Exogenous application of xanthine and uric acid and nucleobase-ascorbate transporter MdNAT7 expression regulate salinity tolerance in apple.

BACKGROUND: Soil salinity is a critical threat to global agriculture. In plants, the accumulation of xanthine activates xanthine dehydrogenase (XDH), which catalyses the oxidation/conversion of xanthine to uric acid to remove excess reactive oxygen species (ROS). The nucleobase-ascorbate transporter (NAT) family is also known as the nucleobase-cation symporter (NCS) or AzgA-like family. NAT is known to transport xanthine and uric acid in plants. The expression of MdNAT is influenced by salinity stress in apple. RESULTS: In this study, we discovered that exogenous application of xanthine and uric acid enhanced the resistance of apple plants to salinity stress. In addition, MdNAT7 overexpression transgenic apple plants showed enhanced xanthine and uric acid concentrations and improved tolerance to salinity stress compared with nontransgenic plants, while opposite phenotypes were observed for MdNAT7 RNAi plants. These differences were probably due to the enhancement or impairment of ROS scavenging and ion homeostasis abilities. CONCLUSION: Our results demonstrate that xanthine and uric acid have potential uses in salt stress alleviation, and MdNAT7 can be utilized as a candidate gene to engineer resistance to salt stress in plants.

Biochemical characterization and structure-function relationship of two plant NCS2 proteins, the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana.

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent KM values were determined with 10.12µM, 4.85µM and 19.95µM, respectively for NAT3 and 1.74µM, 2.44µM and 29.83µM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton-nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1-7 and TM8-14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure-function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.

Endocytosis of nutrient transporters in fungi: The ART of connecting signaling and trafficking.

Plasma membrane transporters play pivotal roles in the import of nutrients, including sugars, amino acids, nucleobases, carboxylic acids, and metal ions, that surround fungal cells. The selective removal of these transporters by endocytosis is one of the most important regulatory mechanisms that ensures a rapid adaptation of cells to the changing environment (e.g., nutrient fluctuations or different stresses). At the heart of this mechanism lies a network of proteins that includes the arrestin-related trafficking adaptors (ARTs) which link the ubiquitin ligase Rsp5 to nutrient transporters and endocytic factors. Transporter conformational changes, as well as dynamic interactions between its cytosolic termini/loops and with lipids of the plasma membrane, are also critical during the endocytic process. Here, we review the current knowledge and recent findings on the molecular mechanisms involved in nutrient transporter endocytosis, both in the budding yeast Saccharomyces cerevisiae and in some species of the filamentous fungus Aspergillus. We elaborate on the physiological importance of tightly regulated endocytosis for cellular fitness under dynamic conditions found in nature and highlight how further understanding and engineering of this process is essential to maximize titer, rate and yield (TRY)-values of engineered cell factories in industrial biotechnological processes.

Conformational transitions of uracil transporter UraA from Escherichia coli: a molecular simulation study.

The Escherichia coli uracil/H + symporter UraA, known as the representative nucleobase/cation symporter 2(NCS2) protein, gets involved in several crucial physiological processes for most living organisms on Earth, such as the uptake of nucleobases and transport of vitamin C. Some experiments proposed a working model to explain proton-coupling and uracil transporting process of UraA on the basis of the crystal structure of NCS2 protein, but the details of conformational changes remained unknown. Thus, in order to make clear conformational changes caused by the protonation and deprotonation process of some conserved proton-coupled residues, the molecular dynamics simulation was used to study the conformation of UraA complexes in different protonation states. The results demonstrated that the protonation of residue Glu241 and Glu290 resulted in the whole conformational transition from the inward-open to the outward-open state. It can be concluded that Glu290 was crucial in a network of hydrogen-bonds in the middle of the core domain involving another essential residue, mainly including tyr288 in TM8, Tyr342, Ser338 in TM12, and the network of hydrogen-bonds was the key to maintain the stability of conformation. Protonation of Glu290 affects the stability of network of H-bond and changed the domains TM3 TM10 TM12. Thus, Glu290 may play a vital role as a 'proton trigger' that affects spatial structural of amino and residues near substrate binding side leading to an outward-open conformation transition.