Since hpGGLT1 plants grown in the presence of 1 mM boric acid or no added boric acid had different phenotypes,we were curious to know if altering the structure and dimerization of RG-II in hpGGLT1 led to changes in other cell wall components. Therefore we determined the monosaccharide composition of destarched leaf AIR from plants grown under different borate concentrations . No significant visible differences were discernible in hpGGLT1 and EV plants grown with 1 mM borate . However, we saw increases in the abundance of several neutral monosaccharides, in particular glucose , in the walls of plants grown with no added borate. No differences in aniline blue staining of the walls of EV and hpGGLT1 lines were observed, suggesting that the increase in non-cellulosic glucose in plants which appear severely stressed is not due to callose deposition . Finally, we performed Saeman hydrolysis of the TFA-resistant AIR to determine the amount of glucose derived from crystalline cellulose. A substantial increase in cellulose-derived glucose was detected in the hpGGLT1 lines grown with no added borate but not in plants grown under high-borate conditions . To further investigate the altered cell wall in the hpGGLT1 plants grown with no added borate, we performed a saccharification assay on the destarched AIR to measure the quantity of enzymatically accessible sugars in this material. Following pre-treatment with hot water,macetas de 5 litros the samples were treated with a commercial saccharification cocktail of GHs, and the amount of reducing sugar released after 72 h was measured.
As expected, given the increase in cell wall glucose , low-borate-grown hpGGLT1 plants had a significant increase in the amount of sugar released compared with EV , a difference that was not seen in high-borate growth conditions .Wall structure is also important for growth under environmental stress Salinity stress weakens the wall, probably by disrupting pectin cross-linking . The FERONIA receptor directly binds pectin, and prevents uncontrolled cell expansion caused by salt stress . Similar to the fer mutant, the root cells of mur1 burst during growth in the presence of salt,suggesting a role for RG-II cross-linking in allowing roots to recover growth. Since hpGGLT1 plants have disrupted RG-II, we predicted that if the FERRONIA model is correct the roots should show a similar loss of wall integrity as mur1 when grown in the presence of salt. Indeed, compared with the wild type, root cells of hpGGLT1 plants burst after salt treatment during the growth recovery phase . However, it is challenging to predict the NDP-sugar transported by these enzymes from amino acid sequences alone. Here, we have provided evidence that GGLT is a GDP-L-galactose transporter, and show that it is required for the production of structurally normal RG-II. Reducing GGLT1 expression led to a decrease in the L-galactose content of RG-II and a reduction in RG-II dimerization and dimer stability. Growth of the silenced plants is rescued by adding additional borate to the growth medium. Thus, suppressing GGLT provides a unique opportunity to investigate the effects of structural changes of RG-II on boron requirements for plant growth.Despite multiple reported failures to heterologously express the GGLT1 protein , we were able to transiently express GGLT1 as a YFP fusion in onion epidermal cells and confirm its predicted localization to the Golgi apparatus. Unfortunately, as others have also reported, we were unable to express GGLT1 in yeast to perform biochemical analysis of in vitro activity. Therefore, we took an in planta approach to determine the function of GGLT1. GDP-L-galactose lacked a reported NST, and in wild-type Arabidopsis L-galactose is present only in the pectic polysaccharide RG-II.
Analysis of RG-II from hpGGLT1 plants showed that there was a reduction in RGII dimer formation, which was correlated with a specific loss of the terminal L-galactose present on side-chain A. We did not detect changes to other biopolymers known to use GDP-mannose, GDP-glucose or GDP-fucose.We propose that GGLT1, and the L-galactose decoration on RG-II, is essential for plant development and reproduction. This is consistent with previous studies showing that L-galactose is present on side-chain A of all RG-IIs analyzed to date , with studies suggesting that RG-II is critical for pollen development, pollen germination and seed development, and that plants with abnormal RGII exhibit major growth defects . When the plants were grown in the presence of boric acid, their reduced growth phenotype was partially rescued. This was also reported for GME-silenced tomato plants . While a reduction in GME expression affects the biosynthesis of all GDP-linked sugars, as well as ascorbate, the authors suggested that it was the loss of RG-II dimerization that was critical , and our study supports this conclusion. It should be noted that the rosette morphology of hpGGLT1 plants is different from that described for mur1 or hpGFT1, which has been suggested to arise from the replacement of RG-II L-fucose with L-galactose, leading to the incomplete formation of side-chain A . Since plants lacking fucose on xyloglucan or arabinogalactan proteins grow normally, the phenotype had been ascribed to reduced RG-II dimerization because of the altered RG-II structure in mur1 and hpGFT1. Our results suggest that either the phenotype is dependent on the exact nature of the RG-II side-chain modification, or that the ‘cabbage-like’ growth habit of mur1 and hpGFT1 results from the loss of fucosylation of another molecule. For example, it has been proposed that fucose is necessary for epidermal growth factor domainactivation of receptor-like kinases , and for promoting the interaction of DELLA with the brassinosteroid pathway .
Boron is an essential micronutrient that is required for normal plant growth and development, and its availability is important for maintaining plant productivity. Too little results in poor plant growth, but too much is toxic. To date, the major described role for boron is to cross-link RG-II . This has been shown to affect the tensile strength and porosity of cell walls. In some species, borate deficiency results in cell wall thickening . In our hands, boron-deficient plants did show an increase in cellulose-derived glucose, as well as some hemicellulose derived sugars, including mannose. A cell wall integrity-sensing pathway responsive to salinity stress, and acting via the receptor kinase FER, has recently been described and is thought to act via interaction with pectin . Here we show that hpGGLT1 plants display a similar salt-specific loss of cell wall integrity as fer and mur1. These data suggest that RG-II cross-linking is directly disrupted by salinity,macetas cultivo or part of a compensatory feedback loop that is necessary to recover wall strength during acclimation. Such a feedback loop has also been reported in other primary cell wall mutants . Transcriptomic data from plants grown under boron deficiency show altered transcript accumulation for polygalacturonases, pectin methylesterases and pectate lyases, all enzymes involved in cell wall remodeling, as well as stress response genes . The hpGGLT1 plants will be a useful tool for investigating this process further. Boron has also been shown to affect the catalytic activities of plasma membrane proteins , control the transcription of specific gene targets and to affect the homeostasis of oxidative compounds that may alter lipid properties . More recently, it was proposed that boron may serve as a potential link between RG-II and GIPCs. These are a heavily glycosylated class of sphingolipids and are major components of the plant plasma membrane. Interestingly, this proposed linkage would provide a physical interaction between the plasma membrane and the cell wall , and is promising avenue for future investigation.Human space travel depends upon the operation of life support systems. In deep space missions, such as the mission to Mars, life support cannot depend upon storage alone, it requires a fully regenerative system as well, i.e. waste must be reclaimed for reuse. A number of solid waste reclamation technologies are under investigation for space applications .
Technologies such as incineration, supercritical water oxidation, steam reformation, and electrochemical oxidation are at various stages of development for use in space. Incineration is perhaps the most promising technology because it rapidly and completely converts the waste to carbon dioxide, water, and minerals. Incineration also lends itself to experiment more affordably than most of the other technologies, and it is already the most thoroughly developed technology for use in a terrestrial environment. The major difficulty with the use of incineration, particularly in a closed environment, is the emission of pollutants that can build up, thus necessitating a flue gas cleanup system. Incineration of the inedible portion of crops and wastes, such as human feces, produces mostly carbon dioxide, water, and ash. However the incineration also produces NOx and SO2; pollutants that need to be removed from flue gas and recovered for reuse. NOx is produced from nitrogen in the waste or fuel and from the nitrogen in the air. Similarly, the sulfur in the waste is converted to SO2 during incineration. To conserve the nutrients for life support, NOx should be converted to N2, NH3, and/or nitrates. The N2 can be used to replace cabin N2 leakage and/or the loss of N2 during combustion, while NH3 and nitrates can be recycled as part of the plant hydroponics nutrient solution. The SO2 can be converted to either elemental sulfur or sulfate because elemental sulfur can be safely stored or converted to sulfate, where sulfate can be recycled as part of the plant hydroponics nutrient solution as well. Many flue-gas clean up technologies have been developed to remove NOx and SO2 from terrestrial incineration . Most of the technologies require expendables, making them unsuitable for a space application. Processes that use catalyst may have problems because catalyst poisoning is an issue that limits the life-span of a catalyst. The poisoning of the catalyst by soot, alkali metals, and chlorides in the flue gases can occur, and wet processes that handle liquids, like using spray absorbers, pose difficulty because of the micro-gravity situation. What also need to be addressed are the issue of safety and energy requirements of the technology. Using potential hazardous high-pressure systems and/or systems that require an excessive amount of electric energy is unwarranted for space missions. In view of the aforementioned constraints and requirements, we are investigating an approach involving the use of rice hulls, an inedible biomass that can be continuously produced in a space vehicle, to clean up flue gas pollutants generated during incineration. We have found that flue gas from the incineration of biomass contains an insignificant amount of SO2, and that most of the sulfur in the biomass has ended up as sulfate in flyash. Presumably, SO2 has reacted with the alkali metal in the biomass, thus, this study focuses on the control of NOx emissions. The approach involves the carbonization of the rice hulls to produce activated carbon for the adsorption of NOx and a subsequent reduction of the adsorbed NOx by carbon to N2. The optimal conditions for the production of activated carbon from rice hulls for the adsorption of NOx has been determined. Parametric studies on the adsorption of NOx by the carbon have been performed. The effectiveness of this approach to control NOx emissions in deep space missions has also been assessed. The activated carbons were characterized by the measurement of their average pore size and surface area. There are three types of pores which developed in the solid: micropores , mesopores , and macropores . The average pore size has an effect on the total surface area that is available for adsorption. The BET surface area and BJH average pore size of activated carbon prepared from rice hulls under different conditions were measured. Temperature and hold time used for activation was varied. As the temperatures varied from 350°C to 800°C and the activation time from 0.5 hr to 5 hrs, the BET and BJH of activated carbon from rice hulls ranged from 76.5 m2 /g to 172.9 m2 /g and from 25.1 to 67.1 Α, respectively. In general, the BET increases with the increase of temperature until about 700°C. Further increases of temperature results in decreases of BET surface area. The BET of rice hulls activated carbon was 76.5 m2 /g, 167.1 m2 /g, 172.9 m2 /g, and 147.9 m 2 /g with an activation temperature of 350°C, 600°C, 700°C, and 800°C, respectively. The time used for activation did not affect the BET surface area substantially under the conditions employed.