Movement of the plant hormone abscisic acid (ABA) within plants has been documented; however, the molecular mechanisms that regulate ABA transport are not fully understood. By using a modified yeast two-hybrid system, we screened Arabidopsis cDNAs capable of inducing interactions between the ABA receptor PYR/PYL/ RCAR and PP2C protein phosphatase under low ABA concentrations. By using this approach, we identified four members of the NRT1/PTR family as candidates for ABA importers. Transport assays in yeast and insect cells demonstrated that at least one of the candidates ABA-IMPORTING TRANSPORTER (AIT) 1, which had been characterized as the low-affinity nitrate transporter NRT1.2, mediates cellular ABA uptake. Compared with WT, the ait1/nrt1.2 mutants were less sensitive to exogenously applied ABA during seed germination and/or postgermination growth, whereas overexpression of AIT1/NRT1.2 resulted in ABA hypersensitivity in the same conditions. Interestingly, the inflorescence stems of ait1/ nrt1.2 had a lower surface temperature than those of the WT because of excess water loss from open stomata. We detected promoter activities of AIT1/NRT1.2 around vascular tissues in inflorescence stems, leaves, and roots. These data suggest that the function of AIT1/NRT1.2 as an ABA importer at the site of ABA biosynthesis is important for the regulation of stomatal aperture in inflorescence stems.guard cells | liquid chromatography-tandem mass spectrometry | water stress

SummaryWe have developed a novel gain-of-function system that we have named the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. About 10 000 independent full-length Arabidopsis cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Each transgenic Arabidopsis contained on average 2.6 cDNA clones and was monitored under various categories such as morphological changes, fertility and leaf color. We found 1487 possible morphological mutants from 15 547 transformants. When 115 pale green T 1 mutants were analyzed, 59 lines represented the mutant phenotypes in more than 50% of the T 2 progeny. Characterization of two leaf color mutants revealed the significance of this approach. We also document mutants from several categories and their corresponding full-length cDNAs.

SUMMARYPlants are able to sense and respond to changes in the balance between carbon (C) and nitrogen (N) metabolite availability, known as the C/N response. During the transition to photoautotrophic growth following germination, growth of seedlings is arrested if a high external C/N ratio is detected. To clarify the mechanisms for C/N sensing and signaling during this transition period, we screened a large collection of FOX transgenic plants, overexpressing full-length cDNAs, for individuals able to continue post-germinative growth under severe C/N stress. One line, cni1-D (carbon/nitrogen insensitive 1-dominant), was shown to have a suppressed sensitivity to C/N conditions at both the physiological and molecular level. The CNI1 cDNA encoded a predicted RING-type ubiquitin ligase previously annotated as ATL31. Overexpression of ATL31 was confirmed to be responsible for the cni1-D phenotype, and a knock-out of this gene resulted in hypersensitivity to C/N conditions during post-germinative growth. The ATL31 protein was confirmed to contain ubiquitin ligase activity using an in vitro assay system. Moreover, removal of this ubiquitin ligase activity from the overexpressed protein resulted in the loss of the mutant phenotype. Taken together, these data demonstrated that CNI1/ATL31 activity is required for the plant C/N response during seedling growth transition.

SummaryTo study the GH3 gene family of Arabidopsis, we investigated a¯anking sequence database of Arabidopsis activation-tagged lines. We found a dwarf mutant, named yadokari 1-D (ydk1-D), that had a T-DNA insertion proximal to a GH3 gene. ydk1-D is dominant and has a short hypocotyl not only in light but also in darkness. Moreover, ydk1-D has a short primary root, a reduced lateral root number, and reduced apical dominance. A GH3 gene, named YDK1, was upregulated in ydk1-D, and YDK1 transgenic plants showed the ydk1-D phenotype. YDK1 gene expression was induced by exogenously applied auxin and regulated by auxinresponse factor (ARF)7. In addition, YDK1 gene expression was downregulated by blue and far-red (FR) lights. Strong promoter activity of YDK1 was observed in roots and¯owers. These results suggest that YDK1 may function as a negative component in auxin signaling by regulating auxin activity.

SummaryIn a screen for morphological mutants from the T 1 generation of approximately 50 000 activation-tagging lines, we isolated four dominant mutants that showed hyponastic leaves, downward-pointing¯owers and decreased apical dominance. We designated them isoginchaku (iso). The iso-1D and iso-2D are allelic mutants caused by activation of the AS2 gene. The T-DNAs were inserted in the 3 H downstream region of AS2. Iso-3D and iso-4D are the other allelic mutants caused by activation of the ASL1/LBD36 gene. These two genes belong to the AS2 family that is composed of 42 genes in Arabidopsis. The only recessive mutation isolated from this gene family was of AS2, which resulted in a leaf morphology mutant. Applying reverse genetics using a database of activation-tagged T-DNA¯anking sequences, we found a dominant mutant that we designated peacock1-D (pck1-D) in which the ASL5/LBD12 gene was activated by a T-DNA. The pck1-D mutants have lost apical dominance, have epinastic leaves and are sterile. These results strongly suggest that activation tagging is a powerful mutant-mining tool especially for genes that make up a gene family.

The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.

The enteric protist parasites Entamoeba histolytica and Entamoeba dispar possess a cysteine biosynthetic pathway, unlike their mammalian host, and are capable of de novo production of L-cysteine. We cloned and characterized cDNAs that encode the regulated enzyme serine acetyltransferase (SAT) in this pathway from these amoebae by genetic complementation of a cysteine-auxotrophic Escherichia coli strain with the amoebic cDNA libraries. The deduced amino acid sequences of the amoebic SATs exhibited, within the most conserved region, 36 -52% identities with the bacterial and plant SATs. The amoebic SATs contain a unique insertion of eight amino acids, also found in the corresponding region of a plasmid-encoded SAT from Synechococcus sp., which showed the highest overall identities to the amoebic SATs. Phylogenetic reconstruction also revealed a close kinship of the amoebic SATs with cyanobacterial SATs. Biochemical characterization of the recombinant E. histolytica SAT revealed several enzymatic features that distinguished the amoebic enzyme from the bacterial and plant enzymes: 1) inhibition by L-cysteine in a competitive manner with L-serine; 2) inhibition by Lcystine; and 3) no association with cysteine synthase. Genetically engineered amoeba strains that overproduced cysteine synthase and SAT were created. The cysteine synthase-overproducing amoebae had a higher level of cysteine synthase activity and total thiol content and revealed increased resistance to hydrogen peroxide. These results indicate that the cysteine biosynthetic pathway plays an important role in antioxidative defense of these enteric parasites.The cysteine biosynthetic pathway plays an important role in incorporation of inorganic sulfur into organic compounds. In bacteria and plants, L-cysteine is the precursor of most sulfurcontaining metabolites including methionine and glutathione. Extracellular sulfate is first imported by specific transporters. Intracellular sulfate is then activated by ATP sulfurylase and adenosine-5Ј-phosphosulfate kinase to form adenosine-5Ј-phosphosulfate and 3Ј-phosphoadenosine 5Ј-phosphosulfate, respectively. These activated sulfates are further reduced to sulfide. Sulfide then reacts with O-acetylserine, which is produced from serine and acetyl-CoA by serine acetyltransferase (SAT, 1 EC 2.3.1.30). This final reaction forming L-cysteine, by transfer of the alanyl moiety of O-acetylserine to sulfide, is catalyzed by L-cysteine synthase (CS; O-acetyl-L-serine (thiol)-lyase, EC 4.2.99.8). In contrast to bacteria and plants, animals are presumed to lack the sulfur assimilation pathway and thus require exogenous methionine as a sulfur source. Biochemical studies using purified (1-4) and recombinant enzymes (5), as well as a genetic approach using a yeast two-hybrid system, revealed that CS and SAT form a heteromeric complex. SAT activity and O-acetylserine availability are the major regulatory factors in the control of the L-cysteine production in plants (6, 7). Cytosolic isoforms of the SAT from Citrullus vulgaris and A...

A novel dominant mutant designated`dwarf in light 1' (d¯1-D) was isolated from screening around 1200 Arabidopsis activation-tagged lines. d¯1-D has a shorter hypocotyl under blue, red and far-red light, but not in darkness. Inhibition of cell elongation in shoots caused an exaggerated dwarf phenotype in the adult plant. The lateral root growth of d¯1-D was inhibited without any reduction of primary root length. The genomic DNA adjacent to the right border of T-DNA was cloned by plasmid rescue. The rescued genomic DNA contained a gene encoding a GH3 homologue. The transcript was highly accumulated in the d¯1-D. The d¯1-D phenotype was con®rmed by over-expression of the gene in the wild-type plant. The d¯1-D showed resistance to exogenous auxin treatment. Moreover, over-expression of antisense DFL1 resulted in larger shoots and an increase in the number of lateral roots. These results indicate that the gene product of DFL1 is involved in auxin signal transduction, and inhibits shoot and hypocotyl cell elongation and lateral root cell differentiation in light.