higher input of mineral dust and black carbon on the glacial and ice sheet surfaces decreases the albedo, causing higher heat absorption and increased melting, which influences the morphology of the ice surface . Bioalbe3− concentrations between the ice-snow interface meltwater and the snowpack [4+ concentrations were variable, possibly indicating microbial activity at the ice-snow interface. The chemical composition of the meltwater that enters the englacial system varies across the ablation season. Whereas meltwater at the beginning of the ablation season is likely to reflect the chemical composition of the snow, microbial processes and particle weathering ensure that meltwater that enters the englacial system later in the season is enriched with carbon, macronutrients  and ions such the dissolved inorganic forms of sulfur  [3− and SO42+, which are abundant in meltwater deriving from supraglacial systems, as terminal acceptors [Snowmelt also enters into the firn aquifer. No studies reporting biological processes in firn aquifers are available. However, there are indications of microbial activity: Holland et al. (2022) observed similar NOsnowpack . However, SO42+) ,149. The, SO42+) . Microbi, SO42+) . These a, SO42+)  or couldcceptors .Methanosphaerula and Methanococcus [In addition to englacial conduits, ice veins, with their high nutrient concentrations, offer a favorable habitat for microbial activity ,152,153,nococcus . Despitenococcus ,151.2 and FeS2), creating environments where redox conditions may vary widely [In subglacial systems, meltwater contacts bedrock  and is consequently enriched in compounds released by rock comminution and dissolution , which then creates carbonic acid in aqueous environments [Depending on the mineral composition of the glacier and ice sheet beds, subsurface meltwater can be influenced by the weathering of pyrite  and/or carbonates  ,158. Theronments . Pyrite ronments ,157. Theronments . The preronments ,160,161,ronments . All theronments ,162,163.2) dissolution is rapid both in oxic and anoxic conditions [2 and Fe3+ can be used as sulfide oxidants [+, Fe(OH)3 (iron (oxyhydr)oxides), and SO42. Fe(OH)3 dissociates to Fe3+ in the acidic subglacial environment (created by a high concentration of H+ due to rock dissolution) [3+, and water react to form Fe2+, SO42−, and H+. Anoxic pyrite dissociation is faster than oxic dissociation because of the higher H+ production, which accelerates rock dissolution and weathering [Thiobacillus and Sideroxydans species, and iron-reducing bacteria, such as Desulfosporosinus, Geobacter and Rhodoferax species [4+ reduction, SO42− reduction, methanogenesis, and nitrification [42− reducing bacteria compete with methanogens for carbon substrates [As in the supraglacial and englacial realms, subglacial microbial communities are largely composed of heterotrophic microorganisms. However, contrary to the supraglacial realm, the primary producers of subglacial communities are chemolithotrophs. These organisms rely on nutrients transported from the glacial surface but also those released by rock weathering, and they accelerate mineral weathering of the glacial bed and chemical transformations within the subglacial system . Biotic nditions ,164, wheoxidants . Sulphidolution) . Anoxic athering ,165. The species ,165,166.fication ,168,169.bstrates .While meltwater that reaches subglacial systems is mostly oxygenated, as it is sourced from well-ventilated environments , oxygen levels in the subglacial environment can significantly vary based on the morphological characteristics of the system and, consequently, on water residence times ,159; wheMost of the studies on glacial microbial communities take place during the summer ablation season at temperate and polythermal glaciers, where geochemical and microbial processes are most active due to the presence of meltwater and nutrients in the system . Consequ2 is released abiotically from rock comminution and can be oxidized in both aerobic and anaerobic conditions, fueling microbial chemolithotrophy [Even in winter, water in ice veins, englacial pockets, and subglacial regions ,19,68 mahotrophy ,178,179.hotrophy .2+ and Fe3+) and iron nanoparticulates oxides and Fe2+-bound compounds), which are bioavailable for microbial uptake [5 cells mL−1 compared to concentrations in supraglacial water which are an order of magnitude lower (with the exception of ice veins where observed concentrations were 106–108 cells mL−1) , where soils show a gradient in texture and chemical characteristics from the ice edge: newly exposed soils are usually characterized by low nutrient levels and little or no vegetation, and are impacted by microorganisms and nutrient from glacial meltwater ,204,205.During the transport in the river, glacial meltwater undergoes chemical modifications. For instance, Fe and DOC are sequestered by precipitation and adsorption during the glacial meltwater transport in rivers and streams in Svalbard . NitrogeSubglacial water from marine-terminating glaciers provides carbon, macronutrients , and micronutrients  to the surrounding marine system ,218,219.2+ and colloidal Fe) [The impact that subglacial discharge has on the coastal system depends on two main factors: the seawater depth at which subglacial water is discharged and the nutrient state of the marine system ,225. Melidal Fe) ,225, andidal Fe) ,226,227.Generally, nutrient export increases with meltwater export to the proglacial systems . This isChanges in water residence times in glaciers could cause drastic shifts in proglacial environments. In particular, a decrease in glacial residence times with a consequent increase of glacial water released to outlet systems is observed due to global warming ,231,232.Supraglacial and subglacial microbial communities have been widely studied and characterized in the last few decades, with many studies showing how biotic and abiotic processes in these systems are linked to global biogeochemical cycles ,34,35. I


Sericinus montela, a globally threatened butterfly species, feeds exclusively on Aristolochia contorta (Northern pipevine). Field surveys and glasshouse experiments were conducted to obtain a better understanding of the relationship between the two species. Interviews with the persons concerned with A. contorta were conducted to collect information about the site management measures. We found that management practices to control invasive species and manage the riverine areas might reduce the coverage of A. contorta and the number of eggs and larvae of S. montela. Our results indicated that the degraded quality of A. contorta may result in a decrease in S. montela populations by diminishing their food source and spawning sites. This study implies that ecological management in the riverine area should be set up to protect rare species and biodiversity. Furthermore, butterflies can serve as indicator species to assess and interpret the status of an ecosystem7. However, butterflies are currently highly threatened due to habitat degradation and loss as a result of human activity10. Humans are encroaching on and destroying wildlife habitats for their own needs at an alarming rate14. Weeds and pests are some of the main targets for elimination in a human-centered ecosystem15. In agroforestry industries, pesticides have been extensively used to control pests on crops, resulting in ecological collapse18. Butterflies are sensitive to human activities20, as they lay their eggs on their host plants or nearby in areas populated by people. The presence, abundance, and vitality of host plants can influence the survival and diversity of butterfly populations21. In the larval stage, butterflies are in danger if host plants are disturbed by human activities such as applying pesticides, mowing, and weeding, or grazing and trampling by livestock22. Understanding the quality of host plants is an important step in estimating the abundance and status of butterflies and establishing management practices for butterfly conservation.Butterflies significantly contribute to ecosystem biodiversitySericinus montela) is designated as a vulnerable species on a regional scale in South Korea according to IUCN criteria23. S. montela is known for the beauty of its unique wings, which have a pair of slender tails elongated from each hindwing25. These butterflies have a life cycle of 36–54 days including the egg stage (5–7 days), larval stage (15–25 days), pupal stage (10–12 days), and adult stage (7–12 days)25. Its larvae are monophagous, feeding exclusively on Northern pipevine (Aristolochia contorta), a perennial herbaceous vine species. This plant is a stem-twiner, but is ground-rooted, non-parasitic, and non-epiphytic26. Usually, A. contorta grows on the edges of forests, rivers, and agricultural fields in East Asia 29. It has low sexual reproductivity and forms small populations30. One of the reasons why A. contorta populations are in decline may be the transition from traditional agricultural practices to modern methods, including the use of pesticides, herbicides, and weeding32. In addition, river improvement works, human-made changes to improve navigation, drainage, irrigation, or flood control, may have also contributed to the decline of its population33. These activities could have disrupted the natural habitat of A. contorta, making it difficult for this species to survive and reproduce in time. The decline of the A. contorta population may result in a decrease of the S. montela population, since S. montela relies on the plant as the only food source for its larvae.The dragon swallowtail butterfly 37, as well as the mitochondrial genome, development, and metapopulation dynamics of the butterfly40. Recently, the optimal habitat of A. contorta29 and the interactive effects of CO2 on A. contorta for S. montela42 were reported. Currently, none of the studies have examined the cumulative effect of anthropogenic disturbances on A. contorta and S. montela beyond one-year life cycle. To address this gap, we investigated the anthropogenic factors that can damage the habitat of A. contorta using a four-year interval field survey. It involved interviews with stakeholders such as government officers, laborers, and farmers who were directly involved in land management practices that may affect the survival of S. montela populations. Furthermore, we performed a glasshouse experiment to examine the effects of the disturbed A. contorta on S. montela. We hypothesized that (1) mowing and pesticides will negatively impact the growth and reproduction of A. contorta, and (2) the population of S. montela will be negatively affected by the decline in growth and reproduction of A. contorta resulting from human activities. By identifying the factors contributing to the population decline of S. montela, this study can suggest management practices that promote the survival and conservation of the species. The findings of this study could be used to develop management strategies that reduce the negative impact of human activities on the habitat of A. contorta and the consequent survival of S. montela.There are studies on the ecological importance of Hibiscus syriacus), a beloved flower in Korea, and some trees, such as the false acacia (Robinia pseudoacaci), and the tree of heaven  were removed, while at JW, the A. contorta habitat was managed under financial support from Pyeongtaek City. A. contorta was considered for removal at GC, where the plant community was separated by a low fence to protect Solomon’s seal (Polygonatum stenophyllum). Selective weeding was performed to remove only invasive plants such as Japanese hop (Humulus japonicus) and bur cucumber (Sicyos angulatus) at YU, but pesticide use was reported by farmers at JP.We interviewed stakeholders and summarized the mowing and weeding information into six categories Table . Toward A. contorta, invasive alien plants were removed through government-led conservation programs at sites PC, MA, JW, GC, and YU; however, the removal involved not only invasive exotic plants but also all other native plants and A. contorta. At site JP, private mowing machines and herbicides were used for road maintenance.According to interviews conducted to investigate the anthropogenic activities affecting A. contorta in 2021 was reduced significantly compared to that observed in 2017 at site PC and GC. In 2021, the coverage were 11.86 ± 0.30% at site PC, 18.50 ± 2.80% at site GC and 32.70 ± 7.67% at site PC, 40.80 ± 8.35% at site GC in 2017  in 2017, but only a few vines were left due to mowing (58.10 ± 11.56 cm) in 2021. The number of leaves per quadrat decreased significantly at sites PC, MA, and GC, but it slightly increased at site JW with an artificial planting area for A. contorta and at site YU, where only invasive species were cut. For instance, at site PC, the number of leaves per quadrat was 5.1, the lowest in 2021, which showed a steep decline from 90 in 2017, while the study site with the most leaves in 2021 was JP (188.3 ± 28.7). A positive correlation was found between the coverage and the number of leaves per quadrat .The coverage of A. contorta displayed higher coverage, height , and number of leaves and flowers in well-maintained areas and lower in areas that had intensive mowing or weeding. Coverage was the highest at site JP (56.43 ± 3.64%), which was newly investigated in 2021, followed by at site YU (34.00 ± 4.66%), JW (29.80 ± 1.36%), MA (19.10 ± 3.45%), GC (18.50 ± 2.80%), and PC (56.43 ± 3.64%) , followed by at site PC (192.86 ± 30.84 cm) and then at site JP (182.86 ± 22.44 cm) which were winding around R. pseudoacacia and Morus alba, respectively , followed by at site JW (172.50 ± 16.09%), YU (47.03 ± 7.76%), MA (32.00 ± 2.84%), GC (21.20 ± 4.35%), and PC (5.00 ± 0.82%) , 32.84 ± 3.12 (PC), and the C/N ratio of leaves was 15.82 ± 0.23 (JP), 16.53 (PC), respectively. Among a total of 120 larvae, 75 died before pupation , eight did not emerge from pupae , while 37 emerged .S. montela responded differently according to the food types (leaves or fruits) and the presence or absence of pesticides  plants had a longer pupal length than those fed with-Pesticides/Fruits (with-P/F). The group fed with-P/L had a longer wingspan than those fed with-Pesticides/Fruits (with-P/F) . In addition, the group fed with-Pesticides food showed higher mortality (80.00%) than those fed on plant matter w/o-Pesticides groups  Fig. . Pupal l/F) Fig. . In weigP/F Fig. . FurtherP/F Fig. . In the sed Fig. . FurtherS. montela depended on the food types and pesticide  can affect the growth of the A. contorta population. Interspecies competition may encourage the deterioration of the growth condition of A. contorta.In the places where mowing did not occur, other factors might have affected A. contorta at site PC showed more vigorous growth than at the other study sites: Flowering and fruiting were completed in early August 2021. In Korea, A. contorta blooms from July to August, and fruits appear from September to October48. Stressed plants eaten by herbivorous insects may engage in rapid flowering and fruiting49. High foraging pressure caused by crowding S. montela larvae may increase the stress of A. contorta individuals. As such, plant stress resulting from insect herbivory could be a factor accelerating the flowering period of plants50. Stress may have stimulated the early flowering and fruiting of plant, so the leaves were wilted early in the A. contorta population at site PC.Unexpectedly, the population of 22. It can be inferred that the larvae at site PC were looking for food due to the lack of fresh leaves by cutting the stem . In this way, the changes in the vegetation of A. contorta may affect the population of S. montela , which may be related to the C/N ratio. The C/N ratio is considered an essential factor in plant–herbivore interaction. The leaves are usually rich in nitrogen, which is beneficial for the development of herbivorous insects62 and may consequently be preferred by herbivores. Furthermore, the high-carbon compounds in fruits significantly reduce the larvae's preference63. Therefore, when larvae ate fruits with higher carbon, they could not grow properly. When leaves are lacking, S. montela may look for some more leaves, requiring more nutrients to reach the adult stage64. However, the larvae are likely to be killed while looking for leaves like at site PC .In this study we investigated whether the types of plant organs the butterflies feed on and the application of pesticides on the food resources can affect the growth of 65. Benfuracarb is a broad-spectrum benzofuranyl methylcarbamate insecticide used for crop protection, and it biochemically inhibits acetylcholinesterase activity66. Buprofezin, a thiadiazine insecticide, is effective against hemipteran pests and other insects67. Buprofezin inhibits chitin synthesis, resulting in molting disruption and abnormal deposition of endocuticles, causing premature death69. They are also highly toxic to target and non-target organisms, including insects, mammals, birds, and aquatic organisms74. The larvae, which had been gaining weight by feeding on with-P/L, suddenly increased their mortality five days after the experiment started . The number of eggs, larvae, and dead larvae of S. montela present on the stem and leaves of A. contorta and on the ground was recorded. Field observations were conducted to record any disturbances on host plants, such as mowing, weeding, trampling, and pesticide application. In association with direct observation, we interviewed laborers, city officials, and local people on the sites to record information on the past and present status of the habitat, threats, and conservation of the study areas.We investigated rea Fig. . Field s48 and Flora of Korea Editorial Committee78. S.-H.P took pictures of all the plants for certification according to the national regulation. There is no regulation for the collection of A. contorta in Korea and A. contorta is regarded as a weed by farmers.We identified plants with illustrated plant books of LeeA. contorta were identified. At site JP, the condition of the A. contorta habitat was mainly suitable for S. montela, but mowing was performed and we captured farmers applying pesticides there. According to the farmers, the pesticides included tricyclazole, benfuracarb, and buprofezin. We collected A. contorta leaves and fruits at site JP for the experiment to investigate the effects of mowing and pesticides on the growth of S. montela. The C/N ratio is related to food quality, which in turn affects S. montela survival rate, and was analyzed with the part of the leaves and fruits using an Elemental Analyzer  by NICEM at Seoul National University. Site PC, whose C/N ratio of leaves and fruits was similar to site JP, was selected as the control. In order to exclude the predecessor feeding experience of the larvae in site PC and JP, 120 third-instar larvae, which have a low mortality rate from other causes22, were taken from site GC. Experimental settings were established in a test room at Seoul National University from August 2021 to September 2021. Three larvae were put into an insect breeding dish (10 cm diameter and 4 cm depth). Four experimental treatments (30 larvae per treatment) were performed with a factorial array of leaves × fruits of A. contorta, and pesticides sprayed × not sprayed. Feeding, molting, and development of larvae were observed in insect breeding dishes at 25 ± 2 °C. Larvae were weighed every 3–5 days, and larval weight change (increase or decrease) compared to their original weight was calculated. The body length of pupae and wingspan were measured using vernier calipers. For eclosions the pupae, glued to sticks were placed in the Butterfly Habitat Terrarium (60 × 60 × 90 cm). The number of deaths from the larval stage and eclosions from the pupae stage were recorded, and mortality rates as well as the number of days S. montela survived were calculated. After eclosions, we observed whether they mated and counted the number of eggs. All the experiments were performed in accordance with relevant guidelines  and safety regulations.Based on field observations, factors that could threaten A. contorta, and the average number of larvae and egg clusters of S. montela in 2017 and 2021, t-test at the 5% significance level were conducted after the homogeneity of variance test using SPSS ver.23.0 software . Correlation analysis was performed to examine the relationships between variables  using SPSS. Pearson's correlation coefficient was used to assess the strength and direction of association between variables. The effects of feeding organs  and the effects of pesticides on morphological and reproductive traits of S. montela were also analyzed with the same process. The interactions between feeding on leaves × fruits and feeding on foods with pesticides × without pesticides were analyzed with a two-way analysis of variance (ANOVA) after homogeneity of variance was confirmed. Duncan’s test was used for post-hoc analysis.To compare the growth and reproductive characteristics of


The cellotriose/CORK1 pathway induces immune responses, including NADPH oxidase-mediated reactive oxygen species production, mitogen-activated protein kinase 3/6 phosphorylation-dependent defense gene activation, and the biosynthesis of defense hormones. However, apoplastic accumulation of cell wall breakdown products should also activate cell wall repair mechanisms. We demonstrate that the phosphorylation pattern of numerous proteins involved in the accumulation of an active cellulose synthase complex in the plasma membrane and those for protein trafficking to and within the trans-Golgi network (TGN) are altered within minutes after cellotriose application to Arabidopsis roots. The phosphorylation pattern of enzymes involved in hemicellulose or pectin biosynthesis and the transcript levels for polysaccharide-synthesizing enzymes responded barely to cellotriose treatments. Our data show that the phosphorylation pattern of proteins involved in cellulose biosynthesis and trans-Golgi trafficking is an early target of the cellotriose/CORK1 pathway.We have recently demonstrated that the cellulose breakdown product cellotriose is a damage-associated molecular pattern (DAMP) which induces responses related to the integrity of the cell wall. Activation of downstream responses requires the Arabidopsis malectin domain-containing CELLOOLIGOMER RECEPTOR KINASE1 (CORK1) Drought, salinity, or toxic compounds force the cells to induce adaptive or compensatory changes, which maintain their architecture and protect the cell; beneficial and pathogenic microbes, insects, and nematodes or physical damage destroy cell wall material, which must be repaired.7 Therefore, cells have developed perception and signaling mechanisms through which they respond to damage of their walls.The shape of a plant cell is determined by its wall, and its integrity is essential for all wall-containing cells. Growth, differentiation, maintenance of the turgor, tropisms, and many other developmental processes require alterations in the cell wall architecture that are orchestrated by signals originating from the plant itself.8 while cellulose is synthesized at the plasma membrane by the cellulose synthase (CESA) complex (CSC).9 The elongating cellulose molecules assemble in the apoplast to form microfibrils with a paracrystalline structure, which associates with hemicellulose, pectin and apoplastic proteins. Breakdown products of the cell wall polysaccharides act as damage-associated molecular patterns  and are recognized by cell surface receptors. Breakdown products of pectin are oligogalacturonides (OGs) which activate the Wall-Associated Kinase 1 (WAK1).12 In rice, mixed-linked β-1,3/1,4-glucans from hemicellulose breakdown, namely 31-β-D-cellobiosyl-glucose and 31-β-D-cellotriosyl-glucose, bind to CERK1, and induce the dimerization of CERK1 and the rice chitin receptor CEBiP.13 Cellooligomers from cellulose breakdown require the malectin-domain (MD) receptor kinase (RK) CORK1 for intracellular signaling.14 Therefore, breakdown products of the three cell wall polysaccharides are recognized by pattern recognition receptors (PRR)s.The major polysaccharides in the plant cell wall are cellulose, hemicellulose, and pectin. Hemicellulose and pectin are made inside the cell at the Golgi apparatus by the coordinated action of many proteinscork1 mutants. Cellulose degradation products (cellooligomers) are generated by plant and microbial enzymes and Aziz et al.15 has already shown that they induce a variety of defense responses in grapevine (Vitis vinifera) cells. Locci et al.16 showed that cellotriose and, to a lesser extent, cellotetraose to cellohexose, induce ROS production, phosphorylation of MAPKs and other proteins, as well as the activation of defense gene expression. Souza et al.17 demonstrated that cellobiose triggers a signaling cascade that shares similarities to responses to well-known elicitors such as chito-oligomers and OGs. In contrast to other known P/DAMPs, cellobiose stimulates neither ROS production nor callose deposition. Transcriptome profiles are very similar after cellobiose and OG treatments.18 Johnson et al.19 showed that cellotriose, induces rapid cytoplasmic Ca2+ elevation in Arabidopsis and tobacco root cells. It acted synergistically with chitin. Induction of the Ca2+ response by cellotriose and activation of the downstream responses requires the poly(A) ribonuclease  which degrades the poly(A) tails of specific mRNAs in roots. Comparison of cellooligomers of different lengths demonstrated that cellotriose is the most active cellooligomer for the induction of downstream responses.19 Thus, evidence for cellooligomer-induced signaling in plants have been reported for several systems. More recently, Tseng et al.1 and Martin-Dacal et al. identified the MD containing LRR receptor kinase CORK1 as cellotriose receptor in Arabidopsis.Here, we investigate early responses which are induced by cellotriose in the roots of wild-type and 17,19 and phosphoproteome pattern in Arabidopsis roots.1 Besides triggering calcium influx, ROS production, electrochemical potentials across the plasma membrane, mitogen-activated protein kinase (MAPK) activation, and defense-related gene expression, previous studies suggest that the cellooligomer application also leads to higher pathogen resistance, similar to observations with pectin and hemicellulose breakdown products.21–25 The defense responses induced by cellooligomers are relatively mild when compared to those induced by the pathogen-associated molecular patterns (PAMPs) chitin or flg22.23 However, in combination with chitin, flg22 or the chitin breakdown product oligogalacturonic acid, synergistic effects on calcium influx, ROS production, and MAPK activation indicate crosstalk between cellooligomer, in particular cellotriose, and PAMP responses.19Cellotriose/cellobiose application to Arabidopsis roots and shoots has profound effects on the expression profile25 and several members are involved in sensing the integrity of the cell wall. While little is known about MD-RKs, the members of the MDL-RKs are better characterized. The MDL-RK FERONIA, e.g., is involved in monitoring cell wall integrity (CWI) and is required for pollen tube development and plant growth.26–28 Its extracellular region interacts with pectin.30 MDL-RKs also interact with other cell-surface PRRs and intracellular nucleotide-binding leucine-rich repeat receptors (NLRs). Rapid alkalinization factor (RALF) peptide ligands, LORELEI-like glycosylphosphatidylinositol-anchored proteins and cell-wall-associated leucine-rich repeat extensions coordinate with MDL-RKs to orchestrate PRR- and NLR-mediated immunity.31 The requirement of the MD-RK CORK1 for many cellooligomer-induced cellular responses suggests that this PRR is also involved in CWI signaling.MD-RKs and MD-like (MDL)-RKs are encoded by a small gene family in Arabidopsis,trans-Golgi network (TGN) compartment. Transport occurs in small CESA compartments  or microtubule-associated CESA compartments , which appear to be specific for CESAs and differ from vesicles involved in sorting and trafficking of other cargos. Six CESA heterotrimers, i.e. 18 CESA proteins, constitute the CSC, a high-order oligomer which can be visualized in scanning electron microscopy as sixfold symmetrical rosettes.32–35 CESA proteins constitute the catalytic core of this complex, and the newly synthesized glucan chains are directly released into the apoplast.36 Cellulose synthesis requires the fully assembled CSC at the plasma membrane, and the complex is stabilized in the plasma membrane by conserved regions and helical exchanges within the transmembrane segments of the individual CESAs.37 CSC forms three channels that are occupied by nascent cellulose polymers. Secretion of the chain into the apoplast steers the polymers to a common exit point which may facilitate protofibril formation. The N-terminal domain of the CESAs assembles into a stalk at the cytoplasmic site of the membrane, which allows interaction with microtubules and associated proteins.38 Amino acids in the N-terminal segment are major targets for phosphorylation,37–42 ubiquitination,43 or acylation,44 which ensure proper exocytosis of the complex to the plasma membrane and its recycling via clathrin-dependent endocytosis.An obvious response to impaired cellulose microfibrils should be the activation of CESA to stimulate cellulose repair. Cellulose, a polymer of long unbranched β-1,4-linked glucan chains, is synthesized by CSCs, which are assembled in the Golgi and secreted to the plasma membrane through the We describe here that the phosphorylation pattern of CESAs, proteins involved in the CESA exocytosis and those facilitating trafficking from the Golgi apparatus to the plasma membrane are early targets of cellotriose signaling. Alterations in the phosphorylation pattern are also observed for proteins involved in CESA internalization from the plasma membrane via clathrin-dependent endocytosis. Since the phosphorylation pattern of enzymes involved in the biosynthesis of hemicellulose and pectin is barely changed, cellotriose appears to trigger preferentially cellulose biosynthesis.cork1-2 insertion mutant line  was obtained from Nottingham Arabidopsis Stock Center (NASC). Homozygous seedlings were crossed to the Columbia wild-type line pMAQ2. The corresponding segregated wild-type and homozygous seedlings from the F3 generation were used for experiments, as earlier described in Tseng et al.1 For the phosphoproteome analysis, cellotriose  was applied to 300 roots from the segregated wild-type and homozygous F3 seedlings at 0 min, or after treatment with either water or 10 µM cellotriose for 5 or 15 min. Samples were immediately frozen in liquid nitrogen until phosphoproteome analysis, as described in detail in.1 The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride) with dataset identifier PXD033224. Data are based on three independent experiments and the statistical analysis is shown in the deposited datasets.The Arabidopsis 1, 4 h, or 8 h 19 after cellotriose application, water was used as control. RNA hybridization was performed according to Agilent’s One-Color Microarray-Based Gene Expression Analysis (cf.19). The accession numbers are given in the publication [for the 1 h time point in1 and the other time points in1]. Statistical tests were performed using R Studio v1.1.463 with R v4.1.2.Expression profiles were obtained with roots 1 h 45 Three distinct CESA proteins are necessary to form a functional complex: CESA1, CESA3, and CESA6-like proteins  are required for primary cell wall synthesis, whereas CESA4, CESA7, and CESA8 are required for secondary cell wall synthesis in Arabidopsis.46–48 The amino acid sequences of the CESAs diverge within their N-terminal cytoplasmic domains which are the major targets for regulation (cf. Introduction). For instance, the well-investigated CESA5 is phosphorylated at four positions in its N-terminal region .49 In our study, cellotriose application altered the phospohorylation pattern of CESA1 and −3 at various serines in their N-terminal cytoplasmic segments . We also observed phosphorylation of CESA4/Ser93 after 15 min, but the results were not significant (http://www.ebi.ac.uk/pride). Since CESA4 was the only detected CSC isoform for cellulose synthesis of the secondary cell wall, cellotriose signaling appears to target primarily CESAs for cellulose of the primary cell wall. The (de-)phosphorylation events at the three CESAs differed for at least one time point between wildtype and cork1 roots: e.g., while all serines are dephosphorylated 15 min after the stimulus in the wild-type, this was not observed in the cork1 mutant. This suggests that these phosphorylation events are controlled by cellotriose signaling via CORK1.Genes for 10 CESA isoforms are present in the Arabidopsis genome.segments . After 540 Genetic studies of CESA1 showed that phosphorylation of Thr166, Ser686, or Ser688 is necessary for tethering between CESA1 and cortical microtubules, whereas phosphorylation of Ser162, Thr165, or Ser167 weakens or impairs this interaction, implying differential regulation of interactions of CESA1 with accessory proteins and thus plasma membrane integration.36 Furthermore, the phosphorylated Ser162 can down-regulate cellulose biosynthesis as it primes the surrounding phosphorylation sites for inhibitory phosphorylation by Brassinosteroid Insensitive 2 (BIN2) protein kinase involved in brassinosteroid signaling.42 Therefore, BIN2, together with other (a) kinase(s), negatively regulates cellulose synthesis by phosphorylating CESA1, consistent with the observation that brassinosteroids inhibit root growth and cell elongation in Arabidopsis.42 Bischoff et al.39 showed that phosphorylation of primary cell wall CESAs is induced by phytochrome. CESA7 phosphorylation causes its degradation via a 26S proteasome-dependent pathway.50 These examples demonstrate that CESA phosphorylations can either promote or inhibit its plasma membrane integration and thus potentially cellulose biosynthesis activity. A comparative analysis of the phosphorylation events at the different amino acids in response to different stimuli may help to define the fate of the phosphorylated CESAs, and to identify the kinases/phosphatases involved in the regulatory circuits.CESA phosphorylations have different effects and occur in response to different stimuli. Mutations of CESA1 phosphorylation sites modulate cell expansion and mobility of CSC.51 and appears to have multiple functions: (a) It participates in the delivery of CSCs from the cortical microtubules to the plasma membrane. During exocytosis, CESA and CSI1 appear first at the plasma membrane, followed by tethering of CSC-containing vesicles to the plasma membrane, which is accompanied by the appearance of Sec5B, an exocyst subunit, and PATROL1.53 Zhu et al.52 proposed that CSI1 plays a role in marking the docking site of CSCs-containing vesicles to the plasma membrane. (b) CSI controls CSC motility at the plasma membrane. CSI1 is still associated with the CSC after its insertion into the plasma membrane, and CSC motility is reduced in the csi1 mutant.54 (c) Under stress, CSC endocytosis generates SmaCCs or MASCs. They are involved in recycling of CSC from and fast recovery of CESAs at the plasma membrane, and these processes require CSI.55 Thus, CSI is a crucial player for CESA activity. It is rapidly phosphorylated at Thr37 within 5 min after cellotriose application to Arabidopsis roots (CESA-Interactive Protein 1 (CSI1) interacts with CESAs and the cortical microtubulesis roots .52 and PATROL1 is also phosphorylated in response to cellotriose application 15 min after the stimulus (56 and the subunits SEC5B (At1g21170), SEC8 (At3g10380) and EXO70D3 (At3g14090) are among the phosphorylated proteins 5 and 15 min after the stimulus. At least for the 15 min time point, all phosphorylations are significantly different from the water control and the cork1 results, indicating again that cellotriose requires CORK1 for signaling  recycling and consequently numerous growth effects.57PATROL1 and the exocyst complex determine the rate of delivery of CSCs to the plasma membranestimulus . The exoignaling . EXO70F158 showed that myosins, in particular myosin XIK, via its globular tail domain (GTD), participates in vesicle tethering during exocytosis through interaction with the exocyst complex. The myosin XIK GTD binds to several exocyst subunits, and inhibition of myosin XIK activity reduced the rate of appearance and lifetime of exocyst complexes at the plasma membrane. Myosin XIK associates with secretory vesicles earlier than exocyst and is required for the efficient localization and normal dynamic behavior of exocyst complex at the plasma membrane tethering site. Already in 2019, Zhang et al.59 showed the importance of these myosins in cellulose production at the cytoskeleton-plasma membrane-cell wall nexus. Myosin XIK is rapidly phosphorylated 5 and 15 min after the cellotriose stimulus and this requires CORK1 , its specific role in the two secretory pathways is not clear. The opposite phosphorylation patterns of components of the exocyst pathway and RABA2A suggest that cellotriose triggers the first and inhibits the second one although this requires further investigation.Finally, the small GTPase RABA2A recruits SNARE proteins to regulate the secretory pathway in parallel with the exocyst complex.es CORK1 . The Ves62 The COMPANION OF CELLULOSE SYNTHASE1 (CC1) and CC2 play a role in localizing CESA to the membrane and controls microtuble dynamics.64 The glycosyl phosphatidylinositol (GPI)-anchored COBRA facilitates cellulose crystallization from the emerging β1–4-glucan chains by acting as a “polysaccharide chaperone”.65 Furthermore, the two plasma membrane-localized proteins SHOU4 and SHOU4-like directly interact with CESAs and negatively affect CSC exocytosis.66 Finally, TRANVIA (TVA) facilitates trafficking of CSCs to the plasma membrane, and tva mutants have defects in CSCs secretion and activity at the plasma membrane.38 From these seven proteins, only the phosphorylation pattern of SHOU4-like and CC1 were altered after the cellotriose treatment. This is interesting, since also Korrigan and CC2 has been shown to be phosphorylated during different developmental processes.49 Two of the three identified phosphorylation sites in SOUL4-like are significantly dephosphorylated (15 min) and one significantly phosphorylated (5 min) by the cellotriose/CORK1 pathway  in wild-type, but not cork1 roots suggests that translocation of preproteins into the ER is rapidly activated by the cellotriose/CORK1 pathway  that are important for cellulose production during CWI stress. 7TMs are associated with guanine nucleotide-binding (G) protein signaling. Unexpectedly, two members of the 7TMs, 7TM1 and 7TM5, localized to the Golgi/TGN where they interacted with G protein components. The authors showed that 7TMs and Gβγ regulated specifically CESA trafficking but did not affect general protein secretion. They hypothesized that the G protein complex could potentially sense the cell wall status via association with receptor-like kinases at the plasma membrane and regulate CSC secretion via the 7TMs at the endomembrane system. Although we did not identify Gβ in our phosphoproteome analyses, 7TM5 (At2g01070), Gα (At2g26300) and Gγ (At3g22942) are phosphorylated after cellotriose application, although not significantly in comparison to the water control or cork1 mutant (http://www.ebi.ac.uk/pride).Recently, McFarlane et al.72 EPSINs are important players in the TGN vesicle formation. EPSIN1 plays an important role in vacuolar trafficking of soluble cargo proteins via interactions with clathrin and clathrin-associated proteins . More recently, Collins et al.74 showed that EPSIN1 also modulates the plasma membrane abundance of the flagellin receptor Flagellin Sensing 2 (FLS2) for effective immune responses. Since the eps1 mutant is impaired in flg22 signaling and showed reduced plasma membrane accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1), EPSIN1 also appears to be involved in protein delivery to the plasma membrane. Lee et al.75 proposed that other EPSIN members in Arabidopsis might have different functions in protein sorting. EPSIN1 is de-phosphorylated 15 min after cellotriose application, and EPSIN2 and EPSIN3 are phosphorylated 5 and 15 min after the stimulus, and the results for the three proteins are significantly different from the cork1 control (http://www.ebi.ac.uk/pride). Although the role of these EPSIN phosphorylations for protein sorting and trafficking of CESA-containing vesicles is not or not well investigated, they are targets of cellotriose/CORK1 signaling. It appears that the signaling path interferes with the dynamics of protein sorting in the TGN.The TGN is a central trafficking hub where secretory, vacuolar, recycling, and endocytic pathways merge.76–84 The function of many proteins in clathrin-mediated endocytosis has been extensively characterized in mammals.85 Although many proteins are conserved in plants,82 only a few components have been associated with CSC endocytosis, recycling, or degradation.Like many PRRs, the CSC is internalized from the plasma membrane via clathrin-dependent endocytosis, although a clathrin-independent pathway has been hypothesized as well.82 eight of them are reversibly phosphorylated in response to cellotriose treatment, although to different extents. This includes the two heavy-chain proteins  and At3g085330 (CHC2), the SH3 domain protein At4g18060, EHD2 (At4g05520), and Dynamin-Related Protein (DRP)2A (At1g10290), which are all dephosphorylated after 15 min. DRP2B (At1g59610) is phosphorylated at 5 min and the clathrin recruiting tPLATE (At3g01780) as well as the SH3 domain protein At1g31440 15 min after the stimulus (http://www.ebi.ac.uk/pride). From the six types of DRPs encoded in the Arabidopsis genome, two of them (DRP1 and DRP2) participate in post-Golgi trafficking (cf.86). In particular, DRP2A and DRP2B function coordinately in multiple pathways of post-Golgi trafficking in a phosphatidylinositol 3- or 4-kinase-dependent manner86 and DRP2B plays a role in flg22-signaling and pattern-triggered immunity in plants.87 The tPLATE complex is recruited to the plasma membrane during the early stages of endocytosis.89 The role of the identified phosphorylation changes in response to cellotriose application is not known but comparison of the responses in wild-type and cork1 roots demonstrates that almost all of them are cellotriose/CORK1-dependent. Furthermore, this appears to be relevant for endocytosis and recycling of the CESAs, since they interact with the Adaptor Protein 2 (AP2)-like and tPLATE complexes.90 None of the four main components constituting the AP2 complex91,92 are phosphorylated in response to cellotriose.At least 28 proteins have been described to be involved in or associated with clathrin-mediated endocytosis in Arabidopsis,86 demonstrated that post-Golgi trafficking is phosphatidylinositol (PI) 3- or 4-kinase-dependent. Several PI 3- and PI 4-kinases (At5g64070 and At1g26270), the PI 3,4- kinase At1g49340 and the PI 3,4,5-trisphosphate 3-phosphatase (At3g19420) are (de-) phosphorylated 5 or 15 min after the cellotriose stimulus (http://www.ebi.ac.uk/pride). Fujimoto et al.93 performed inhibitor studies and showed that PI 3-kinases are mainly controlling endocytosis of CESAs while PI 4-kinases are involved in exocytosis. The PI 4 kinase At5g64070 is required for proper organization of the TGN and post-Golgi secretion in root hairs. Furthermore, the PI 4 kinase activity is Ca2+-dependent.94 However, no clear conclusions can be drawn from the phosphorylation pattern observed for this kinase. However, PIP 4 kinase At1g26270, PIP 3/4 kinase At1g49340 and PIP 3 phosphatase At3g19420 exhibit rapid changes in their phosphorylation patterns in response to cellotriose application . However, the stimuli and kinases that induce the phosphorylation events are still poorly understood. Besides signals deriving from developmental programs, receptor kinases located at or in the plasma membrane are likely candidates to regulated cellulose biosynthesis in response to external signals. CWI signaling was often associated with Catharanthus roseus receptor (like) kinases with MD or MDL domains,101–105 among them is FERONIA104 with its extracellular domain that is recognized by pectic homogalacuronan complexes in the presence of Ca2+.29 Furthermore, genetic screens identified THESEUS1, another MDL-containing receptor kinase, to be involved in CWI signaling.103 WAK1 and so far uncharacterized members of the WAK family interact with pectin in the presence of Ca2+.107 Furthermore, MIK2 (a LRR-receptor kinase MALE DISCOVERER1-INTERACTING RECEPTOR-LIKE KINASE2) might operate upstream of THESEUS1 and alters expression profiles in a CESA6-dependent manner in Arabidopsis.108 In addition, FEI1 and FEI2, leucine-rich repeat receptor kinases and SOS5 (SALT OVERLY SENSITIVE5) are involved in cellulose biosynthesis and have been proposed to transduce apoplastic signals into appropriate changes in the cell wall architecture.109 Tseng et al.110 recently demonstrated that CORK1 is an important MD-RK for many downstream responses associated with CWI signaling in response to cellotriose/cellobiose in the apoplast. Here, we propose that (de-)phosphorylation of crucial proteins involved in cellulose synthesis and those involved in cellular protein trafficking are fast responses to cellotriose. cork1 mutant roots, indicating that the receptor is involved in cellotriose signaling. However, some of the responses are not CORK1-dependent suggesting that cellotriose may also activate other pathways or require additional proteins besides CORK1. Another interesting observation is that several proteins are rapidly phosphorylated after one time, and no longer phosphorylated or even dephosphorylated at the other time point. Apparently, the response is highly dynamic, and without knowing the exact role of the phosphorylation targets of the individual proteins, an interpretation of the results is purely speculative. The model tries to summarize major proteins identified in this study, which are targeted by cellotriose. It may provide a basis for future studies on signals, which regulate cellulose synthase, and intracellular protein sorting . We propose that cellotriose signaling rapidly interferes with the central trafficking hub where secretory, vacuolar, recycling, and endocytic pathways merge.