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The effects of cadmium stress on the growth and physiological characteristics of Sassafras tzumu Hemsl . were studied in pot experiments. Five Cd levels were tested [CT(Control Treatment) : 0 mg/kg, Cd5: 5 mg/kg, Cd20: 20 mg/kg, Cd50: 50 mg/kg, and Cd100: 100 mg/kg]. The growth and physiological characteristics of the sassafras seedlings in each level were measured. The results showed that soil Cd had negative influences on sassafras growth and reduced the net growth of plant height and the biomass of leaf, branch and root. Significant reductions were recorded in root biomass by 18.18%(Cd5), 27.35%(Cd20), 27.57%(Cd50) and 28.95%(Cd100). The contents of hydrogen peroxide decreased first then increased while malondialdehyde showed the opposite trend with increasing cadmium concentration. Decreases were found in hydrogen peroxide contents by 10.96%(Cd5), 11.82%(Cd20) and 7.02%(Cd50); increases were found in malondialdehyde contents by 15.47%(Cd5), 16.07%(Cd20) and 7.85%(Cd50), indicating that cadmium stress had a certain effect on the peroxidation of the inner cell membranes in the seedlings that resulted in damage to the cell membrane structure. Superoxide dismutase activity decreased among treatments by 17.05%(Cd5), 10,68%(Cd20), 20.85%(Cd50) and 8.91%(Cd100), while peroxidase activity increased steadily with increasing cadmium concentration; these results suggest that peroxidase is likely the main protective enzyme involved in the reactive oxygen removal system in sassafras seedlings. Upward trends were observed in proline content by 90.76%(Cd5), 74.36%(Cd20), 99.73%(Cd50) and 126.01%(Cd100). The increase in proline content with increasing cadmium concentration indicated that cadmium stress induced proline synthesis to resist osmotic stress in the seedlings. Compared to that in CT, the soluble sugar content declined under the different treatments by 32.84%(Cd5), 5.85%(Cd20), 25.55%(Cd50) and 38.69%(Cd100). Increases were observed in the soluble protein content by 2.34%(Cd5), 21.36%(Cd20), 53.15%(Cd50) and 24.22%(Cd100). At different levels of cadmium stress, the chlorophyll content in the seedlings first increased and then decreased, and it was higher in the Cd5 and Cd20 treatments than that in the CT treatment. These results reflected that cadmium had photosynthesis-promoting effects at low concentrations and photosynthesis-suppressing effects at high concentrations. The photosynthetic gas exchange parameters and photosynthetic light-response parameters showed downward trends with increasing cadmium concentration compared with those in CT; these results reflected the negative effects of cadmium stress on photosynthesis in sassafras seedlings.

L-Proline is a multifunctional amino acid that plays an essential role in primary metabolism and physiological functions. Proline is oxidized to glutamate in the mitochondria and the FAD-containing enzyme proline oxidase (PO) catalyzes the first step in L-proline degradation pathway. Alterations in proline metabolism have been described in various human diseases, such as hyperprolinemia type I, velo-cardio-facial syndrome/Di George syndrome, schizophrenia and cancer. In particular, the mutation giving rise to the substitution Leu441Pro was identified in patients suffering of schizophrenia and hyperprolinemia type I. Here, we report on the expression of wild-type and L441P variants of human PO in a U87 glioblastoma human cell line in an attempt to assess their effect on glutamate metabolism. The subcellular localization of the flavoenzyme is not altered in the L441P variant, for which specific activity is halved compared to the wild-type PO. While this decrease in activity is significantly less than that previously proposed, an effect of the substitution on the enzyme stability is also apparent in our studies. At 24 hours of growth from transient transfection, the intracellular level of proline, glutamate, and glutamine is decreased in cells expressing the PO variants as compared to control U87 cells, reaching a similar figure at 72 h. On the other hand, the extracellular levels of the three selected amino acids show a similar time course for all clones. Furthermore, PO overexpression does not modify to a significant extent the expression of GLAST and GLT-1 glutamate transporters. Altogether, these results demonstrate that the proline pathway links cellular proline levels with those of glutamate and glutamine. On this side, PO might play a regulatory role in glutamatergic neurotransmission by affecting the cellular concentration of glutamate.

The pseudoacacia species (Robinia pseudoacacia L.) are flowering plants that produce nectar and can be the sources of unifloral honey. Robinia is native to North America and is invasive in several European countries. The aim of this work was to determine and compare the physicochemical parameters, proline content, and antioxidant capacity of pseudoacacia honey collected from different locations in two central European countries (Hungary and Slovakia). The botanical origin of each honey sample was verified by melissopalynological analysis. The color intensity was determined using the Pfund scale. The antioxidant activity was determined with different spectrophotometric methods (DPPH, ABTS, and FRAP). The content of the total polyphenols, flavonoids, phenolic acids, and proline was quantified using spectrophotometric methods. The electrical conductivity, refractive index, and optical rotation were analyzed according to European Pharmacopoeia, 12th edition. Our study shows that the antioxidant capacity, and proline and phenolic content of unifloral pseudoacacia honeys can vary according to the geographical origin, polyphenol content, and the pollen profile in honey samples.

Is it possible to characterize the types of honey based on their chemical composition, their content of bioactive substances, and their physicochemical properties? The objective of this study was a comparative analysis of four types of honey from the Carpathian Foothills area, located in south-east Poland, based on the content of the main phenolic acids and proline, the mineral composition, and selected physicochemical properties. Most analyses, such as those of phenolic acids, sugars, and proline content, in honey samples were performed using chromatographic methods. These experiments demonstrated that honeydew honeys were the richest in phenolic acids, minerals, as well as oligosaccharides, compared to other honeys. Dark-colored honeys were characterized by the highest proline content. The dominant elements in all types of honey were potassium and calcium. The results of the present study show that analyses of specific phenolic acids, minerals, proline, and sugar content, in combination with chemometrics analysis, may successfully differentiate between the biological origins of honey samples and allow the preliminary verification of the samples before performing time-consuming pollen analysis.

T cell–antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling. TCR signaling initiates a signaling cascade involving the kinases Lck and Zap70 and the adaptor LAT. Weiss and colleagues discover a proline-rich motif in LAT, which facilitates interactions among Lck, LAT and Zap70 for efficient TCR signaling.

Proline, a stress marker, is routinely quantified by a protocol that essentially uses hazardous toluene. Negative impacts of toluene on human health prompted us to develop a reliable alternate protocol for proline quantification. Absorbance of the proline-ninhydrin condensation product formed by reaction of proline with ninhydrin at 100 °C in the reaction mixture was significantly higher than that recorded after its transfer to toluene, revealing that toluene lowers sensitivity of this assay. λ max of the proline-ninhydrin complex in the reaction mixture and toluene were 508 and 513 nm, respectively. Ninhydrin in glacial acetic acid yielded higher quantity of the proline-ninhydrin condensation product compared to ninhydrin in mixture of glacial acetic acid and H 3 PO 4 , indicating negative impact of H 3 PO 4 on proline quantification. Further, maximum yield of the proline-ninhydrin complex with ninhydrin in glacial acetic acid and ninhydrin in mixture of glacial acetic acid and H 3 PO 4 was achieved within 30 and 60 min, respectively. This revealed that H 3 PO 4 has negative impact on the reaction rate and quantity of the proline-ninhydrin complex formed. In brief, our proline quantification protocol involves reaction of a 1-ml proline sample with 2 ml of 1.25 % ninhydrin in glacial acetic acid at 100 °C for 30 min, followed by recording absorbance of the proline-ninhydrin condensation product in the reaction mixture itself at 508 nm. Amongst proline quantification protocols known till date, our protocol is the most simple, rapid, reliable, cost-effective, and eco-friendlier.

WRKY transcription factors (TFs) have been mainly associated with plant defense, but recent studies have suggested additional roles in the regulation of other physiological processes. Here, we explored the possible contribution of two related group III WRKY TFs, WRKY70 and WRKY54, to osmotic stress tolerance. These TFs are positive regulators of plant defense, and co-operate as negative regulators of salicylic acid (SA) biosynthesis and senescence. We employed single and double mutants of wrky54 and wrky70, as well as a WRKY70 overexpressor line, to explore the role of these TFs in osmotic stress (polyethylene glycol) responses. Their effect on gene expression was characterized by microarrays and verified by quantitative PCR. Stomatal phenotypes were assessed by water retention and stomatal conductance measurements. The wrky54wrky70 double mutants exhibited clearly enhanced tolerance to osmotic stress. However, gene expression analysis showed reduced induction of osmotic stress-responsive genes in addition to reduced accumulation of the osmoprotectant proline. By contrast, the enhanced tolerance was correlated with improved water retention and enhanced stomatal closure. These findings demonstrate that WRKY70 and WRKY54 co-operate as negative regulators of stomatal closure and, consequently, osmotic stress tolerance in Arabidopsis, suggesting that they have an important role, not only in plant defense, but also in abiotic stress signaling.

Drought-induced water stress affects the productivity of the Vanilla planifolia Jacks. ex Andrews crop. In vitro culture technique is an effective tool for the study of water stress tolerance mechanisms. This study aimed to evaluate the morphological, physiological and biochemical response of V. planifolia under in vitro water stress conditions induced with polyethylene glycol (PEG). In vitro regenerated shoots of 2 cm in length were subjected to different concentrations of PEG 6000 (0, 1, 2 and 3% w/v) using Murashige and Skoog semi-solid culture medium. At 60 days of culture, different growth variables, dry matter (DM) content, chlorophyll (Chl), soluble proteins (SP), proline (Pro), glycine betaine (GB), stomatal index (SI) and open stomata (%) were evaluated. Results showed a reduction in growth, Chl content, SP, SI and open stomata (%) with increasing PEG concentration, whereas DM, Pro and GB contents rose with increasing PEG concentration. In conclusion, PEG-induced osmotic stress allowed describing physiological and biochemical mechanisms of response to water stress. Furthermore, the determination of compatible Pro and GB osmolytes can be used as biochemical markers in future breeding programs for the early selection of water stress tolerant genotypes.

PurposeTo explain the effects of the osmolyte proline on the protein-protein interactions (PPI), viscosity and stability of highly concentrated antibody solutions in contrast to other neutral osmolytes.MethodsThe viscosity of ~225 mg/mL mAb solutions was measured with proline, glycine and trehalose as a function of pH and co-solute concentration up to 1.3 M. The stability was assessed via turbidity as well as size exclusion chromatography after 4 weeks storage at 40°C. The PPI strength was assessed qualitatively via the high concentration diffusion rate by dynamic light scattering.ResultsIncreasing proline significantly reduced the mAb viscosity and increased the colloidal stability at pH 6, but not at pH 5 further from the mAb pI. In contrast, glycine and trehalose did not improve the viscosity nor stability. The normalized diffusion coefficient at high concentration, which is inversely proportional to the attractive PPI strength, increased with proline concentration but decreased with increasing glycine.ConclusionsProline demonstrated greater efficacy for improving mAb viscosity and stability in contrast to glycine and trehalose due to its amphipathic structure and partial charge on the pyrrolidine side chain. These properties likely allow proline to screen the attractive electrostatic and hydrophobic interactions that promote self-association and high viscosities. Binary proline-histidine formulations also demonstrated greater viscosity reduction effects than histidine alone at the same total co-solute concentration, while maintaining a lower total solution osmolarity.

Background Proline-rich extension-like receptor protein kinases (PERKs) are an important class of receptor kinases located in the plasma membrane, most of which play a vital role in pollen development. Results Our study identified 25 putative PERK genes from the whole Brassica rapa genome (AA). Phylogenetic analysis of PERK protein sequences from 16 Brassicaceae species divided them into four subfamilies. The biophysical properties of the BrPERK s were investigated. Gene duplication and synteny analyses and the calculation of Ka / Ks values suggested that all 80 orthologous/paralogous gene pairs between B. rapa and A. thaliana , B. nigra and B. oleracea have experienced strong purifying selection. RNA-Seq data and qRT-PCR analyses showed that several BrPERK genes were expressed in different tissues, while some BrPERK s exhibited high expression levels only in buds. Furthermore, comparative transcriptome analyses from six male-sterile lines of B. rapa indicated that 7 BrPERK genes were downregulated in all six male-sterile lines. Meanwhile, the interaction networks of the BrPERK genes were constructed and 13 PERK coexpressed genes were identified, most of which were downregulated in the male sterile buds. Conclusion Combined with interaction networks, coexpression and qRT-PCR analyses, these results demonstrated that two BrPERK genes, Bra001723.1 and Bra037558.1 (the orthologs of AtPERK6 (AT3G18810)), were downregulated beginning in the meiosis II period of male sterile lines and involved in anther development. Overall, this comprehensive analysis of some BrPERK genes elucidated their roles in male sterility.