Biochemical and morphometric parameters of the liver.

RAW264.7 cells were seeded at a density of 5 × 106 cells per well in a six-well plate (3 ml RPMI-10% fetal bovine serum [FBS] with penicillin-streptomycin) and allowed to adhere for 24 h. Cells were then infected with conidia at a multiplicity of infection (MOI) of 10:1. The plates were centrifuged at 800 × g for 5 min to bring the conidia to the bottoms of the wells. After 4 h of coincubation at 37°C and 5% CO2, cells and conidia were scraped from the wells and fixed with 4% paraformaldehyde for 30 min. The cell pellets were washed twice and resuspended in PBS plus 0.1% Tween 80. Samples were then analyzed with a FACScalibur flow cytometer (BD Biosciences, Mississauga, Ontario, Canada). Analysis was done using the FlowJo v8.8.6 program (Tree Star, Ashland, OR).

The biosynthesis of trehalose phosphate.

The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively.

The biosynthesis of trehalose in the locust fat body.

Future research on abiotic stress response should acknowledge the growing impact of a constantly changing environment that causes a huge biomass yield loss even for the most adapted crops (; ). Those environmental conditions challenge more than ever agricultural industry due to low producing soils and difficult growing conditions. Therefore the optimization of transgenic plants is now considered as one effective strategy to combat expected food shortage. For this reason, we need crops with genetic and epigenetic prosperities already adapted to local environmental conditions. These varieties should have specific characteristics including high leaf area index, little need for fertilization and a broad resistance pattern.

The biosynthesis of trehalose phosphate.

The high expression of trehalase in the stomata connects trehalose metabolism with stomatal regulation (). The responsible mechanism is not yet known, but seems to involve ABA, as ABA-induced closing of stomata depends on the expression of AtTRE1 (). Other studies have also correlated trehalose metabolism to stomata, as ABA induces TPS1 expression in stomata and tppg mutants are insensitive to ABA-induced stomatal closure (; ). In addition, the promoter of AtTRE1 is predicted to contain a putative binding site for regulation by ABA () which is supported by the need for AtTRE1 in ABA-induced closing of the stomata. Stomatal opening and closing is a complex process, which is mediated by many factors, including ABA. Moreover, timing (night/day) and environmental factors (stress/non-stress) also influence stomatal movements. Differential water potential causing opening and closing of stomata is achieved by the exit of different ions, mainly potassium and nitric oxide and malate (; ; ). Furthermore, a role for sugar sensing in stomatal movements is very likely as elevated expression levels of hexokinase (HXK) in guard cells causes accelerated stomatal closure and this closure is induced by sugar and ABA, indicating a sucrose-regulated feedback inhibition mechanism (). have demonstrated that during the day sucrose stimulates stomatal closure via HXK and ABA. They hypothesize that the sucrose exported from source cells enters the apoplastic space before it is loaded into the phloem. This apoplastic raise in sucrose reaches the guard cells via the transpiration stream where sucrose is channeled via transporters located in the plasma membrane into the cytosol. Here it is degraded into its hexose components that are recognized by HXK. This recognition by HXK would trigger in association with ABA a reduction in stomatal aperture. We have found that TRE is required for ABA induced stomatal closure (). We hypothesize that the knockout of TRE1 increases the apoplastic concentration of trehalose and that trehalose might have a higher affinity for the sucrose transporters. By this, trehalose would block the plasma membrane transporters for sucrose transport and hence preventing/lowering the HXK signal and the ABA-mediated stomatal closure. OX of TRE would reduce the apoplastic trehalose content and by this enhance the sucrose transport and stimulate stomatal closure via HXK and ABA. Kelly et al. hypothesized that overexpression of TRE1 leads to an increased glucose monomer concentration that should stimulate the closure response of stomata to ABA ().

The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose.

of a functional trehalose biosynthetic ..

The toxicity of trehalose feeding to plants in high concentrations has been linked to an over-accumulation of T6P, through the regulation of starch metabolism (; ). These findings have now been linked to a transcription factor bZIP11 (basic region /leucine zipper motif) as bZIP11 overexpression plants show insensitivity toward supplied trehalose (). Since SnRK1 (sucrose non-fermenting-related kinase-1, a kinase acting as energy sensor) overexpression similarly circumvent growth arrest on trehalose and SnRK1 is postulated to be inhibited by T6P, it might be tempting to speculate a connection between T6P, SnRK1, and bZIP11 to explain the resulting toxicity of trehalose (; ). Furthermore, feeding trehalose was initially linked to starch metabolism via redox activation of AGPase (adenine-di-phosphate glucose pyrophosphorylase; ) but more evidence points toward a deteriorated starch breakdown that affects starch levels (). Indeed, an ethanol-induced overexpression of TPS (OtsA) failed to connect directly elevated T6P with a change in the redox status of AGPase (). Therefore, the change in redox status of AGPase might be an indirect or even independent consequence, possibly in response to sucrose. In fact, studies have shown the connection between T6P and sucrose in A. thaliana seedlings (; ). A recent review by specifically deals with the discussion on the connections between T6P, SnRK1, sucrose, and starch.

The biosynthesis of trehalose Phosphate

Plants may encounter external trehalose in cases where plant pathogens or mycorrhizal fungi come into contact with the plant. To understand how plants may react to this, several studies were conducted where trehalose was added to seedlings or adult plants. Trehalose treatment has been shown to induce both biotic and abiotic stress-related genes (). Interestingly, using lower concentrations of trehalose (30 mM instead of 100 mM) together with 1% sucrose showed actually more down-regulated abiotic stress associated genes [e.g., peroxidase 2 (PRXR2)] than upregulated ones (). Apart from the different trehalose concentrations used, these results may also be explained by the use of different DNA microarray providers.

Building a kinetic model of trehalose biosynthesis in ..

During infection, A. fumigatus is exposed to conditions of both oxidative stress and nutrient depletion. We found that A. fumigatus conidia deficient in tpsA and tpsB had increased susceptibility to severe oxidative shock with hydrogen peroxide as well as delayed germination, suggesting that this strain might be impaired in virulence. Surprisingly, we found that the trehalose-deficient ΔtpsAB mutant strain did not display a reduction in virulence in a murine model of invasive aspergillosis and was in fact hypervirulent. Mice infected with the ΔtpsAB mutant strain had a shorter survival and a higher fungal burden than mice infected with the wild-type parent strain. Furthermore, the higher fungal burden was associated with higher levels of pulmonary inflammation as measured by total pulmonary myeloperoxidase. Complementation of the ΔtpsAB mutant with a single copy of tpsA resulted in lower fungal burdens and a prolongation of survival to wild-type levels. These results contrast sharply with the role of trehalose-6-phosphate synthases in other fungi such as C. albicans and C. neoformans, in which Tps activity is required for normal virulence (, ).