1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana.

A gene-trapping vector carrying a GUS/Luciferase dual reporter gene was developed to establish an efficient and convenient screening system for T-DNA-based gene trapping in plants. A key feature of this gene trap scheme is to place two different types of reporters, luciferase (Luc) and beta-glucuronidase (GUS), as a fusion protein within a trapped gene to probe the activity of the gene. Luc is then utilized as a non-invasive, vital and highly sensitive screening reporter to identify trapped lines, including direct screening of the trapped lines from the primary T-DNA mutant pools. GUS is utilized as a histochemical assay reporter to analyze detailed cellular expression patterns. Transgenic expression studies in Arabidopsis showed that this fusion reporter protein retains functional enzyme activity for both GUS and Luc. Using this system in Arabidopsis, we were able to identify 3,737 trapped lines from 26,900 individual T-DNA insertion lines. Sequence determination of the T-DNA insertion loci in the genome of 78 trapped lines identified GUS/Luc fusions with 27 annotated Arabidopsis genes which included a subset of transcription factors, protein kinases, regulatory proteins and metabolic enzymes. Of these, particular expression patterns of four tagged genes were further confirmed by analyzing putative promoter regions of the corresponding wild-type genes. Furthermore, the protein stability of the GUS/Luc fusion reporter was controlled by application of luciferase substrate (luciferin), overcoming the excessive stability problem of GUS that causes misrepresentation of the transcriptional activity of a promoter. These results demonstrate the utility of the GUS/Luc dual reporter system as a gene trap reporter for studying plant genome function and also as a convenient dual reporter system for study of gene expression.

Genome-wide screening of Saccharomyces cerevisiae revealed that signaling pathways related to the alkaline pH stress contribute to resistance to plant antimicrobial peptide, Pn-AMP1. Plant antimicrobial peptides (AMPs) are considered to be promising candidates for controlling phytopathogens. Pn-AMP1 is a hevein-type plant AMP that shows potent and broad-spectrum antifungal activity. Genome-wide chemogenomic screening was performed using heterozygous and homozygous diploid deletion pools of Saccharomyces cerevisiae as a chemogenetic model system to identify genes whose deletion conferred enhanced sensitivity to Pn-AMP1. This assay identified 44 deletion strains with fitness defects in the presence of Pn-AMP1. Strong fitness defects were observed in strains with deletions of genes encoding components of several pathways and complex known to participate in the adaptive response to alkaline pH stress, including the cell wall integrity (CWI), calcineurin/Crz1, Rim101, SNF1 pathways and endosomal sorting complex required for transport (ESCRT complex). Gene ontology (GO) enrichment analysis of these genes revealed that the most highly overrepresented GO term was "cellular response to alkaline pH". We found that 32 of the 44 deletion strains tested (72 %) showed significant growth defects compared with their wild type at alkaline pH. Furthermore, 9 deletion strains (20 %) exhibited enhanced sensitivity to Pn-AMP1 at ambient pH compared to acidic pH. Although several hundred plant AMPs have been reported, their modes of action remain largely uncharacterized. This study demonstrates that the signaling pathways that coordinate the adaptive response to alkaline pH also confer resistance to a hevein-type plant AMP in S. cerevisiae. Our findings have broad implications for the design of novel and potent antifungal agents.

Composting earthworms of the genus Eisenia play an important role in soil ecosystems. However, taxonomic classification of this genus, especially the sibling species Eisenia fetida and Eisenia andrei, is complicated because of their morphological similarity. In this study, we assessed the utility of the complete mitochondrial genome (mitogenome) for identification and differentiation of the two species. The complete mitogenomes of E. andrei and E. fetida were 15,714 and 16,560 bp, respectively. They contained 37 genes, comprising 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and a putative non-coding region, as observed in other earthworms. Sequence comparisons based on the complete nucleotide sequences excluding the non-coding region showed 85.8% similarity, whereas the predicted amino acid sequences of the 13 PCGs were 92.7% similar between the two species. In particular, distinct features were found in the non-coding regions of the mitogenomes. They include a control region associated with putative mitogenome replication and an extended sequence. The extended sequence showed significant differences between the two species and other known earthworm species, suggesting its potential as a feasible molecular marker for species identification. Phylogenetic analysis of the 36 mitogenomes of earthworm species corroborated the monophyly of the genus Eisenia and the taxonomic distinctness of the sibling species pair, E. fetida and E. andrei.

No abstract