Gas exchange and leaf metabolism of irrigated maize at different growth stages

Abstract

Net ecosystem exchange (NEE), leaf gas exchange and biochemical traits were investigated in an irrigated maize crop grown under Mediterranean conditions. Sub-optimal irrigation water supply determined a drought stress during the early vegetative growth stage (45–49 days after swing) that decreased NEE. Drought, in the late vegetative stage, also caused a reduction of leaf gas exchange. In the latter period, proline, glycine and serine, as well as sucrose leaf contents increased, while starch, proteins and glucose contents decreased. In the early reproductive stage, the crop experienced a longer dry spell that induced a reduction in canopy as well as in leaf gas exchanges, while protein and free amino acid contents decreased with respect to the late vegetative stage. Both ecophysiological and biochemical data demonstrate a good capacity of cultivar Pioneer PR32D99 to endure the environmental stress, related to Mediterranean summer drought, leading to an elevated dry matter yield at harvest. Photosynthetic apparatus appeared fairly resistant to soil water shortage due likely to the increased leaf content of organic solutes, such as amino acids and soluble sugars.     

Improvements in single-use bioreactor film material composition leads to robust and reliable Chinese hamster ovary cell performance

Single-use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO-K1, and CHO-DP12, cultured in media conditioned in an older single-use bioreactor (SUB) film (F-1) and a newer generation film (F-2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F-1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F-2 film. Proteomic profiling of CHO cells cultured in the F-1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F-2 films when compared to F-1 films including bDtBPP, potentially explaining improved CHO cell growth in F-2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess.     

Polymorphism of a new Ty1-copia retrotransposon in durum wheat under salt and light stresses

Long terminal repeat retrotransposons are the most abundant mobile elements in the plant genome and play an important role in the genome reorganization induced by environmental challenges. Their success depends on the ability of their promoters to respond to different signaling pathways that regulate plant adaptation to biotic and abiotic stresses. We have isolated a new Ty1-copia-like retrotransposon, named Ttd1a from the Triticum durum L. genome. To get insight into stress activation pathways in Ttd1a, we investigated the effect of salt and light stresses by RT-PCR and S-SAP profiling. We screened for Ttd1a insertion polymorphisms in plants grown to stress and showed that one new insertion was located near the resistance gene. Our analysis showed that the activation and mobilization of Ttd1a was controlled by salt and light stresses, which strengthened the hypothesis that stress mobilization of this element might play a role in the defense response to environmental stresses.     

A novel alpha-D-galactosynthase from Thermotoga maritima converts beta-D-galactopyranosyl azide to alpha-galacto-oligosaccharides

The large-scale production of oligosaccharides is a daunting task, hampering the study of the role of glycans in vivo and the testing of the efficacy of novel glycan-based drugs. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, are becoming important chemo-enzymatic tools for the production of oligosaccharides. However, while β-glycosynthase can be produced with a rather well-established technology, examples of α-glycosynthases are thus far limited only to enzymes from glycoside hydrolase 29 (GH29), GH31 and GH95 families. α-L-Fucosynthases from GH29 use convenient glycosyl azide derivatives as a strategic alternative to glycosyl fluoride donors. However, the general applicability of this method to other α-glycosynthases is not trivial and remains to be confirmed. Here, β-D-galactopyranosyl azide was converted to α-galacto-oligosaccharides with good yields and high regioselectivity, catalyzed by a novel α-galactosynthase based on the GH36 α-galactosidase from the hyperthermophilic bacterium Thermotoga maritima. These results open a new avenue to the practical synthesis of biologically interesting α-galacto-oligosaccharides and demonstrate more widespread use of β-glycosyl-azide as donors, confirming their utility to expand the repertoire of glycosynthases.     

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol x g(-1) FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol x g(-1) FW at the end of the night, which rose to 108 pmol x g(-1)FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 microM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.     

Structural characterization of the O-chain polysaccharide from an environmentally beneficial bacterium Pseudomonas chlororaphis subsp. aureofaciens strain M71

Pseudomonas chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo Fusarium oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot. Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.). Strain M71 ability to persist on the tomato rhizosphere and on the aerial part of cypress plants could be related to the nature of the lipopolysaccharides (LPS) present on the outer membrane and in particular to the O-specific polysaccharide.A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide from P. chlororaphis subsp. aureofaciens strain M71. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as the following linear trisaccharide.     

Structural determination of the O-specific polysaccharide from Aeromonas hydrophila strain A19 (serogroup O:14) with S-layer

Bacteria belonging to the genus Aeromonas are Gram-negative mesophilic and essentially ubiquitous in the microbial biosphere; moreover they are considered very important pathogens in fish and responsible for a great variety of human infections.The virulence of Gram-negative bacteria is often associated with the structure of lipopolysaccharides, which consist of three regions covalently linked: the glycolipid (lipid A), the oligosaccharide region (core region) and the O-specific polysaccharide (O-chain, O-antigen).The O-chain region seems to play an important role in host-pathogen interaction. In the case of Aeromonas hydrophila the majority of pathogenic strains belongs to serogroups O:11, O:16, O:18 and O:34. In this paper, we report the complete structure of the O-chain of A. hydrophila strain A19 (serogroup O:14), a pathogenic strain isolated from European eels, which showed high virulence when tested in trout or mice. Dried cells were extracted by the PCP (phenol/chloroform/petroleum ether) method obtaining the lipopolysaccharide. After mild acid hydrolysis the lipid A was removed by centrifugation and the obtained polysaccharide was fully characterized by means of chemical analysis and one- and two-dimensional NMR spectroscopy. All the data collected are directed towards the following structure: