Reactive oxygen species (ROS) mediate abscisic acid (ABA) signaling in guard cells. To dissect guard cell ABA-ROS signaling genetically, a cell type-specific functional genomics approach was used to identify 2 MAPK genes, MPK9 and MPK12, which are preferentially and highly expressed in guard cells. To provide genetic evidence for their function, Arabidopsis single and double TILLING mutants that carry deleterious point mutations in these genes were isolated. RNAi-based gene-silencing plant lines, in which both genes are silenced simultaneously, were generated also. Mutants carrying a mutation in only 1 of these genes did not show any altered phenotype, indicating functional redundancy in these genes. ABA-induced stomatal closure was strongly impaired in 2 independent RNAi lines in which both MPK9 and MPK12 transcripts were significantly silenced. Consistent with this result, mpk9-1/ 12-1 double mutants showed an enhanced transpirational water loss and ABA-and H2O2-insensitive stomatal response. Furthermore, ABA and calcium failed to activate anion channels in guard cells of mpk9-1/12-1, indicating that these 2 MPKs act upstream of anion channels in guard cell ABA signaling. An MPK12-YFP fusion construct rescued the ABA-insensitive stomatal response phenotype of mpk9-1/12-1, demonstrating that the phenotype was caused by the mutations. The MPK12 protein is localized in the cytosol and the nucleus, and ABA and H2O2 treatments enhance the protein kinase activity of MPK12. Together, these results provide genetic evidence that MPK9 and MPK12 function downstream of ROS to regulate guard cell ABA signaling positively.abscisic acid ͉ anion channels ͉ protein kinase ͉ reactive oxygen species ͉ stomata

Cellular membranes act as signaling platforms and control solute transport. Membrane receptors, transporters, and enzymes communicate with intracellular processes through protein-protein interactions. Using a split-ubiquitin yeast two-hybrid screen that covers a test-space of 6.4 × 10(6) pairs, we identified 12,102 membrane/signaling protein interactions from Arabidopsis. Besides confirmation of expected interactions such as heterotrimeric G protein subunit interactions and aquaporin oligomerization, >99% of the interactions were previously unknown. Interactions were confirmed at a rate of 32% in orthogonal in planta split-green flourescent protein interaction assays, which was statistically indistinguishable from the confirmation rate for known interactions collected from literature (38%). Regulatory associations in membrane protein trafficking, turnover, and phosphorylation include regulation of potassium channel activity through abscisic acid signaling, transporter activity by a WNK kinase, and a brassinolide receptor kinase by trafficking-related proteins. These examples underscore the utility of the membrane/signaling protein interaction network for gene discovery and hypothesis generation in plants and other organisms.

Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs) out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway-compatible vector. The mating-based split ubiquitin system was used to screen for potential protein–protein interactions (pPPIs) among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases (RLKs), 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions, and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 386) pPPIs between 179 proteins, yielding a scale-free network (r2 = 0.863). Eighty of 142 transmembrane RLKs tested positive, identifying 3 homomers, 63 heteromers, and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs) had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G-protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

Clostridium acetobutylicum and Clostridium aminovalericum, both obligatory anaerobes, grow normally after growth conditions are changed from anoxic to microoxic, where the cells consume oxygen proficiently. In C. aminovalericum, a gene encoding a previously characterized H 2 O-forming NADH oxidase, designated noxA, was cloned and sequenced. The expression of noxA was strongly upregulated within 10 min after the growth conditions were altered to a microoxic state, indicating that C. aminovalericum NoxA is involved in oxygen metabolism. In C. acetobutylicum, genes suggested to be involved in oxygen metabolism and genes for reactive oxygen species (ROS) scavenging were chosen from the genome database. Although no clear orthologue of C. aminovalericum NoxA was found, Northern blot analysis identified many O 2 -responsive genes (e.g., a gene cluster [CAC2448 to CAC2452] encoding an NADH rubredoxin oxidoreductase-A-type flavoprotein-desulfoferrodoxin homologue-MerR family-like protein-flavodoxin, an operon [CAC1547 to CAC1549] encoding a thioredoxin-thioredoxin reductase-glutathione peroxidase-like protein, an operon [CAC1570 and CAC1571] encoding two glutathione peroxidase-like proteins, and genes encoding thiol peroxidase, bacterioferritin comigratory proteins, and superoxide dismutase) whose expression was quickly and synchronously upregulated within 10 min after flushing with 5% O 2. The corresponding enzyme activities, such as NAD(P)H-dependent peroxide (H 2 O 2 and alkyl hydroperoxides) reductase, were highly induced, indicating that microoxic growth of C. acetobutylicum is associated with the expression of a number of genes for oxygen metabolism and ROS scavenging.Bacteria belonging to the genus Clostridium are classified as obligatory anaerobes (26,62) and are widely used in the field of solvent fermentation, biodegradation, and microbial energy production. Oxygen has a crucial effect on the growth of clostridia, but the mechanisms of growth inhibition, as well as the existence of O 2 metabolic systems, remain unknown. Some hypotheses to explain aerobic growth inhibition in anaerobes were proposed, such as the possibility that oxygen attacks oxygen-sensitive enzymes causing metabolic cessation or that anaerobes lack the ability to decompose active oxygen species, such as catalase, which cause irreversible oxidative damage to DNA and lipid molecules (2,27,49,63,67). O'Brien and Morris proposed that NAD(P)H oxidation systems react with oxygen to cause oxidation of the electron donor, i.e., NAD(P)H, which is required for the central pathway for anaerobic metabolism; this then leads to the eventual inability of clostridia to maintain their internal redox balance (51, 55). However, many questions remain about the mechanisms of aerobic growth inhibition in clostridia (50).Most Clostridium species do not form colonies in the presence of 1% oxygen (2, 62); however, they can accept microoxic conditions when grown in liquid medium (32-35, 39, 51, 55). Based on physiological examination, clostridia possess systems to met...

We investigated the effects of O 2 on Bifidobacterium species using liquid shaking cultures under various O 2 concentrations. Although most of the Bifidobacterium species we selected showed O 2 sensitivity, two species, B. boum and B. thermophilum, demonstrated microaerophilic profiles. The growth of B. bifidum and B. longum was inhibited under high-O 2 conditions accompanied by the accumulation of H 2 O 2 in the medium, and growth was restored by adding catalase to the medium. B. boum and B. thermophilum grew well even under 20% O 2 conditions without H 2 O 2 accumulation, and growth was stimulated compared to anoxic growth. H 2 O-forming NADH oxidase activities were detected dominantly in cell extracts of B. boum and B. thermophilum under acidic reaction conditions (pH 5.0 to 6.0).Although anaerobes are defined as being unable to grow in the presence of O 2 , their degree of O 2 sensitivity exhibits wide variation (2,3,10,11,14,15,16,17,18,20,21,25,28). The genus Bifidobacterium is a well-investigated anaerobe known to be beneficial to human health. Its sensitivity to O 2 causes a loss of viability during manufacture and storage as well as after incorporation into the human body (36). The O 2 sensitivity differs among strains and species (13). de Vries and Stouthamer (7) classified Bifidobacterium species into three categories according to their sensitivities to O 2 . They proposed that some O 2 -sensitive species produce H 2 O 2 through NADH oxidase activity. Since then, there have been several approaches taken to investigate bifidobacterial oxidative growth inhibition (1,6,9,32,33,37); however, the mechanism of growth inhibition under oxic conditions remains unclear.In this study, microaerophilic Bifidobacterium species were found. The main objectives of the present study were to (i) determine growth characteristics with respect to O 2 using several Bifidobacterium species, (ii) determine the factor responsible for aerobic growth inhibition using O 2 -sensitive species, (iii) investigate the metabolic properties of O 2 -sensitive and microaerophilic Bifidobacterium species under oxic growth conditions, and (iv) investigate the properties of O 2 reduction systems that should differ between O 2 -sensitive and microaerophilic Bifidobacterium species.Effect of O 2 on the growth of Bifidobacterium species. Bifidobacterium species are classified as typical anaerobic bacteria; however, their differing degrees of O 2 sensitivity in liquid shaking culture are not well characterized. We selected several species by referring to reports concerning the physiological effects of O 2 . B. bifidum, B. longum, B. breve, and B. infantis were selected from among strains often used in milk products and intestinal probiotics. B. asteroides is reported to possess catalase (13, 30). B. indicum has characteristics similar to those of B. asteroides but shows catalase activity only when hemin is added to the medium (13, 30). B. boum, B. globosum, and B. thermophilum are reported to form colonies under atmospheric conditions of 90% air...