Arabidopsis thaliana proliferating cell nuclear antigen has several potential sumoylation sites.

Proliferating cell nuclear antigen (PCNA) is post-translationally modified in yeast and animal cells. Major studies carried out in the last decade have focused on the role of sumoylated and ubiquitinated PCNA. Using different approaches, an interaction between plant PCNA and SUMO both in vivo and in bacteria has been demonstrated for the first time. In addition, identical sumoylation patterns for both AtPCNA1 and 2 were observed in bacteria. The plant PCNA sumoylation pattern has been shown to differ significantly from that of Saccharomyces cerevisiae. This result contrasts with a common opinion based on previous structural analysis of yeast, human, and plant PCNAs, which treats PCNA as a highly conserved protein even between species. Analyses of AtPCNA post-translational modifications using different SUMO proteins (SUMO1, 2, 3, and 5) revealed similar modification patterns for each tested SUMO protein. Potential target lysine residues that might be sumoylated in vivo were identified on the basis of in bacteria AtPCNA mutational analyses. Taken together, these results clearly show that plant PCNA is post-translationally modified in bacteria and may be sumoylated in a plant cell at various sites. These data open up important new perspectives for further detailed studies on the role of PCNA sumoylation in plant cells.

ATP-dependent but proton gradient-independent polyphosphate-synthesizing activity in extraradical hyphae of an arbuscular mycorrhizal fungus.

Arbuscular mycorrhizal (AM) fungi benefit their host plants by supplying phosphate obtained from the soil. Polyphosphate is thought to act as the key intermediate in this process, but little is currently understood about how polyphosphate is synthesized or translocated within arbuscular mycorrhizas. Glomus sp. strain HR1 was grown with marigold in a mesh bag compartment system, and extraradical hyphae were harvested and fractionated by density gradient centrifugation. Using this approach, three distinct layers were obtained: layers 1 and 2 were composed of amorphous and membranous materials, together with mitochondria, lipid bodies, and electron-opaque bodies, and layer 3 was composed mainly of partially broken hyphae and fragmented cell walls. The polyphosphate kinase/luciferase system, a highly sensitive polyphosphate detection method, enabled the detection of polyphosphate-synthesizing activity in layer 2 in the presence of ATP. This activity was inhibited by vanadate but not by bafilomycin A(1) or a protonophore, suggesting that ATP may not energize the reaction through H(+)-ATPase but may act as a direct substrate in the reaction. This report represents the first demonstration that AM fungi possess polyphosphate-synthesizing activity that is localized in the organelle fraction and not in the cytosol or at the plasma membrane.

Myrosinases, TGG1 and TGG2, redundantly function in ABA and MeJA signaling in Arabidopsis guard cells.

Thioglucoside glucohydrolase (myrosinase), TGG1, is a strikingly abundant protein in Arabidopsis guard cells. We investigated responses of tgg1-3, tgg2-1 and tgg1-3 tgg2-1 mutants to abscisic acid (ABA) and methyl jasmonate (MeJA) to clarify whether two myrosinases, TGG1 and TGG2, function during stomatal closure. ABA, MeJA and H(2)O(2) induced stomatal closure in wild type, tgg1-3 and tgg2-1, but failed to induce stomatal closure in tgg1-3 tgg2-1. All mutants and wild type showed Ca(2+)-induced stomatal closure and ABA-induced reactive oxygen species (ROS)production. A model is discussed in which two myrosinases redundantly function downstream of ROS production and upstream of cytosolic Ca(2+) elevation in ABA and MeJA signaling in guard cells.

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells.

Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.